Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes.
Histones are the fundamental components of the nucleosome. Physiologically relevant variation is introduced into this structure through chromatin remodeling, addition of covalent modifications, or replacement with specialized histone variants. The histone H3 family contains an evolutionary conserved variant, H3.3, which differs in sequence in only five amino acids from the canonical H3, H3.1, and was shown to play a role in the transcriptional activation of genes. Histone H3.3 contains a serine (S) to alanine (A) replacement at amino acid position 31 (S31). Here, we demonstrate by both MS and biochemical methods that this serine is phosphorylated (S31P) during mitosis in mammalian cells. In contrast to H3 S10 and H3 S28, which first become phosphorylated in prophase, H3.3 S31 phosphorylation is observed only in late prometaphase and metaphase and is absent in anaphase. Additionally, H3.3 S31P forms a speckled staining pattern on the metaphase plate, whereas H3 S10 and H3 S28 phosphorylation localizes to the outer regions of condensed DNA. Furthermore, in contrast to phosphorylated general H3, H3.3 S31P is localized in distinct chromosomal regions immediately adjacent to centromeres. These findings argue for a unique function for the phosphorylated isoform of H3.3 that is distinct from its suspected role in gene activation.
Hake
Sandra B
SB
Laboratory of Chromatin Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Garcia
Benjamin A
BA
Kauer
Monika
M
Baker
Stephen P
SP
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Allis
C David
CD
D000595:Amino Acid Sequence; D002460:Cell Line; D002503:Centromere / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D020732:Cytogenetic Analysis; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008677:Metaphase / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D006657:Histones; D012694:Serine