Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.
5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.
Department of Molecular Medicine, Ewha Womans University Medical School, 911-1, Mok-6-dong, Yangchun-gu, Seoul, 158-710, South Korea.
Ji-Young
JY
Choi
Sangmi
S
Lee
Soojin
S
Hwang
Sangmee Ahn
SA
Jo
Miji
M
Kim
Young Ju
YJ
Kim
Myung-Geol
MG
Pang
Inho
I
Jo
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000633:toxicity; D018899:CpG Islands / Q000187:drug effects; D000077209:Decitabine; D000071221:Enhancer of Zeste Homolog 2 Protein; D044127:Epigenesis, Genetic / Q000187:drug effects; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D007985:Leydig Cells / Q000473:pathology; D008297:Male; D051379:Mice; D063151:Polycomb Repressive Complex 2 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012098:Reproduction / Q000187:drug effects; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism*
C494820:Bad protein, mouse; D006657:Histones; D051025:bcl-Associated Death Protein; D000077209:Decitabine; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D063151:Polycomb Repressive Complex 2; D001374:Azacitidine