Histone deacetylase (HDAC) inhibitors have been considered as anti-leukemic agents but have shown poor efficacy in clinical trials. In this study, we investigated the immediate transcriptional response to the HDAC inhibitor SAHA (vorinostat) in healthy CD34⁺ blood stem/progenitor cells and myeloblasts from patients with primary acute myeloid leukemia (AML) carrying TET2 and NPM1 mutations. We found that although healthy CD34⁺ and AML cells differed substantially at the transcriptional level, they responded very similarly to 10-minute SAHA treatment. HDAC inhibition led to a global increase in histone acetylation; however, only 150 to 250 genes were upregulated. These were involved in oxidative stress, metabolism, chromatin regulation, cell cycle control, and cell death, and the vast majority was upregulated in both healthy and AML cells. Upregulated genes were more highly acetylated compared with average expressed genes and had higher levels of promoter-proximal paused RNA polymerase II (Pol II) before treatment. Upon HDAC inhibition, upregulated genes increased BRD4 occupancy the most and released paused Pol II into transcription elongation. Our results suggest that the immediate effect of HDAC inhibition is to trigger release of paused Pol II into elongation. We speculate that the similar transcriptional response in healthy and leukemic cells may contribute to the poor efficacy of HDAC inhibitors in patients with hematologic malignancies.

Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. Here we show that specific snoRNAs are required for the activation of immune response genes and for survival during viral infections in Drosophila melanogaster. We have studied snoRNA:U3:9B, a chromatin-associated snoRNA that binds to a large number of protein coding genes, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B and study its function in vivo. SnoRNA:U3:9B-deficient larvae are viable but failed to develop into pupae when challenged by expression of a Sindbis virus replicon. SnoRNA:U3:9B is localized to immune genes in vivo and the chromatin decompaction and gene activation typically observed at immune genes following infection are abolished in snoRNA:U3:9B-deficient larvae, which suggests that this snoRNA acts locally to regulate chromatin accessibility. Mechanistically, snoRNA:U3:9B is required for the recruitment of the chromatin remodeler Brahma to a set of target immune genes. In summary, these results uncover an antiviral defense mechanism that relies on a snoRNA for the recruitment of a chromatin remodeling factor to immune genes to facilitate immune gene activation.

Immediately after fertilization, the genome is transcriptionally quiescent. Maternally encoded pioneer factors reprogram the chromatin state and facilitate transcription of the zygotic genome. In Drosophila, transcription is initiated by the pioneer factor Zelda. While Zelda-occupied sites are enriched with histone acetylation, a post-translational mark associated with active cis-regulatory regions, the functional relationship between Zelda and histone acetylation remained unclear. We show that Zelda-mediated recruitment of the histone acetyltransferase CREB-binding protein (CBP) is essential for zygotic transcription. CBP catalytic activity is necessary for the release of RNA polymerase II (RNA Pol II) into elongation and for embryonic development. However, CBP also activates transcription independent of acetylation through RNA Pol II recruitment. Neither CBP-mediated acetylation nor CBP itself is required for the pioneering function of Zelda. Our data suggest that pioneer-factor-mediated recruitment of CBP is a conserved mechanism required to activate zygotic transcription but is separable from the function of pioneer factors in restructuring chromatin accessibility.

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.

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.

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.

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.

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.

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.

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.