Intron-containing mRNAs are cotranscriptionally spliced and assembled into messenger ribonucleoprotein (mRNP) particles, a process monitored by surveillance pathways. Here, we combined biochemical and structural approaches to elucidate the mechanisms by which mRNPs are sorted between two opposing fates: nuclear degradation and cytoplasmic export. While the human GANP-PCID2 complex is known to connect mRNPs to nuclear export, our data indicate that the LENG8-PCID2 complex operates as an mRNP decay connector, coupling nuclear mRNPs to the RNA-degrading exosome via the PAXT adaptor complex. Both recognize the mRNP component UAP56, but LENG8-PCID2 uniquely associates with early splicing factors through a direct interaction with U1A and RRP1B. Similarly, the Thp3-Csn12 ortholog in budding yeast couples the early splicing factors Mud2-Bbp with the nuclear exosome. The spliceosome-exosome mRNP decay pathway we uncovered reveals molecular principles that remain strikingly conserved across evolution, despite the fundamental differences in splicing and decay between humans and budding yeast.

Polycomb group proteins maintain gene expression patterns established during early development, with Polycomb repressive complex 2 (PRC2) methyltransferase being a key regulator of cell differentiation, identity, and plasticity. Consequently, extensive somatic mutations in PRC2, including gain or loss of function (GOF or LOF), are observed in human cancers. The regulation of chromatin structure by PRC2 is critically dependent on its enhancer of zeste homolog 2 (EZH2) subunit, which catalyzes the methylation of histone H3 lysine 27 (H3K27). Recent structural studies of PRC2 revealed extensive conformational changes in the noncatalytic EZH2 N-terminal SANT1-binding domain (SBD) during PRC2 activation, though the functional significance remains unclear. Here, we investigated how the SBD regulates PRC2 function. The domain is highly conserved in metazoans and dispensable for PRC2 assembly and chromatin localization yet is required for genome-wide histone H3K27 methylation. Furthermore, we show that an intact SBD is necessary for the proliferation of EZH2-addicted lymphomas, and its deletion in the presence of EZH2 GOF mutations inhibits cancer cell growth. These observations provide new insights into the regulation of PRC2 activity in normal development and malignancy.

The precise spatiotemporal regulation of developmental genes is required for proper organogenesis. Engrailed-1 (En1) is essential for dorsal-ventral patterning during mouse limb development from embryonic day 9.5 (E9.5) to E11.5. Previously, we identified the long noncoding RNA locus Maenli, which drives limb-specific En1 expression at E9.5. Here, we uncover two intergenic enhancer elements, LSEE1&2, that are essential for En1 expression at E10.5 and E11.5. We demonstrate that Maenli and LSEE1&2 coordinate two transcriptional waves of En1 expression that are essential for establishing dorsal-ventral limb identity. The early wave, controlled by Maenli, is required for ventral ectoderm and apical ectodermal ridge specification, while the late wave, associated with LSEE1&2, is critical for dorsal-ventral ectoderm specification in mouse limbs. Loss of Maenli versus LSEE1&2 results in distinct developmental phenotypes by altering the spatiotemporal expression of key genes governing axial patterning pathways. Our study underscores how temporal cis-regulatory elements modulate lineage specification and developmental patterning through shaping spatiotemporal gene function during development, representing potential candidates underpinning isolated subphenotypes.

Transforming growth factor β (TGF-β) signaling is a highly pleiotropic pathway with an important role in development, homeostasis, and cancer. Chromatin regulators contribute to the regulation of TGF-β-responsive transcription. The requirement of subunits of the MLL3/MLL4 histone methyltransferase complexes for TGF-β responses has been reported. However, their exact roles are not fully understood. To investigate the functions of these complexes, we employed CRISPR/Cas9 genome editing to inactivate the KMT2C/MLL3 or KMT2D/MLL4 genes in human diploid epithelial cells. Time-course RNA-seq experiments revealed the requirement of MLL4 but not of MLL3 for TGF-β transcriptional responses. CUT&RUN experiments showed that MLL4 binding increases after TGF-β treatment and is especially enriched at AP-1 transcription factor binding sites. Interestingly, TGF-β-induced chromatin binding of MLL4 correlates with increases in H3K27ac but not in H3K4me1 modifications. Furthermore, TGF-β treatment sets off SMAD2-induced JUNB expression, which forms a feed-forward loop with MLL4. By inhibiting the activities of AP-1, the BAF chromatin remodeler, or the CBP/p300 histone acetyltransferase, we found that AP-1 binding and these chromatin regulators are all necessary for TGF-β induction of MLL4 binding and transcriptional activation of its genomic targets. Taken together, our study reveals distinctive roles for the MLL3 and MLL4 paralogs in the transcriptional response to TGF-β. In contrast to MLL3, MLL4 forms a feed-forward loop of JUNB, the BAF complex, and CBP/p300 to sustain transcription activation by TGF-β.

Medulloblastoma (MB) groups 3 and 4 arise from human-specific developmental contexts that remain experimentally inaccessible, limiting mechanistic insight into tumor initiation. We generated cerebellar organoids (CbOs) from expanded potential stem cells (EPSCs) and performed integrated single-cell transcriptomic and DNA methylation analyses, revealing discrete human rhombic lip progenitor populations whose developmental programs align with group 3- and group 4-associated lineages. Using c-MYC overexpression as an initiating oncogenic event, we demonstrated that these lineage-restricted progenitors are susceptible to neoplastic transformation, generating tumors with group 3 MB molecular features in vitro and in vivo. We further established a CbO-MB coculture system that preserves malignant and nonmalignant cellular compartments, hence enabling interrogation of tumor-microenvironment interactions and providing simultaneous readouts of antitumor efficacy and cerebellar toxicity upon drug treatment. Receptor-ligand modeling in this system identifies TGFβ paracrine signaling as a microenvironmental pathway supporting MB growth, which we validated pharmacologically. Further analyses of cocultured MB cells revealed a myogenic differentiation program enriched in MYC-driven MB and recurrent disease, which is associated with poorer prognosis. Together, this work establishes a collection of CbO models for studying group 3/4 MB initiation, growth, and microenvironmental dependencies and provides a tractable platform for further mechanistic and therapeutic investigation of these tumors.

DUX4 initiates the first wave of zygotic genome activation (ZGA) in the human embryo, and its misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). However, other factors that help regulate ZGA-like transcription in human development and disease are still not fully understood. Here we identify Krüppel-like factor 18 (KLF18) as a component of a DUX4 feed-forward network that is necessary for expression of KLF17 and a subset of DUX4-regulated totipotency-associated genes. We mapped the genome-wide binding profile of KLF18 downstream from DUX4 and showed that its activity is influenced by DUX4 and chromatin accessibility. We found that in contrast to the rodent ortholog, human KLF18 has a predicted β-solenoid structure composed of a variable number of tandem repeats (VNTR) with multiple different structural variants in the human population. Expression of different KLF18 variants in combination with DUX4 showed similar transcriptional activity, whereas assessment of KLF18 structural variants in individuals with FSHD1 showed inconsistent association with disease severity that requires further study as a modifier locus. Together, these findings establish the polymorphic transcription factor KLF18 as a critical component of a DUX4-initiated feed-forward network and a candidate locus for human diversity.

FoxA transcription factors pattern gut tissue across animal phylogeny. Beyond their early patterning function, little is known about whether they control the terminal differentiation and/or function of the fully mature enteric nervous system, the intrinsic nervous system of the gut. We show here that the expression and function of the sole Caenorhabditis elegans FoxA homolog, PHA-4, reach beyond its previously described pioneer factor roles in patterning the foregut. Through the engineering of neuron-specific cis-regulatory alleles, Cre-mediated cell-specific knockouts, and degron-mediated, temporally controlled PHA-4/FoxA removal in postmitotic neurons, we found that PHA-4/FoxA is required not only to initiate the terminal differentiation program of foregut-associated enteric neurons but also to maintain their functional properties throughout the life of the animal. Moreover, we discovered novel sites of expression of PHA-4/FoxA in extrapharyngeal enteric neurons that innervate the hindgut (AVL and DVB), a GABAergic interneuron that controls foregut function during sleep (RIS), and a peptidergic neuron (PVT) that we implicate here in controlling defecation behavior. We show that while PHA-4/FoxA is not required for the developmental specification of AVL, DVB, RIS, and PVT, it is required to enable these neurons to control enteric functions. Taken together, PHA-4/FoxA is the only transcription factor known to date that is expressed in and required for the proper function of all distinct types of enteric neurons in a nervous system.

The unfolded protein response (UPR) was discovered in budding yeast as a mechanism that allows cells to adapt to endoplasmic reticulum (ER) stressors. Although the UPR is not thought to be necessary for cellular fitness of wild-type cells in the absence of stress, we found that UPR deficiency led to poor growth in cycling mitotic yeast cells. This led to pervasive adaptive aneuploidy of specific chromosomes that was seen in divergent strain backgrounds, indicating an important basal role for this pathway that was missed by studies of the most common laboratory-derived strains. Aneuploid UPR-deficient cells grew better than euploid UPR-deficient cells but exhibited heightened general proteostatic stress, a hallmark of aneuploidy in wild-type cells. Modulation of key genes involved in ER proteostasis that were encoded on aneuploid chromosomes could phenocopy the effects of aneuploidy, indicating that the reason UPR-deficient cells become aneuploid is to counteract protein folding stress in the ER. Proteomic analyses indicate that expression of a small subset of stress-induced UPR targets is supported by basal UPR activity, including the chaperone Kar2/BiP. Together, our results reveal an unexpected role for the UPR in baseline ER folding that is important enough to safeguard cellular fitness that cells tolerate the substantial proteostatic costs that result from aneuploidy to counteract its loss.

MyoD is well known for its ability to reprogram a broad range of cell types into myogenic cells and for its pioneer function in activating the myogenic program during muscle development and regeneration. The basic helix-loop-helix (bHLH) protein achieves this by directly binding to E-boxes in DNA and recruiting proteins like histone acetyltransferases and the SWI/SNF chromatin remodeling complex. Interestingly, Nicoletti and colleagues (doi:10.1101/gad.352708.125) report in this issue of Genes & Development an unexpected finding; namely, that MyoD can also act as a repressor. This repressive activity is E-box-independent, meaning that MyoD can be indirectly recruited to distinct sites in chromatin. Transcription factor motifs enriched at these sites correspond to E2F, NF-Y, and Jun/Fos motifs. The genes that are repressed by this noncanonical MyoD function control nonmyogenic fates and participate in cell cycle regulation as well as proliferation. At such sites, MyoD binding is associated with chromatin compaction and repression of transcription.

The exon junction complex (EJC) has roles in mRNA export and cytoplasmic quality control. However, the EJC is recruited to pre-mRNA by the spliceosome prior to the completion of splicing. When splicing is cotranscriptional, the EJC is deposited on nascent RNA early during synthesis, raising the question of whether the EJC regulates downstream RNA processing. Here we show, using long-read sequencing, that degron-mediated depletion of EJC component EIF4A3 leads to skipping of neighboring pairs of two or more exons on the same mRNA molecule. These data suggest that the entire "exon block" requires the EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. In our working model, block exons are first spliced together to form a larger EJC-marked exon that promotes surrounding splicing events. Strikingly, analysis of 480 RNA binding protein knockdowns across two different human cell lines revealed block exons that are dependent on other splicing factors, indicating that coordinated splicing of adjacent exons is a general mechanism, of which the EJC is the dominant regulator. Cell type-specific coordinated splicing of adjacent exon pairs has been observed before. Here we identify the EJC as the main protein factor massively regulating this novel splicing mechanism in trans.