Purification and characterization of C-terminal truncated forms of histone H2A in monocytic THP-1 cells.
Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14kDa) and ubiquitinated H2A (23kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A(1-87) and 12-kDa H2A(1-114), suggesting that both were produced by limited proteolysis of intact H2A(1-129). The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A(1-87) in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A(1-114) was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) but not with carbobenzoxy-l-isoleucyl-gamma-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.
Minami
Jiro
J
Department of Biochemistry 1, Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.
Takada
Koji
K
Aoki
Katsuhiko
K
Shimada
Yohta
Y
Okawa
Yutaka
Y
Usui
Noriko
N
Ohkawa
Kiyoshi
K
D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007976:Leupeptins; D009000:Monocytes / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014452:Ubiquitins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D000970:Antineoplastic Agents; D002273:Carcinogens; D002843:Chromatin; D006657:Histones; D007976:Leupeptins; D014452:Ubiquitins; C021645:chromatin conjugate protein A24; D014212:Tretinoin; D013755:Tetradecanoylphorbol Acetate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde