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

Transcription regulation across levels of chromatin organization

With advancements in high-throughput sequencing and high-resolution microscopy techniques, the significance of genome organization in transcription regulation is rapidly unveiling. Nonetheless, we are yet far from completely understanding this key relationship. In this thesis, I utilize some of the cutting-edge high-throughput sequencing techniques along with the power of Drosophila as a model to understand some of the molecular mechanisms active at various levels of chromatin organization that influence gene expression. At the level of DNA sequence, our analysis revealed occupancy of CBP/p300, a transcription co-activator and histone acetyltransferase at insulator regions genome wide, we also identified a novel role of CBP as a barrier to heterochromatin spreading at these insulator regions. To gain insight into transcription regulation at level of nucleosomes, we perturbed histone modifications either chemically, by inhibiting histone deacetylases (HDACs), or genetically, by creating Drosophila mutants for the H3K14 residue.  We found that the immediate transcriptional response to HDAC inhibition is only up-regulation (96 genes). Moreover, our results suggest that histone acetylation stimulates transcription by releasing promoter-proximal paused Pol II into elongation. In another study we discovered that acetylation of histone 3 lysine 14, H3K14ac, decorates some genes that lack canonical histone marks, thereby constituting a unique chromatin state. Further, Drosophila mutants expressing only H3K14R histones revealed that this residue is vital for expression of these genes, for animal survival and for developmental patterning. To further understand gene regulation during tissue specification in early embryo development, we utilized dorsoventral (DV) patterning as a model along with PRO-seq, ATAC-seq and ChIP-seq. We identified zygotically transcribed DV patterning genes that are spatially and temporally resolved. Most interestingly, the DV patterning genes are all highly paused and promoter proximal paused Pol II is released into active elongation in a tissue-specific manner. Finally our single cell RNA-seq (scRNA-seq) and HiC data in DV mutants revealed that differential regulation of gene expression occurs independently of differences in higher-order chromatin organization. Collectively, we provide evidence that transcription is being modulated at various levels of chromatin organization and our results suggest that histone modifications but not higher order chromatin organization influence transcriptional output.Med framsteg inom high-throughput sekvensering och högupplösta mikroskopitekniker uppenbaras betydelsen av genomets organisation för transkriptionsreglering i snabb takt. Ändå är vi ännu långt ifrån att helt och hållet att förstå detta viktiga förhållande. I denna avhandling använder jag några banbrytande high-throughput sekvenseringstekniker tillsammans med Drosophila som en kraftfull modell för att förstå några av de molekylära mekanismer som är aktiva vid olika nivåer av kromatinorganisering och som påverkar genuttryck. På DNA-sekvensnivå visade vår analys att CBP/p300, en transkriptions-coaktivator och histonacetyltransferas, intar insulator områden över hela genomet. Vi identifierade också en ny roll för CBP som en barriär mot heterokromatinspridning vid dessa insulator-regioner. För att få insikt i transkriptionsreglering på nukleosomnivå så rubbade vi histonmodifieringar antingen kemiskt, genom att hämma histondeacetylaser (HDAC), eller genetiskt genom att skapa Drosophila mutanter för H3K14. Vi fann att det omedelbara transkriptionella svaret på HDAC-hämning endast omfattar uppreglering (96 gener). Dessutom tyder våra resultat på att histonacetylering stimulerar transkription genom att frisätta promotor-proximal pausad Pol II till elongering. I en annan studie upptäckte vi att acetylering av histon 3 lysin 14, H3K14ac, dekorerar vissa gener som saknar typiska histonmodifieringar och därmed utgör ett unikt kromatintillstånd. Vidare avslöjade Drosophila mutanter som endast uttryckte H3K14R histoner att denna aminosyra är avgörande för uttryck av dessa gener, för djurets överlevnad och för mönsterbildande under djurets utveckling. För att ytterligare förstå genreglering under specificering av olika vävnader i tidig embryoutveckling använde vi dorsoventral (DV) mönsterbildning som modell tillsammans med PRO-seq, ATAC-seq och ChIP-seq. Vi identifierade zygotiskt transkriberade DV-mönstergener som kunde separeras rumsligt och över tid. Det mest intressanta är att DV-mönstergenerna är högt pausade och att promotor-proximal pausad Pol II frigörs till aktiv elongering på ett vävnadsspecifikt sätt. Slutligen uppenbarade vår enkelcells RNA-seq (scRNA-seq) och HiC-data i DV-mutanter att differentiell reglering av genuttryck sker oberoende av skillnader i högre nivåer av kromatinorganisation. Sammantaget tillhandahåller vi bevis på att transkription moduleras vid olika nivåer av kromatinorganisation och våra resultat tyder på att histonmodifieringar, men inte högre nivåer av kromatinorganisation, påverkar gentranskription

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

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.Erratum to: CBP binding outside of promoters and enhancers in Drosophila melanogasterEpigenetics &amp; Chromatin 2016 9:3810.1186/s13072-015-0042-4</p

Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Background: During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results: The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions: Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model

Global loss of cellular m 6 A RNA methylation following infection with different SARS-CoV-2 variants

International audienceInsights into host–virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N 6 -Methyladenosine modification (m 6 A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m 6 A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m 6 A in cellular RNAs, whereas m 6 A is detected abundantly in viral RNA. METTL3, the m 6 A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m 6 A than did the B.1 and B.1.1.7 variants. We also observed a loss of m 6 A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m 6 A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m 6 A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m 6 A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m 6 A-dependent manner