Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide 1,2 has successfully identified specific subtypes of regulatory elements 3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb-Response Elements 4, chromatin states 5, transcription factor binding sites (TFBS) 6–9, PolII regulation 8, and insulator elements 10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome based on more than 300 chromatin immuno-precipitation (ChIP) datasets for eight chromatin features, five histone deacetylases (HDACs) and thirty-eight site-specific transcription factors (TFs) at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and we validated a subset of predictions for promoters, enhancers, and insulators in vivo. We also identified nearly 2,000 genomic regions of dense TF binding associated with chromatin activity and accessibility. We discovered hundreds of new TF co-binding relationships and defined a TF network with over 800 potential regulatory relationships.

The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. Here, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, we provide evidence for the independence of chromatin organization and dorsoventral gene expression. We define tissue-specific enhancers and link them to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. Our results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active.

Small differences in the levels of an extracellular signaling molecule can specify cell fate during development. Threshold responses are often determined at the level of transcription. Cell-specific and spatially localized patterns of gene expression depend on combinations of sequence-specific activators and repressors that bind to extensive cis-regulatory regions. Different mechanisms for integrating this complex regulatory information are discussed, particularly the role of coregulatory proteins, which are recruited to the DNA template by sequence-specific transcription factors. Recent studies suggest that a growing set of coactivators and corepressors mediate communication between diverse upstream regulatory proteins and the core RNA polymerase II transcription complex.

Abstract. Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAFIs), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAFIs, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAFI63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.T hE initiation of transcription by all three eukaryotic RNA polymerases depends on the assembly of specific transcription factors at the corresponding promoters, and substantial progress has been recently made in the definition of these molecules (for recent reviews see Reeder, 1990; Gabrielson and Sentenac, 1991;Drapkin et al., 1993). A particularly well-studied transcription factor is the TATA-binding protein (TBP) 1. Although TBP was originally thought to be a specific component of the RNA polymerase II transcription machinery, recent evidence indicates that it is also required for transcription by RNA polymerases I and III (for review see Hernandez, 1993;Goodrich and Tjian, 1994). In the case of RNA polymerase II promoters containing a TATA sequence, TBP acts as a DNA-binding protein, whereas in the TATA-less promoters from RNA polymerases II, I, and III, TBP

Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent.The Ada2 protein was first described for yeast, in which its inactivation could relieve the toxicity caused by overexpression of the strong transcriptional activation domain in the viral VP16 protein (6). This provided one of the earliest pieces of evidence for the existence of transcriptional adapters or coactivators. Ada2 proteins were subsequently shown to exist in protein complexes containing the Gcn5 histone acetyltransferase (23). Histone acetylation has been linked to transcriptional activation for a long time, given the observation that actively transcribed regions tend to be hyperacetylated, whereas transcriptionally silent regions generally are hypoacetylated (3). With the identification of the first histone acetyltransferase (HAT) as the Tetrahymena homolog of Gcn5 (13) and the subsequent demonstration that other transcriptional regulators, such as the coactivators p300 and CBP, steroid receptor coactivators, and the TFIID subunit TAF1, also possess intrinsic HAT activity, the link between acetylation and transcription was firmly established (12). In addition, an important role for histone acetylation in DNA replication and repair is emerging (27,38).Gcn5 is evolutionarily conserved and is present in several multiprotein complexes, such as the SAGA and ADA complexes in yeast and the PCAF, STAGA, and TFTC complexes in mammals (17). In yeast, Gcn5 is contained in a trimeric subcomplex with the Ada2 and Ada3 proteins within SAGA and ADA complexes (21,24). Ada2 binds to Gcn5, and deletion of Ada2 results in a loss of Gcn5 from bot...