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. 2010 May 25;107(21):9671-6.
doi: 10.1073/pnas.1000401107. Epub 2010 May 10.

KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation

Datsun A Hsia  1 , Clifford G Tepper, Mamata R Pochampalli, Elaine Y C Hsia, Chie Izumiya, Steve B Huerta, Michael E Wright, Hong-Wu Chen, Hsing-Jien Kung, Yoshihiro Izumiya
Affiliations

KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation

Datsun A Hsia et al. Proc Natl Acad Sci U S A. .

Abstract

Localized chromatin modifications of histone tails play an important role in regulating gene transcription, and aberration of these processes leads to carcinogenesis. Methylated histone lysine residues, a key player in chromatin remodeling, are demethylated by the JmjC class of enzymes. Here we show that JMJD5 (now renamed KDM8), a JmjC family member, demethylates H3K36me2 and is required for cell cycle progression. Chromatin immunoprecipitation assays applied to human genome tiling arrays in conjunction with RNA microarray revealed that KDM8 occupies the coding region of cyclin A1 and directly regulates transcription. Mechanistic analyses showed that KDM8 functioned as a transcriptional activator by inhibiting HDAC recruitment via demethylation of H3K36me2, an epigenetic repressive mark. Tumor array experiments revealed KDM8 is overexpressed in several types of cancer. In addition, loss-of-function studies in MCF7 cells leads to cell cycle arrest. These studies identified KDM8 as an important cell cycle regulator.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
KDM8 H3K36me2 demethylation in MCF7 breast cancer cells. (A) KDM8 and H321A, an inactive enzymatic mutant, were induced to express as Flag fusion proteins. Indirect immunofluorescence with antibodies against Flag (red staining) and methylated H3K36me1, H3K36me2, or H3K36me3 (green staining) was used to analyze in vivo substrate specificity of KDM8. DAPI staining (blue) indicates nuclei location in each field. Cells overexpressing KDM8 (arrows) showed significant loss of H3K36me2 staining, which was dependent on the active JmjC domain, and not observed in the H321A overexpressed cells. (B) Quantitative analysis of Dox-induced KDM8/H321A MCF7 H3K36me2 stained cells from 10 random fields. Eighty percent of induced KDM8 cells showed diminished H3K36me2, whereas 60% of H321A-induced cells exhibited increases in H3K36me2 staining. Percentage of cells that exhibited no change in H3K36me2 is not shown. (C) Histones extracted from Dox-induced KDM8/H321A MCF7 were analyzed by Western blotting with antibodies against H3K36me2 and H3K9me3. A decreased signal in H3K36me2 was observed in KDM8 overexpressed cells (lane 2 vs. lane 1), which was dependent on the active JmjC domain (inactive mutant lanes 3–4). (D) MS analysis of observed in vitro KDM8 demethylase activity. Reactions using H3K36me2 peptide combined with either GST KDM8 101-C WT or GST-KDM8 101-C H321A show 14 Da shift in WT reactions only (asterisk).
Fig. 2.
Fig. 2.
KDM8 is overexpressed in tumors and required for breast cancer cell proliferation. A quantitative examination of KDM8 expression in cancer cells was performed. (A) Western blot analysis showed significantly higher expression of endogenous KDM8 in a panel of breast cancer cell lines compared to primary HMECs. (B) Immunohistochemistry KDM8 staining of malignant breast cancer tumors (M) and patient-matched adjacent normal tissues (N). (C) Higher-magnification view of respective malignant and normal IHC staining of KDM8. (Scale bar, 10 μm.) (D) Intensity of staining was scored on a scale of lowest (0) to highest (3+). (E) (i) Loss of KDM8 contributes to a decrease in cell growth. Two KDM8 knockdown clones (shKDM8 no. 1, no. 5) or control cells (shControl) were maintained in culture for 5 d. Cell numbers were counted at indicated time points in triplicate. Growth data presented are means ± SD. from three independent experiments. Immunoblotted lysates showed decreased expression of KDM8 in knockdown lines (Upper). (ii) Enzymatic activity is required for MCF7 proliferation. KDM8 WT or catalytic inactive mutant (H321A) expression was induced (Dox) and cell growth was examined by counting cells for 6 d. Overexpression of KDM8 WT increased cell proliferation compared to noninduced control, whereas induction of mutant decreased cell proliferation. (F) Flow cytometry analysis of KDM8 shRNA MCF7 cells stained with PI identified 40% of total cells arrested in G2/M phase compared with 20% in control shRNA cells.
Fig. 3.
Fig. 3.
KDM8 increases cyclin A1 gene and protein expression through binding in the coding region. (A) Endogenous KDM8 recruitment sites in MCF7 cells were mapped using ChIP-on-chip analysis. KDM8-associated DNA was enriched by ChIP with a KDM8-specific antibody and subsequently analyzed with Affymetrix Human Promoter 1.0R tiling arrays as described in SI Materials and Methods. CisGenome software was used for peak detection and annotation of genomic regions bound by KDM8. The figure demonstrates that endogenous KDM8 is recruited to the cyclin A1 coding region and most prominently to exon 2. The binding profile along the CCNA1 locus is depicted based on magnitude of enrichment at each location, which is signified by the height of each bar. Location of primer pairs are indicated in the panel by letters and black bars. (B) Quantitative real-time PCR confirming the binding of endogenous KDM8 in the cyclin A1 gene. (C) Quantitative RT-PCR analysis of cyclin A1 in Dox-inducible Flag-KDM8/H321A MCF7 cells. Induction of KDM8 increased cyclin A1 expression. (D) Immunoblotting analysis of MCF10A cells transfected with KDM8 siRNA and scramble siRNA shows that knockdown of KDM8 leads to a decrease in cyclin A expression and increases the amount of H3K36me2.
Fig. 4.
Fig. 4.
KDM8 mediated demethylation of H3K36me2 leads to a decrease in HDAC1 recruitment in the cyclin A1 coding region. (A) ChIP analysis was performed to examine KDM8 effects on H3K36me2 and HDAC1 recruitment to the coding region of the cyclin A1 gene with inducible cells. ChIP assays were performed with indicated antibodies. The results were normalized to IgG controls and shown as an average fold change in enrichment. Data are presented as a mean ± SD from three independent experiments. (B). ChIP analysis was performed similar to A in the MCF7 cells that had stable knockdown of KDM8 with the indicated antibodies. (C). Immunoblot analysis of KDM8 knockdown MCF7 cells. The lysates from the KDM8 knockdown and control cells were probed with the antibodies as indicated.
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