Demethylation of RB Regulator MYPT1 by Histone Demethylase LSD1 Promotes Cell Cycle Progression in Cancer Cells

Cho, Hyun Soo; Suzuki, Takehiro; Dohmae, Naoshi; Hayami, Shinya; Unoki, Motoko; Yoshimatsu, Masanori; Toyokawa, Gouji; Takawa, Masashi; Chen, Taiping; Kurash, Julia K.; Field, Helen I.; Ponder, Bruce A.J.; Nakamura, Yusuke; Hamamoto, Ryuji

doi:10.1158/0008-5472.can-10-2446

Cited by 195 publications

(145 citation statements)

References 24 publications

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“…The substrate was incubated with the purified fusion protein of GST-JmjC domain of JMJD3 in demethylation buffer (20 mM TrisHCl pH 7.3, 150 mM NaCl, 50 μM (NH 4 )2Fe(SO 4 )2+6(H 2 O), 1 mM α-ketoglutarate and 2 mM ascorbic acid) for 4 h at 37°C. 36,41 Substrate was reacted in a total volume of 40 μl of reaction mixture. The reaction was stopped with SDS loading buffer, and western blotting analysis was performed.…”

Section: Discussionmentioning

confidence: 99%

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

et al. 2015

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Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the nonhistone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV 12 , and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly. Cellular senescence is an irreversible process of cell cycle arrest. The senescent cells remain metabolically active but are unable to express genes required for cell proliferation. 1,2 The known causes of cellular senescence include telomere shortening, oxidative stress, DNA damage and hyperoncogenic signaling. 3 H-RasV 12 has been used as a model to induce senescence in normal cells. [4][5][6] Senescent cells are typically characterized by a large flat morphology and the expression of a senescence-associated β-galactosidase activity (SA-β-gal), and nuclei of senescence cells may remodel to form the heterochromatin structures termed the senescenceassociated heterochromatin foci (SAHF). 7 SAHF are condensed regions of DNA that correlate with transcriptionally inactive sites. 8 These heterochromatin foci are hallmarked by H3K9me3 and the incorporation of heterochromatin protein HP1, macroH2A, PML (promyelocy leukemia protein) and HMGA1 (high mobility group AT-hook 1). 7,9-11 Recently, it has been shown that repressive markers, such as H3K9me3, H3K9me2 and H3K27me3, are rearranged into the nonoverlapping structural layers in SAHF. 12-14 Changes of heterochromatin organization generate a repressive chromatin environment that prevents transcription of genes that promote growth, thereby stabilize the state of senescence. 7,15 The retinoblastoma (RB) tumor suppressor is an important senesc...

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Section: Discussionmentioning

confidence: 99%

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

et al. 2015

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“…Forty-eight hours after transfection, cultured cells were fixed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at room temperature for 30 minutes and permeabilized with 0.5% Triton X-100 in PBS (Sigma). Fixed cells were blocked with 5% bovine serum albumin in PBS for 1 hour and incubated with primary antibodies overnight at 4 C. Then they were incubated with Alexa Fluor-conjugated second antibodies (Molecular Probes; Invitrogen) and observed using a Leica confocal microscopy (23).…”

Section: Immunocytochemistrymentioning

confidence: 99%

The Histone Demethylase JMJD2B Plays an Essential Role in Human Carcinogenesis through Positive Regulation of Cyclin-Dependent Kinase 6

Toyokawa

Cho

Iwai

et al. 2011

Cancer Prevention Research

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Histone methyltransferases and demethylases are known to regulate transcription by altering the epigenetic marks on histones, but the pathologic roles of their dysfunction in human diseases, such as cancer, still remain to be elucidated. Herein, we show that the histone demethylase JMJD2B is involved in human carcinogenesis. Quantitative real-time PCR showed notably elevated levels of JMJD2B expression in bladder cancers, compared with corresponding nonneoplastic tissues (P < 0.0001), and elevated protein expression was confirmed by immunohistochemistry. In addition, cDNA microarray analysis revealed transactivation of JMJD2B in lung cancer, and immunohistochemical analysis showed protein overexpression in lung cancer. siRNA-mediated reduction of expression of JMJD2B in bladder and lung cancer cell lines significantly suppressed the proliferation of cancer cells, and suppressing JMJD2B expression lead to a decreased population of cancer cells in S phase, with a concomitant increase of cells in G 1 phase. Furthermore, a clonogenicity assay showed that the demethylase activity of JMJD2B possesses an oncogenic activity. Microarray analysis after knockdown of JMJD2B revealed that JMJD2B could regulate multiple pathways which contribute to carcinogenesis, including the cell-cycle pathway. Of the downstream genes, chromatin immunoprecipitation showed that CDK6 (cyclin-dependent kinase 6), essential in G 1 -S transition, was directly regulated by JMJD2B, via demethylation of histone H3-K9 in its promoter region. Expression levels of JMJD2B and CDK6 were significantly correlated in various types of cell lines. Deregulation of histone demethylation resulting in perturbation of the cell cycle, represents a novel mechanism for human carcinogenesis and JMJD2B is a feasible molecular target for anticancer therapy. Cancer Prev Res; 4(12); 2051-61. Ó2011 AACR.

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“…For example, this general, transferable concept is at the basis of the histone deacetylase inhibitors recently introduced for treatment of certain types of lymphomas (1,2). Lysine-specific demethylase-1 (LSD1 or KDM1A) is overexpressed in many solid tumors such as breast, colon, neuroblastoma, bladder, small cell lung, blood, and prostate cancers (3)(4)(5)(6)(7)(8)(9)(10)(11) and plays an important role in leukemia (12), attracting steadily increasing attention as a major target for epigenetic drug discovery (13,14).…”

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confidence: 99%

LSD1/CoREST is an allosteric nanoscale clamp regulated by H3-histone-tail molecular recognition

Baron

Vellore

2012

Proc. Natl. Acad. Sci. U.S.A.

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The complex of lysine-specific demethylase-1 (LSD1/KDM1A) with its corepressor protein CoREST is an exceptionally relevant target for epigenetic drugs. Here, we provide insight into the local and global changes of LSD1/CoREST conformational dynamics that occur upon H3 binding on the basis of a total cumulative time of one microsecond molecular dynamics simulation. The LSD1/CoREST complex functions as an allosteric nanoscale-binding clamp, which is regulated by substrate binding. In the unbound state, LSD1/CoR-EST reversibly visits clamp states that are more open or significantly more closed compared with the available X-ray crystal structures. The Lys triad of residues Lys355, Lys357, and Lys359 gates the entrance of the H3 pocket. H3 binding shifts the pocket breathing dynamics toward open, higher-volume states while reducing the overall flexibility of the LSD1/CoREST nanoscale clamp. We show that the H3 pocket is an allosteric site for the regulation of the rotation of the amino oxidase domain with respect to the Tower domain. The allosteric mechanism relies on the specific reduction of nanoscale domain rotation upon local H3-tail binding. Instead, clamp opening/closing motions that do not involve domain rotation only reduce in amplitude yet are dominant in the bound state. Overall, our data suggest that the H3 binding pocket is a central target site to (i) switch off LSD1 amino oxidase activity, thus H3-tail demethylation; (ii) block the competitive binding of transcription factors; and (iii) prevent chromatin anchoring to LSD1/CoREST. This study underscores the importance of receptor flexibility for future epigenetic drug discovery.chromatin remodeling | epigenetics | protein dynamics | computer simulation | pharmacology E pigenetic drugs have promising, potential advantages compared with other cancer treatments (e.g., radiotherapy or DNA-modifying drugs), because they could act on replication and gene expression by modulating DNA access without altering the sequence and structure of DNA itself. Therefore, epigenetic pharmacology holds great promise to reduce the side effects that typically limit chemotherapy and antineoplastic efficacy. For example, this general, transferable concept is at the basis of the histone deacetylase inhibitors recently introduced for treatment of certain types of lymphomas (1, 2). Lysine-specific demethylase-1 (LSD1 or KDM1A) is overexpressed in many solid tumors such as breast, colon, neuroblastoma, bladder, small cell lung, blood, and prostate cancers (3-11) and plays an important role in leukemia (12), attracting steadily increasing attention as a major target for epigenetic drug discovery (13,14).LSD1 associated to its corepressor protein CoREST catalyzes the oxidative, specific demethylation of the H3 histone N-terminal (H3) mono-and di-methylated Lys4 residue, using flavin adenosine dinucleotide (FAD) as a cofactor (15-21). LSD1/CoREST structural biology architecture is summarized in Fig. 1 on the basis of various studies (21-26). Recent X-ray crystallography experiments sho...

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Demethylation of RB Regulator MYPT1 by Histone Demethylase LSD1 Promotes Cell Cycle Progression in Cancer Cells

Cited by 195 publications

References 24 publications

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

The Histone Demethylase JMJD2B Plays an Essential Role in Human Carcinogenesis through Positive Regulation of Cyclin-Dependent Kinase 6

LSD1/CoREST is an allosteric nanoscale clamp regulated by H3-histone-tail molecular recognition

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