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

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

<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