H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization.

Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance

PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels

PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels

Loss of Suv39h enzymes affect H3K56me3.

<p>IF microscopy of wild type (WT), Suv39h double-null (Suv39h DKO) and Suv4-20h double-null (Suv4-20h DKO) MEF cells using various H3K56 (A) and H3K9 (B) methyl-specific antibodies (Ab-Cy3, red) and DAPI (DNA, blue). Scale bar  = 5 µm. (C) Immunoblots using acid extracted histones from HeLa Kyoto (positive control), wild type MEF, Suv39h DKO and Suv4-20h DKO cells. Blots were incubated with αH3K56me3 (left, top) or αH3K9me3 (right, top) antibodies, respectively. Blots shown at the bottom were incubated with αH4 to ensure equal loading.</p

H3K56me3 is conserved in <i>Caenorhabditis elegans</i>.

<p>Shown are representative IF microscopy pictures from adult <i>C. elegans</i> hermaphrodite tissues. In all images H3K56me3 is shown in green, H3K9me3 in red, and DAPI (DNA) in blue. Scale bar  = 5 µm. A) H3K56me3 co-localizes with H3K9me3 in the early germline, late pachytene and in a 100-cell embryo (top picture). Interestingly, although H3K56me3 and H3K9me3 are both present in oocytes, only H3K56me3, but not H3K9me3, staining could be observed in sperm. (bottom, split channels) (B) H3K56me3 and H3K9me3 co-localize throughout all stages of mitosis.</p

Determination of αH3K56me3 specificity and suitability in diverse applications.

<p>(A) Immunoblot peptide competition experiment. αH3K56me3 antibody was preincubated with competitor peptides before addition to immunoblots containing recombinant H3 protein (R) or acid extracted HeLa Kyoto histones (H) (top). Ponceau staining (bottom) serves as loading control. (B) IF microscopy peptide competition experiment. αH3K56me3 antibody (green) was preincubated with competitor peptides before addition to fixed HeLa Kyoto cells. DAPI (blue) stains DNA. Scale bar  = 5 µm. (C) Spot-blot with different concentrations (5–1000 ng) of H3 peptides to determine αH3K56me3-binding affinities. (D) Immunoblot of sequential tryptic digest of HeLa Kyoto-derived mononucleosomes using αH3K56me3 (top), αH3K9me3 (middle) and αH3 (bottom). FL  =  full-length histone H3, GD  =  N-terminally deleted globular domain of histone H3.</p

<i>C. elegans</i> RNAi screen to identify H3K56me3-specific KMTs.

<p>Shown are representative IF images from adult <i>C. elegans</i> hermaphrodite somatic intestinal nuclei following RNAi treatment. H3K56me3 (left) or H3K9me3 (right) staining is shown in green and DAPI (DNA) is shown in blue. CAPG-1 co-staining was used as a staining control (data not shown). Results show that <i>met-1</i> and <i>met-2</i> depletion severely affect both H3K56me3 and H3K9me3, while reduction of additional KMTs (<i>set-6, set-25</i> and <i>set-32</i>) has a stronger effect on H3K56me3 levels compared to H3K9me3. Scale bar  = 5 µm.</p