Project description:Recombinant antibodies to histone post-translational modifications (PTMs), with their essentially infinite renewability, could fundamentally eliminate a major source of low reproducibility in epigenetics research. Here, we report new recombinant antibodies to trimethylated Lys4 and Lys9, respectively, on histone H3. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications, including ChIP. These results demonstrate the feasibility of generating recombinant antibodies to a range of histone marks, which will accelerate epigenetics research.
Project description:Recombinant antibodies to histone post-translational modifications (PTMs), with their essentially infinite renewability, could fundamentally eliminate a major source of low reproducibility in epigenetics research. Here, we report new recombinant antibodies to trimethylated Lys4 and Lys9, respectively, on histone H3. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications, including ChIP. These results demonstrate the feasibility of generating recombinant antibodies to a range of histone marks, which will accelerate epigenetics research. We characterized recombinant antibodies with native ChIP using HEK293 cells followed by deep sequencing.
Project description:We characterize the epigenome of the human malaria vector Anopheles gambiae in midgut cells by mapping the distribution and levels of two post-translational histone modifications, H3K27ac and H3K27me3. These histone profiles were then correlated with levels of gene expression obtained by RNA-seq. ChIP-seq and RNA-seq were performed on adult female A. gambiae midguts. RNA-seq was performed on adult female A. gambiae salivary glands.
Project description:Post-translational modifications (PTMs) on histone proteins regulate genome accessibility and are frequently studied using chromatin immunoprecipitation (ChIP). In ChIP, an antibody putatively specific towards a histone PTM is used to map its genomic locations. ChIP experiments assume perfect antibody-epitope specificity, an assumption previously shown to be problematic, largely through peptide array studies. Among the most well-studied histone PTMs are the mono-, di-, and tri-methylation states of histone H3 lysine 4 (H3K4). While each state has been ascribed different biological functions, the methylation state specificity of antibodies used in these studies has not been systematically interrogated. Here, we use internally calibrated ChIP (ICeChIP) to comprehensively define the specificities of 52 commercially available antibodies marketed to distinguish the three methylation states of H3K4, allowing identification of both high- and low-specificity antibodies. We then conduct ICeChIP-seq with 18 such antibodies of varying specificity. We further find that the sum of H3K4me1 and H3K4me2 across enhancers contacting a promoter correlates strongly with gene expression for all genes, including housekeeping genes, and note that use of low-quality antibodies yields materially different biological interpretations. These results illustrate the way by which variable specificity of commercial antibodies contributes to the “reproducibility crisis” in biological research and demonstrates the need to carefully validate antibodies with techniques appropriate for the intended applications.
Project description:Post-translational modifications of histones determines cell lineage- or signal-specific gene expression. Depending on the type and combination of modifications, histones bind to functionally distinct effector proteins ('readers') that control gene activation or silencing. The current pharmacological modulation of the epigenome aims to control gene expression by regulation of the enzymes that catalyze post-translational histone modifications. Here we present a novel pharmacological approach that targets gene expression by interfering with the function of histone ?readers?. We describe the impact of a synthetic compound that selectively occupies the acetylated histone-binding pocket of the Bromodomain and Extra Terminal domain (BET) family of proteins and prevents their interaction with acetylated histones. The bromodomain blocking compound suppresses the expression of a specific subset of key inflammatory genes in activated macrophages and confers protection against LPS-induced septic shock in vivo. Our findings suggest that small molecules specifically targeting histone 'readers' can serve as a new generation of drugs to treat immune diseases. Microarray, ChIP-qPCR and ChIP-seq examination of control, 1H LPS stimulated bone-marrow-derived macrophages in the presence/absence of acetylated histone mimic in mouse.
Project description:We have performed ChIP-seq of H3K27ac and H3K4me3 histone post-translational modifications at three stages during induced neural differentiation (ESCs, neural progenitors and neurons). This data has allowed us to identify candidate enhancer-like signatures in the genome, which we have defined as regions that are enriched in H3K27ac and depleted of H3K4me3.
Project description:Enhancers are fundamental to gene regulation. Post-translational modifications by the small ubiquitin-like modifiers (SUMO) modify chromatin regulation enzymes, including histone acetylases and deacetylases. However, it remains unclear whether SUMOylation regulates enhancer marks, acetylation at the 27th lysine residue of the histone H3 protein (H3K27Ac). We hypothesize that SUMOylation regulates H3K27Ac. To test this hypothesis, we performed genome-wide ChIP-seq analyses. We discovered that knockdown (KD) of the SUMO activating enzyme catalytic subunit UBA2 reduced H3K27Ac at most enhancers. Bioinformatic analysis revealed that TFAP2C-binding sites are enriched in enhancers whose H3K27Ac was reduced by UBA2 KD. ChIP-seq analysis in combination with molecular biological methods showed that TFAP2C binding to enhancers increased upon UBA2 KD or inhibition of SUMOylation by a small molecule SUMOylation inhibitor. However, this is not due to the SUMOylation of TFAP2C itself. Proteomics analysis of TFAP2C interactome on the chromatin identified histone deacetylation (HDAC) machinery. TFAP2C KD reduced HDAC binding to chromatin and increased H3K27Ac marks at enhancer regions, suggesting that TFAP2C is involved in recruiting HDAC. Taken together, our findings provide important insights into regulation of enhancer marks by SUMOylation.
Project description:Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions as cells transit the cell cycle, but how remains unclear. Here, we use ChIP-seq in cell-cycle sorted cultured cells to define histone modification patterns through the cell cycle.
Project description:OCI-AML3 Acute myeloid leukemia cell line was used for ChIP-sequencing profiling of H3K4me3, H3K4me1, H3K9ac and H3K27ac histone post-translational modifications to identify active promoter and enhancer regions.