FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16.
The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.
Heo
Kyu
K
Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA.
Kim
Hyunjung
H
Choi
Si Ho
SH
Choi
Jongkyu
J
Kim
Kyunghwan
K
Gu
Jiafeng
J
Lieber
Michael R
MR
Yang
Allen S
AS
An
Woojin
W
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006609:High Mobility Group Proteins; D006657:Histones; D009707:Nucleosomes; D020033:Protein Isoforms; D011994:Recombinant Proteins; C073537:SSRP1 protein, human; C489489:SUPT16H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D000244:Adenosine Diphosphate; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases