Tissue-specific enhancer repression through molecular integration of cell signaling inputs

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.

Oncoprotein Networks

The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes.

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Pias3-Dependent SUMOylation Directs Rod Photoreceptor Development

The Evolution of Gene Regulation Underlies a Morphological Difference between Two Drosophila Sister Species

The ability of B cells to express immunoglobulins with identical antigen specificity but different effector functions results from their capacity to undergo class switch recombination (CSR). This process consists of an intrachromosomal DNA recombination event that involves repetitive switch (S) regions located 5′ of each CH gene, except Cδ, and results in the juxtaposition of different downstream CH genes to the expressed VDJ gene (reviewed in Ref. 1). The S region involved in recombination is not targeted at random. Instead, CSR appears to be directed by T-helper-cell-derived cytokines in conjunction with the regulation of B-cell proliferation and differentiation. Thus, human B cells activated by CD40 cross-linking in the presence of IL-4 or IL-13 switch from IgM to IgE (2, 3). Molecular analysis has shown that cytokine-dependent induction of CSR to a particular CH gene almost invariably correlates with the transcriptional activation of the same gene in germline (i.e., unrearranged) configuration. All GL transcripts share the same overall structure, and originate from TATAA-less promoters a few kilobases upstream of the S region, proceed through short noncoding exons (IH exons), the S regions and CH exons, and are polyadenylated near the normal poly(A) site for mature IgH mRNAs. The IH exon is then spliced to the normal acceptor site in the first exon of the CH gene. During CSR, the region containing the GL promoter and the IH exon is deleted as part of a switch circle. GL transcripts are “sterile”; i.e., they do not appear to be translated into protein, because they contain stop codons in all reading frames (reviewed in Ref. 1). Targeted mutagenesis experiments have indicated that integrity of the GL transcription units (promoters and/or exons), rather than transcription through the S regions, is essential for CSR (4–8). More recently, splicing of primary GL transcripts has been shown to constitute an essential step in CSR activation (8–10). This finding implies that a spliced-out RNA species may be an integral part of the CSR enzymatic machinery, and/or some of the factors responsible for RNA splicing may also be involved in DNA recombination (see below). Supporting the hypothesis of a functional connection between RNA splicing and CSR are the identification of proteins involved in RNA processing as components of S region DNA-binding complexes (11, 12) and the precedent of yeast factors involved in both splicing and meiotic recombination (13). Involvement of the splicing machinery is also suggested from studies on the distribution of CSR junctions in CH12F3-2 cells that undergo efficient cytokine-dependent CSR in vitro (14). CSR junctions were found not only within but also outside the S region. However, they were rarely found within the I exon, suggesting that breakpoints of CSR are restricted to the intron region of GL transcription. If processing of tran-scripts and recombination of DNA are really coupled as they appear to be (see below), CSR would be a very unique system with unprecedented character (15). The full extent to which GL transcripts participate in CSR remains unclear, but their role is currently being reinterpreted. For a number of years, the invariable association between GL transcription and isotype switching was taken to support the accessibility model, according to which chromatin opening coupled with transcription allows the CSR recombinase to access the target DNA. However, this model could not account for isotype switching induced by stimuli (e.g., glucocorticoids) that do not affect GL transcription (16). In addition, it was convincingly argued that, since GL transcripts must be spliced together for CSR to occur, they cannot be simply involved in chromatin remodeling (17). Indeed, recent evidence suggests that the choice of antibody class in CSR may be regulated by GL transcription through the formation of RNA/DNA hybrids that select the sites of recombination. This view is supported by biochemical experiments demonstrating that transcripts of the S regions in immunoglobulin genes remain associated with the template DNA after transcription in vitro (18). Mouse and human S regions contain G-rich sequence repeats. The G-rich transcribed RNA appears to form a stable hybrid with the C-rich DNA template, leaving the other DNA strand (the nontemplate strand) on its own in a single-stranded state (19). Most importantly, after B cells from mouse spleen are induced to undergo CSR in vivo, RNA/DNA hybrids form within the S regions of the DNA. These RNA/DNA hybrids appear to be mechanistically important for CSR, because mice transgenic for ribonuclease H (an enzyme that specifically snips the RNA in the hybrids) showed a marked reduction in hybrid formation and, in parallel, lower IgG levels and inhibition of CSR in their splenic B cells (20). What structural features of the S region are required for formation of the stable hybrid during transcription remains to be established. Although mouse and human S regions are G-rich, and runs of guanosines are known to form unusual structures, this may not be a crucial feature because the S region upstream of the Ig Cμ gene in Xenopus is A-T rich (21). It is possible that the stretches of purine-pyrimidine asymmetry, the repeated sequences, and the numerous palindromes (sequences that read identically in both directions) in the S regions of mouse, man, and Xenopus are essential for CSR. How RNA/DNA hybrids regulate CSR is also unclear, but it is likely that they serve as the recognition target for a CSR mach-inery still uncharacterized. Splicing may be required to stabilize the hybrids by removing extra nucleotides incapable of participating in their formation (17). The counterintuitive conclusion warranted by these data is that the intronic region spliced out of the primary GL transcript, rather than the processed IH-CH transcript itself, may represent what is required to target recombinase activity to the appropriate S region. The current model for the control of CSR to IgE through GL transcription is presented in Fig. 1. In the absence of cytokine stimulation and CD40 engagement, a naive B cell expresses IgM and has the IgH locus in GL (unrearranged) configuration. Even under these conditions, Iμ-Cμ GL transcripts are constitutively expressed (step 1). After stimulation with IL-4 or IL-13 (and possibly with the cooperation of CD40 cross-linking), the ε GL promoter is activated (see below), and transcription is initiated (step 2). Primary ε GL transcripts contain the Iε exon, Sε, and Cε (step 3), and share the overall structure, albeit not the sequence, of primary μGL transcripts. Upon splicing of the primary GL transcripts (step 4), mature IH-CH transcripts are released, whereas the spliced intron corresponding to S region sequences remains associated with template DNA. The resulting stable RNA/DNA hybrid represents the target for a still unidentified endonuclease that might initiate CRS. Joining of the two S regions involved in CSR, followed by DNA repair mediated by a number of factors, including Ku proteins (22) and DNA mismatch repairing enzymes (23), results in the formation of a hybrid Sμ/Sε region, bringing Cε in close proximity to the rearranged VDJ complex. The excised DNA forms a circle and is rapidly degraded (step 5). Induction of class switch recombination to IgE through GL transcription. The model is extensively discussed in the text. The different steps in CSR are labeled in blue. RNA is indicated in red. 1) Constitutive expression of primary μ GL transcripts; 2) cytokine (CD40)-dependent activation of the ε GL promoter; 3) cytokine-dependent expression of primary ε GL transcripts; 4) splicing of primary GL transcripts, release of mature I-CH GL transcripts, and formation of stable hybrids between the S region and the intron of GL transcripts; the hybrids target the switch recombinase to the appropriate S region; 5) Sμ/Sε recombination and formation of circles containing the excised DNA. ss: splice site. The postulated mechanisms through which S region transcription and splicing may cooperate to target CSR reiterate the critical role of cytokine-dependent induction of GL transcription in determining the isotype specificity of switching. Thus, it is necessary to understand how individual cytokines activate specific GL promoters in order to understand how the isotype specificity of CSR is achieved. Full activation of the human ε GL promoter in response to IL-4 and CD40 engagement requires a constellation of nuclear factors that are either constitutively expressed or recruited to the promoter by specific activating signals. In the last few years, our group has performed a systematic characterization of the proximal region of the human ε germline promoter by electrophoretic mobility shift assays (EMSA) and reporter analysis. EMSA analysis provides information on the number and affinity of specific binding sites for nuclear proteins in a DNA fragment. The specificity of protein–DNA interactions is assessed with oligonucleotide competitors to compare the affinities of different DNA sequences for the protein(s) of interest. Another useful feature is the ability to identify the protein(s) involved in the formation of the complex of interest, using antibodies to specific transcription factors in an attempt to modify the mobility of the protein/DNA complex, or to prevent its formation. Once a protein binding to a given DNA motif has been identified by the pattern of oligonucleotide competition and antibody-mediated supershifting, the original probe may be tested for its ability to bind the transcription factors of interest expressed in recombinant form. Reporter assays, on the other hand, allow the characterization of promoter sequences essential to drive transcription of a reporter construct with the same stimuli known to activate the endogenous gene. The functional role played by individual transcription factors in regulating promoter activity can be assessed with constructs that contain mutations shown to abrogate binding of specific nuclear proteins. The human ε GL promoter is exquisitely IL-4/IL-13-inducible. Promoter response to Th2 cytokines appears to rely critically on a highly conserved sequence 5′ to the major initiation sites for ε GL transcripts. This region (position −109/−80 in Fig. 2) represents an IL-4RE that binds STAT6 and recombinant CAAT enhancer-binding (C/EBP) proteins, and is sufficient to confer IL-4 inducibility on a heterologous promoter in transient transfection studies (24, 25). The proximal human ε GL promoter. Position +1 corresponds to the major ε RNA start site in BL-2 cells (65). Binding sites for transcription factors identified in the promoter are boxed and labeled. The IL-4RE and its boundaries are indicated by dashed line and arrowheads. STAT6, whose essential role in IL-4-dependent ε GL transcription (26) and IgE switching (27–30) is well established, belongs to the recently identified family of signal transducers and activators of transcription (STAT) (31). Binding of IL-4 to its receptor (IL-4R) leads to activation by tyrosine phosphorylation of two receptor-associated cytoplasmic tyrosine kinases, Janus kinase (JAK)3 and JAK1 (reviewed in Ref. 32). These kinases are believed to induce rapidly phosphorylation of critical tyrosine residues in the intracellular domain of the IL-4Rα chain. These phosphorylated tyrosine residues provide docking sites for the latent cytosolic transcription factor STAT6. STAT6 binds to these docking sites via its SH2 domain, and is in turn tyrosine-phosphorylated by the receptor-associated JAKS. Phosphorylated STAT6 then homodimerizes, translocates to the nucleus, and binds to the promoter of a number of genes, contributing to the activation of transcription (33). STAT6 preferentially binds dyad symmetric half-sites separated by four base pairs (TTC-N4-GAA) (34). DNA binding specificity is localized to a region of 180 amino acids at the N terminal side of the SH3 domain (35). The discovery of JAKs and STATs provided an explanation for the apparent paradox that the IL-4R, as well as several other cytokine receptors, lacks kinase domains, and yet couples ligand binding to tyrosine phosphorylation. While the −109/−80 region functions as an IL-4RE, IL-4 inducibility was surprisingly lost in constructs containing a slightly shorter sequence (position −105/−83), even though this region contained intact binding sites for both STAT6 and C/EBP (36). This discrepancy raised the possibility that deletions introduced at the two ends of the IL-4RE in the −105/−83 construct destroyed unidentified binding sites for nucleoprotein(s) important in the regulation of ε GL promoter activity. Indeed, we have recently been able to show that the IL-4RE contains at its ends two motifs that strongly resemble the consensus binding site for the Myb family of transcription factors (PyAACG/TG) (37), and bind endogenously expressed Myb proteins. The myb1 site overlaps the 5′ half of the putative C/EBP site, whereas myb2 overlaps the 3′ half of the STAT6 element (Monticelli et al., submitted for publication) (Fig. 2). The presence of binding sites for Myb proteins at the two ends of the human IL-4RE in the ε promoter may provide a mechanistic explanation for the long-sought link between germline transcription, switch recombination, and B-cell proliferation (38). Indeed, full expression of the transactivation potential of two myb family members, A- and B-Myb, requires unmasking of their transactivation domain through phosphorylation of the carboxy-terminal regulatory domain (39, 40). Phosphorylation appears to be mediated by cyclin A/cdk2 and cyclin E/cdk2 kinases, which are specifically expressed at the G1/S transition (40–42). Thus, the carboxy-termini of A- and B-Myb act as cell-cycle sensors. Furthermore, RNA for A- and B-myb is expressed in a cell cycle-dependent manner during late G1 and early S-phase (41, 43). This dual control mechanism ensures that maximal A- and B-Myb activity peaks in the G1/S phase. Myb binding may also contribute to the early steps of ε GL gene activation. Indeed, recent data indicate that both Myb and STAT6 proteins interact with the coactivators p300 and CBP, thereby recruiting histone acetyltransferase activity to their cognate sites (44, 45). Thus, binding of STAT6 and Myb to adjacent sites in the IL-4RE may play a key role in chromatin remodeling, as well as in the interaction with the basal transcription machinery. STAT6-binding sites share homology with the DNA recognition motif for BCL-6, a transcriptional repressor expressed at high levels in germinal center B cells (46). Indeed BCL-6, a POZ/zinc-finger protein, has been shown to bind the STAT6 site in the CD23b promoter and block IL-4-dependent transcription of this gene in transient transfection assays (47). Furthermore, it has been proposed that BCL-6-mediated repression through the STAT6 site may contribute to silencing the IL-4-inducible ε GL promoter under basal conditions (48). This hypothesis would be consistent with the finding that mutations of the STAT6 recognition element in the ε GL promoter may result in markedly increased levels of basal transcription (49, 50), suggesting a disruption in the recruitment of a repressor to these sites. We showed that BCL-6 is expressed in the BL-2 cell line used in our experiments. However, this factor appeared to play no role in the regulation of transcription from the human proximal ε GL promoter. Indeed, EMSA analysis failed to show BCL-6 binding to the STAT6 site; furthermore, mutations of the STAT6 element in the ε GL reporter construct abrogated STAT6 binding but had no effect on the basal activity of the reporter gene in transient transfection assays. It is noteworthy that the recognition sites for BCL-6 and STAT6 are similar but not necessarily identical. Unlike the STAT6 elements in the proximal region of the ε promoter, an optimal BCL-6 site defined in vitro (46) and the one found in the CD23b promoter (51) contain three, not four, nucleotides between the two palidromic half-sites. It is possible that endogenous BCL-6 binds with high affinity only to elements that contain a 3-bp spacer, i.e., to sites recognized by most STAT proteins. In contrast, STAT6 is known to bind sites with 3-bp spacers, but, in addition, has a unique ability to interact with 4N sites (34, 35). This would imply that BCL-6 may interact with some but not all STAT6-binding sites, but at the same time could modulate events triggered by other STAT proteins as well. The region immediately downstream of the IL-4RE contains two sites that bind NF-κB/Rel proteins with comparable affinity (Fig. 2) and appear to be essential for promoter function (see below). The upstream site (NF-κB1) bound essentially p50/p65 heterodimers. c-Rel binding was also detectable in cells stimulated with IL-4 and/or anti-CD40 mAb. The downstream NF-κB site (NF-κB2) bound the same NF-κB/Rel family members. In addition, we detected p50 homodimers in extracts from cells treated with anti-CD40 mAb, with or without IL-4, and p50/RelB heterodimers in untreated and IL-4-stimulated cells. Perhaps the most interesting outcome of the NF-κB studies was the demonstration that NF-κB/Rel proteins directly associate with IL-4-inducible STAT6, and synergize with it in activating IL-4-dependent transcription (see below). Similar findings were reported for the murine ε and γ1 GL promoters, in which a STAT6 element is located upstream of two or three NF-κB binding sites (52–54).EMSA analysis with a probe (position −97/−57) that spans both the STAT6 and the NF-κB1 elements revealed three major specific complexes in cells treated with IL-4, with or without anti-CD40 mAb (Fig. 3). Complex 2 was IL-4-inducible, and contained STAT6, because it was specifically competed by an oligonucleotide (STAT6, −97/−72) containing the STAT6 binding site, but not by an NF-κB consensus sequence (κBH2), nor by an unrelated oligonucleotide (COUP). Furthermore, complex 2 was completely supershifted by an anti-STAT6 antiserum. Complex 3 contained NF-κB/Rel proteins, because it was specifically competed by κBH2, but not by STAT6. Complex 3 was supershifted by an anti-NF-κB p50 antiserum and inhibited by an antiserum to p65, but was unaffected by addition of anti-STAT6 antiserum. Most importantly, an additional band (complex 1) was detected. Complex 1 contained both STAT6 and NF-κB nucleoproteins, because it was specifically competed by STAT6 and κBH2 oligonucleotides, and supershifted or inhibited by antisera to STAT6 and NF-κB family members (p65, p50, and c-Rel). These data show that, consistent with findings in the murine model (55), STAT6 and NF-κB physically associate when binding to their adjacent cognate sites in the human ε GL promoter. Association between NF-κB/Rel and STAT6 proteins. Nuclear extracts (5 µg) from serum-starved BL-2 cells incubated in the presence or absence of IL-4 (100 U/ml) and/or anti-CD40 mAb (5 µg/ml) for 30 min were analyzed by EMSA, using as probe an oligonucleotide spanning both the STAT6 and NF-κB1 sites in the ε GL promoter (position −97/−57). Extracts from BL-2 cells stimulated with IL-4+anti-CD40 mAb were preincubated with rabbit antibodies (2 µg/reaction) specific for individual NF-κB/Rel proteins or unrelated factors for 30 min on ice before adding the probe. Competitors were added at 100-fold molar excess. Specific complexes containing STAT6 and/or NF-κB proteins are indicated by arrows. The B-cell-specific activator protein (BSAP, Pax-5) is a homeodomain-class transcription factor specifically expressed in the B-cell lineage from pro-B to mature B cells, but not in plasma cells (56). Binding sites for BSAP have been found in various promoters of B-cell-related genes (e.g., CD19, VpreB1, λ5, and blk), including the Ig gene locus (reviewed in Ref. 57). BSAP binds to a highly conserved region immediately upstream of the major Iε transcription initiation site in both murine (58) and human (59) B cells (Fig. 2). Notably, among the factors that regulate ε GL transcription, BSAP is unique in that it is specifically expressed in B cells. GL transcription through the immunoglobulin locus is a B-cell-specific event as well. Thus, it is tempting to speculate that BSAP may be involved in the tissue-specific activation of immunoglobulin GL promoters, several of which contain BSAP-binding sites. Having identified a set of transcription factors that bind the human ε GL promoter, we then assessed the functional role played by these nucleoproteins in regulating promoter ativity. For this purpose, luciferase reporter constructs containing the human ε GL promoter (either wild-type or mutated at specific binding sites) were transiently transfected into BL-2 cells. Reporter activity was measured in cells left untreated or stimulated with IL-4 and/or anti-CD40 mAb. As shown in Fig. 4, the wild-type promoter was activated 3.6-fold by IL-4, 2.3-fold by CD40 engagement, and 31.6-fold by a combination of the two signals. Thus, CD40 cross-linking enhanced IL-4-dependent transcription by 8.7-fold, pointing to a strong synergism between the two Reporter analysis of the proximal ε GL promoter. BL-2 cells were transiently with the reporter constructs and an control using and into that were incubated in the presence of IL-4 (100 anti-CD40 mAb (5 or a combination of the two stimuli activity was assessed and for transfection and protein in transfected cells were as induction represents the of luciferase activity of between treated and untreated cells. The results show the of at three each performed in introduced in the reporter constructs had but different on the activation of the promoter in response to IL-4 and/or CD40 of the STAT6 and NF-κB1 binding sites had on promoter i.e., they both the response to IL-4 and the of ε transcription and of the site IL-4 only and the synergism between IL-4 and CD40 by only that NF-κB proteins bound downstream may not be involved in the interaction with STAT6. of the BSAP-binding site strongly both the activation and the of ε GL promoter activity of both NF-κB sites, with or without the BSAP abrogated not only CD40 but also IL-4-dependent even though the STAT6 element was left intact not The reporter construct containing mutations of the STAT6 and NF-κB1 and/or sites was from the not results indicate that regulation of human ε GL transcription involves complex interactions between factors recruited through different NF-κB/Rel proteins cooperate with STAT6 for the activation of IL-4-dependent human ε GL transcription. NF-κB activity was critical not only for the of ε GL transcription, but also for IL-4 in the absence of antibody-mediated CD40 are likely to the association between NF-κB/Rel proteins and STAT6. Upon interaction with STAT6 appears to undergo critical and functional DNA-binding affinity is and, most importantly, ability is induced (35). Thus, interactions are necessary for the activation of transcription from promoters containing both cognate sites. Notably, activation of STAT6 nor NF-κB requires protein the of the signal to the More recently, it has been shown that the upstream NF-κB site overlaps with a of DNA binding of both and NF-κB in of IL-4 inducibility of IgE GL promoter constructs This result suggested that as well as NF-κB factors, may cooperate with STAT6 to IL-4 induction of the ε GL promoter. BSAP may contribute to IgE switching not only through activation of the ε GL promoter, but also through regulation of GL transcription. BSAP inhibited GL transcription and switching to in murine cells, but enhanced ε GL transcription and switching to IgE The of BSAP on ε and GL transcription the ability of this factor to activate or transcription, on the protein(s) with which it at a particular binding site and the affinity for its different binding During a B-cell BSAP its activator functions but is of its repressor This of BSAP was shown to be regulated by a mechanism activator motifs for BSAP had a binding affinity than repressor of activator and repressor showed that the of the rather than the BSAP as an activator or repressor The role of C/EBP proteins in regulating human ε GL transcription through the putative site in the IL-4RE remains sites for C/EBP and STAT6 proteins are adjacent or in the promoters of genes, this was to serve a functional Although both factors can bind DNA would stabilize STAT6 thereby activating IL-4-dependent transcription Indeed, the 5′ of the IL-4RE binds recombinant However, we found no evidence for interactions with endogenous members of this family in BL-2 cells. of IgE stimulation with IL-4 and a CD40 ligand was normal in mice Ig ε GL transcription in this model was not specifically these findings imply that may not be necessary for IgE switching in In factors contribute to the regulation of ε GL transcription binding the proximal region of the promoter. It be interesting to additional regulatory elements in the region 5′ to the analyzed in the as well as interactions the Furthermore, it be important to compare binding and functional of the ε and GL promoters, since they both to IL-4 and CD40 but show of activation in

Update on epigenetics in allergic disease

Physiological studies confirm improvement of memory by Olibanum, a resin from Boswellia species, while little is known about the molecular mechanism by which it affects memory performance. Two master transcription factors, CREB-1 and CREB-2, regulate downstream memory-related genes expression, leading to the long-term memory potentiation. This study addresses the effects of Beta-boswellic acid (β-BA), the main ingredient of Olibanum, and ethanolic extract of the resin from Boswellia serrata on the expression of CREB-1 and CREB-2 genes in B65 cell line. B65 cells were treated with β-BA or ethanolic extract of Olibanum in different doses and time intervals and the cell viability/toxicity was measured by MTT assay. Total RNA was extracted from the treated and untreated control cells and cDNA was synthesized. The expression levels of CREB-1 and CREB-2 genes were quantified by Real-time PCR. MTT assays revealed 50% inhibitory concentrations of 42.05, 29.63, and 21.78 μg/mL for ethanolic extract of Olibanum and 89.54, 44.05, and 21.12 µM for β-BA at 24, 48, and 72 h time intervals respectively. Both β-BA and ethanolic extract of Olibanum altered CREB-1 and CREB-2 gene expression levels in time-dependent but not in dose-dependent way. However, β-BA showed stronger and more stable effects. The expression levels of the both genes followed an alternate upregulation and downregulation pattern, but in opposite directions, in response to the both solutions with the progress of time. These results suggest that Olibanum possibly improves memory performance, at least partially, by regulating the levels of CREB-1 and CREB-2 transcription factors via positive/negative-feedback loops.

During development, neuronal identity is determined by a combination of numerous transcription factors. However, the mechanisms of synergistic action of these factors in transcriptional regulation and subsequent cell fate specification are largely unknown. In this study, we identified a novel gene, Corl1, encoding a nuclear protein with homology to the Ski oncoprotein. Corl1 was highly selectively expressed in the central nervous system (CNS). In the embryonic CNS, Corl1 was expressed in a certain subset of postmitotic neurons generated posterior to the midbrain-hindbrain border. In the developing spinal cord, Corl1 was selectively expressed in the dorsal horn interneurons where a homeodomain transcription factor, Lbx1, is required for proper specification. Corl1 was localized in a nuclear dot-like structure and interacted with general transcriptional corepressors. In addition, Corl1 showed transcriptional repression activity in the GAL4-fusion system, indicating its involvement in the regulation of transcriptional repression. Furthermore, Corl1 interacted with Lbx1 and cooperatively repressed transcription, suggesting that it acts as a transcriptional corepressor for Lbx1 in regulating cell fate determination in the dorsal spinal cord. Corl1 corepressor activity did not depend on Gro/TLE activity, and Gro/TLE also functioned as a corepressor for Lbx1. Thus, Lbx1 can select two independent partners, Corl1 and Gro/TLE, as corepressors. Identification of a novel transcriptional corepressor with neuronal subtype-restricted expression might provide insights into the mechanisms of cell fate determination in neurons.

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation. Editor's evaluation Authors provide valuable evidence identifying a lncRNA transcribed specifically in the pDC subtype from the +32Kb promoter region which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. With convincing methodology, they provide in-depth analysis about the possible role of lncIrf8, and its promoter region and cross-talk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region that is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. The work will be of interest to immunologists working on immune cell development. https://doi.org/10.7554/eLife.83342.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Lineage-determining transcription factors (TF) are master regulators of gene programs that frequently initiate self-reinforcing loops by autoactivation. TF autoactivation is important for cells to pass restriction points during development (referred to as points of no return) and to enforce cellular identity. Molecular circuitries of autoactivation have been studied for several TF, such as GATA-binding factor 1 (GATA1), PU.1 (encoded by Spi1), CCAAT enhancer-binding protein α and ε (C/EBPα and ε; Graf and Enver, 2009; Loughran et al., 2020; Nishimura et al., 2000; Okuno et al., 2005; Theilgaard-Mönch et al., 2022). A further example is interferon regulatory factor 8 (IRF8), which shows autoactivation in cooperation with basic leucine zipper ATF-like transcription factor 3 (BATF3; Anderson et al., 2021; Grajales-Reyes et al., 2015). An important principle in nature is negative feedback control to avoid signal overshooting and toxicity. Negative feedback control applies also to lineage-determining TF; however, there is a paucity on our knowledge of the molecular mechanisms involved. IRF8 is a hematopoietic TF positioned at the center of the regulatory gene network for dendritic cell (DC) development (Anderson et al., 2021; Belz and Nutt, 2012; Chauvistré and Seré, 2020; Kim et al., 2020; Lin et al., 2015; Nutt and Chopin, 2020; Tamura et al., 2015; Verlander et al., 2022). IRF8 is a member of the interferon regulatory factor (IRF) family of TF. Initially members of this TF family were found to mediate the induction of interferon induced genes, but are now known to serve diverse functions in regulating the immune system (Honda and Taniguchi, 2006; Tamura et al., 2008). Irf8 knockout mice show abnormal development of classical DC type 1 (cDC1) and plasmacytoid DC (pDC) (Durai et al., 2019; Schiavoni et al., 2002; Sichien et al., 2016; Tsujimura et al., 2003). Irf8 is prominently upregulated during DC development by autoactivation (Grajales-Reyes et al., 2015; Lin et al., 2015), yet how Irf8 autoactivation is controlled and eventually limited, and the epigenetic mechanisms involved is largely unknown. Irf8 expression in hematopoietic cells is induced and maintained by enhancers located at –50 kb,+32 kb,+41 kb and +56 kb relative to Irf8 transcription start site (TSS) (Anderson et al., 2021; Bagadia et al., 2019; Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013). Enhancers are cis-regulatory sequences with multiple TF binding sites that cooperatively bind TF and thereby activate transcription, as demonstrated by many studies including our work (Davidson et al., 1986; Long et al., 2016; Wildeman et al., 1986; Zenke et al., 1986). Enhancers regulate complex gene networks and can also produce non-coding RNA, referred to as enhancer RNA (eRNA). eRNA serve as an indicator for enhancer activity and some eRNA have an activity on their own and act in cis or trans to regulate cell fate decisions (Sartorelli and Lauberth, 2020; Statello et al., 2021). Enhancer-associated long non-coding RNA (lncRNA) represent a class of lncRNA transcribed from active enhancers. Thus, eRNA and enhancer-associated lncRNA provide opportunities to detect enhancer activity and to investigate enhancer function. DC are highly specialized immune cells that play a critical role in regulating innate and adaptive immune responses (Cabeza-Cabrerizo et al., 2021). DC develop from hematopoietic stem cells (HSC) via successive steps of lineage commitment and differentiation. More specifically, HSC develop into multipotent progenitors (MPP) that are committed to DC restricted common DC progenitors (CDP) and differentiate into classic DC (cDC) type 1 and type 2 (cDC1 and cDC2, respectively) and pDC (Anderson et al., 2021; Cabeza-Cabrerizo et al., 2021; Ginhoux et al., 2022; Nutt and Chopin, 2020; Rodrigues and Tussiwand, 2020). pDC were recently also shown to develop from lymphoid progenitors (Dress et al., 2019; Rodrigues et al., 2018; Rodrigues and Tussiwand, 2020). Differential expression of Irf8 regulates DC and monocyte specification in a dose-dependent manner (Cytlak et al., 2020; Murakami et al., 2021). Irf8 expression starts at the CDP stage, and is high in pDC and cDC1, which is attributed to the autoactivation of Irf8 during DC subsets specification (Grajales-Reyes et al., 2015; Lin et al., 2015). Interestingly, IRF8 can act as a transcriptional activator or repressor in hematopoiesis by interacting with different partner TF and binding to specific DNA sequences (Tamura et al., 2015). As an activator, IRF8 binds to its own promoter in DC differentiation, which is considered as the autoactivation capacity of Irf8 (Grajales-Reyes et al., 2015; Lin et al., 2015). For instance, IRF8 interacts with partner TF, such as PU.1, to initiate Irf8 autoactivation at the CDP stage (Grajales-Reyes et al., 2015). Inversely, IRF8 inhibits C/EBPα activity in neutrophil differentiation (Kurotaki et al., 2014). IRF8 also represses C/EBPβ to generate and maintain DC lineage-specific enhancer landscapes (Bornstein et al., 2014). In addition, IRF8 is important for the Myc-Mycl transition in DC differentiation (Anderson lll et al., 2021). IRF8 represses Myc expression in progenitors, while IRF8 at high levels interacts with PU.1 and drives Mycl expression (Anderson lll et al., 2021). All this emphasizes the central position of IRF8 in coordinating the gene network that regulates DC differentiation and function. During DC differentiation, the Irf8 gene locus shows high epigenetic dynamics, including histone modifications and TF binding identified by ChIP-seq (Chauvistré and Seré, 2020; Durai et al., 2019; Grajales-Reyes et al., 2015; Lin et al., 2015), chromatin accessibility measured by ATAC-seq (Kurotaki et al., 2019; Li et al., 2019), and three-dimensional chromatin structure remodeling determined by chromosome conformation capture (3 C) (Kurotaki et al., 2022; Schönheit et al., 2013). All this emphasizes the impact of epigenetic regulators on Irf8 gene activity in DC differentiation. Notably, Irf8 is flanked by multiple enhancers at –50 kb,+32 kb,+41 kb, and +56 kb that regulate Irf8 expression in hematopoietic cells (Anderson et al., 2021; Murakami et al., 2021). These four enhancers were found to be driven by PU.1, BATF3, E proteins and Runt-related transcription factor (RUNX)-core binding factor beta (CBFβ) (RUNX-CBFβ), respectively (Bagadia et al., 2019; Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013). Chromatin conformation, particularly enhancer promoter interactions, provides a platform for TF-driven gene regulation and serves as a driving force for cell-fate determinations (Misteli and Finn, 2021; Oudelaar and Higgs, 2021; Stadhouders et al., 2019). Schönheit et al. demonstrated Irf8 promoter interactions with its upstream enhancers by quantitative 3 C (Schönheit et al., 2013). In this study PU.1 was found to regulate chromatin remodeling between the –50 kb enhancer and the Irf8 promoter in myeloid differentiation. In a recent study Kurotaki et al., 2022 determined the higher-order chromatin structure in DC progenitors, cDC1 and cDC2 on a genome-wide scale by Hi-C. In this study, reorganization of chromatin conformation at DC-specific gene loci was observed during cDC differentiation, and IRF8 was found to promote chromatin activation in DC progenitors leading to cDC lineage-specific gene expression. However, high resolution maps of the physical chromatin interactions of the Irf8 promoter with upstream and downstream enhancers in the full complement of DC subsets, including pDC, are required for understanding Irf8 regulation during DC differentiation. Frequently, chromatin data, including ATAC-seq and/or ChIP-seq data, are used to identify regulatory elements of gene transcription. Here we embarked on a different approach and searched for lncRNA, which by themselves might have regulatory functions or are indicative of enhancer activity. We identified a novel lncRNA transcribed from the Irf8 +32 kb enhancer, which is specifically expressed in pDC, referred to as lncIrf8. We found that the lncIrf8 promoter element but not lncIrf8 itself impacts pDC and cDC1 development. Thus, lncIrf8 acts as an indicator for the Irf8 +32 kb enhancer activity. Importantly, our study revealed a previously unrecognized negative feedback loop of Irf8 in DC differentiation. Irf8 first activates its expression by autoactivation via the +32 kb enhancer and second limits its own expression through the lncIrf8 promoter element in the +32 kb enhancer. Results lncIrf8 marks a pDC-specific Irf8 enhancer element Irf8 expression in DC development is subject to complex epigenetic regulation. Here, we used an integrated approach with RNA-seq, ATAC-seq, ChIP-seq and Capture-C to track the dynamics of gene expression, histone modification and chromatin conformation in the sequel MPP, CDP, pDC, cDC1, and cDC2 (Figure 1, Figure 1—figure supplements 1 and 2). Figure 1 with 3 supplements see all Download asset Open asset Irf8 epigenetic signatures and promoter-enhancer interaction maps during DC differentiation. (A) Gene expression and epigenetic signatures of Irf8 downstream region in MPP, CDP, pDC, all cDC, cDC1, and cDC2 are visualized by IGV browser. Gene expression was measured by RNA-seq, chromatin accessibility by ATAC-seq, H3K27ac and IRF8 binding by ChIP-seq. Positions of Irf8 3’ end, Irf8 enhancers, pDC specific lncIrf8 and cDC1 specific Tcons_00190258 lncRNA are indicated. For RNA-seq - and +strands are shown. Scale bar: 5 kb. (B) Physical interactions of Irf8 promoter with flanking sequences in MPP, CDP, pDC, cDC1, and cDC2 by nuclear-titrated (NuTi) Capture-C (turquoise), and CTCF binding by ChIP-seq in DC (Garber et al., 2012). Mean numbers of unique interactions normalized to a 300 kb region around the Irf8 promoter viewpoint (green triangle) and scaled by a factor of 1,000,000 are shown (n=2). The orientations of CTCF binding are indicated with blue and red arrows. Tcons_00190258 refers to the cDC1-specific lncRNA shown in (A). Scale bar: 100 kb. (C) Comparations of the chromatin interactions with Irf8 promoter in pDC, cDC1 and cDC2. Differential tracks were created by subtraction of the mean normalized tracks of (B). Pairwise comparisons are shown and color coded. Turquoise and orange tracks represent specific interactions with the Irf8 promoter in the indicated cell types. Scale bar: 100 kb. Purple bars and lines indicate the position of flanking enhancers relative to Irf8 TSS. The purple bars from left to right represent –50 kb, –34 kb, –26 kb, –16 kb,+27 kb,+32 kb,+38 kb,+41 kb,+47 kb,+56 kb and +62 kb enhancer, respectively (panels B and C). Irf8 +32 kb enhancer is highlighted by purple box. We performed de novo transcript assembly of the RNA-seq data and detected two previously unknown transcripts without coding potential downstream of Irf8: a pDC specific lncRNA (Tcons_00190250) in the following referred to as lncIrf8 and a cDC1 specific lncRNA (Tcons_00190258; Figure 1A and Figure 1—figure supplement 1). lncIrf8 and Tcons_00190258 show the same expression pattern in pDC and cDC1, respectively, in BM and spleen (Figure 1—figure supplement 3), as revealed by reanalyzing scRNA-seq and bulk RNA-seq data (Pang et al., 2022; Rodrigues et al., 2018). lncIrf8 is transcribed within an enhancer region located 32 kb downstream of the Irf8 TSS labeled by H3K27ac and H3K4me1 and occupied by DC differentiation-associated TF, such as IRF8 and PU.1 (Figure 1A and Figure 1—figure supplement 1). This region is largely devoid of H3K9me3, a chromatin modification frequently associated with heterochromatin, indicating an open chromatin configureuration in DC (Figure 1—figure supplement 1). In addition, sequences of this region have been implicated in DC development and referred to as +32 kb enhancer (Durai et al., 2019). Thus, we proceeded to study lncIrf8 in detail. ATAC-seq analysis revealed further details of the lncIrf8 region in CDP, pDC, cDC1 and cDC2 (Figure 1A, Figure 2A and Figure 1—figure supplement 1). In cDC1 the prominent ATAC-seq and IRF8 peaks mark the cDC1 specific +32 kb enhancer (Durai et al., 2019). In pDC the ATAC-seq peak is smaller and shifted further towards downstream but aligns well with the valley in the prominent H3K27ac peak. This ATAC-seq peak marks the lncIrf8 promoter and aligns with p300 (Durai et al., 2019) and H3K4me3 (Figure 2A and Figure 1—figure supplement 1). All this indicates that this chromatin region is open and transcriptionally active in pDC, enabling lncIrf8 transcription. Figure 2 with 4 supplements see all Download asset Open asset IncIrf8 promoter KO compromises pDC and cDC1 development in vitro. (A) Genomic anatomy of lncIrf8 locus determined by 3’ and 5’ RACE PCR. Blue box, exon 2 and 3 (48 bp and 468 bp, respectively). The 1010 bp intron and polyA tail are shown. Data of RNA-seq, ATAC-seq, ChIP-seq of H3K27ac (enhancer mark) and H3K4me3 (active promoter mark, near TSS) are visualized by IGV browser for the indicated cell populations (pDC, all cDC, cDC1 and cDC2). Grey box, lncIrf8 promoter KO region; open box, cDC1 specific +32 kb enhancer by Durai et al., 2019. Irf8 +32 kb enhancer based on the H3K27ac enhancer mark is indicated with a purple line. Scale bar: 1 kb. (B) Gene expression of lncIrf8 and Irf8 in lncIrf8 promoter KO and control at day 0, 5, and 7 of Flt3L directed DC differentiation. Gene expression was determined by RT-qPCR and normalized to GAPDH. n=4. (C) Representative flow cytometry analysis of Flt3L directed DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control (Lutz et al., 2022; Xu et al., 2022). pDC, all cDC, cDC1, and cDC2 were gated as in Figure 2—figure supplement 1E and are shown. Bar diagrams depict quantification of pDC, cDC1 and cDC2 normalized to living single cells on DC differentiation day 0, 3, 5, 7, and 9. n=6–7. (D) Representative phase-contrast microscopy images of lncIrf8 promoter KO HoxB8 MPP and control on day 7 of Flt3L directed DC differentiation. Scale bar: 200 μm. (E) Representative flow cytometry analysis of spontaneous DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control with growth factors but without E2 (Lutz et al., 2022; Xu et al., 2022) at day 8. Gr1+ monocytes and CD11c+ DC are shown. Quantification of Gr1+ monocytes of living single cells on day 3, 6, 8, and 10 of spontaneous DC differentiation. n=6, lncIrf8 promoter KO; n=4, control. Empty gRNA vector or non-targeting gRNA vector HoxB8 MPP were used as controls. Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of lncIrf8 promoter KO and control without deletion. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled. Next, we determined the chromatin conformation of the Irf8 locus and the lncIrf8 region. We generated interaction profiles by nuclear-titrated (NuTi) Capture-C in MPP, CDP, pDC, cDC1, and cDC2 (Figure 1—figure supplement 2A and B) using Irf8 promoter as viewpoint. The Irf8 promoter shows multiple interactions with regions spanning up to ~100 kb upstream and downstream of Irf8 (Figure 1B and Figure 1—figure supplement 1). In pDC, the Irf8 promoter interactions are stronger with the upstream sequences than with downstream sequences (Figure 1C and Figure 1—figure supplement 2C). In cDC1 Irf8 promoter interactions are more confined to the regions downstream of Irf8 compared to MPP, CDP and pDC (Figure 1C and Figure 1—figure supplement 2C). This suggests that upstream and downstream sequences of Irf8 gene are involved in differentially regulating Irf8 expression and controlling the development of pDC and cDC1, respectively. The CCCTC-binding factor (CTCF) is important for regulation of chromatin conformation through loop extrusion (Sanborn et al., 2015) and we therefore visualized CTCF binding sites in the Irf8 locus in DC (Garber et al., 2012). Interestingly, most of the Irf8 flanking enhancers (Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013) are located within convergent CTCF binding sites upstream and downstream of the Irf8 gene (Figure 1B and Figure 1—figure supplement 1). There are also multiple interactions within this region without convergent CTCF binding sites, suggesting interactions with Irf8 promoter in a CTCF independent manner, such as by TF binding, active histone modifications and gene transcription (Figure 1B and Figure 1—figure supplement 1; Owens et al., 2022). Surprisingly, in pDC H3K27ac at the lncIrf8 promoter is high, but this locus shows less interactions with the Irf8 promoter in pDC compared to CDP, cDC1 and cDC2 (Figure 1C and Figure 1—figure supplement 2C). In addition, in pDC the IRF8 protein occupancy at the lncIrf8 promoter is low and much higher in cDC (Figure 1A and Figure 1—figure supplement 1; Durai et al., 2019; Grajales-Reyes et al., 2015). These observations warrant further studies and we thus proceeded to investigate the lncIrf8 locus in detail. lncIrf8 promoter KO compromises pDC and cDC1 development First, we annotated lncIrf8. Our de-novo transcript assembly of RNA-seq data revealed different isoforms of lncIrf8, with the most prominent isoform comprising exon 2 and 3 (Figure 1A, Figure 2A and Figure 1—figure supplement 1). Additionally, 3' end and 5' end RACE PCR confirmed the anatomy of this lncIrf8 isoform: two exons, one intron, and a polyA tail (Figure 2A). As expected lncIrf8 is not conserved across species (data not shown), which is in line with the general characteristics of lncRNA. Then second, we deleted 300 bp in the lncIrf8 promoter by CRISPR/Cas9 editing in conditionally immortalized HoxB8 MPP (Figure 2A and Figure 2—figure supplement 1A–D). The lncIrf8 promoter is located in the Irf8 +32 kb enhancer region and is in close proximity to the cDC1 specific +32 kb enhancer (Durai et al., 2019; Figure 2A and Figure 2—figure supplement 2). The 300 bp deletion comprises the H3K4me3 promoter mark and is confined to open chromatin identified by ATAC-seq and positioned in the valley of the H3K27ac mark (Figure 2A). Additionally, it contains binding sites for IRF8, PU.1, and BATF3 TF, which are important for DC development (Figure 2A and Figure 2—figure supplement 2B). Generation of a precise deletion requires clonal cell populations, which is hardly achieved in somatic cells due to their limited lifespan. Therefore, we developed a Mx-Cas9-GFP system of conditionally immortalized HoxB8 MPP, which upon differentiation faithfully recapitulate DC development (Figure 2—figure supplement 1A, B; Xu et al., 2022). HoxB8 MPP were obtained from bone marrow of Mx-Cas9-GFP mice by infection with the estrogen (E2) inducible These MPP an and potential and into all DC subsets in and in et al., 2022). of gRNA lncIrf8 promoter in Mx-Cas9-GFP HoxB8 MPP and induction of by interferon generated lncIrf8 promoter KO of with were further studied and to DC differentiation (Figure Figure 2—figure supplement and Figure 2—figure supplement lncIrf8 promoter KO lncIrf8 expression during DC differentiation compared to control without deletion (Figure 2B). Surprisingly, Irf8 expression was also which points to a cross-talk of the lncIrf8 promoter element with the Irf8 lncIrf8 promoter KO also impacts DC subsets, CD11c+ pDC and cDC subsets cDC1 and cDC2 were (Figure and Figure 2—figure supplement of pDC and cDC1 were while cDC2 were (Figure 2C). lncIrf8 promoter KO cDC2 and some cells and were more compared to control without which multiple DC subsets (Figure and Figure 2—figure supplement lncIrf8 promoter KO also the differentiation of progenitors upon E2 from (Figure 1—figure supplement 2A and Figure 2—figure supplement lncIrf8 promoter KO a in cells compared to control (Figure 2—figure supplement lncIrf8 promoter KO higher of Gr1+ monocytes (Figure Figure 2—figure supplement and of all DC subsets CD11c+ pDC, cDC1, and cDC2 (Figure 2—figure supplement compared to control without deletion. The lncIrf8 promoter element is in close proximity to the cDC1 specific +32 kb enhancer previously in mice by Durai et al., and thus we generated the cDC1 specific +32 kb enhancer KO following the same as for the lncIrf8 promoter of clones with of cDC1 +32 kb enhancer were to DC differentiation and further (Figure 2—figure supplement to lncIrf8 promoter cDC1 specific +32 kb enhancer KO lncIrf8 expression and also Irf8 expression during DC differentiation (Figure 2—figure supplement The cDC1 +32 kb enhancer KO also the of cDC1 in Flt3L directed DC differentiation, while cDC2 were (Figure 2—figure supplement of pDC were also at day however, this was not These observations are in line with studies in mice that cDC1 +32 kb enhancer KO cDC1 differentiation and left pDC and cDC2 largely (Durai et al., 2019; Murakami et al., 2021). In addition, cDC1 +32 kb enhancer KO higher of Gr1+ monocytes upon spontaneous DC differentiation by of E2 (Figure 2—figure supplement and thus a as the lncIrf8 promoter KO upon spontaneous DC differentiation (Figure the novel of the lncIrf8 promoter we proceeded to investigate the impact of the lncIrf8 promoter KO on DC differentiation in in We lncIrf8 promoter KO and control HoxB8 MPP into mice (Figure supplement DC in bone marrow and spleen were by flow cytometry on day 7 and cell (Figure 3, Figure supplement 1A, In bone lncIrf8 promoter KO cells into Gr1+ and of all DC subsets were observed on day 7 for lncIrf8 promoter KO cells compared to control (Figure In of cell populations from lncIrf8 promoter KO and control were to bone including of all DC subsets for lncIrf8 promoter KO (Figure HoxB8 cells were largely at day cell (Figure and Figure Figure 3 with 1 supplement see all Download asset Open asset lncIrf8 promoter KO comprises pDC and cDC1 development in upon cell (A) Representative flow cytometry analysis of lncIrf8 promoter KO and control HoxB8 MPP in BM at day 7 cell details see Figure supplement 1A, cell populations were gated from cells and Gr1+ pDC, cDC1 and cDC2 are shown. Quantification of Gr1+ CD11c+ pDC, cDC1, and cDC2 of living single cells in BM on day 7 and cell Representative flow cytometry analysis of lncIrf8 promoter KO and control HoxB8 MPP in spleen at day 7 cell was as in (A). Quantification of Gr1+ CD11c+ pDC, cDC1 and cDC2 on day 7 and cell Data represent mean ± SD from 3 to 4 *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled. Thus, lncIrf8 promoter KO pDC and cDC1 development both in and in lncIrf8 acts as an indicator of Irf8 +32 kb enhancer activity in pDC of lncIrf8 promoter and thus of lncIrf8 expression pDC and cDC1 development in and in The lncIrf8 promoter is located within Irf8 +32 kb enhancer (Durai et al., 2019) and thus it was important to lncIrf8 itself a role in regulating pDC and cDC1 development. this we lncIrf8 in MPP and lncIrf8 in lncIrf8 promoter KO MPP and its impact on DC development (Figure 4 and Figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset lncIrf8 pDC and cDC1 development and B) of lncIrf8 in HoxB8 MPP and of and A polyA signal for transcription was at the 3’ end of lncIrf8 and (C) Gene expression of lncIrf8 and Irf8 in and HoxB8 MPP on day 0, 3, 5, and 7 of Flt3L directed DC differentiation Gene expression was by RT-qPCR and normalized to GAPDH. (D) Representative flow cytometry of DC subsets at day 5 and of Flt3L directed DC differentiation of and HoxB8 Quantification of pDC and cDC1 of living single cells on Flt3L directed DC differentiation day 0, 3, 5, 7, and is shown. for pDC and cDC1 was as in Figure 2—figure supplement (E) of DC subsets of (D) at day 0, 3, 5, 7, and of DC differentiation. high and low Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of and *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled. lncIrf8 was into a polyA An signal et al., 2015) was at the 3’ end of lncIrf8 to avoid transcripts than lncIrf8 Figure The vector was used as control. lncIrf8 clones were generated by (Figure supplement and to DC differentiation. As expected lncIrf8 expression was in cells compared to while there were no in Irf8 expression between the two during DC differentiation (Figure there were no in the of pDC, cDC1, and cDC2 between cells and controls (Figure and Figure supplement indicating that lncIrf8 no on Irf8 expression and DC differentiation. further this we performed a lncIrf8 in lncIrf8 promoter KO lncIrf8 was in lncIrf8 promoter KO MPP by vector and cells were to DC differentiation (Figure supplement lncIrf8 RNA was expressed and cells in response to Flt3L

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation–deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis. Editor's evaluation In this revised and important study, the authors investigate the roles of histone deacetylases in the spatial epigenetic regulation of zygotic gene expression during embryo gastrulation. They provide convincing evidence for HDAC1 binding to genes around the timing of large-scale genome activation, and that inhibition of histone acetylation blocks gastrulation, blurring cell lineage integrity, tied to both positive and negative regulatory effects on transcription in space and time. The research reveals new insight on the role of histone acetylation-deacetylation in dynamics in epigenetic control of gene expression and cell fate determination during early tissue patterning in embryogenesis. https://doi.org/10.7554/eLife.79380.sa0 Decision letter Reviews on Sciety eLife's review process Introduction A fundamental question in early development is the mechanism of zygotic genome activation (ZGA), which requires the degradation of maternal mRNAs and the activation of embryonic transcription (Blitz and Cho, 2021, Tadros and Lipshitz, 2009). During ZGA, the embryonic genome undergoes a dramatic reprogramming of gene expression, which is also accompanied by remodeling of the embryonic epigenome. Post-translational modifications to histones are a major epigenetic regulation influencing chromatin structure and thus play a central role in ZGA. Histone acetylation appears during the onset of both minor and major ZGA waves in many species. In Drosophila, histone acetylation occurs at mitotic cycle 8 on a few early zygotic genes (Li et al., 2014). miR430, the first zygotically active gene, is marked by H3K27ac in 64-cell staged zebrafish embryos (Chan et al., 2019). Genome-wide H3K27ac is detected at mid-blastula shortly after the onset of ZGA in Xenopus (Gupta et al., 2014). In mice, the zygotic genome is increasingly marked by H3K27ac from immature and metaphase II oocytes to 2-cell-stage embryos (Dahl et al., 2016). Despite these findings, there remain several major questions. How is the interplay of enzymes regulating histone acetylation deployed in developing embryos? What is the role of observed histone acetylation on gene expression? How are the spatial and temporal patterns of histone acetylation established during ZGA? Histone acetylation occurs on the ε-amino group of the lysine residues within N-terminal tails of all four core histones (Inoue and Fujimoto, 1969; Seto and Yoshida, 2014). Acetylation is a reversible process that is directly catalyzed by opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In addition, HATs and HDACs can also regulate the acetylation of lysine residues on non-histone proteins (Choudhary et al., 2009). Histone acetylation is often associated with active gene transcription because the acyl groups neutralize the positive charge on the lysine residues, thereby reducing the affinity of histones to DNA (Wang et al., 2000; Anderson et al., 2001); it also serves as a binding platform for bromodomain (BRD) proteins which scaffold and stimulate the transcriptional machinery (Hassan et al., 2007; Filippakopoulos et al., 2012). The balance between HATs and HDACs directly shapes histone acetylation landscapes and subsequently affects transcriptomes. HDACs are critical epigenetic regulators because they reset chromatin states by returning acetylated lysine residues on histones to the basal state, which can subsequently be subjected to alternative modifications such as methylation. HDACs are grouped into four classes based on phylogenetic conservation. Class I (HDAC1, 2, 3, 8), Class II (HDAC4, 5, 6, 7, 9, 10), and Class IV (HDAC11) HDACs are zinc dependent and are related to yeast Rpd3, Had1, and Hos3, respectively; Class III (SIRT1, 2, 3, 4, 5, 6, 7) HDACs, also known as Sirtuins, are NAD+ dependent and are related to yeast Sir2 (Gregoretti et al., 2004; Milazzo et al., 2020). HDACs are well-characterized negative regulators of gene expression during development. For example, Hdac1 silences homeotic genes in cooperation with Polycomb group repressors in Drosophila (Chang et al., 2001). In zebrafish, Hdac1 represses Notch targets during neurogenesis (Cunliffe, 2004; Yamaguchi et al., 2005). In Xenopus, HDAC activity suppresses Vegt-induced ectopic mesoderm in ectoderm lineages (Gao et al., 2016), represses multi-lineage marker genes at blastula (Rao and LaBonne, 2018), and desensitizes dorsal Wnt signaling at late blastula (Esmaeili et al., 2020). Conversely, HDACs can also positively regulate gene expression. For instance, inhibition of HDAC activities rapidly down-regulates some genes in yeast, suggesting an activator function of HDACs (Bernstein et al., 2000). Genetic deletions or pharmacological application of HDAC inhibitors in cell lines results in both up- and down-regulation of genes (Reid et al., 2005; Zupkovitz et al., 2006; Meganathan et al., 2015). Furthermore, genome-wide studies showed that HDACs occupy genomic loci of active genes, and their binding correlates with gene activities (Kurdistani et al., 2002; Wang et al., 2002; Wang et al., 2009; Kidder and Palmer, 2012). These seemingly opposing functions of HDACs raise an important question as to the exact roles of HDACs on chromatin states and transcriptomes in developing embryos. In this study, we focus on the role of Hdac1 in regulating the zygotic epigenome and transcriptome during Xenopus germ-layer specification coinciding with ZGA. Current evidence in Xenopus as well as in other non-mammalian systems suggests that the early embryonic genome is rather naive and that major chromatin modifications occur at or after ZGA (Bonn et al., 2012; Vastenhouw et al., 2010; Gupta et al., 2014; van Heeringen et al., 2014; Hontelez et al., 2015). Thus, the system allows us to probe the earliest establishment of histone acetylation and dissect the link between actions of Hdac1, the zygotic histone acetylome, and zygotic transcriptome during the first cell lineage segregation event. Here, we report that the major Hdac1 binding to the embryonic genome occurs at blastula; the binding of Hdac1 during this stage is dependent on maternal factors. We highlight a dual function model for Hdac1. First, Hdac1 keeps inactive chromatin free of histone acetylation, preventing gene misactivation in respective germ layers. Second, Hdac1 participates in dynamic histone acetylation–deacetylation cycles on active chromatin, sustaining the expression of genes that are enriched in respective germ layers. Taken together, our study reveals a coordinated spatial and temporal regulation by Hdac1 during ZGA. Results Hdac1 binds to the genome progressively during blastula and onward To identify the major functional candidates of HDACs during the early Xenopus embryogenesis, we examined the temporal RNA expression profiles (Owens et al., 2016) of HDAC family members (Class I, II, and III HDACs) from the zygote to the beginning of the neurula stage in Xenopus tropicalis. The RNA expression level of hdac1 is the highest among all HDACs examined, followed by hdac2 (Figure 1—figure supplement 1A). Both Hdac1 and Hdac2 proteins are present in the unfertilized egg to the mid gastrula stage, and the overall expression levels of Hdac1 and Hdac2 are relatively constant during this period (Figure 1A). These data reveal that Hdac1/2 are the major maternally endowed HDACs functioning during this window of development. Figure 1 with 1 supplement see all Download asset Open asset Hdac1 binds to the genome gradually during early Xenopus development. (A) Western blot analyses showing Hdac1 and Hdac2 proteins over a time course of early development. β-Tubulin is used as a loading control. (B) Venn diagram comparing Hdac1 irreproducibility discovery rate (IDR) peaks among examined stages. The sums of peaks in st8 and st9 are smaller due to instances where more than one peak from st8 or st9 overlaps the same single st10.5 peak. (C) Clustered heatmap showing Hdac1 ChIP-seq signals at each stage over a window of 5 kb centered on the summit of all Hdac1 IDR peaks in descending order. (D) The expression levels (TPM, transcripts per million) of genes associated with Hdac1 clusters at different developmental periods. (E) Distributions of Hdac1 IDR peaks at each stage across five defined genomic features. The promoter is defined as −1 kb to +100 bp from TSS (transcription start site) while the TTS (transcription termination site) is defined as −100 bp to +1 kb from TTS. (F) Distributions of Hdac1 ChIP-seq signals within the intervals of 5 kb upstream of gene model 5′ ends, gene bodies (normalized for length), and 5 kb downstream of gene model 3′ ends at each stage. The signal of st9 input DNA ChIP-seq is used as a negative control. Y-axis values represent reads quantified by bins per million (BPM) at a bin size of 50 bp. (G) ChIP-qPCR showing Hdac1 enrichment on selected genomic regions (nine positive regions: alkbh2, fgf5, foxi4.2, gdnf, hhex, miR428a, not, snai1, and sp8; two negative regions: hspa4 and klf11) at indicated stages of early embryogenesis. Figure 1—source data 1 Western blot analyses showing Hdac1 and Hdac2 proteins during Xenopus embryogenesis. https://cdn.elifesciences.org/articles/79380/elife-79380-fig1-data1-v2.zip Download elife-79380-fig1-data1-v2.zip Since Hdac1 modulates various aspects of transcriptional regulation and the chromatin landscape, we examined genome-wide Hdac1-binding patterns during early germ-layer development (Figure 1—figure supplement 1B) using chromatin immunoprecipitation (ChIP) assays followed by sequencing (ChIP-seq). A set of high-confidence peaks at each stage were obtained using irreproducibility discovery rate (IDR) analysis (Li et al., 2011) from two biologically independent samples (Figure 1—figure supplement 1C). Hdac1 binds to 1340 regions at the mid-blastula (st8), 5136 regions at the late blastula (st9), and 22,681 regions at the early gastrula (st10.5) stages (Figure 1B). Overall, a minority of Hdac1 peaks are unique to each of the blastula stages (Clusters a and b) and a majority of Hdac1 peaks are present across multiple stages (Cluster c) and at the early gastrula stage (Cluster d) (Figure 1C, Figure 1—figure supplement 1D). The expression levels of zygotic genes associated with Cluster c Hdac1 peaks are higher than those associated with Clusters a, b, and d, suggesting that the genes associated with sustained Hdac1 binding are more active during gastrulation (Figure 1D). Similarly, Hdac2 gradually accumulates on the embryonic genome (Figure 1—figure supplement 1E). Interestingly, 99% of Hdac1 peaks at stage 9 and 97% of Hdac1 peaks at stage 10.5 overlap with Hdac2 peaks (Figure 1—figure supplement 1F, G). When the levels of Hdac2 peaks associated with Hdac1 were compared to Hdac2 peaks , Hdac1 peaks display a significantly higher Hdac2 peak enrichment (Figure 1—figure supplement 1H). Together, we revealed that Hdac1/2 are progressively directed to the genome during early development. Since a majority of Hdac1-bound peaks are similarly bound by Hdac2, we focus on Hdac1 peaks in subsequent analyses. To investigate the differences of Hdac1 genomic occupancy across early stages, we examined various genomic features of regions bound by Hdac1 at each stage. The majority of Hdac1 peaks are found in either intergenic or intronic regions; a minor fraction of Hdac1 peaks is present within exons or transcriptional termination sites. A notable observation is the increased Hdac1 binding to more promoter regions over developmental times (Figure 1E). We then analyzed the distribution of Hdac1 across three stages along the gene bodies of genes bound by Hdac1. A higher enrichment of Hdac1 binding is located near the promoters of genes as development proceeds (Figure 1F). Importantly, the timing of Hdac1-binding accumulation coincides with the mid-blastula stage (st8) (Figure 1G), suggesting that Hdac1 is involved in the epigenetic regulation of these genes during ZGA. Blastula Hdac1 binding is maternally instructed Since Hdac1 binds progressively to the genome during ZGA, we investigated the contribution of zygotic factors to the recruitment of Hdac1 at the late blastula stage. We injected α-amanitin, which blocks both transcription initiation and elongation (Chafin et al., 1995), to block embryonic transcription (Figure 2—figure supplement 1A). A high Pearson correlation between α-amanitin-injected and uninjected embryos is observed genome-wide or at all Hdac1 IDR peaks (Figure 2A), which is higher than the Pearson correlation representing the batch effect when compared to a different batch of same staged embryos (WT) (Figure 2—figure supplement 1B). There is no significant signal difference between α-amanitin-injected and uninjected embryos at all Hdac1 IDR peaks (Figure 2—figure supplement 1C, H). We conclude that Hdac1 binding is independent of zygotic transcription at the late blastula stage, suggesting the importance of maternal factors in Hdac1 recruitment. Figure 2 with 1 supplement see all Download asset Open asset Maternal factors instruct Hdac1 recruitment during blastula stages. (A) Pairwise Pearson correlation analyses comparing ChIP-seq signals of st9 Hdac1 irreproducibility discovery rate (IDR) peaks between α-amanitin-injected and uninjected embryos across the genome (left) and among Hdac1 IDR peaks (right). (B) Motif analyses of st9 Hdac1 peaks (500 bp centered on IDR peak summit). Motif sequence to the corresponding factor is retrieved from JASPAR. Hey1 is an example of TF motif with low significance. (C) Clustered heatmap depicting st9 Foxh1 and Sox3 ChIP-seq signals in a window of 10 kb centered on st9 Hdac1 IDR peaks with descending order. (D) ChIP-seq signal enrichment of Hdac1, Foxh1, and Sox3 within the intervals of 5 kb upstream of gene model 5′ ends, gene bodies (normalized for length), and 5 kb downstream of gene model 3′ ends of st9 Hdac1 associated genes. The signal of st9 input DNA ChIP-seq is used as a negative control. Y-axis values represent reads quantified by bins per million (BPM) at a bin size of 50 bp. (E) St9 sequential ChIP-qPCR analyses for Foxh1 and Hdac1 co-bound regions and Sox3 and Hdac1 co-bound regions. anti-HA is used as a negative control. The error bars represent the variation from two technical replicates. (F) ChIP-qPCR analysis of Hdac1 peaks that are also Foxh1/Sox3 co-bound after Foxh1 and/or Sox3 depletion. Genomic loci associated with klf11 (no Hdac1 signals), erfl and insm2 (Hdac1 signals without Foxh1 or Sox3 signals) are negative controls. The error bars represent the variation from two technical replicates. Student’s t-test is used to calculate p-values over Hdac1 enrichment of uninjected embryos. * is for p< 0.05** is for p< 0.01. To identify maternal factors that facilitate the recruitment of Hdac1 to the genome, we performed a de novo motif search of the DNA sequences of 5136 st9 Hdac1peaks. Sox, Foxh1, and Pou motifs are found to be the most frequent maternal TF motifs (Figure 2B, Supplementary file 1). We thus compared the genomic binding profiles of Hdac1 to two maternal TFs, Foxh1 (Charney et al., 2017) and Sox3. A majority of Hdac1-bound regions (Cluster 1) overlaps with both Foxh1- and Sox3-bound regions while only a small fraction of Hdac1-bound regions (Cluster 2) overlaps the binding of either Foxh1, or Sox3, or neither (Figure 2C). More than 80% of Hdac1 peaks overlap with Foxh1 or Sox3 peaks (Figure 2—figure supplement 1D, E). A positive correlation between Hdac1 binding and Foxh1/Sox3 binding is observed at all Hdac1 IDR peaks (Figure 2—figure supplement 1F, G). We noted frequent overlapping binding of Hdac1 with Foxh1/Sox3 (Figure 2—figure supplement 1I, J), and highly enriched signals of Hdac1, Foxh1, and Sox3 present around promoters of genes (Figure 2D). All these observations suggest a role for Foxh1 and Sox3 in Hdac1 recruitment. We confirmed the co-occupancy of Hdac1 with each of Foxh1 and Sox3 TFs on the same DNA molecules using sequential ChIP-qPCR (Figure 2E, Figure 2—figure supplement 1K, L). Depletion of Foxh1, Sox3, or both TFs by morpholino injections showed a reduced binding of Hdac1 around Foxh1/Sox3 co-occupied genomic regions (Figure 2F). Hence, we propose that Foxh1 and Sox3 maternal TFs facilitate Hdac1 recruitment during ZGA. Hdac1 binds to genomic regions with distinct epigenetic signatures To further characterize regions bound by Hdac1 across early germ-layer development, we examined epigenetic signatures (Gupta et al., 2014; Hontelez et al., 2015; Charney et al., 2017) on Hdac1 peaks across various stages. Ep300 binding (a HAT), which catalyzes the acetylation of histone, is observed in Hdac1 peaks (Clusters b–d) from the late blastula and onward where RNA polymerase II signals also emerge (Figure 3—figure supplement 1A). This indicates that Hdac1 and Ep300 share similar binding profiles on many transcriptionally active genes. We next surveyed several histone acetylation modifications in regions bound by Hdac1 (Figure 3A). Consistent with the overlapping binding of Ep300, Hdac1 peaks display signals of H3K9ac (Clusters c and d), H3K18ac (Clusters b–d), H3K27ac (Clusters b–d), and pan-H3 lysine acetylation (pan-H3Kac) (Clusters b–d). We then examined several histone methylation modifications that are associated with gene activation (Figure 3—figure supplement 1B). H3K4me1, a primed enhancer mark (Creyghton et al., 2010), and H3K4me3, an active promoter mark (Heintzman et al., 2007), display signals at Hdac1-bound regions (Clusters c and d); H3K36me3, a transcription elongation mark (Kolasinska-Zwierz et al., 2009), displays minimal signals at any Hdac1-bound regions. These observations reveal that Hdac1 binds to genomic regions with active epigenetic signatures. Figure 3 with 1 supplement see all Download asset Open asset Hdac1 binds to cis-regulatory modules (CRMs) containing functionally distinct histone modifications. Clustered heatmaps showing signals from several stages of various (A) histone acetylation marks and (B) repressive histone methylation marks on Hdac1 peaks. Each cluster corresponds to the same regions present in Figure 1C. (C) Venn diagram illustrating Hdac1 peaks overlapping with H3K27me3 and H3K27ac peaks from both st9 and st10.5 combined. (D) Clustered heatmaps depicting signals of H3K27me3 and H3K27ac on combined st9 and st10.5 Hdac1 peaks. Clusters denote the same genomic regions in C. Numbers on the right side indicate the total number of regions in each cluster. The signals are shown in a window of 5 kb centered on the summits of Hdac1 peaks presented in descending order of track signal intensities within each cluster. Hdac1 resets the state of acetylated histones by removing acetyl groups, and thus can facilitate the formation of repressive chromatin. Here, we analyzed several histone methylation modifications associated with gene repression (Figure 3B). Hdac1 binds to genomic regions mostly free of H3K9me2, H3K9me3, or H4K20me3. All three modifications are known to mark constitutive heterochromatin denoting gene-poor areas consisting of tandem repeats (Richards and Elgin, 2002). In addition, a fraction of Hdac1-bound regions (Clusters c and d) display signals of H3K27me3 (Figure 3B), which is known to mark facultative heterochromatin consisting of developmental-cue silenced genes (Trojer and Reinberg, 2007). These observations suggest that Hdac1 binds to facultative heterochromatic regions facilitating the repression of genes. Since the majority of Hdac1 peaks (Clusters b–d) are marked by both active and repressive epigenetic signatures, we wonder how Hdac1 functions in epigenetically distinct genomic loci. We compared peaks between two functionally opposing histone modifications H3K27ac and H3K27me3 to Hdac1 peaks, then subdivided these Hdac1 peaks into four clusters representing functionally distinct (Figure Figure 3—figure supplement 1C). Cluster I Hdac1 peaks marked by both H3K27ac and H3K27me3 (Figure that both H3K27ac and H3K27me3 are on the same lysine we that these regions are marked in space due to cell present in the Hdac1 Cluster I peaks are to as Cluster II Hdac1 peaks marked by only H3K27me3 that these regions are associated with inactive developmental genes (Figure Hdac1 Cluster II peaks represent repressive Cluster III Hdac1 peaks marked by only suggesting that these are active Cluster IV Hdac1 peaks with neither H3K27ac H3K27me3 modifications (Figure genomic loci marked with two distinct modifications (Figure 3—figure supplement the expression levels of genes bound by Hdac1 II, and are higher than that of genes and (Figure 3—figure supplement suggesting that Hdac1 binding correlates with transcriptional activity of the genes. Together, we show that Hdac1-bound CRMs are to distinct epigenetic which differential HDAC activity is for differential germ-layer histone A major function of HDACs is to the of acetyl groups from We that Hdac1 regulates histone acetylation of four different Hdac1 Clusters (Clusters in Figure To this we embryos with a used A et al., beginning at the stage and followed the development to stages. with are at gastrula (Figure We observed the of dorsal suggesting that the but not the initiation of gastrulation is A Class I HDAC et al., also a similar (Figure supplement 1A). We first showed that levels of Hdac1 and Hdac2 are not by (Figure supplement 1B). Hdac1 ChIP-seq display high Pearson on embryos with control or at st9 (Figure supplement and st10.5 (Figure supplement which is further by higher Pearson than the batch effect (Figure supplement differential peak analysis on Hdac1 IDR peaks in embryos with control or showed any differential Hdac1 signals peaks or at st9 (Figure supplement and st10.5 (Figure supplement 1H). These results suggest that of early Xenopus embryos not the recruitment of Hdac1 to the To the of HDAC activity inhibition by we surveyed histone acetylation modifications by increased levels of and H3K27ac are observed in embryos (Figure and LaBonne, 2018), and are not detected during this stage of development. These data indicate that HDAC activity is to the level of histone acetylation during gastrulation. Figure with 3 see all Download asset Open asset Hdac1 maintains differential between germ layers. (A) with A gastrulation the dorsal side containing the early (B) Western blot analyses showing various histone acetylation modifications by HDAC is used as a loading control. (C) Clustered heatmap depicting signals of at st10.5 in embryos and The signals are shown in a window of 5 kb centered on the summits of combined and peaks presented in descending order within each cluster. (D) representing of signals found at Hdac1 cis-regulatory clusters in Figure A Hdac1 peak is to it overlaps with either or peaks (Cluster or in a Hdac1 peak is to it overlaps with peaks between and (Cluster A in not overlap with any peak. (E) signals across Hdac1 clusters in Figure in or the differences of signals between and genomic regions are used as the negative control. (F) of signals at Hdac1 clusters in Figure in or the level of (G) of signals in Cluster I of Hdac1 clusters in Figure for each spatial effect size by Figure data 1 Western blot analyses showing various histone acetylation modifications after HDAC Download that Hdac1 Clusters (Clusters in Figure are subjected to both active and repressive epigenetic modifications in different germ we compared the of the (pan-H3Kac) between two distinct germ the and the We performed ChIP-seq at early gastrula (st10.5) on and from embryos with either or control (Figure supplement A of high-confidence peaks from each reveals that a majority of are marked in and germ layers (Figure Figure supplement We observed the signal of in genomic regions (Cluster A in Figure is significantly higher than those of or genomic regions (Clusters and in Figure which also coincides with higher gene expression levels (Figure supplement 2C). To the differential states and the differential gene expression profiles between the two germ we high enrichment peaks to the genes within 10 kb and compared the expression levels of these genes in each germ (Blitz et al., the expression level of genes enriched with are higher in than (Figure supplement and the expression levels of genes enriched with are higher in than (Figure supplement genes with expression signals between germ layers (Figure supplement 2F). These results that the histone acetylation coincides with the and expression of To the role of Hdac1 in regulating histone acetylation states of clusters (Clusters in Figure we first examined the distribution of in these Hdac1 Consistent with both CRMs (Cluster I, both H3K27ac and and active CRMs (Cluster only but not repressive CRMs (Cluster II, only are marked by of marks on or active CRMs are either or (Figure suggesting that these CRMs are active in germ layers. We then

Microorganisms commonly use ‘two-component’ signaling systems for sensing environmental conditions, with members being present in nearly all bacterial and archaeal genomes in different numbers. Prototypical two-component systems are comprised of a sensory histidine kinase and a response regulator protein that is phosphorylated by the kinase. The regulator typically acts as a transcription factor regulating gene expression. Due to their prevalence in microorganisms, a basic understanding of the principles of two-component systems provides insights into mechanisms of prokaryotic signaling circuits. Apart from a few studies performed in vitro, the signaling properties of a whole bacterial two-component network in vivo remains largely unclear. Using a system level approach, we characterized the localization of signaling proteins and the interactions between pathways on different levels in the model bacterium Escherichia coli. We constructed a comprehensive library of fluorescent protein fusions to kinases and regulators from two-component systems, and applied fluorescence microscopy to analyze their spatial organization within the cell. We found that most of the sensors exhibit a intrinsic tendency for cluster formation, which might provide a scaffold for information processing and insulation against detrimental noise from other sources. We used in vivo FRET microscopy tools to study protein interactions in a systematic way. For kinases, we could confirm the formation of sensor dimers or higher order oligomers in vivo in most of the cases. For the sensor BaeS, we characterized the change of sensor confirmation upon stimulation. Additionally, we identified a few pathways exhibiting interconnections on the sensor level and between sensors and non-cognate regulators, providing an additional layer of information processing. We could confirm the physiological relevance of some of these pathway interconnections on the gene expression level, using flow cytometry measurements of a fluorescent promotor library. Moreover, using this library we found most of the reported stimuli not affecting two-component gene expression. Taken together, our data provide a deeper understanding of cellular organization within and interconnections between different two-component systems in E. coli. The results of this study can broaden the understanding of cell signaling principles in bacteria.