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J. Biol. Chem., Vol. 282, Issue 19, 14262-14271, May 11, 2007

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Identification of Histone Demethylases in Saccharomyces cerevisiae^*

Shengjiang Tu¹, Esther M. M. Bulloch¹, Lanhao Yang, Chen Ren, Wei-Chieh Huang, Pang-Hung Hsu^¶, Chein-Hung Chen^¶, Chung-Lin Liao^¶, Hui-Ming Yu^¶, Wan-Sheng Lo², Michael A. Freitas³, and Ming-Daw Tsai^¶||4

From the Departments of Chemistry and ^||Biochemistry, The Ohio State University, Columbus, Ohio 43210, the Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, 100, Taiwan, and ^¶Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5, gis1, rph1, jhd1, and jhd2 (yjr119c)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1 and jhd2 strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1 and jhd1 strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

Received for publication, October 23, 2006 , and in revised form, February 15, 2007.

^* This work was supported by funding from the Genomics Research Center of Academia Sinica (to M.-D. T.) and by National Institutes of Health Grants CA69472 (to M.-D. T.) and CA110496 (to M. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed. E-mail: sunnylo{at}ha.mc.ntu.edu.tw. ³ To whom correspondence may be addressed. E-mail: Freitas.5{at}osu.edu. ⁴ To whom correspondence may be addressed. Tel.: 614-292-3080; Fax: 614-292-1532; E-mail: Tsai.7{at}osu.edu.

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