07-327 Sigma-AldrichAnti-acetyl-Histone H4 (Lys5) Antibody

Histone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits.We report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-R) are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. They contribute to many vascular-related pathologies, and hence VEGF-targeted therapies have been widely sought after. In this study, the authors investigated the ability of promoter-targeted small hairpin RNAs (shRNAs) to regulate VEGF-A, VEGF-C and VEGF-R1 in different cell lines. The authors identified shRNAs that can upregulate hVEGF-C at both the mRNA and protein levels, and differentially regulate hVEGF-A depending on the cell type. Likewise, the authors identified shRNA that downregulated VEGF-R1 gene expression. Hence, promoter-targeted shRNAs can affect endogenous gene expression not only bimodally, but also differentially in a cell-type specific manner. Importantly, all three genes tested were regulated by at least one shRNA, supporting the idea that nuclear RNA interference is a widespread phenomenon. The level of regulation across the panel of shRNAs varied maximally from a 2.2-fold increase to a 4-fold decrease. This level of change should be useful in fine-tuning and modulating target gene expression, which for potent molecules, such as VEGF-A and VEGF-C, can be very beneficial. These promoter-targeted shRNAs may facilitate the design and development of targeted, context-dependent strategies for both pro- and antiangiogenic therapies for the treatment of vascular-related pathologies.

During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.

Cell growth is attuned to nutrient availability to sustain homeostatic biosynthetic processes. In unfavorable environments, cells enter a nonproliferative state termed quiescence but rapidly return to the cell cycle once conditions support energetic needs. Changing cellular metabolite pools are proposed to directly alter the epigenome via histone acetylation. Here we studied the relationship between histone modification dynamics and the dramatic transcriptional changes that occur during nutrient-induced cell cycle reentry from quiescence in the yeast Saccharomyces cerevisiae. SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry showed that histone methylation-in contrast to histone acetylation-is surprisingly static during quiescence exit. Chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) revealed genome-wide shifts in histone acetylation at growth and stress genes as cells exit quiescence and transcription dramatically changes. Strikingly, however, the patterns of histone methylation remain intact. We conclude that the functions of histone methylation and acetylation are remarkably distinct during quiescence exit: acetylation rapidly responds to metabolic state, while methylation is independent. Thus, the initial burst of growth gene reactivation emerging from quiescence involves dramatic increases of histone acetylation but not of histone methylation.

Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.

Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (Zea mays L. ssp. parviglumis and Zea nicaraguensis) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome.

Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation.

We describe the genome-wide distributions and temporal dynamics of nucleosomes and post-translational histone modifications throughout the maternal-to-zygotic transition in embryos of Drosophila melanogaster. At mitotic cycle 8, when few zygotic genes are being transcribed, embryonic chromatin is in a relatively simple state: there are few nucleosome free regions, undetectable levels of the histone methylation marks characteristic of mature chromatin, and low levels of histone acetylation at a relatively small number of loci. Histone acetylation increases by cycle 12, but it is not until cycle 14 that nucleosome free regions and domains of histone methylation become widespread. Early histone acetylation is strongly associated with regions that we have previously shown to be bound in early embryos by the maternally deposited transcription factor Zelda, suggesting that Zelda triggers a cascade of events, including the accumulation of specific histone modifications, that plays a role in the subsequent activation of these sequences.

Histone acetylation has been implicated in learning and memory in the brain, however, its function at the level of the genome and at individual genetic loci remains poorly investigated. This study examines a key acetylation mark, histone H4 lysine 5 acetylation (H4K5ac), genome-wide and its role in activity-dependent gene transcription in the adult mouse hippocampus following contextual fear conditioning.Using ChIP-Seq, we identified 23,235 genes in which H4K5ac correlates with absolute gene expression in the hippocampus. However, in the absence of transcription factor binding sites 150 bp upstream of the transcription start site, genes were associated with higher H4K5ac and expression levels. We further establish H4K5ac as a ubiquitous modification across the genome. Approximately one-third of all genes have above average H4K5ac, of which ~15% are specific to memory formation and ~65% are co-acetylated for H4K12. Although H4K5ac is prevalent across the genome, enrichment of H4K5ac at specific regions in the promoter and coding region are associated with different levels of gene expression. Additionally, unbiased peak calling for genes differentially acetylated for H4K5ac identified 114 unique genes specific to fear memory, over half of which have not previously been associated with memory processes.Our data provide novel insights into potential mechanisms of gene priming and bookmarking by histone acetylation following hippocampal memory activation. Specifically, we propose that hyperacetylation of H4K5 may prime genes for rapid expression following activity. More broadly, this study strengthens the importance of histone posttranslational modifications for the differential regulation of transcriptional programs in cognitive processes.

Recent studies suggest a hierarchical model in which lineage-determining factors act in a collaborative manner to select and prime cell-specific enhancers, thereby enabling signal-dependent transcription factors to bind and function in a cell-type-specific manner. Consistent with this model, TLR4 signaling primarily regulates macrophage gene expression through a pre-existing enhancer landscape. However, TLR4 signaling also induces priming of ∼3,000 enhancer-like regions de novo, enabling visualization of intermediates in enhancer selection and activation. Unexpectedly, we find that enhancer transcription precedes local mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). H3K4 methylation at de novo enhancers is primarily dependent on the histone methyltransferases Mll1, Mll2/4, and Mll3 and is significantly reduced by inhibition of RNA polymerase II elongation. Collectively, these findings suggest an essential role of enhancer transcription in H3K4me1/2 deposition at de novo enhancers that is independent of potential functions of the resulting eRNA transcripts.