The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells

Abstract

The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.

Figure 1

MMSET induces global changes in histone methylation. (A) Schematic representation of MMSET main isoforms showing the regions where the shRNA were designed. (Right) Cells containing the inducible shRNA were grown in the presence of doxycycline for 7 days. After 7 days, cell were washed and incubated for 7 more days without doxycycline to restore MMSET expression. Nuclear extracts were immunoblotted with anti-MMSET antibody. (B-C) Nuclear extracts from the knockdown and knockout systems were immunoblotted with the indicated antibodies. MMSET depletion induces methylation of H3K27me2/3 and decreases methylation of H3K36me2/3. (D) MMSET repletion induces global changes in chromatin. The MMSET knockout cell line was infected with retrovirus containing a vector control (lane 1), wild-type MMSET II (lane 2), or 2 different SET domain mutants (lanes 3-4). Nuclear extracts were immunoblotted with the indicated antibodies. Restoration of wild-type and active mutant of MMSET decreases methylation of H3K27me2/3 and increases methylation of H3K36me2/3. Repletion with the enzymatically dead mutant form of MMSET shows the same methylation pattern as the vector control. (E) MMSET expression affects chromatin structure. MNase assay in knockdown system. Lane 1: molecular weight marker; lanes 2-3: negative control with no enzyme. The presence of MMSET correlates with a more open chromatin state (lane 4). When MMSET is blocked, the chromatin closes, and it is not accessible for the digestion by MNase (lane 5). Re-expression of MMSET upon doxycycline removal opens the chromatin (lane 6).

Figure 2

t(4;14)-positive cells have a different histone methylation pattern than t(4;14)-negative cells. (A) Nuclear extracts from 9 t(4;14)-positive cells and 7 t(4;14)-negative cells were immunoblotted with the indicated antibodies. MMSET expression correlates with higher H3K36me2 and lower H3K27me3 marks. (B) Intact mass profiles of histones H3.1, H3.2, and H3.3 from 4 multiple myeloma cell lines. H929 and LP1 are t(4;14)+ and show a global increase in H3.1 and H3.2 methylation compared with the t(4;14)− KMS12 and MM.M1 cells. The [M+18H]¹⁸⁺ species are shown. Distinct peaks correspond to different numbers of methyl or acetyl groups (14 and 42 Da). The 6 methyl equivalent is labeled for each histone variant. (C) K36 methylation levels were determined from y₁₇⁴⁺, y₁₈⁴⁺, and y₁₉³⁺ from collision-induced dissociation (CID) of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. (D) K36 dimethylation is reported on by y₁₉³⁺ ions (717.42 m/z) from CID of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. Peak intensity (arbitrary units) is shown in the top right corner for each mass spectrum. (E) K27 methylation levels were determined from y₂₄⁴⁺, y₂₇⁴⁺, y₃₅⁶⁺, and y₃₆⁶⁺ for K36+K27 from CID of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. Values for K27 methylation were deconvoluted from K36+K27 and K36 data using a system of inequalities (Mathematica). The methylation levels, with SEM, are shown in supplemental Table 1.

Figure 3

MMSET alters cell growth, cell cycle, and apoptosis in multiple myeloma cells. (A) MMSET affects growth of multiple myeloma cells. Cloned KMS11 cells harboring inducible shRNA were grown in the presence of doxycycline, and cells were counted at the indicated time points. Fresh media with or without doxycycline was added at days 7, 10, and 13 to allow continued cell growth. Two different clones are shown in the graphs. Knockdown using the C-terminal shRNA shows a drastic change in growth behavior. (B) MMSET alters adhesion of multiple myeloma cells. Knockdown cells where grown in the presence of doxycycline for 7 days, and pictures were imaged as described in “Adhesion assay.” (C) MMSET depletion induces apoptosis. Knockdown cells were grown in the presence of doxycycline, and annexin V staining was performed as described in “Cell cycle analysis and apoptosis assay.” (D) MMSET induces cell cycle arrest. Cell-cycle profiles of knockdown cells grown in the presence of doxycycline for 7 days were assessed after a 30-minute BrdU pulse as described in “Cell cycle analysis and apoptosis assay.” MMSET knockdown decreases the percentage of cells in S-phase, and this effect is more pronounced using the C-terminal shRNA. (E) MMSET induces proliferation. Knockout cells infected with vector control, wild-type, and mutants of MMSET were grown, and cell counts were performed over a period of 2 weeks. Cells containing wild-type and active mutant (F1177A) of MMSET proliferate faster than cells infected with vector control or the enzymatically dead MMSET (Y1118A).

Figure 4

Identification of MMSET target genes by gene expression array. (A) Comparison of gene expression dataset from N-terminal knock down and MMSET II add back (t test paired; > 1.5-fold, P < .05). (B) Comparison of gene expression dataset from knockout cell line repleted with wild-type, active mutant, or dead mutant forms of MMSET (> 1.5-fold, P < .05). (C) Validation of MMSET target genes by real-time PCR. A representative experiment showing the regulation of JAM2 and GLS2 genes in the presence or absence of MMSET using the knockdown, knockout, and repletion systems. (D) Classification of MMSET target genes by Ontology analysis was performed using DAVID Ontology and Ingenuity Pathway Analysis. MMSET regulates genes mainly related with apoptosis, cell cycle, and DNA repair.

Figure 5

MMSET binding across genes overlaps with high levels of H3K36me2 and low levels of H3K27me3. Chromatin of MMSET shRNA-inducible cells grown with or without doxycycline for 7 days was immunoprecipitated with anti-MMSET, anti-H3K27me3, anti-H3K36me2, or anti-H3K36me3 antibodies. Immunoglobulin G was used as negative control (not shown). Eluted DNA was PCR amplified with primers designed across JAM2 (A) and GLS2 (B) loci.