Histone H2B Phosphorylation in Mammalian Apoptotic Cells

Abstract

Histone phosphorylation was investigated in several mammalian cells undergoing apoptosis (human HL-60 and HeLa, mouse FM3A and N18 cells, and rat thymocytes). Among the four nucleosomal core histones (H2A, H2B, H3, and H4), H2B, which is not usually phosphorylated in quiescent or growing cells, was found to be phosphorylated after treatment with various apoptotic inducers. The H2B was phosphorylated around the time when nucleosomal DNA fragmentation was initiated and, like this fragmentation, was completely blocked with Z-Asp-CH 2 -DCB, an inhibitor of ICE or ICE-like caspase. The involved single phosphopeptide of H2B proved to be phosphorylatable in vitro with a protein kinase C, and the site Ser-32 was tentatively identified. Despite typical apoptotic chromatin condensation, the H3 phosphorylation was at a low level, and the sites where phosphorylation did occur did not include any mitosis-specific phosphopeptides. Phosphorylation of H4 was increased, but the other two histone proteins (H1 and H2A) were not appreciably changed. These observations imply that 1) H2B phosphorylation occurs universally in apoptotic cells and is associated with apoptosis-specific nucleosomal DNA fragmentation, 2) chromatin condensation in apoptosis occurs by a different biochemical mechanism from those operating during mitosis or premature chromosome condensation, and 3) this unique phosphorylation of H2B is a useful biochemical hallmark of apoptotic cells.

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K+ ions

“…3a). Previous studies have shown that histone H2B is specifically phosphorylated during apoptosis in intact cells [24,25], however it is unknown if Ca 2+ treatment of isolated nuclei can also promote this apoptoticspecific phosphorylation of H2B. Figure 3b shows that H2B is phosphorylated in thymocytes induced to undergo apoptosis by dexamethasone, whereas H2B in isolated nuclei treated with 1 mM Ca 2+ was not phosphorylated, despite the occurrence of degraded DNA.…”

“…During analysis of histone H2B phosphorylation, thymocytes were treated with 1 μM dexamethasone for 4 h and nuclei were incubated with CaCl 2 and harvested. Histones were extracted [24] and western blotted against anti-phospho S14H2B (Upstate) [25].…”

Autoimmune cytopenias associated with autoantibodies to nuclear envelope polypeptides

“…An appealing hypothesis could be that anti-lamin Abs are generated as a secondary response to apoptotic cells, exhibiting phosphorylated cleaved lamins [41,42]. Such a response to phosphorylated nucleoproteins in apoptotic cells as well as defects in the clearing of these apoptotic cells are often advocated in the recent models of autoimmunity [43][44][45].…”

Chemical “Diversity” of Chromatin Through Histone Variants and Histone Modifications

“…Histone phosphorylation has been linked to diverse biological functions, including chromosome condensation (H1 and H3 phosphorylation; reviewed in [162,163]), transcriptional regulation (H3 phosphorylation; reviewed in [164,165]), DNA damage repair (H2A.X phosphorylation; [97,166,167]), and even apoptosis (H2B phosphorylation; [168][169][170][171]). Phosphorylation adds negative charges to the modified protein, and similar to histone acetylation, phosphorylation can decrease the association of the modified histones with DNA.…”