Functional Interplay between Histone Demethylase and Deacetylase Enzymes

Min Lee

Christopher Wynder

Daniel Bochar

Mohamed Hakimi

Neil Cooch

Ramin Shiekhattar

The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104

^{Corresponding author. Mailing address: The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-3896. Fax: (215) 898-3986. E-mail:}gro.ratsiw@rattahkeihs.

^{Present address: The University of Michigan, 3301E MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0606.}

^{Present address: CNRS, UMR5163, Université Joseph Fourier, 38700 La Tronche, France.}

^{Present address: N3108, Wyeth Pharmaceuticals and Research Headquarters, 500 Arcola Road, Collegeville, PA 19426.}

Received 2006 Apr 26; Revised 2006 May 30; Accepted 2006 Jun 7.

Abstract

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.

Abstract

In eukaryotes, the nucleosome serves as the in vivo target of multiple chromatin-modifying enzymes (17). Strikingly, tail domains of histones as well as the nucleosome core domain are subject to several posttranslational modifications, including methylation, acetylation, phosphorylation, and ubiquitylation (33). Such histone modifications are hypothesized as “histone code” to modulate many cellular processes by recruiting regulatory transcription complexes and changing gene expression (16).

In particular, histone methylation was considered irreversible. However, recent studies have revealed that histone methylation can be reversed by several histone demethylases, including PAD4/PADI4, BHC110/LSD1, and JmjC domain-containing demethylases (6, 24, 31, 34-36). PAD4/PADI4 has been reported to convert monomethyl arginine to citrulline by demethylimination. The JmjC domain-containing demethylases contain a JmjC domain responsible for their enzymatic activity and demethylate mono-, di- or trimethylated lysines by a hydroxylation-based mechanism (31, 35, 36). BHC110 demethylates mono- and dimethyl histone H3 lysine 4 (H3K4) and belongs to a family of FAD-dependent polyamine oxidases which use molecular oxygen as an electron acceptor to oxidize an amine group (24). In addition, we and others have shown that CoREST, the corepressor of REST (RE1-silencing transcription factor) protein, mediates nucleosomal demethylation by BHC110 (18, 26). Surprisingly, androgen receptor was recently reported to alter BHC110 enzymatic activity, leading to the demethylation of histone H3 lysine 9 (20).

BHC110 has been isolated as a component of many multiprotein complexes (1, 9, 12, 14, 15, 25, 30). One such complex, termed BHC (BRAF-HDAC complex), mediates the repression of neuron-specific genes (1, 12). Importantly, we have shown that such corepressor complexes share two enzymatic core subunits: histone deacetylase (HDAC1/2) and BHC110 (13). Here we provide evidence that the enzymatic activities of the histone demethylase and the deacetylase are intimately linked. Such cross talk between the two enzymes is seen only when nucleosomal substrates are used and is mediated through different domains of the CoREST protein. Importantly, we show that due to such functional connections, HDAC inhibitors diminish histone demethylation. Given that some HDAC inhibitors have reached clinical trials (21), these findings not only reveal a second mechanism of action for HDAC inhibitors but also point to the future potential of histones demethylase inhibitors as therapeutics for cancer.

Acknowledgments

We thank Gail Mandel for a CoREST cDNA construct.

R.S. was supported by NIH grant R01 GM 61204.

Acknowledgments

REFERENCES

References

1. Ballas, N., E. Battaglioli, F. Atouf, M. E. Andres, J. Chenoweth, M. E. Anderson, C. Burger, M. Moniwa, J. R. Davie, W. J. Bowers, H. J. Federoff, D. W. Rose, M. G. Rosenfeld, P. Brehm, and G. Mandel. 2001. Regulation of neuronal traits by a novel transcriptional complex. Neuron31:353-365. [[PubMed]
2. Barak, O., M. A. Lazzaro, W. S. Lane, D. W. Speicher, D. J. Picketts, and R. Shiekhattar. 2003. Isolation of human NURF: a regulator of Engrailed gene expression. EMBO J.22:6089-6100.
3. Binda, C., A. Coda, R. Angelini, R. Federico, P. Ascenzi, and A. Mattevi. 1999. A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Structure Fold Des.7:265-276. [[PubMed]
4. Binda, C., M. Li, F. Hubalek, N. Restelli, D. E. Edmondson, and A. Mattevi. 2003. Insights into the mode of inhibition of human mitochondrial monoamine oxidase B from high-resolution crystal structures. Proc. Natl. Acad. Sci. USA100:9750-9755.
5. Chopin, V., R. A. Toillon, N. Jouy, and X. Le Bourhis. 2004. P21(WAF1/CIP1) is dispensable for G1 arrest, but indispensable for apoptosis induced by sodium butyrate in MCF-7 breast cancer cells. Oncogene23:21-29. [[PubMed]
6. Cuthbert, G. L., S. Daujat, A. W. Snowden, H. Erdjument-Bromage, T. Hagiwara, M. Yamada, R. Schneider, P. D. Gregory, P. Tempst, A. J. Bannister, and T. Kouzarides. 2004. Histone deimination antagonizes arginine methylation. Cell118:545-553. [[PubMed]
7. Ding, Z., L. L. Gillespie, and G. D. Paterno. 2003. Human MI-ER1 alpha and beta function as transcriptional repressors by recruitment of histone deacetylase 1 to their conserved ELM2 domain. Mol. Cell. Biol.23:250-258.
8. Dover, J., J. Schneider, M. A. Tawiah-Boateng, A. Wood, K. Dean, M. Johnston, and A. Shilatifard. 2002. Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J. Biol. Chem.277:28368-28371. [[PubMed]
9. Eimer, S., B. Lakowski, R. Donhauser, and R. Baumeister. 2002. Loss of spr-5 bypasses the requirement for the C. elegans presenilin sel-12 by derepressing hop-1. EMBO J.21:5787-5796.
10. Forneris, F., C. Binda, M. A. Vanoni, E. Battaglioli, and A. Mattevi. 2005. Hum. histone demethylase LSD1 reads the histone code. J. Biol. Chem.280:41360-41365. [[PubMed]
11. Guenther, M. G., O. Barak, and M. A. Lazar. 2001. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol.21:6091-6101.
12. Hakimi, M. A., D. A. Bochar, J. Chenoweth, W. S. Lane, G. Mandel, and R. Shiekhattar. 2002. A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc. Natl. Acad. Sci. USA99:7420-7425.
13. Hakimi, M. A., Y. Dong, W. S. Lane, D. W. Speicher, and R. Shiekhattar. 2003. A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes. J. Biol. Chem.278:7234-7239. [[PubMed]
14. Humphrey, G. W., Y. Wang, V. R. Russanova, T. Hirai, J. Qin, Y. Nakatani, and B. H. Howard. 2001. Stable histone deacetylase complexes distinguished by the presence of SANT domain proteins CoREST/kiaa0071 and Mta-L1. J. Biol. Chem.276:6817-6824. [[PubMed]
15. Jarriault, S., and IGreenwald. 2002. Suppressors of the egg-laying defective phenotype of sel-12 presenilin mutants implicate the CoREST corepressor complex in LIN-12/Notch signaling in C. elegans. Genes Dev.16:2713-2728. [Google Scholar]
16. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science293:1074-1080. [[PubMed]
17. Kornberg, R. D., and Y. Lorch. 1999. Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell98:285-294. [[PubMed]
18. Lee, M. G., C. Wynder, N. Cooch, and R. Shiekhattar. 2005. An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature437:432-435. [[PubMed]
19. Lo, W. S., L. Duggan, N. C. Emre, R. Belotserkovskya, W. S. Lane, R. Shiekhattar, and S. L. Berger. 2001. Snf1—a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science293:1142-1146. [[PubMed]
20. Metzger, E., M. Wissmann, N. Yin, J. M. Muller, R. Schneider, A. H. Peters, T. Gunther, R. Buettner, and R. Schule. 2005. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature437:436-439. [[PubMed]
21. Monneret, C. 2005. Histone deacetylase inhibitors. Eur. J. Med. Chem.40:1-13. [[PubMed]
22. Nakano, K., T. Mizuno, Y. Sowa, T. Orita, T. Yoshino, Y. Okuyama, T. Fujita, N. Ohtani-Fujita, Y. Matsukawa, T. Tokino, H. Yamagishi, T. Oka, H. Nomura, and T. Sakai. 1997. Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. J. Biol. Chem.272:22199-22206. [[PubMed]
23. Ng, H. H., R. M. Xu, Y. Zhang, and K. Struhl. 2002. Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J. Biol. Chem.277:34655-34657. [[PubMed]
24. Shi, Y., F. Lan, C. Matson, P. Mulligan, J. R. Whetstine, P. A. Cole, and R. A. Casero. 2004. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell119:941-953. [[PubMed]
25. Shi, Y., J. Sawada, G. Sui, B. Affar el, J. R. Whetstine, F. Lan, H. Ogawa, M. P. Luke, and Y. Nakatani. 2003. Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature422:735-738. [[PubMed]
26. Shi, Y. J., C. Matson, F. Lan, S. Iwase, T. Baba, and Y. Shi. 2005. Regulation of LSD1 histone demethylase activity by its associated factors. Mol. Cell19:857-864. [[PubMed]
27. Solari, F., A. Bateman, and J. Ahringer. 1999. The Caenorhabditis elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning. Development126:2483-2494. [[PubMed]
28. Sowa, Y., T. Orita, S. Minamikawa-Hiranabe, T. Mizuno, H. Nomura, and T. Sakai. 1999. Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor. Cancer Res.59:4266-4270. [[PubMed]
29. Sun, Z. W., and C. D. Allis. 2002. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature418:104-108. [[PubMed]
30. Tong, J. K., C. A. Hassig, G. R. Schnitzler, R. E. Kingston, and S. L. Schreiber. 1998. Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex. Nature395:917-921. [[PubMed]
31. Tsukada, Y., J. Fang, H. Erdjument-Bromage, M. E. Warren, C. H. Borchers, P. Tempst, and Y. Zhang. 2006. Histone demethylation by a family of JmjC domain-containing proteins. Nature439:811-816. [[PubMed]
32. Utley, R. T., T. A. Owen-Hughes, L. J. Juan, J. Cote, C. C. Adams, and J. L. Workman. 1996. In vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement. Methods Enzymol.274:276-291. [[PubMed]
33. Vaquero, A., A. Loyola, and D. Reinberg. 2003. The constantly changing face of chromatin. Sci. Aging Knowledge Environ.2003:RE4. [[PubMed]
34. Wang, Y., J. Wysocka, J. Sayegh, Y. H. Lee, J. R. Perlin, L. Leonelli, L. S. Sonbuchner, C. H. McDonald, R. G. Cook, Y. Dou, R. G. Roeder, S. Clarke, M. R. Stallcup, C. D. Allis, and S. A. Coonrod. 2004. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science306:279-283. [[PubMed]
35. Whetstine, J. R., A. Nottke, F. Lan, M. Huarte, S. Smolikov, Z. Chen, E. Spooner, E. Li, G. Zhang, M. Colaiacovo, and Y. Shi. 2006. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell125:467-481. [[PubMed]
36. Yamane, K., C. Toumazou, Y. I. Tsukada, H. Erdjument-Bromage, P. Tempst, J. Wong, and Y. Zhang. 2006. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell125:483-495. [[PubMed]
37. Zhang, Y., H. H. Ng, H. Erdjument-Bromage, P. Tempst, A. Bird, and D. Reinberg. 1999. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev.13:1924-1935.