Histone lysine methacrylation is a dynamic post-translational modification regulated by HAT1 and SIRT2

Histone lysine crotonylation is a posttranslational modification with demonstrated functions in transcriptional regulation. Here we report the discovery of a new type of histone posttranslational modification, lysine methacrylation (Kmea), corresponding to a structural isomer of crotonyllysine. We validate the identity of this modification using diverse chemical approaches and further confirm the occurrence of this type of histone mark by pan specific and site-specific anti-methacryllysine antibodies. In total, we identify 27 Kmea modified histone sites in HeLa cells using affinity enrichment with a pan Kmea antibody and mass spectrometry. Subsequent biochemical studies show that histone Kmea is a dynamic mark, which is controlled by HAT1 as a methacryltransferase and SIRT2 as a de-methacrylase. Altogether, these investigations uncover a new type of enzyme-catalyzed histone modification and suggest that methacrylyl-CoA generating metabolism is part of a growing number of epigenome-associated metabolic pathways.

Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This is an essential step towards the accuracy of cell division and therefore must be precisely regulated in both mitosis and meiosis. One of the main centromeric regulatory signaling pathways is the Haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. In mitosis, Haspin kinase phosphorylates H3 at threonine 3 (H3T3ph), the essential histone mark that recruits the CPC whose catalytic component is Aurora B kinase. To date, no data has yet been presented about the action of the centromeric Haspin-H3T3ph-CPC pathway in mammalian male meiosis. We have analyzed the consequences of Haspin chemical inhibition in cultured spermatocytes using LDN-192960. Our in vitro studies suggest that Haspin kinase activity is required for proper chromosome congression during both meiotic divisions and for the recruitment of phosphorylated Aurora B at meiotic centromeres. These results have been confirmed by the characterization of the meiotic phenotype of the genetic mouse model Haspin-/-, which displays similar defects. In addition, our work demonstrates that the absence of H3T3ph histone mark does not alter SGO2 localization to meiotic centromeres. These results add new and relevant information regarding the regulation of centromere function during meiosis.

Insights into Enhancer RNAs: Biogenesis and Emerging Role in Brain Diseases

Enhancers are cis-acting elements that control the transcription of target genes and are transcribed into a class of noncoding RNAs (ncRNAs) termed enhancer RNAs (eRNAs). eRNAs have shorter half-lives than mRNAs and long noncoding RNAs; however, the frequency of transcription of eRNAs is close to that of mRNAs. eRNA expression is associated with a high level of histone mark H3K27ac and a low level of H3K27me3. Although eRNAs only account for a small proportion of ncRNAs, their functions are important. eRNAs can not only increase enhancer activity by promoting the formation of enhancer-promoter loops but also regulate transcriptional activation. Increasing numbers of studies have found that eRNAs play an important role in the occurrence and development of brain diseases; however, further research into eRNAs is required. This review discusses the concept, characteristics, classification, function, and potential roles of eRNAs in brain diseases.

A Key Silencing Histone Mark on Chromatin Is Lost When Colorectal Adenocarcinoma Cells Are Depleted of Methionine by Methionine γ-Lyase

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.

ULI-NChIP assay protocol

This protocol presents ULI-NChIP-seq (ultra-low-input micrococcal nuclease-based native ChIP-seq) assay to generate high quality and complexity genome-wide histone mark profiles from rare oocytes andembryos populations. The procedure of ULI-NChIP-seq assay typically consists of five parts including Binding antibodies to magnatic beads, Chromatin shearing and nuclear membrane solubilization, Magnetic immunoprecipitation, Washes and DNA isolation. Sample preparation involves to remove the zona Pellucida of oocyte and polar body to avoid the genomic contamination of polar bodies.

Epigenetic effects induced by the ectopic expression of Pax7 in 3T3-L1

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

The Functions and Mechanisms of PR-DUB in Malignancy

The interplay between cancer genome and deregulated epigenomic control is critical for cancer initiation and progression.ASXL1(Additional Sex combs-like 1) is frequently mutated in tumors especially myeloid malignancies. However, there remains a debate whether the mutations are loss or gain-of-function. Mechanistically, ASXL1 forms a complex with BAP1 for the erasure of mono-ubiquitylation at lysine 119 on Histone H2A (H2AK119ub1), a well-known histone mark associated with transcription repression. Unexpectedly, this de-ubiquitylation complex has been genetically defined as a Polycomb Repressive complex though the regulatory mechanisms are elusive. In this review, we will discuss about the functions of ASXL1 in malignancies and reconcile seemingly paradoxical effects of ASXL1 or BAP1 loss on transcription regulation.

Nucleosomal Asymmetry Shapes Histone Mark Binding and Promotes Poising at Bivalent Domains

SummaryPromoters of developmental genes in embryonic stem cells (ESCs) are marked by histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in an asymmetric nucleosomal conformation, with each sister histone H3 carrying only one mark. These bivalent domains are thought to poise genes for timely activation upon differentiation. Here we show that asymmetric bivalent nucleosomes recruit repressive H3K27me3 binders but fail to enrich activating H3K4me3 binders, despite presence of H3K4me3, thereby promoting a poised state. Strikingly, the bivalent mark combination further attracts chromatin proteins that are not recruited by each mark individually, including the histone acetyltransferase complex KAT6B (MORF). Knockout of KAT6B blocks neuronal differentiation, demonstrating that bivalency-specific readers are critical for proper ESC differentiation. These findings reveal how histone mark bivalency directly promotes establishment of a poised state at developmental genes, while highlighting how nucleosomal asymmetry is critical for histone mark readout and function.