Histone Variant H3.3 as a Transcription Rheostat in the Immune System

Weill Medical Coll Of Cornell Univ, New York NY

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Abstract

In the previous award period, we identified a critical function for the histone variant H3.3 and its phosphorylation in the rapid transcription of inflammatory genes across immune cells and especially in macrophages and germinal center B cells. Surprisingly, we also discovered that H3.3 expression (encoded by the two identical paralogs H3f3a and H3f3b) is dynamically regulated during inflammation in both human and mouse. With these genes among the most differentially expressed in inflammatory and disease states, it led us to predict that their modulation may act as a tunable regulator of immune responses. In support of this, we find that H3.3 levels are rate-limiting for stimulation-induced transcription and that inflammatory gene transcription titrates with graded expression of H3.3. Here, we test the hypothesis that H3.3 dosage acts as a rheostat for stimulation induced transcription across immune cells and their activation states. In Aim 1 we will define the impact of H3.3 dosage on inflammatory gene transcription, immune cell function, and defense to infection, using genetic mouse models with varying levels of H3.3 (from 0 to 4 copies) and competitive mixed bone marrow chimeras, in vitro transcriptomic and epigenomic studies, and carefully selected in vivo infection models (Listeria monocytogenes, Aspergillus fumigatus, and Plasmodium chabaudi). With these approaches, we will define the impact of H3.3 titration and its expression from the H3f3a and H3f3b loci on immune cell differentiation, activation, and pathogen control. We will identify which genes, and functional categories of genes, are most sensitive to H3.3 dosage. To understand the regulatory logic of H3.3 expression tuning, we will identify the gene enhancers and transcription factors that control cell-type-specific and stimulation- responsive expression of H3f3a and H3f3b. We apply innovative computational prediction of enhancers from single cell combined transcriptomic and epigenomic datasets and have identified putative enhancers at the H3f3a and H3f3b loci that we will functionally assess using genetic mouse models and in vitro enhancer perturbation assays (CRISPRi). In Aim 2 we will dissect the molecular mechanisms by which H3.3 regulates transcription, including its function in RNA polymerase II pause-release, co-transcriptional splicing, and establishing the histone post-translational modification landscape. To understand H3.3-dependent transcription processes we apply precision run-on sequencing (PRO-seq) and chromatin immunoprecipitation with exonuclease digestion (ChIP-exo) to map RNA polymerase II and other transcription regulatory factors at nucleotide resolution, and we quantify histone post-translational modifications that occur on or require H3.3. Finally, in Aim 3 we apply an innovative H3.3 mutagenesis screen to identify functional residues and modifications on H3.3. This screen will be performed in primary macrophages, enabling us to assess the impact of each mutation on inflammatory gene expression and cell activation, and will generate a first-of-its- kind functional roadmap of histone modifications in mammalian cells.