Variant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Belongs to the histone H3 family.
Expressed throughout the cell cycle independently of DNA synthesis.
Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me). Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription. Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters. Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5 (H3K4me), Lys-37 and Lys-80. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me), which are linked to gene repression, are underrepresented. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin. Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin. Phosphorylation on Ser-32 (H3S31ph) is specific to regions bordering centromeres in metaphase chromosomes. Ubiquitinated. Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination.
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab5169 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
All batches of ab5169 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - phospho S28 peptide (ab5499), indicating that this antibody specifically recognises the Histone H3 - phospho S28 modification.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (phospho S28) antibody (ab5169)The image was submitted as part of a review by Krik McManus, University of British Columbia
Indian Muntjac (top panel) and HeLa cells (bottom panel) immunofluorescently labelled with ab5169 (green) at a working dilution of 1/5000. The DNA is counterstained with DAPI and is shown in blue in the top panel and red in the bottom panel. This antibody gives a characteristic staining pattern for Histone H3 (phospho S28) whereby the signal increases in intensity during late G2 and continues to increase until metaphase. Upon entry into anaphase the signal begins to decrease until reaching basal levels by early G1. 100x magnification.
Western blot - Anti-Histone H3 (phospho S28) antibody (ab5169)Image courtesy of Richelle Sopko, Harvard University, U.S.A.
All lanes : Anti-Histone H3 (phospho S28) antibody (ab5169) at 1/1000 dilution
Lane 1 : Wild type 0-4 hour old fruit fly embryos. Lane 2 : 0-4 hour old fruit fly embryos expressing wee RNAi
Secondary All lanes : Donkey anti-rabbit IgG, Horseradish Peroxidase-L at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Exposure time: 10 minutes
0-4hr old wee shRNA embryos (lane 2) should display elevated phH3Ser28 levels relative to 0-4hr old EGFP shRNA embryos (lane 1). Blocked with 10% BSA.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (phospho S28) antibody (ab5169)This image was submitted as part of a review by Kirk McManus, University of British Columbia
In situ peptide competition was perfomed on paraformaldehyde-fixed HeLa cells. Four 25µl aliquots were made, to which 7.5µg (1.5µl) of no peptide, H3 unmodified peptide (ab2623), H3 phospho S10 peptide (ab11477) or H3 phospho S28 peptide (ab5499) was added, mixed by vortexing and incubated for 1 hour at room temperature. Cells on glass coverslips were fixed with 4% paraformaldehyde (10min) and gently washed twice with PBS, then permeabilized with 0.5% Triton X-100 in PBS (10min) and gently washed three times with PBS. The cells were immunofluorescently labeled with either the peptide-competed antibody or the control antibody (i.e. no peptide) for 30min at room temperature, washed briefly with PBS containing 0.1% Triton X-100 (1 min) and twice with PBS. The cells were then incubated with an appropriate dilution of a secondary antibody at room temperature for 30min, rinsed as above and mounted using a 90% glycerol in PBS mount
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Bhattacharya S et al. Brief Communication: Featured Article: Histone H2A mono-ubiquitination and cellular transformation are inversely related in N-nitrosodiethylamine-induced hepatocellular carcinoma. Exp Biol Med (Maywood)241:1739-44 (2016).
Read more (PubMed: 27190257) »