Beyond histones: the elusive substrates of chromatin regulators.

Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many additional, nonhistone substrates, making it difficult to ascribe the most relevant modification. In this issue of Genes & Development, Crain and colleagues (doi:10.1101/gad.351698.124) have combined a powerful histone replacement system with mutational analysis of a chromatin regulator and a chromatin reader in Drosophila melanogaster Importantly, they discovered that genes controlled by the histone 4 lysine 20 (H4K20) methyltransferase Set8 and the protein recognizing H4K20 monomethylation, L(3)mbt, differ substantially from those affected by mutation of H4K20 itself. This demonstrates that H4K20 is not the key substrate for Set8 but that methylation of other, unidentified proteins mediates its effects on transcription.

Enhancer-promoter communication in Drosophila developmental gene transcription.

Enhancers play an essential role in gene regulation by receiving cues from transcription factors and relaying these signals to modulate transcription from target promoters. Enhancer-promoter communications occur across large linear distances of the genome and with high specificity. The molecular mechanisms that underlie enhancer-mediated control of transcription remain unresolved. In this review, we focus on research in Drosophila uncovering the molecular mechanisms governing enhancer-promoter communication and discuss the current understanding of developmental gene regulation. The functions of protein acetylation, pausing of RNA polymerase II, transcriptional bursting, and the formation of nuclear hubs in the induction of tissue-specific programs of transcription during zygotic genome activation are considered.

Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network.

BACKGROUND: Formation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning of the Drosophila embryo. This involves interactions between transcriptional enhancers and promoters in a chromatin context, but how the chromatin landscape influences transcription is not fully understood. RESULTS: Here we comprehensively resolve differential transcriptional and chromatin states during Drosophila DV patterning. We find that RNA Polymerase II pausing is established at DV promoters prior to zygotic genome activation (ZGA), that pausing persists irrespective of cell fate, but that release into productive elongation is tightly regulated and accompanied by tissue-specific P-TEFb recruitment. DV enhancers acquire distinct tissue-specific chromatin states through CBP-mediated histone acetylation that predict the transcriptional output of target genes, whereas promoter states are more tissue-invariant. Transcriptome-wide inference of burst kinetics in different cell types revealed that while DV genes are generally characterized by a high burst size, either burst size or frequency can differ between tissues. CONCLUSIONS: The data suggest that pausing is established by pioneer transcription factors prior to ZGA and that release from pausing is imparted by enhancer chromatin state to regulate bursting in a tissue-specific manner in the early embryo. Our results uncover how developmental patterning is orchestrated by tissue-specific bursts of transcription from Pol II primed promoters in response to enhancer regulatory cues.

Separation of transcriptional repressor and activator functions in Drosophila HDAC3.

The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex, and its canonical function is in transcriptional repression, but it can also activate transcription. Here, we show that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhances repressor activity beyond wild type in the early embryo. We conclude that the crucial functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.

Inhibition of mitochondrial transcription by the neurotoxin MPP.

The neurotoxin MPP+ triggers cell death of dopamine neurons and induces Parkinson's disease symptoms in mice and men, but the immediate transcriptional response to this neurotoxin has not been studied. We therefore treated human SH-SY5Y cells with a low dose (0.1 mM) of MPP+ and measured the effect on nascent transcription by precision run-on sequencing (PRO-seq). We found that transcription of the mitochondrial genome was significantly reduced already after 30 min, whereas nuclear gene transcription was unaffected. Inhibition of respiratory complex I by MPP+ led to reduced ATP production, that may explain the diminished activity of mitochondrial RNA polymerase. Our results show that MPP+ has a direct effect on mitochondrial function and transcription, and that other gene expression or epigenetic changes induced by this neurotoxin are secondary effects that reflect a cellular adaptation program.

p300/CBP sustains Polycomb silencing by non-enzymatic functions.

Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here, we show that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

Transgenerational effects of gamma radiation dose and dose rate on Drosophila flies irradiated at an early embryonal stage.

Ionizing radiation (IR) kills cells mainly through induction of DNA damages and the surviving cells may suffer from mutations. Transgenerational effects of IR are well documented, but the exact mechanisms underlying them are less well understood; they include induction of mutations in germ cells and epigenetic inheritance. Previously, effects in the offspring of mice and zebrafish exposed to IR have been reported. A few studies also showed indications of transgenerational effects of radiation in humans, particularly in nuclear power workers. In the present project, short- and long-term effects of low-dose-rate (LDR; 50 and 97 mGy/h) and high-dose-rate (HDR; 23.4, 47.1 and 495 Gy/h) IR in Drosophila embryos were investigated. The embryos were irradiated at different doses and dose rates and radiosensitivity at different developmental stages was investigated. Also, the survival of larvae, pupae and adults developed from embryos irradiated at an early stage (30 min after egg laying) were studied. The larval crawling and pupation height assays were applied to investigate radiation effects on larval locomotion and pupation behavior, respectively. In parallel, the offspring from 3 Gy irradiated early-stage embryos were followed up to 12 generations and abnormal phenotypes were studied. Acute exposure of embryos at different stages of development showed that the early stage embryo is the most sensitive. The effects on larval locomotion showed no significant differences between the dose rates but a significant decrease of locomotion activity above 7 Gy was observed. The results indicate that embryos exposed to the low dose rates have shorter eclosion times. At the same cumulative dose (1 up to 7 Gy), HDR is more embryotoxic than LDR. We also found a radiation-induced depigmentation on males (A5 segment of the dorsal abdomen, A5pig-) that can be transmitted up to 12 generations. The phenomenon does not follow the classical Mendelian laws of segregation.

The control of transcriptional memory by stable mitotic bookmarking.

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. We show that, during mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we report that GAF binding establishes competence for rapid activation upon mitotic exit.

Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning.

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.

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation.

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Release of promoter-proximal paused Pol II in response to histone deacetylase inhibition.

A correlation between histone acetylation and transcription has been noted for a long time, but little is known about what step(s) in the transcription cycle is influenced by acetylation. We have examined the immediate transcriptional response to histone deacetylase (HDAC) inhibition, and find that release of promoter-proximal paused RNA polymerase II (Pol II) into elongation is stimulated, whereas initiation is not. Although histone acetylation is elevated globally by HDAC inhibition, less than 100 genes respond within 10 min. These genes are highly paused, are strongly associated with the chromatin regulators NURF and Trithorax, display a greater increase in acetylation of the first nucleosomes than other genes, and become transcriptionally activated by HDAC inhibition. Among these rapidly up-regulated genes are HDAC1 (Rpd3) and subunits of HDAC-containing co-repressor complexes, demonstrating feedback regulation upon HDAC inhibition. Our results suggest that histone acetylation stimulates transcription of paused genes by release of Pol II into elongation, and that increased acetylation is not a consequence of their enhanced expression. We propose that HDACs are major regulators of Pol II pausing and that this partly explains the presence of HDACs at active genes.

Gene activation by dCas9-CBP and the SAM system differ in target preference.

Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in Drosophila cells. Our results suggest that different rate-limiting steps in the transcription cycle are affected by dCas9-CBP and the SAM system, and that comparing these activators may be useful for mechanistic studies of transcription as well as for increasing the number of hits in genome-wide overexpression screens.

CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II.

Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.

Atrophin controls developmental signaling pathways via interactions with Trithorax-like.

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.

Erratum to: CBP binding outside of promoters and enhancers in Drosophila melanogaster.

[This corrects the article DOI: 10.1186/s13072-015-0042-4.].

H2A deubiquitinases UBP12/13 are part of the Arabidopsis polycomb group protein system.

Polycomb group (PcG) proteins form an epigenetic memory system in plants and animals, but interacting proteins are poorly known in plants. Here, we have identified Arabidopsis UBIQUITIN SPECIFIC PROTEASES (USP; UBP in plant and yeasts) 12 and 13 as partners of the plant-specific PcG protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1). UBP12 binds to chromatin of PcG target genes and is required for histone H3 lysine 27 trimethylation and repression of a subset of PcG target genes. Plants lacking UBP12 and UBP13 developed autonomous endosperm in the absence of fertilization. We have identified UBP12 and UBP13 as new proteins in the plant PcG regulatory network. UBP12 and UBP13 belong to an ancient gene family and represent plant homologues of metazoan USP7. We have found that Drosophila USP7 shares a function in heterochromatic gene repression with UBP12/13 and their homologue UBP26. In summary, we demonstrate that USP7-like proteins are essential for gene silencing in diverse genomic contexts.

Initiation of diverse epigenetic states during nuclear programming of the Drosophila body plan.

Epigenetic patterns of histone modifications contribute to the maintenance of tissue-specific gene expression. Here, we show that such modifications also accompany the specification of cell identities by the NF-κB transcription factor Dorsal in the precellular Drosophila embryo. We provide evidence that the maternal pioneer factor, Zelda, is responsible for establishing poised RNA polymerase at Dorsal target genes before Dorsal-mediated zygotic activation. At the onset of cell specification, Dorsal recruits the CBP/p300 coactivator to the regulatory regions of defined target genes in the presumptive neuroectoderm, resulting in their histone acetylation and transcriptional activation. These genes are inactive in the mesoderm due to transcriptional quenching by the Snail repressor, which precludes recruitment of CBP and prevents histone acetylation. By contrast, inactivation of the same enhancers in the dorsal ectoderm is associated with Polycomb-repressed H3K27me3 chromatin. Thus, the Dorsal morphogen gradient produces three distinct histone signatures including two modes of transcriptional repression, active repression (hypoacetylation), and inactivity (H3K27me3). Whereas histone hypoacetylation is associated with a poised polymerase, H3K27me3 displaces polymerase from chromatin. Our results link different modes of RNA polymerase regulation to separate epigenetic patterns and demonstrate that developmental determinants orchestrate differential chromatin states, providing new insights into the link between epigenetics and developmental patterning.

CBP binding outside of promoters and enhancers in Drosophila melanogaster.

BACKGROUND: CREB-binding protein (CBP, also known as nejire) is a transcriptional co-activator that is conserved in metazoans. CBP plays an important role in embryonic development and cell differentiation and mutations in CBP can lead to various diseases in humans. In addition, CBP and the related p300 protein have successfully been used to predict enhancers in both humans and flies when they occur with monomethylation of histone H3 on lysine 4 (H3K4me1). RESULTS: Here, we compare CBP chromatin immunoprecipitation sequencing data from Drosophila S2 cells with modENCODE data and show that CBP is bound at genomic sites with a wide range of functions. As expected, we find that CBP is bound at active promoters and enhancers. In addition, we find that the strongest CBP sites in the genome are found at Polycomb response elements embedded in histone H3 lysine 27 trimethylated (H3K27me3) chromatin, where they correlate with binding of the Pho repressive complex. Interestingly, we find that CBP also binds to most insulators in the genome. At a subset of these, CBP may regulate insulating activity, measured as the ability to prevent repressive H3K27 methylation from spreading into adjacent chromatin. CONCLUSIONS: We conclude that CBP could be involved in a much wider range of functions than has previously been appreciated, including Polycomb repression and insulator activity. In addition, we discuss the possibility that a common role for CBP at all functional elements may be to regulate interactions between distant chromosomal regions and speculate that CBP is controlling higher order chromatin organization.

Gene expression profiling of brakeless mutant Drosophila embryos.

The transcriptional co-regulator Brakeless performs many important functions during Drosophila development, but few target genes have been identified. Here we use Affymetrix microarrays to identify Brakeless-regulated genes in 2-4 h old Drosophila embryos. Robust multi-array analysis (RMA) and statistical tests revealed 240 genes that changed their expression more than 1.5 fold. We find that up- and down-regulated genes fall into distinct gene ontology categories. In our associated study [2] we demonstrate that both up- and down-regulated genes can be direct Brakeless targets. Our results indicate that the co-repressor and co-activator activities of Brakeless may result in distinct biological responses. The microarray data complies with MIAME guidelines and is deposited in GEO under accession number GSE60048.