The Histone Methyltransferase MMSET Regulates Class Switch Recombination

Huadong Pei, Xiaosheng Wu, Tongzheng Liu, Kefei Yu, Diane F. Jelinek and Zhenkun Lou
J Immunol January 15, 2013, 190 (2) 756-763; DOI: https://doi.org/10.4049/jimmunol.1201811

Abstract

Wolf–Hirschhorn syndrome (WHS) is a genetic disease with characteristic facial features and developmental disorders. Of interest, loss of the MMSET gene (also known as WHSC1) is considered to be responsible for the core phenotypes of this disease. Patients with WHS also display Ab deficiency, although the underlying cause of this deficiency is unclear. Recent studies suggest that the histone methyltransferase activity of MMSET plays an important role in the DNA damage response by facilitating the recruitment of 53BP1 to sites of DNA damage. We hypothesize that MMSET also regulates class switch recombination (CSR) through its effect on 53BP1. In this study, we show that MMSET indeed plays an important role in CSR through its histone methyltransferase activity. Knocking down MMSET expression impaired 53BP1 recruitment as well as the germline transcription of the Igh switch regions, resulting in defective CSR but no effect on cell growth and viability. These results suggest that defective CSR caused by MMSET deficiency could be a cause of Ab deficiency in WHS patients.

Introduction

Human Ab molecules are comprised of H chains and L chains with both constant and V regions that are encoded by different genes. During early B cell development in primary lymphoid organs, the genes encoding the H chain and L chain V regions are assembled from component V, D, and J gene segments by the process of V(D)J recombination (1). The productive assembly of H chains and L chains generates B cells that express IgM on its surface. These B cells subsequently migrate to the secondary lymphoid organs such as spleen and lymph nodes, where upon encountering Ag they can switch expression of Ig class (isotype) from IgM to IgG, IgE, or IgA to mediate different isotype-specific effector functions essential for normal immunity. This process requires an additional round of genetic alteration, termed class switch recombination (CSR) (2–5).

CSR is initiated by activation-induced cytidine deaminase (AID), which deaminates deoxycytidines on both DNA strands within Igh switch (S) regions (5, 6). The deaminated S regions are then processed by proteins of the base excision repair pathway (e.g., including uracil DNA glycosylase, APE1) and mismatch repair pathway (e.g., including Msh2/Msh6, Mlh1/Pms2, Exo1) to ultimately generate DNA double-stranded breaks (DSBs) (7). Finally, the AID-induced DSBs are repaired predominantly by the nonhomologous end-joining pathway. In nonhomologous end-joining–deficient cells, DSBs are repaired by an alternative end-joining pathway (8–10). Of interest, we have recently reported that B cells can be induced to acquire heightened DNA repair activity upon receipt of signals from CD4 T cells. We termed this process somatic hyperrepair, which we think is necessary to protect the well-being of B cells during the processes of somatic hypermutation and CSR (11).

Proteins required for CSR include Ku, DNA-PK, ATM, Mre11-Rad50-Nbs1, γH2AX, RNF8, 53BP1, MDC1, and XRCC4-ligase IV (12–19). All of these proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur. Among these proteins, 53BP1 deficiency affects CSR the most, as CSR is reduced >90% in cultured 53BP1−/− splenic B cells relative to wild-type (WT) B cells (20–22). This is not due to decreased cell proliferation or reduced germline transcripts (GLTs) at the switch regions. 53BP1-deficient cells do not have a dramatic increase in general chromosome instability, unlike ATM−/− and H2AX−/− cells, but a much higher proportion of the chromosomal aberrancies in 53BP1−/− cells involve the Igh locus, suggesting that 53BP1 has a distinct role at this locus (22).

53BP1 is also a well-known mediator of the cellular response to DNA damage (20, 22). 53BP1 localizes to DNA breaks via two mechanisms, one by interaction with methylated histone H4 Lys20 (H4K20), and another via ubiquitylation of H2A-type histones mediated by a novel E3 ubiquitin ligase, RNF8. We and others recently found that the histone methyltransferase MMSET could affect H4K20 methylation and thereby 53BP1 recruitment at the sites of DNA damage (23, 24). Interestingly, loss of the MMSET gene at chromosome 4p is linked to Wolf–Hirschhorn syndrome (WHS) (25, 26). MMSET is considered one of the causative genes because this gene is deleted in every known case of WHS (25). WHS is a genetic disease with characteristic craniofacial features and developmental disorders, including microcephaly, growth and mental retardation, muscle hypotonia, seizures, and congenital heart defects (25). MMSET+/− mice show varying degrees of congenital heart disease, growth retardation, and craniofacial defects similar to those seen in WHS patients (27). Patients with WHS also display Ab deficiency, especially IgA and IgG isotypes, although the underlying cause of this deficiency is unclear (28). Based on our recent finding of a role for MMSET in the DNA damage response, the goal of this study was to test the hypothesis that MMSET is important for CSR.

Materials and Methods

In vitro cell culture

CH12F3 cells and the subclone C2 were maintained in vitro and CSR assays were performed as previously described (29). In brief, cells were maintained in RPMI 1640 supplemented with 10% FBS, 10 mM 2-ME, and 5% NCTC-109 (Invitrogen). The subclone C2 cells were used in most of the experiments. For the CSR assay, cells were stimulated with 250 ng/ml recombinant murine CD40L (PeproTech) (or 2 μg/ml functional grade purified anti-murine CD40 Ab; BD Biosciences), 10 ng/ml recombinant murine IL-4 (R&D Systems), and 1 ng/ml recombinant human TGF-β (R&D Systems) (CIT) for 72 h and then analyzed by flow cytometry.

Small interfering RNA, short hairpin RNA lentiviral infection and Ab information

To knockdown 53BP1 in CH12F3-C2 B cells, we used small interfering RNA against 53BP1 that was previously described (30). Two independent murine 53BP1- targeting sequences were used: 5′-TGGTCATCCAATGGCTAC-3′ and 5′-GCCAGGTTCTGGAAGAAGA-3′. Lentiviral short hairpin RNA (shRNA) constructs for MMSET and nonsilencing negative control shRNA were purchased from Origene (catalog no. TF517851). The target sequences for murine MMSET are 5′-TGGATATTTGAGAAGAGCCTTGTTGCTTT-3′ and 5′-ATGTCAATAGAGGAGCGGAAAGCCAAATT-3′. For viral infection, CH12F3-C2 cells were transduced with lentivirus by spin inoculation at 800 × g for 30 min at room temperature in the presence of 8 μg/ml polybrene. Cells were then incubated for 3 d, after which positively transduced clones were obtained by puromycin selection. For growth curve analysis, CH12F3-C2 cells were diluted to a concentration of 1 × 105 cells/ml and aliquoted in triplicate in T25 cell culture flasks. At various time points, the numbers and viabilities were analyzed on a Vi-CELL analyzer (Beckman Coulter).

Abs used in this study include the following: anti-γH2AX (07-164; Upstate Biotechnology), anti-53BP1 (NB100-304; Novus Biologicals), anti-H4K20me2 (39173; Active Motif), anti-H3K4me3 (17-614; Upstate Biotechnology), anti-H3K36me2 (07-369; Upstate Biotechnology), anti-MMSET (ab75359; Abcam), and anti-AID (4959; Cell Signaling Technology).

Flow cytometric analyses

For analyzing CSR from IgM (IgM+/IgA−) to IgA (IgM−/IgA+), CH12F3-C2 cells were intracellularly stained with PE-conjugated anti-murine IgA clone 1144-2 (12-5994-82; eBioscience), using Cytofix/Cytoperm and Perm/Wash buffers (BD Biosciences), and assessed for membrane IgM expression using FITC-conjugated anti-murine IgM (11-5890-82; eBioscience). Cells were then analyzed on a FACSCalibur (BD Biosciences) and the data were analyzed using FlowJo software (Tree Star).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (23, 31). Briefly, 2 × 106 cells were fixed by adding formaldehyde (1% final concentration) for 10 min at room temperature and reactions were quenched by adding glycine (final concentration 0.125 M). Cell lysates were sonicated to reduce the DNA length, and the soluble chromatin fraction was obtained after centrifugation. This fraction was diluted in ChIP dilution buffer (2 ml) and precleared using protein A-agarose slurry (Upstate Biotechnology). Precleared lysates were incubated with 4 μg anti-γH2AX, anti-53BP1, anti-H4K20me2, anti-H3K4me3, anti-H3K36me2/3, or IgG overnight at 4°C and complexes were recovered using protein G-Sepharose (Amersham Biosciences). Precipitates were washed several times with high-salt washing buffer and two times with lithium chloride washing buffer. The bound immunocomplex was then reverse cross-linked in elution buffer by heating at 65°C for 10–12 h. Samples were treated with RNase A and proteinase K, respectively, and DNA was ethanol precipitated after a phenol chloroform extraction. Precipitated DNA was dissolved in 50 μl TE and subjected to PCR with or without serial dilution using the following chromatin immunoprecipitation primers: Sμ1 forward, 5′-GCTTCTAAAATGCGCTAAACTGAGGTGATT-3′, reverse, 5′-GTTTAGCTCTATTCAACCTAG-3′; Sμ2 forward, 5′-AAAGAGACATTTGTGTGTCTTTGAGTACCG-3′, reverse, 5′-ATTGGTTAACAGGCAACATTTTTCTTTTAC-3′; Sμ3 forward, 5′-GCTAAACTGAGGTGATTACTCTGAGGTAAG-3′, reverse, 5′-GTTTAGCTTAGCGGCCCAGCTCATTCCAGT-3′; Cμ forward, 5′-CTGTCGCAGAGATGAACCCCA-3′; reverse, 5′-ATCCTTTGTTCTCGATGGTCACCGG-3′; Sα forward, 5′-GTGATTCAGGGAGCAAGAGC-3′, reverse, 5′-TCTAGCCTGGGAGTCTCCTG-3′.

Sequencing of switch junctions

Cμ–Cα switch junction sequences from parental as well as MMSET knockdown CH13F3 cells were amplified by PCR. The PCR products were then subcloned and sequenced. Specifically, genomic DNAs, isolated from ∼105 CIT-induced CH12F3 cells by proteinase K digestion and ethanol precipitation, were amplified by PCR with primers M1 (5′-TAGTAAGCGAGGCTCTAAAAAGCAT-3′) and A1 (5′-CAGCAGTGAGTTTAACAATCC-3′) and nested PCR with primers M2 (5′-GCTTGAGCCAAAATGAAGTAGACT-3′) and A2 (5′-CCTCAGTGCAACTCTATCTAGGTC T-3′). The final PCR products were then subcloned into pCRII TOPO TA vector (Invitrogen) and subsequently sequenced. A total of 20 sequences from MMSET-depleted cells and 22 sequences from parental cells were analyzed. Switch junction sequences were aligned and analyzed for microhomology using the MACAW program (National Institutes of Health). The sizes of sequence microhomology of the two groups were compared for statistical significance using an unpaired two-tailed t test.

Quantitative PCR

Quantitative PCR (qPCR) was performed on a 7500 RT-PCR System (Applied Biosystems) using the SYBR Green detection system with the following program: 95°C for 5 min, 1 cycle; 95°C for 45 s; and 62°C for 45 s, 40 cycles. As an internal control for the normalization of the specific fragments amplified, a locus outside the region of the DSB was amplified, in this case FKBP5, using the input control sample as template. The internal control (FKBP5) primers were as follows: forward, 5′-CAGTCAAGCAATGGAAGAAG-3′, reverse, 5′-CCCGTGCCACCCCTCAGTGA-3′.

After qPCR amplification, the FKBP5 input controls for non-CIT treatment (no class switch) and CIT treatment (class switch) were used to normalize the unswitched and switched samples, respectively. After normalization, the relative levels of the indicated proteins on a specific Igh region were calculated by comparison of unswitched and switched samples to their respective IgG controls. Each experiment was independently performed at least three times and the SEM values were calculated from at least three independent experiments.

RT-PCR analysis

Total RNA was extracted from CH12F3 cells using a PARIS kit (Applied Biosystems). Quantitative RT-PCR was performed using a Brilliant II SYBR Green qRT-PCR Master Mix kit (Agilent Technologies). The primers for RT-PCR analysis of GLTs are as follows: μGLT forward, 5′-CTCTGGCCCTGCTTATTGTTG-3′, reverse, 5′-AATGGTGCTGGGCAGGAAGT-3′; αGLT forward, 5′-CCAGGCATGGTTGAGATAGAGATAG-3′, reverse, αGLT reverse: 5′-GAGCTGGTGGGAGTGTCAGTG-3′.

Results

MMSET binds to regions of the Igh locus and H4K20me2 increases during CSR

Histone posttranslational modifications such as H2A phosphorylation, as well as H4k20 and H3K4 methylation, have been shown to be important for CSR (32–36). To determine whether the histone methyltransferase activity of MMSET is involved in CSR, we used the CH12F3-C2 cell line capable of switching from IgM to IgA expression upon stimulation with CIT (29). As shown in Fig. 1A, 3 d after stimulation with CIT, ∼21% of the CH12F3 cells underwent CSR from IgM to IgA as indicated by the loss of IgM and simultaneous gain of IgA expression.

FIGURE 1.
FIGURE 1.

Induction of H4K20 methylation and recruitment of MMSET to regions of the Igh locus during CSR. (A) Flow cytometry analysis of CH12F3 cells stimulated with CIT for 3 d and stained with anti-IgM and anti-IgA Abs. Simultaneous gain of IgA and loss of IgM expression signifies an effective CSR. Numbers indicate the percentage of total live IgA+ CH12F3 B cells. (B) MMSET ChIP assay for regions of the Igh locus (Sα, left panel; Cμ, middle panel; and Sμ1, right panel) of the CH12F3 cell line with or without CIT stimulation. qPCR analyses of the indicated ChIP samples were performed, where the y-axis represents the relative enrichment of the indicated proteins compared with the IgG control (after normalized with a PCR internal control to a locus other than the Igh region). Three independent experimental replicates were performed for each experiments (±SEM, n = 3). (C) H4K20me2 and H3K36me2 ChIP assay for the indicated regions (Sα, left panel; Cμ, middle panel; and Sμ1, right panel) of the Igh locus in the CH12F3 cell line stimulated with or without CIT for 24 h. (D) Schematic diagram of the position of the PCR products for the ChIP assays employed in this study. (E) ChIP assay of γH2AX at non-Ig gene loci (GADPH and FKBP51) was performed after 24 h CIT stimulation of CH12F3-C2 cells.

We next tested whether MMSET localizes to the S regions following CSR induction. After CSR induction with CIT, chromatin was immunoprecipitated from the cells using Abs directed against MMSET and qPCR was used to determine the relative abundance of MMSET at Igh switch regions, whereas conventional PCR gave a visual representation of the relative accumulation of these proteins at the DSB sites. As shown in Fig. 1B and Supplemental Fig. 1A, similar to 53BP1, MMSET accumulated at the Igh Sμ1, Cμ, and Sα regions during CSR. Because MMSET is a histone methyltransferase, we examined histone methylation at H3K36 and H4K20 during CSR. Using a ChIP assay, we observed that dimethylated H3K36 (H3K36me2) and H4K20 (H4K20me2) increased after CSR induction at Igh switch regions, as did the γH2AX signal (Fig. 1C, Supplemental Fig. 1B). This CIT-induced accumulation was specific to the regions of the Igh locus because non-Ig genes, such as GADPH and FKBP51, were not coimmunoprecipitated by Abs against γH2AX (Fig. 1E). These results imply that MMSET may play a role in CSR.

MMSET facilitates CSR

Although B cells from MMSET-deficient adult mice would be ideal for testing our hypothesis, MMSET-null mice are embryonic or neonatal lethal (27). Therefore, we alternatively used the CH12F3 murine lymphoma cell line to study the role of MMSET in CSR (29). Cells were infected with lentivirus encoding different shRNAs specific for MMSET, and MMSET knockdown cells were isolated using puromycin selection. Cells depleted of 53BP1 were used as a positive control. Compared with cells infected with a scrambled, nonsilencing shRNA construct (control), MMSET knockdown cells showed a significant reduction in class switching efficiency (40 and 62% decrease using MMSET sh2 and sh3, respectively) (Fig. 2A, Supplemental Fig. 2A). To further determine whether the MMSET methyltransferase activity is required for these processes, we mutated the critical residue (F1117) required for MMSET methyltransferase activity (23). We reintroduced shRNA-resistant human WT MMSET or MMSET-F1117A to cells stably transfected with MMSET shRNA. As shown in Supplemental Fig. 2A, whereas MMSET-WT restored class switch efficiency, MMSET-F1117A did not.

FIGURE 2.
FIGURE 2.

MMSET facilitates CSR. (A) CH12F3 cells were infected with lentivirus expressing the indicated shRNA, and CSR was assessed using flow cytometry analysis of IgM and IgA expression following CIT. Numbers indicate the percentage of total live IgA+ B cells. The right panels show the knockdown efficiency of 53BP1and MMSET by Western blot. Data are representative of two independent experiments. (B) Evaluation of the effect of MMSET expression on cell growth. Cell numbers of CH12F3 cells expressing either control shRNA or different MMSET-specific shRNAs were counted on 3 consecutive days. (C) Evaluation of the effect of MMSET expression on cellular viability of preswitched CH12F3 cells. Similar to the experiments in (B), cell viability of CH12F3 cells expressing either control shRNA or different MMSET-specific shRNAs were analyzed on 3 consecutive days. (D) Evaluation of the effect of MMSET expression on cell viability of postswitched CH12F3 cells. Class-switched IgA+/IgM− CH12F3 cells expressing either control or MMSET-specific shRNA were sorted and cell viability was analyzed. (E) Western analysis of AID expression in CH12F3 cells pre- and post-MMSET knockdown by specific shRNAs. (F) ChIP analysis of γH2AX at the Sμ1 region and Sα region of Igh locus in CH12F3 cells transfected with the indicated shRNAs, with or without CIT stimulation. Three independent experimental replicates were performed for each experiment (±SEM, n = 3).

It is possible that downregulation of MMSET could affect cell growth or viability, which could account for the decrease in class switching efficiency. We next tested whether MMSET affects cell proliferation. As shown in Fig. 2B and 2C, cells depleted of MMSET using shRNAs displayed growth curves and overall cell viability comparable to that of control cells before CSR induction. Cells depleted of MMSET also showed similar cell viability to that of parental cells after CSR (Fig. 2D), suggesting that impaired proliferation is not a primary reason for the dramatic decrease of CSR in MMSET-deficient cells.

AID is essential for CSR, and AID expression is rapidly induced upon CIT induction (1). Because MMSET has been shown to play a role in gene expression (27, 37, 38), it is also possible that MMSET knockdown affected AID expression and hence CSR efficiency. However, knockdown of MMSET had no detectable effect on AID expression at the protein level (Fig. 2E). The production of DNA DSBs at the μ region of the Igh locus was also not significantly affected, but DNA DSBs at the Sα region were dramatically decreased, as indicated by the γH2AX level (Fig. 2F, Supplemental Fig. 2B). The latter observation might reflect the possibility that the transcriptional level of these two regions is differently regulated by MMSET as we describe below.

MMSET regulates 53BP1 recruitment to regions of the Igh locus

The inability to complete CSR is frequently associated with accumulation of IgH-associated breaks, and 53BP1 is essential for repair of Igh breaks (22). Because MMSET regulates 53BP1 recruitment to DSB sites in other somatic cells (23, 24), we hypothesized that MMSET regulates CSR in B cells through its effects on 53BP1. To test whether MMSET regulates 53BP1 recruitment and function during CSR, we examined the accumulation of 53BP1 at the Igh switch regions by ChIP coupled with a qPCR assay. Indeed, we found that downregulation of MMSET significantly decreased CIT-induced recruitment of 53BP1 to the Sμ region, but not the level of γH2AX, which lies upstream of MMSET in the DNA repair pathway (Fig. 3A, 3B). Because H4K20me2 is affected by MMSET and is required for 53BP1 recruitment during the DNA damage response, we next examined the level of H4K20me2 at the Igh switch regions. As shown in Fig. 3B and Supplemental Fig. 3A and 3B, knockdown of MMSET dramatically decreased H4K20me2 levels at the Igh switch regions.

FIGURE 3.
FIGURE 3.

MMSET regulates H4k20 methylation in regions of the IgH locus and 53BP1 recruitment. (A and B) ChIP analysis of the indicated proteins at the Igh-Sμ1 switch region in CH12F3 cells transfected with the indicated shRNA with or without CIT stimulation. Right panels, qPCR analysis of ChIP samples from left panels, where the y-axis represents the relative enrichment of the indicated proteins compared with the IgG control. Three independent experimental replicates were performed for each experiment (±SEM, n = 3).

MMSET affects DNA end joining of the Igh switch regions

Because 53BP1 is critical for end joining at the Igh locus during CSR, MMSET could also affect the end joining of broken switch regions through its effect on 53BP1. To directly assay for alterations in DNA repair, we analyzed switch recombination junctions from successfully class-switched IgA+ B cells. We found that Sμ–Sα junctions were significantly different between control and MMSET-deficient cells. In control cells, >65% of the switch junctions analyzed were direct joins, with not >10% of junctions displaying 1 nt microhomology and only 20% of junctions showing 2 nt microhomology (Fig. 4A). In contrast, in the MMSET-depleted cells, we observed that ∼20% of the junctions had >10 nt microhomology and another 20% had >5 nt microhomology. Lastly, <10% were direct joins (Fig. 4A). These results suggest that the nonhomologous end-joining pathway is defective in MMSET-depleted cells, and DNA end joining during CSR resembles that observed in 53BP1-deficient cells (39).

FIGURE 4.
FIGURE 4.

MMSET regulates DNA end joining and transcription of the Igh switch region. (A) Effect of MMSET on microhomology of Cμ–Cα junctions. Percentage breakdown of clones based on the number of overlapping nucleotides (n = 20 for MMSET depleted, n = 22 for parental cells) is shown. (B) RT-qPCR analysis of spliced Igh-Sα switch transcripts in CH12F3 B cells stimulated under the indicated conditions. Data are representative of three independent experiments. (C) RT-qPCR analysis of spliced Igh-Sμ1 switch transcripts in CH12F3 B cells stimulated under the indicated conditions. Data are representative of three independent experiments. (D) CH12F3 cells were transfected with the indicated shRNA and treated with or without CIT. ChIP analysis was then performed for the indicated histone modifications by the Sα primer (left panel) or Sμ1 primer (right panel) pair. The y-axis represents the relative enrichment of the indicated proteins compared with the IgG control. Data are representative of three independent experiments. (±SEM, n = 3).

MMSET regulates transcription of the Igh switch region

MMSET may also modulate gene transcription by regulating H3K36 methylation and H4K20 methylation (23, 27, 37, 38). Previous studies have established that transcription of the Igh switch regions could regulate class switch efficiency (2, 5, 34, 35, 40). To investigate whether MMSET regulates transcription of the Igh switch regions, we measured the levels of germline transcripts that were spliced to join the initiating exon located 5′ of the switch region to the constant exons located 3′ of the switch regions from stimulated B cells. We found that MMSET-depleted cells had reduced levels of the Cα germline transcript, but had near-normal Cμ germline transcripts (Fig. 4B, 4C). This is consistent with the lower levels of H4K4me3, another histone marker linked to transcription initiation regulation at the Igh-Sα region but not at the Igh-Sμ region when MMSET was depleted (Fig. 4D). We additionally found that both H3K36me2 and H4K20me2 decreased at both Igh-Sα and Igh-Sμ regions in the absence of MMSET (Fig. 4D, Supplemental Fig. 3B). Therefore, in addition to the reduced end-joining capability, the decreased transcription of Cα germline transcript at the Igh regions in MMSET-depleted cells may also contribute to the reduced levels of CSR.

Discussion

Recent findings, especially studies linking 53BP1 to CSR, suggest that the DNA damage response plays an important role in CSR. Therefore, knowledge gained in DNA damage response pathways has provided many insights into the molecular mechanism of CSR. Similar to the DNA damage response pathway, the induction of DSBs during CSR initiated an ATM-dependent signaling pathway. ATM phosphorylates H2AX, and phosphorylated H2AX (γH2AX) amplifies the DNA damage signal, in part, by recruiting MDC1 and the E3 ubiquitin ligase RNF8 to promote H2A/H2AX ubiquitination critical for subsequent accumulation of 53BP1 to DSBs (13, 15, 20, 21). In support of this model, ATM-, H2AX-, MDC1-, and RNF8-deficient mice all exhibit defects in CSR due to DSB repair defects (13, 15, 21). Our recent finding that MMSET regulates 53BP1 accumulation through histone methylation at DSBs prompted us to investigate whether MMSET plays a role in CSR. Our study indicates a significant contribution of MMSET to the CSR reaction. We found that following CSR induction, histone H3K36 and H4K20 methylation are increased in the Igh switch regions. Depletion of MMSET decreases these histone modifications and further attenuates 53BP1 accumulation in these Igh switch regions, resulting in significant defects in CSR.

We found that MMSET not only regulates 53BP1 recruitment and subsequent DNA end joining, but also regulates the germline transcription of Sα. Germline transcription at the Igh locus, known to be critical for CSR, is also defective in MMSET-deficient cells (Fig. 4B). Because 53BP1 is not required for germline transcription at the Igh locus (20, 21), these results suggest that, additionally, MMSET regulates a 53BP1-independent function. MMSET deficiency only affects the downstream α germline transcript, but it has no effect on levels of the μ germline transcript. This is reminiscent of the change of H3K4me3 in PTIP−/− cells (34). Thus PTIP-driven changes in H3K4me3 levels can regulate the α germline transcript, but they have no effect on levels of the μ germline transcript. These observations make it likely that MMSET regulates germline transcription through modulation of H3K36 and H4K20 methylation. Several studies suggest the primary methyltransferase activity of MMSET is toward H3K36, but not H4K20 (27, 41, 42). We and others also observed changes of H4K20 methylation during DNA damage responses (23, 24). It is possible that MMSET regulates H4K20 methylation indirectly. Another possibility is that MMSET specificity could change at the sites of DNA damage, given that the specificity of MMSET is substrate dependent (41). Interestingly, H3K4 methylation also decreased in the Igh-Sα region in MMSET-deficient cells. We therefore suggest there is crosstalk between these histone posttranslational modifications in the Igh-Sα region but not in Igh-Sμ region and this is consistent with previous reports (34,38) . Future studies are required to elucidate how MMSET regulates germline transcription at the switch region

Of great interest, MMSET was named as such due to its overexpression as a result of a chromosomal translocation (4, 14) in a subset of patients with multiple myeloma, a malignancy of post-CSR plasma cells. Therefore, it is tempting to speculate that aberrant CSR may be directly involved in the initiation of oncogenic transformation of multiple myeloma. Consistent with this speculation, many multiple myeloma signature translocations involve IgH loci, further suggesting their CSR connection/origin.

Finally, our studies also provide new insights into the etiology of WHS. Although it has long been recognized that WHS patients display Ab deficiency, especially IgA and IgG isotypes, the underlying causes of this deficiency, however, have remained unclear (25, 28). Our study showing that MMSET, a gene often deleted in WHS, regulates CSR provides one possible mechanism for Ab deficiency observed in these patients.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Tasuku Honjo for providing the CH12F3 cell line.

Footnotes

  • H.P., T.L., and X.W. performed experiments, analyzed data, and wrote the paper; and K.Y., D.F.J., and Z.L. designed research, analyzed data, and wrote the paper.

  • This work was supported by the Richard Schulze Family Foundation and by National Institutes of Health Grants CA130996 and CA108961 (to Z.L.) and R01 CA136591 (to D.F.J.). This work was also supported by the Fellowship Award from the Fraternal Order of Eagles and specific start-up funding from the Beijing Proteome Research Center (to H.P.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AID
    activation-induced cytidine deaminase
    ChIP
    chromatin immunoprecipitation
    CIT
    CD40L, IL-4, and TGF-β
    CSR
    class switch recombination
    DSB
    double-stranded break
    GLT
    germline transcript
    H4K20
    histone H4 Lys20
    qPCR
    quantitative PCR
    shRNA
    short hairpin RNA
    S region
    switch region
    WHS
    Wolf–Hirschhorn syndrome
    WT
    wild-type.

  • Received June 29, 2012.
  • Accepted November 11, 2012.

References

  1. ↵
    1. Jung D.,
    2. C. Giallourakis,
    3. R. Mostoslavsky,
    4. F. W. Alt
    . 2006. Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24: 541–570.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Chaudhuri J.,
    2. U. Basu,
    3. A. Zarrin,
    4. C. Yan,
    5. S. Franco,
    6. T. Perlot,
    7. B. Vuong,
    8. J. Wang,
    9. R. T. Phan,
    10. A. Datta,
    11. et al
    . 2007. Evolution of the immunoglobulin heavy chain class switch recombination mechanism. Adv. Immunol. 94: 157–214.
    OpenUrlCrossRefPubMed
    1. Papavasiliou F. N.,
    2. D. G. Schatz
    . 2002. Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109(Suppl): S35–S44.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Peled J. U.,
    2. F. L. Kuang,
    3. M. D. Iglesias-Ussel,
    4. S. Roa,
    5. S. L. Kalis,
    6. M. F. Goodman,
    7. M. D. Scharff
    . 2008. The biochemistry of somatic hypermutation. Annu. Rev. Immunol. 26: 481–511.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Stavnezer J.,
    2. J. E. Guikema,
    3. C. E. Schrader
    . 2008. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26: 261–292.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Di Noia J. M.,
    2. M. S. Neuberger
    . 2007. Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76: 1–22.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Saribasak H.,
    2. D. Rajagopal,
    3. R. W. Maul,
    4. P. J. Gearhart
    . 2009. Hijacked DNA repair proteins and unchained DNA polymerases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 364: 605–611.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Pan-Hammarström Q.,
    2. A. M. Jones,
    3. A. Lähdesmäki,
    4. W. Zhou,
    5. R. A. Gatti,
    6. L. Hammarström,
    7. A. R. Gennery,
    8. M. R. Ehrenstein
    . 2005. Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells. J. Exp. Med. 201: 189–194.
    OpenUrlAbstract/FREE Full Text
    1. Franco S.,
    2. M. M. Murphy,
    3. G. Li,
    4. T. Borjeson,
    5. C. Boboila,
    6. F. W. Alt
    . 2008. DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination. J. Exp. Med. 205: 557–564.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Yan C. T.,
    2. C. Boboila,
    3. E. K. Souza,
    4. S. Franco,
    5. T. R. Hickernell,
    6. M. Murphy,
    7. S. Gumaste,
    8. M. Geyer,
    9. A. A. Zarrin,
    10. J. P. Manis,
    11. et al
    . 2007. IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449: 478–482.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Wu X.,
    2. R. C. Tschumper,
    3. A. Gutierrez Jr..,
    4. S. A. Mihalcik,
    5. G. S. Nowakowski,
    6. D. F. Jelinek
    . 2010. Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells. PLoS ONE 5: e15549.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Dinkelmann M.,
    2. E. Spehalski,
    3. T. Stoneham,
    4. J. Buis,
    5. Y. Wu,
    6. J. M. Sekiguchi,
    7. D. O. Ferguson
    . 2009. Multiple functions of MRN in end-joining pathways during isotype class switching. Nat. Struct. Mol. Biol. 16: 808–813.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Li L.,
    2. M. J. Halaby,
    3. A. Hakem,
    4. R. Cardoso,
    5. S. El Ghamrasni,
    6. S. Harding,
    7. N. Chan,
    8. R. Bristow,
    9. O. Sanchez,
    10. D. Durocher,
    11. R. Hakem
    . 2010. Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer. J. Exp. Med. 207: 983–997.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Lähdesmäki A.,
    2. A. M. Taylor,
    3. K. H. Chrzanowska,
    4. Q. Pan-Hammarström
    . 2004. Delineation of the role of the Mre11 complex in class switch recombination. J. Biol. Chem. 279: 16479–16487.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Ramachandran S.,
    2. R. Chahwan,
    3. R. M. Nepal,
    4. D. Frieder,
    5. S. Panier,
    6. S. Roa,
    7. A. Zaheen,
    8. D. Durocher,
    9. M. D. Scharff,
    10. A. Martin
    . 2010. The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination. Proc. Natl. Acad. Sci. USA 107: 809–814.
    OpenUrlAbstract/FREE Full Text
    1. Reina-San-Martin B.,
    2. H. T. Chen,
    3. A. Nussenzweig,
    4. M. C. Nussenzweig
    . 2004. ATM is required for efficient recombination between immunoglobulin switch regions. J. Exp. Med. 200: 1103–1110.
    OpenUrlAbstract/FREE Full Text
    1. Reina-San-Martin B.,
    2. M. C. Nussenzweig,
    3. A. Nussenzweig,
    4. S. Difilippantonio
    . 2005. Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1. Proc. Natl. Acad. Sci. USA 102: 1590–1595.
    OpenUrlAbstract/FREE Full Text
    1. Reina-San-Martin B.,
    2. S. Difilippantonio,
    3. L. Hanitsch,
    4. R. F. Masilamani,
    5. A. Nussenzweig,
    6. M. C. Nussenzweig
    . 2003. H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197: 1767–1778.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Santos M. A.,
    2. M. S. Huen,
    3. M. Jankovic,
    4. H. T. Chen,
    5. A. J. López-Contreras,
    6. I. A. Klein,
    7. N. Wong,
    8. J. L. Barbancho,
    9. O. Fernandez-Capetillo,
    10. M. C. Nussenzweig,
    11. et al
    . 2010. Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J. Exp. Med. 207: 973–981.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Manis J. P.,
    2. J. C. Morales,
    3. Z. Xia,
    4. J. L. Kutok,
    5. F. W. Alt,
    6. P. B. Carpenter
    . 2004. 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5: 481–487.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Bothmer A.,
    2. D. F. Robbiani,
    3. N. Feldhahn,
    4. A. Gazumyan,
    5. A. Nussenzweig,
    6. M. C. Nussenzweig
    . 2010. 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination. J. Exp. Med. 207: 855–865.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Ward I. M.,
    2. B. Reina-San-Martin,
    3. A. Olaru,
    4. K. Minn,
    5. K. Tamada,
    6. J. S. Lau,
    7. M. Cascalho,
    8. L. Chen,
    9. A. Nussenzweig,
    10. F. Livak,
    11. et al
    . 2004. 53BP1 is required for class switch recombination. J. Cell Biol. 165: 459–464.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Pei H.,
    2. L. Zhang,
    3. K. Luo,
    4. Y. Qin,
    5. M. Chesi,
    6. F. Fei,
    7. P. L. Bergsagel,
    8. L. Wang,
    9. Z. You,
    10. Z. Lou
    . 2011. MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. Nature 470: 124–128.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Hajdu I.,
    2. A. Ciccia,
    3. S. M. Lewis,
    4. S. J. Elledge
    . 2011. Wolf-Hirschhorn syndrome candidate 1 is involved in the cellular response to DNA damage. Proc. Natl. Acad. Sci. USA 108: 13130–13134.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Bergemann A. D.,
    2. F. Cole,
    3. K. Hirschhorn
    . 2005. The etiology of Wolf-Hirschhorn syndrome. Trends Genet. 21: 188–195.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Stec I.,
    2. T. J. Wright,
    3. G. J. van Ommen,
    4. P. A. de Boer,
    5. A. van Haeringen,
    6. A. F. Moorman,
    7. M. R. Altherr,
    8. J. T. den Dunnen
    . 1998. WHSC1, a 90 kb SET domain-containing gene, expressed in early development and homologous to a Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical region and is fused to IgH in t(4;14) multiple myeloma. Hum. Mol. Genet. 7: 1071–1082.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Nimura K.,
    2. K. Ura,
    3. H. Shiratori,
    4. M. Ikawa,
    5. M. Okabe,
    6. R. J. Schwartz,
    7. Y. Kaneda
    . 2009. A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome. Nature 460: 287–291.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Hanley-Lopez J.,
    2. L. L. Estabrooks,
    3. R. Stiehm
    . 1998. Antibody deficiency in Wolf-Hirschhorn syndrome. J. Pediatr. 133: 141–143.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Nakamura M.,
    2. S. Kondo,
    3. M. Sugai,
    4. M. Nazarea,
    5. S. Imamura,
    6. T. Honjo
    . 1996. High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int. Immunol. 8: 193–201.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Tripathi V.,
    2. T. Nagarjuna,
    3. S. Sengupta
    . 2007. BLM helicase-dependent and -independent roles of 53BP1 during replication stress-mediated homologous recombination. J. Cell Biol. 178: 9–14.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Begum N. A.,
    2. K. Kinoshita,
    3. N. Kakazu,
    4. M. Muramatsu,
    5. H. Nagaoka,
    6. R. Shinkura,
    7. D. Biniszkiewicz,
    8. L. A. Boyer,
    9. R. Jaenisch,
    10. T. Honjo
    . 2004. Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch. Science 305: 1160–1163.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Stanlie A.,
    2. M. Aida,
    3. M. Muramatsu,
    4. T. Honjo,
    5. N. A. Begum
    . 2010. Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination. Proc. Natl. Acad. Sci. USA 107: 22190–22195.
    OpenUrlAbstract/FREE Full Text
    1. Schotta G.,
    2. R. Sengupta,
    3. S. Kubicek,
    4. S. Malin,
    5. M. Kauer,
    6. E. Callén,
    7. A. Celeste,
    8. M. Pagani,
    9. S. Opravil,
    10. I. A. De La Rosa-Velazquez,
    11. et al
    . 2008. A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes Dev. 22: 2048–2061.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Daniel J. A.,
    2. M. A. Santos,
    3. Z. Wang,
    4. C. Zang,
    5. K. R. Schwab,
    6. M. Jankovic,
    7. D. Filsuf,
    8. H. T. Chen,
    9. A. Gazumyan,
    10. A. Yamane,
    11. et al
    . 2010. PTIP promotes chromatin changes critical for immunoglobulin class switch recombination. Science 329: 917–923.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Schwab K. R.,
    2. S. R. Patel,
    3. G. R. Dressler
    . 2011. Role of PTIP in class switch recombination and long-range chromatin interactions at the immunoglobulin heavy chain locus. Mol. Cell. Biol. 31: 1503–1511.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Oda H.,
    2. M. R. Hübner,
    3. D. B. Beck,
    4. M. Vermeulen,
    5. J. Hurwitz,
    6. D. L. Spector,
    7. D. Reinberg
    . 2010. Regulation of the histone H4 monomethylase PR-Set7 by CRL4Cdt2-mediated PCNA-dependent degradation during DNA damage. Mol. Cell 40: 364–376.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Marango J.,
    2. M. Shimoyama,
    3. H. Nishio,
    4. J. A. Meyer,
    5. D. J. Min,
    6. A. Sirulnik,
    7. Y. Martinez-Martinez,
    8. M. Chesi,
    9. P. L. Bergsagel,
    10. M. M. Zhou,
    11. et al
    . 2008. The MMSET protein is a histone methyltransferase with characteristics of a transcriptional corepressor. Blood 111: 3145–3154.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Martinez-Garcia E.,
    2. R. Popovic,
    3. D. J. Min,
    4. S. M. Sweet,
    5. P. M. Thomas,
    6. L. Zamdborg,
    7. A. Heffner,
    8. C. Will,
    9. L. Lamy,
    10. L. M. Staudt,
    11. et al
    . 2011. The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells. Blood 117: 211–220.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Difilippantonio S.,
    2. E. Gapud,
    3. N. Wong,
    4. C. Y. Huang,
    5. G. Mahowald,
    6. H. T. Chen,
    7. M. J. Kruhlak,
    8. E. Callen,
    9. F. Livak,
    10. M. C. Nussenzweig,
    11. et al
    . 2008. 53BP1 facilitates long-range DNA end-joining during V(D)J recombination. Nature 456: 529–533.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Ramiro A. R.,
    2. P. Stavropoulos,
    3. M. Jankovic,
    4. M. C. Nussenzweig
    . 2003. Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat. Immunol. 4: 452–456.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Li Y.,
    2. P. Trojer,
    3. C. F. Xu,
    4. P. Cheung,
    5. A. Kuo,
    6. W. J. Drury III.,
    7. Q. Qiao,
    8. T. A. Neubert,
    9. R. M. Xu,
    10. O. Gozani,
    11. D. Reinberg
    . 2009. The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate. J. Biol. Chem. 284: 34283–34295.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Kuo A. J.,
    2. P. Cheung,
    3. K. Chen,
    4. B. M. Zee,
    5. M. Kioi,
    6. J. Lauring,
    7. Y. Xi,
    8. B. H. Park,
    9. X. Shi,
    10. B. A. Garcia,
    11. et al
    . 2011. NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming. Mol. Cell 44: 609–620.
    OpenUrlCrossRefPubMed
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