Cis-Regulatory Elements in Plant Development, Adaptation, and Evolution.

Correct patterning of the antero-posterior axis of the embryonic trunk is dependent on spatiotemporally restricted Hox gene expression. In this study, we identified components of the Hoxd4 P1 promoter directing expression in neurally differentiating retinoic acid-treated P19 cells. We mapped three nucleosomes that are subsequently remodeled into an open chromatin state upon retinoic acid-induced Hoxd4 transcription. These nucleosomes spanned the Hoxd4 transcriptional start site in addition to a GC-rich positive regulatory element located 3' to the initiation site. We further identified two major cis-acting regulatory elements. An autoregulatory element was shown to recruit HOXD4 and its cofactor PBX1 and to positively regulate Hoxd4 expression in differentiating P19 cells. Conversely, the Polycomb group (PcG) protein Ying-Yang 1 (YY1) binds to an internucleosomal linker and represses Hoxd4 transcription before and during transcriptional activation. Sequential chromatin immunoprecipitation studies revealed that the PcG protein MEL18 was co-recruited with YY1 only in undifferentiated P19 cells, suggesting a role for MEL18 in silencing Hoxd4 transcription in undifferentiated P19 cells. This study links for the first time local chromatin remodeling events that take place during transcriptional activation with the dynamics of transcription factor association and DNA accessibility at a Hox regulatory region.

The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.

Brassinosteroids (BRs) regulate a diverse spectrum of processes during plant growth and development and modulate plant physiology in response to environmental fluctuations and stress factors. Thus, the BR signaling regulators have the potential to be targeted for gene editing to optimize the architecture of plants and make them more resilient to environmental stress. Our understanding of the BR signaling mechanism in monocot crop species is limited compared to our knowledge of this process accumulated in the model dicot species - Arabidopsis thaliana. A deeper understanding of the BR signaling and response during plant growth and adaptation to continually changing environmental conditions will provide insight into mechanisms that govern the coordinated expression of the BR signaling genes in rice (Oryza sativa) which is a model for cereal crops. Therefore, in this study a comprehensive and detailed in silico analysis of promoter sequences of rice BR signaling genes was performed. Moreover, expression profiles of these genes during various developmental stages and reactions to several stress conditions were analyzed. Additionally, a model of interactions between the encoded proteins was also established. The obtained results revealed that promoters of the 39 BR signaling genes are involved in various regulatory mechanisms and interdependent processes that influence growth, development, and stress response in rice. Different transcription factor-binding sites and cis-regulatory elements in the gene promoters were identified which are involved in regulation of the genes’ expression during plant development and reactions to stress conditions. The in-silico analysis of BR signaling genes in O. sativa provides information about mechanisms which regulate the coordinated expression of these genes during rice development and in response to other phytohormones and environmental factors. Since rice is both an important crop and the model species for other cereals, this information may be important for understanding the regulatory mechanisms that modulate the BR signaling in monocot species. It can also provide new ways for the plant genetic engineering technology by providing novel potential targets, either cis-elements or transcriptional factors, to create elite genotypes with desirable traits.

BackgroundLaccases (LACs) are vital plant growth and development enzymes, participating in lignin biopolymerization and responding to stress. However, the role of LAC genes in plant development as well as stress tolerance, is still not well understood, particularly in sesame (Sesamum indicum L.), an important oilseed crop.ResultsIn this study, 51 sesame LAC genes (SiLACs) were identified, which were unevenly distributed across different chromosomes. The phylogeny of Arabidopsis LAC (AtLACs) subdivided the SiLAC proteins into seven subgroups (Groups I-VII), of which Group VII contained only sesame LACs. Within the same subgroup, SiLACs exhibit comparable structures and conserved motifs. The promoter region of SiLACs harbors various cis-acting elements that are related to plant growth, phytohormones, and stress responses. Most SiLACs were expressed in the roots and stems, whereas some were expressed specifically in flowers or seeds. RNA-seq analysis revealed that 19 SiLACs exhibited down-regulation and three showed up-regulation in response to drought stress, while 15 SiLACs were down-regulated and four up-regulated under salt stress. Additionally, qRT-PCR analysis showcased that certain SiLAC expression was significantly upregulated as a result of osmotic and salt stress. SiLAC5 and SiLAC17 exhibited the most significant changes in expression under osmotic and salt stresses, indicating that they may serve as potential targets for improving sesame resistance to various stresses.ConclusionsOur study offers a thorough comprehension of LAC gene structure, classification, evolution, and abiotic stress response in sesame plants. Furthermore, we provide indispensable genetic resources for sesame functional characterization to enhance its tolerance to various abiotic stresses.

Depression is a serious mental illness and a prevalent condition with multiple aetiologies. The impact of the current therapeutic strategies is limited and the pathogenesis of the illness is not well understood. According to previous studies, depression onset is influenced by a variety of environmental and genetic factors, including chronic stress, aberrant changes in gene expression, and hereditary predisposition. Transcriptional regulation in eukaryotes is closely related to chromosome packing and is controlled by histone post-translational modifications. The development of new antidepressants may proceed along a new path with medications that target epigenetics. Histone deacetylase inhibitors (HDACis) are a class of compounds that interfere with the function of histone deacetylases (HDACs). This review explores the relationship between HDACs and depression and focuses on the current knowledge on their regulatory mechanism in depression and the potential therapeutic use of HDACis with antidepressant efficacy in preclinical research. Future research on inhibitors is also proposed and discussed.

The biological interpretation of the genome starts from transcription, and many different signaling pathways are integrated at this level. Transcription factors play a central role in the transcription process, because they select the down-stream genes and determine their spatial and temporal expression. In higher eudicot species around 2000 specific transcription factors are present, which can be classified into families based on conserved common domains. The MADS-box transcription factor family is an important family of transcription regulators in plants and genetic studies revealed that members of this family are involved in various developmental processes, like floral induction, floral organ formation and fruit development. In contrast to this wealth of information concerning MADS-box gene functions, the molecular mode of action of the encoded proteins is far from completely understood. Biochemical and yeast η-hybrid experiments performed in the past showed that MADS-box proteins are able to interact mutually, and based on these findings a hypothetical quaternary model has been proposed as molecular working mechanism. According to this model two MADS-box protein dimers assemble into a higher order complex, which binds DNA and regulates target gene expression. Although, this molecular mechanism sounds plausible, it still lacks evidence from in vivo studies. In this study we investigated physical interactions among members of the Petunia hybrida and Arabidopsis thaliana MADS-box transcription factor families in living plant cells. For this purpose, sophisticated micro-spectroscopy techniques have been implemented and in addition, some novel fluorescent-protein-based tools were developed. The first chapter gives an introduction about the dynamic transcriptional process and describes our current knowledge about transcriptional regulation in eukaryotes. The central question of this chapter is how transcription factors are able to find their specific binding sites (ci's-elements) within the huge genome. The various mechanisms, such as "looping" and "sliding", that have been proposed are discussed, as well as the relevance of direct interactions between transcription factors for the control of gene expression. In a first attempt to detect protein interactions in living cells, we transiently expressed combinations of petunia MADS-box transcription factors labeled with different color variants of the Green Fluorescent Protein (GFP) in leaf protoplasts (Chapter 2). Subsequently, the transfected protoplasts were analyzed by means of FRET-FLIM (Fluorescence Resonance Energy Transfer - Fluorescence Lifetime Imaging) to identify specific dimerization. In addition, we have obtained indirect evidence for higher-order complex formation of the petunia MADS-box proteins FLORAL BINDING PROTEIN2 (FBP2), FBP11, and FBP24 in living cells. Similar kind of analyses for Arabidopsis MADS-box proteins involved in petal and stamen development revealed clear differences in interaction affinities in vivo and furthermore, many homodimers were identified that could not be detected by yeast-based systems in the past (Chapter 3). This result demonstrated the robustness of the FRET-FLIM approach. Based on our observations, we hypothesize that 'partner selectivity' plays an important role in complex formation at particular developmental stages. To study differences in interaction affinity and selectivity and the consequences for complex formation in more detail, a novel method was developed (Chapter 4). The technique, designated "Competition-FRET", allows the verification of competition effects between proteins, and furthermore, it may provide information about the formation of higher-order complexes between different proteins under study. The developed method was implemented to investigate in depth the preference for homo- or heterodimer interactions of the Arabidopsis MADS-box proteins AGAMOUS (AG) and SEPALLATA3 (SEP3). The detection of interactions in living cells by FRET as it has been done in the studies described above demands a sophisticated microscopy set-up, and therefore, we decided to test and implement an alternative and theoretically simple technique (Chapter 5). This method for the in vivo detection of protein-protein interaction is called BiFC (Bimolecular Fluorescence Complementation), or "Split-YFP". In this system, a fluorescent molecule is split into two inactive domains and these two non-fluorescent parts are fused to the proteins under study. Only upon interaction of the two protein partners the two non-fluorescent parts of the fluorescent molecule are brought into close proximity, which enables the recovery of fluorescence. We used the EYFP (Enhanced Yellow Fluorescence Protein) molecule as fluorescent molecule and were able to detect the interaction between AG and SEP3 in nuclei of Arabidopsis leaf protoplasts. Techniques like this and FRET-FLIM allow the analyses of interactions between proteins in living cells, but give no information about the size of the formed complexes. To get a first indication about the stoichiometry of protein complexes, we monitored the diffusion time of in vitro synthesized AG-EYFP and SEP3-EYFP fusion proteins by means of FCS (Fluorescence Correlation Spectroscopy). From these experiments described in Chapter 6, we could speculate that SEP3 is present as a dimer and also as a higher order complex, whilst AG on its own is able to assemble into larger complexes. The diffusion time of the product formed upon co-translation of both AG and SEP3, suggests that a multimenc protein complex with a high molecular weight is formed upon interaction between AG and SEP3. Even though FCS is a powerful technique, these interpretations should be taken cautiously, mainly because these experiments were done in vitro instead of in living cells. Finally, in the last chapter we discuss the various methods that have been implemented and developed to monitor protein-protein interactions and complex formation of MADS-box transcription factors in living plant cells. Furthermore, we made a first step to monitor interactions in intact tissues under endogenous expression levels, and the preliminary results obtained from these in planta FRET-FLIM measurements are discussed.

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

BackgroundAxis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra.ResultsTo gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity.ConclusionsThe activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Understanding plant responses to developmental and environmental cues is crucial for studying morphological divergence and local adaptation. Gene expression changes, governed by cis-regulatory modules (CRMs) including enhancers, are a major source of plant phenotypic variation. However, while genome-wide approaches have revealed thousands of putative enhancers in mammals, far fewer have been identified and functionally characterized in plants. This review provides an overview of how enhancers function to control gene regulation, methods to predict DNA sequences that may have enhancer activity, methods utilized to functionally validate enhancers and the current knowledge of enhancers in plants, including how they impact plant development, response to environment and evolutionary adaptation.

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

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Nature provides all necessary components for healthy growth and development of plants in the form of air, water, light, nutrients, and soil. Any imbalance in the environmental harmony may cause stress to them. Stresses encountered by plants can broadly be categorized into biotic and abiotic stresses. Biotic stresses are mainly caused by pathogens and herbivory, whereas abiotic stresses include the threat imposed by drought, salinity, and extremes of temperature, heavy metals, and pollution. Drought stress is a major cause of yield instability in crops across diverse eco-geographic regions worldwide. A variety of biochemical, molecular, and physiological changes are manifested by plants in response to drought stress. The cellular abscisic acid (ABA) concentration increases on water deficit leading to the activation of a number of stress-responsive genes and the patterns of expression of these genes are very complex, with some genes being induced early while others respond slowly. In general, drought-responsive genes respond to salt and cold stresses as well as to exogenous ABA treatment. However, there are several genes, which express themselves in an ABA-independent manner suggesting that both ABA-dependent and -independent signal transduction cascades exist for drought stress perception, response, and adaptation. Drought stress response and adaptation in plants involves an array of pathways for signal perception, transduction, gene expression and synthesis of proteins, and other stress metabolites. Drought-responsive genes can mainly be classified into two groups. First group constitutes genes whose products provide osmotolerance and protection to plants thus directly functioning in tolerance to stress, while the second group includes genes playing a role in signal transduction as well as regulation of gene expression. This chapter summarizes the complex molecular mechanisms of drought stress response and adaptation in plants, highlighting the transcriptional regulation of stress-responsive gene expression. It also focuses on the recent advances in analyzing various stress-responsive pathways with prime emphasis on ABA-dependent and -independent pathways.

RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms (1–3). Regulation of splicing occurs through differential selection of splice sites in a splicing reaction, which results in variation in the abundance of isoforms and/or splicing events. However, genomic determinants that influence splice-site selection remain largely unknown. While traditional approaches for analyzing splicing rely on quantifying variant transcripts (i.e. isoforms) or splicing events (i.e. intron retention, exon skipping etc.) (4), recent approaches focus on analyzing complex/mutually exclusive splicing patterns (5–8). However, none of these approaches explicitly measure individual splice-site usage, which can provide valuable information about splice-site choice and its regulation. Here, we present a simple approach to quantify the empirical usage of individual splice sites reflecting their strength, which determines their selection in a splicing reaction. Splice-site strength/usage, as a quantitative phenotype, allows us to directly link genetic variation with usage of individual splice-sites. We demonstrate the power of this approach in defining the genomic determinants of splice-site choice through GWAS. Our pilot analysis with more than a thousand splice sites hints that sequence divergence in cis rather than trans is associated with variations in splicing among accessions of Arabidopsis thaliana. This approach allows deciphering principles of splicing and has broad implications from agriculture to medicine.

CREB1-binding protein (CBP) and p300 are believed to participate in the activities of hundreds of different transcription factors (see Fig.1). Current models suggest that the binding of these coactivators to transcription factor activation domains positions histone acetyltransferases (HATs) near specific nucleosomes in target gene promoter regions (for review, see Ref. 1Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1406) Google Scholar). Interactions with components of the general transcriptional machinery, such as TFIID, TFIIB, and the RNA polymerase II holoenzyme (RNAPII) have also been suggested to contribute to CBP/p300 function. The simultaneous interaction of multiple transcription factors with CBP/p300 has been proposed to contribute to transcriptional synergy. Conversely, competition for CBP/p300 binding has been suggested to mediate some examples of signal-induced transcriptional repression. An overview of CBP/p300 in cellular growth and differentiation has recently been published (2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar), but many questions regarding their role in transcriptional regulation remain unanswered. This review deals with some of the more controversial aspects of CBP/p300 function. In particular, we will ask whether CBP and p300 have distinct functions, review the evidence for their regulation by phosphorylation, and ask whether they function primarily by acetylating histones or other proteins. We will also revisit the evidence for the role of CBP/p300 as transcriptional “integrators.” Finally, we will attempt to localize CBP/p300 function within the complex series of processes involved in transcriptional activation. Although CBP and p300 are highly related and share many functional properties, there is evidence that these factors are not really interchangeable. Subtle differences in the expression of CBP and p300 during development (3Partanen A. Motoyama J. Hui C.C. Int. J. Dev. Biol. 1999; 43: 487-494PubMed Google Scholar) may explain why knockouts of the two coactivators in mice result in somewhat distinct phenotypes. For example, heterozygosity for CBP causes certain hematological defects and a predisposition to cancer that is not seen in mice lacking one allele of p300 (4Kung A.L. Rebel V.I. Bronson R.T. Ch'ng L.E. Sieff C.A. Livingston D.M. Yao T.P. Genes Dev. 2000; 14: 272-277PubMed Google Scholar). Studies of specific transcription factor pathways provide additional evidence of differences between the functions of CBP and p300. For example, fibroblasts derived from homozygous p300 knockouts are defective for retinoic acid receptor but not CREB signaling (5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Similarly, ribozyme-mediated ablation of p300, but not CBP, blocks the retinoic acid receptor response (6Kawasaki H. Eckner R. Yao T.P. Taira K. Chiu R. Livingston D.M. Yokoyama K.K. Nature. 1998; 393: 284-289Crossref PubMed Scopus (302) Google Scholar). Other differential functions of CBP and p300 have been revealed by their distinct interactions with viral transforming proteins. For example, the Kaposi sarcoma-associated herpesvirus protein vIRF has been reported to be stimulated by CBP and repressed by p300 (7Jayachandra S. Low K.G. Thlick A.E., Yu, J. Ling P.D. Chang Y. Moore P.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11566-11571Crossref PubMed Scopus (87) Google Scholar). Distinct roles for CBP and p300 have also been suggested in the differentiation of muscle and F9 teratocarcinoma cells (8Puri P.L. Sartorelli V. Yang X.J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 9Ugai H. Uchida K. Kawasaki H. Yokoyama K.K. J. Mol. Med. 1999; 77: 481-494Crossref PubMed Scopus (31) Google Scholar). On the other hand, homozygous mutations in CBP and p300 both result in lethality and a similar constellation of phenotypic defects (4Kung A.L. Rebel V.I. Bronson R.T. Ch'ng L.E. Sieff C.A. Livingston D.M. Yao T.P. Genes Dev. 2000; 14: 272-277PubMed Google Scholar, 5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Moreover, CBP/p300 double heterozygotes are invariably lethal, suggesting that functions of CBP and p300 must overlap, at least to some degree. This complexity is not shared by simpler metazoans, such as Drosophila andCaenorhabditis elegans, which express only a single isoform of CBP/p300 (reviewed in Ref. 2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). Although cell cycle-dependent phosphorylation of p300 was reported almost a decade ago (10Yaciuk P. Carter M.C. Pipas J.M. Moran E. Mol. Cell. Biol. 1991; 11: 2116-2124Crossref PubMed Scopus (92) Google Scholar), it is still not entirely clear how phosphorylation regulates CBP/p300 function. In large part, this lack of understanding is because of the fact that the specific phosphorylation sites in CBP/p300 have never been precisely identified. Phosphorylation of p300 and CBP by cyclin E/Cdk2 was reported by Perkins et al. (11Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar) and Ait-Si-Ali et al. (12Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar), respectively. In the case of p300, cyclin E/Cdk2 was shown to negatively regulate coactivator function in a manner that can be blocked by the cyclin-dependent kinase inhibitor, p21. In this model, p21 was proposed to participate in a positive feedback loop, whereby activators such as p53, which depend upon p300 for function, induce p21, which then alleviates the block in p300 action mediated by cyclin E/Cdk2. In contrast, cyclin E/Cdk2 was reported to increase the intrinsic HAT activity of CBP, potentially activating expression of S-phase genes that are repressed in early G1(12Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar). Because the phosphorylation sites in p300 and CBP have not been mapped, however, it may be premature to conclude that the two coactivators are differentially regulated by cyclin-dependent kinases. CBP and p300 both contain a consensus protein kinase A (PKA) site adjacent to their third zinc finger domains, and several groups have proposed that phosphorylation by PKA may contribute to CBP/p300 regulation. For example, Xu et al. (13Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (248) Google Scholar) have argued that phosphorylation of CBP is responsible for the PKA-mediated augmentation of the transcription factor Pit-1. This is an intriguing model because Pit-1 itself cannot be phosphorylated by PKA. Using microinjection assays, these workers demonstrated that the activation of Pit-1 by PKA was lost in the presence of CBP containing a point mutation at the consensus PKA site. This model was not confirmed by Zanger et al. (14Zanger K. Cohen L.E. Hashimoto K. Radovick S. Wondisford F.E. Mol. Endocrinol. 1999; 13: 268-275PubMed Google Scholar), however, and Swope et al. (15Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) have suggested that the PKA-responsive domain in CBP resides near its amino terminus. At this point, the mechanism of PKA activation of CBP/p300 remains enigmatic. Other kinases proposed to regulate CBP/p300 function include calcium/calmodulin (CaM) kinase IV, MAPK, and pp90Rsk. Although several reports suggested that CBP cannot mediate its transcriptional functions in the absence of CaM kinase IV stimulation (16Chawla S. Hardingham G.E. Quinn D.R. Bading H. Science. 1998; 281: 1505-1509Crossref PubMed Scopus (379) Google Scholar, 17Hardingham G.E. Chawla S. Cruzalegui F.H. Bading H. Neuron. 1999; 22: 789-798Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 18Hu S.C. Chrivia J. Ghosh A. Neuron. 1999; 22: 799-808Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), later studies showed that recruitment of CBP by itself was sufficient for transcriptional activation (19Cardinaux J.R. Notis J.C. Zhang Q. Vo N. Craig J.C. Daniel F.M. Brennan R.G. Goodman R.H. Mol. Cell. Biol. 1999; 20: 1546-1552Crossref Scopus (155) Google Scholar, 20Du K. Asahara H. Jhala U.S. Wagner B.L. Montminy M. Mol. Cell. Biol. 2000; 20: 4320-4327Crossref PubMed Scopus (90) Google Scholar). It remains possible, however, that phosphorylation of CBP/p300 by CaM kinase IV could contribute to signaling by augmenting the transcriptional response. Phosphorylation and activation of CBP by MAPK was first reported by Janknecht and Nordheim (21Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar). Activation of MAPK through the Ras pathway by insulin or nerve growth factor was reported to recruit pp90Rsk to the third zinc finger domain of CBP in a manner that prevents the binding of essential CBP effectors such as RNAPII (22Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Interestingly, modulation of CBP by pp90Rsk does not appear to require its catalytic kinase activity. This inhibitory effect of pp90Rsk has not been seen by other investigators, however, so it is possible that it is cell type-specific (23Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1085) Google Scholar). Clearly, the understanding of CBP/p300 regulation by phosphorylation remains a major topic for future study. In addition to their intrinsic acetyltransferase functions, CBP and p300 are known to associate with additional HATs, including P/CAF, SRC-1, and p/CIP. Why so many different HATs are required for transcriptional regulation is unknown, but the answer may lie in the differing preferences of these enzymes for free histones as compared with nucleosomes and their distinct targets within the histone substrates (for review, see Ref. 24Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6623) Google Scholar). Although it has been suggested that the HAT domains in CBP/p300 are highly related to those in P/CAF and GCN5 (25Martinez-Balbas M.A. Bannister A.J. Martin K. Haus-Seuffert P. Meisterernst M. Kouzarides T. EMBO J. 1998; 17: 2886-2893Crossref PubMed Scopus (227) Google Scholar), the primary sequences of these domains are actually quite different. Moreover, these differences are significant enough to allow the development of specific inhibitors of the P/CAF and CBP/p300 enzymatic activities (26Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ait S. Khalil E.M. Vassilev A. Wolffe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; 5: 589-595Abstract Full Text Full Text PDF PubMed Google Scholar). Kraus et al. (27Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar) have shown that the p300-mediated activation of estrogen receptor (ER) function on reconstituted chromatin depends upon the intrinsic acetyltransferase activity of the coactivator, demonstrating that this enzymatic function is essential in the context of chromatin. Because histone acetylation is not required for transcription of naked DNA templates, these results imply that some component of chromatin is the acetylation target. These conclusions are supported by the results of Ludlam et al., 2W. Ludlam, R. H. Goodman, and S. Smolik, submitted for publication. which show that flies containing an acetyltransferase-deficient form of CBP are incapable of activating specific target genes in vivo. Although confirming the importance of the CBP enzymatic function, these studies do not identify the acetylation target. Recent studies have shown that the HAT activity of CBP/p300 is directed toward nucleosomes through interactions with the histone chaperone, RbAp 48 (28Zhang Q. Vo N. Goodman R.H. Mol. Cell. Biol. 2000; 20: 4970-4978Crossref PubMed Scopus (54) Google Scholar). Moreover, Ito et al. (29Ito T. Ikehara T. Nakagawa T. Kraus W.L. Muramatsu M. Genes Dev. 2000; 14: 1899-1907PubMed Google Scholar) have found that histone acetylation by p300 facilitates the transfer of H2A-H2B from nucleosomes to the chaperone protein NAP-1. In this model, the recruitment of p300 and the subsequent histone acetylation follow a chromatin remodeling step mediated by ATP-dependent proteins in the ISWI family. These results are consistent within vivo chromatin immunoprecipitation experiments in yeast showing that the association of SWI/SNF components on the HO promoter is required for the subsequent HAT recruitment (30Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 31Krebs J.E. Kuo M.H. Allis C.D. Peterson C.L. Genes Dev. 1999; 13: 1412-1421Crossref PubMed Scopus (251) Google Scholar). Whether the release of H2A-H2B results from the acetylation of these proteins directly or whether other nucleosomal components are the primary targets of the acetyltransferases remains to be determined. Acetylation of transcription factors (through FAT, factoracetyltransferase activities) by CBP/p300 may provide an equally important mode of regulation. First identified in the context of the tumor suppressor p53 (32Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2177) Google Scholar), acetylation of transcription factors has been increasingly recognized as a mechanism of gene regulation. In some instances, acetylation has clearly been shown to increase the binding of transcription factors to DNA (32Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2177) Google Scholar). In most cases, however, the mechanism of activation is unknown. Recent evidence suggests that coactivator acetyltransferases might also serve to disrupt activator and repressor complexes. For example, Evans and co-workers (33Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar) have shown that the recruitment of p300 to the ligand-activated ER leads to the acetylation of ACTR (an associated acetyltransferase), disruption of the ACTR-p300-ER complex, and the termination of transcription. Another possibility, consistent with the multistep model of transcription proposed by Roeder (reviewed in Refs.34Roeder R.G. Chambon P. Fukasawa T. Kornberg R. Coath C. Transcription Regulation in Eukaryotes. Human Frontiers Science Program, Strausbourg, France1999: 106-121Google Scholar and 35Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), is that CBP/p300-mediated acetylation of the complex may promote the transition from a CBP/p300-dependent to a mediator-dependent stage of transcription (see below). A converse mechanism was proposed by Zhang et al. 3Zhang, Q., Yao, H., Vo, N., and Goodman, R. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97,14323–14328. In these studies, interaction of the histone deacetylase-binding corepressor, CtBP (carboxyl-terminal bindingprotein), to a variety of transcriptional repressors was shown to be blocked by acetylation of the CtBP interaction sites. In this instance, as in the classical histone acetylation model, acetylation is proposed to activate transcription by disrupting protein complexes involved in repression. Paradoxically, acetylation by CBP can also cause transcriptional repression in some systems. For example, in flies, CBP has been shown to inhibit wingless signaling by acetylating the Drosophila homologue of the high mobility group protein, LEF/TCF-1 (36Waltzer L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (326) Google Scholar). Acetylation of a specific residue in LEF/TCF-1 is believed to block the binding of the coactivator β-catenin/Armadillo, one of the intermediates in the wingless signaling pathway. In support of this model, CBP loss-of-function mutants have been found to suppress the effects of an Armadillo mutation. It is somewhat surprising that CBP and p300, which mediate the activities of so many different transcription factors, might be present in the cell at limiting concentrations. Nonetheless, there is considerable evidence that this is the case. Even discounting experiments involving transcription factor overexpression, which would perhaps be expected to exceed the capacity of the endogenous CBP/p300, studies have shown that relatively small decreases in the concentrations of coactivator are deleterious. For example, in the human Rubinstein-Taybi syndrome, loss of a single CBP allele results in severe developmental defects (37Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C. Masuno M. Tommerup N. van Ommen G.J. Goodman R.H. Peters D.J. Bruening M.H. Nature. 1995; 376: 348-351Crossref PubMed Scopus (1024) Google Scholar). The idea that CBP/p300 levels are limiting is also supported by tissue culture experiments, as exemplified by the studies of Hottiger et al. (38Hottiger M.O. Felzien L.K. Nabel G.J. EMBO J. 1998; 17: 3124-3134Crossref PubMed Scopus (127) Google Scholar), which examined the ability of interferon-α (IFN-α) to inhibit tumor necrosis factor-α-stimulated human immunodeficiency virus gene expression. This inhibition was shown to be mediated by competition between STAT-2 (stimulated by IFN-α) and the p65 subunit of NF-κB (stimulated by tumor necrosis factor-α) for a shared binding site within the first zinc finger domain of CBP/p300. It is not certain that two transcription factors must compete for the same binding site to be mutually antagonistic, however. If CBP/p300 levels are truly limiting, it is possible that they could be directed toward specific genes to the exclusion of others. Testing this hypothesis will require the use of experimental paradigms that do not involve the overexpression of exogenous transcription factors. The idea that CBP/p300 contributes to transcriptional synergy is probably best supported by studies of the IFN-β enhanceosome (see below), but other complex promoters have also been shown to contain binding sites for multiple CBP/p300-interacting transcription factors. (Indeed, given the large number of factors that bind CBP/p300, it is difficult to imagine a promoter where this would not be the case.) Nonetheless, although transcriptional synergy through CBP/p300 is an appealing model, it has not been shown conclusively that these coactivators interact with multiple transcription factors simultaneously. In addition, although the recruitment of coactivators to the enhanceosome appears to be required for synergistic activation, tethering CBP/p300 to the promoter through a heterologous DNA-binding domain is not sufficient. As suggested by Merika et al.(39Merika M. Williams A.J. Chen G. Collins T. Thanos D. Mol. Cell. 1998; 1: 277-287Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), the activation domains of the individual transcription factors comprising the enhanceosome may contribute critical interactions with basal factors. CBP/p300 may only participate in a step in the transcriptional It may be equally important for CBP/p300 to be by other factors, such as the complex, for transcription to In support of this Kraus and Kadonaga W.L. Kadonaga J.T. Genes Dev. 1998; PubMed Scopus Google Scholar) have demonstrated that although both the ER and p300 are for transcriptional from chromatin templates, only the ER is required for processes are regulated through the interactions of a large number of complexes. of basal transcription factors at the promoter the result of these Regulation is by additional components such as and complexes with specific and are complexes that promote the recruitment of coactivators and the RNAPII complex to sites of transcription. In one example, of the enhanceosome recruitment of factors, and to in the IFN-β promoter to interaction between the binding proteins and the RNAPII complex M.G. Lin T. Mol. Cell. 1998; 1: Full Text Full Text PDF PubMed Scopus Google Scholar, T.K. T. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The critical role of CBP/p300 in this context is to promote the of the and complex to multiple of transcription J. Merika M. N. Chen G. Thanos D. EMBO J. 1999; PubMed Scopus Google Scholar). of CBP/p300 from this complex the of transcription J. K. Thanos D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). CBP/p300 may also participate in IFN-β gene transcription by acetylating its for DNA and disrupting the enhanceosome J. Merika M. N. Chen G. Thanos D. EMBO J. 1999; PubMed Scopus Google N. Merika M. J. K. Chen G. Thanos D. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus (305) Google Scholar, M. J.E. M. Vassilev A. Nakatani Y. Martin B. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Whether actually on the HAT activity of CBP/p300 has not been however. Recent evidence demonstrating an HAT activity in one of the DNA-binding proteins found in the IFN-β suggests that the CBP/p300 HAT function could be H. L. Chiu R. K. Taira K. Nakatani Y. Yokoyama K.K. Nature. 2000; PubMed Scopus Google Scholar). complexes provide the step in the activation to the recruitment of the general transcriptional These and share a of components (reviewed in R.E. Nature. 1999; PubMed Scopus Google Scholar and 35Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The between these complexes and CBP/p300 have not been entirely however. the first of the to be does not contain CBP/p300 and HAT activity H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, M. S. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). with its absence of associated HAT activities and with its to the yeast coactivator functions with activators on naked DNA templates, additional functions with chromatin remain to be In contrast, and transcription has been on chromatin templates, the presence of some components in the with DNA the association or of CBP/p300 other with and complexes P.A. Zhou S. S. W. R. Nature. 1999; PubMed Scopus Google Scholar, C. B.D. V. M. H. P. Nature. 1999; PubMed Scopus Google Scholar). components also additional essential functions with chromatin templates, a for additional interactions for of the complex in this is the role of CBP/p300 in and CBP/p300 transcriptional activators to the complex, or is its role to in a manner that the to function at a subsequent stage of of the regarding the association of CBP/p300 with complexes may from the different to these was a functional H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and identified through their binding to transcription factors C. B.D. V. M. H. P. Nature. 1999; PubMed Scopus Google Scholar). It is that the proteins to and interact with CBP/p300 and the in a mutually This would be consistent with the multistep interaction model proposed by Roeder S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). In this model, transcription factors have the capacity to interact with both CBP/p300 and the but the interactions might be nucleosomal have been by CBP/p300. Nonetheless, it may be premature to conclude that do not contain et al. Y. J. Kornberg Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) have that the yeast interactions with nucleosomes and a that nucleosomal histone at least in yeast does not contain complexes do have intrinsic HAT activity. These studies the of whether the complexes might also contain associated HATs or whether this activity must be by a distinct complex containing CBP/p300. In the step model of transcriptional coactivator HATs such as CBP/p300 are in the and of the basal transcription how CBP/p300 the for subsequent in the transcriptional remains to be determined. the activation, and termination of CBP/p300 functions will on how cells use transcriptional complexes to mediate specific to cellular We and W. Kraus for protein protein histone acetyltransferase RNA polymerase II holoenzyme protein kinase A protein kinase factor acetyltransferase estrogen receptor interferon-α protein protein

Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by posttranslational modification of histones.The regulation of histone acetyltransferases (HATs) and histone deacetylases(HDACs) is one of important post translational modification (PTMs).Aspergillus flavus is the main source of aflatoxin,the most important mycotoxins in the world and food supplies.HDACs can play an important role in secondary metabolites production and normal vegetative growth.The focus of our work was on underastanding the possible effect of HDAC inhibitors ,SAHA, on growth, aflatoxin synthesis, conidiation and morphology in A.flavus.The result indicate that SAHA inhibits the ability to produce aflatoxin B1 and also causes a “fluffy” phenotype.This change was dose-dependent and not hereditary.Three HDACs genes were founded in this fungi ,HosA,HosB and Rpd1 that have conserved domains like other fungi .This results suggest that HDACs have important roles in A. flavus and HDACi could prove to be a valid approach for selective treatment of infection cause by Aspergillus species and other fungi and be a good subject for epigenetic remodeling in fungi .