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

Two Classes of Dosage Compensation Complex Binding Elements along Caenorhabditis elegans X Chromosomes ▿ †

Dosage compensation equalizes X-linked gene products between the sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both X chromosomes in XX animals and halves the transcription from each. The DCC is recruited to the X chromosomes by a number of loci, rex sites, and is thought to spread from these sites by an unknown mechanism to cover the rest of the chromosome. Here we describe a novel class of DCC-binding elements that we propose serve as “way stations” for DCC binding and spreading. Both rex sites and way stations comprise strong foci of DCC binding on the native X chromosome. However, rex sites maintain their ability to bind large amounts of DCC even on X duplications detached from the native X, while way stations do not. These results suggest that two distinct classes of DCC-binding elements facilitate recruitment and spreading of the DCC along the X chromosome

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

<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

Jmjd2E demethylase affects H3K56me3.

<p>(A) IF microscopy of HeLa Kyoto cells transfected with mJmjd2E-GFP (green, left) or jmjc-domain mutated mJmjd2E-GFP (mutant, green, right) and stained with various H3K56 and H3K9 PTM-specific antibodies (red) and DAPI (DNA, blue). Arrows indicate transfected GFP-positive cells. Scale bar  = 10 µm. See also Figure S2A for IF results of cells transfected with other GFP-tagged mJMJD2 family members (mJmjd2a-d). (B) List of PTMs analyzed in IF after expression of mJmjd2E in HeLa Kyoto cells indicating changes in fluorescence intensities. See also Figure S2B for examples of IF results summarized in this table. (C) qPCR analysis with cDNAs from different human cell lines and tissues using primer pair specific for human Jmjd2E (hKDM4DL). Data were normalized to HPRT1 and GAPDH expression levels. (D) IF microscopy of HeLa Kyoto cells transfected with human GFP-hKDM4L (green) and stained with various H3K56 and H3K9 methyl-specific antibodies (red) and DAPI (DNA, blue). Arrows indicate transfected and GFP-positive cells. Scale bar  = 10 µm.</p

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

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

A Deletion at the Mouse Xist Gene Exposes Trans-effects That Alter the Heterochromatin of the Inactive X Chromosome and the Replication Time and DNA Stability of Both X Chromosomes

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of γ-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes