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L3MBTL1 Polycomb Protein, a Candidate Tumor Suppressor in Del(20Q12) Myeloid Disorders, Is Essential for Genome Stability

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L3MBTL1 Polycomb Protein, a Candidate Tumor Suppressor in Del(20Q12) Myeloid Disorders, Is Essential for Genome Stability L3MBTL1 polycomb protein, a candidate tumor suppressor in del(20q12) myeloid disorders, is essential for genome stability Nadia Gurvicha, Fabiana Pernaa, Andrea Farinab, Francesca Vozaa, Silvia Menendeza, Jerard Hurwitzb, and Stephen D. Nimera,1 aMolecular Pharmacology and Chemistry Program and bMolecular Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Contributed by Jerard Hurwitz, November 17, 2010 (sent for review August 2, 2010) The l3mbtl1 gene is located on the long arm of chromosome 20 erate mutations and/or chromosomal translocations that can (q12), within a region commonly deleted in several myeloid malig- promote tumorigenesis (9). DNA breaks resulting from replica- nancies. L3MBTL1 is a human homolog of the Drosophila polycomb tive stress trigger an ATM/ATR-dependent DNA damage re- L(3)MBT tumor suppressor protein and thus a candidate tumor sup- sponse (DDR), which prevents the proliferation of cells with pressor in del(20q12) myeloid disorders. We used the loss-of-func- damaged DNA by inducing either cell cycle arrest or apoptosis. tion approach to explore the possible tumor suppressive mecha- An activated DDR is present in precancerous lesions from tis- nism of L3MBTL1 and found that depletion of L3MBTL1 from sues of different origins, and many overexpressed oncogenes human cells causes replicative stress, DNA breaks, activation of cause replicative stress and activation of the DDR. When cou- the DNA damage response, and genomic instability. L3MBTL1 pled with mutations in checkpoint and/or DNA repair genes, interacts with Cdc45, MCM2-7 and PCNA, components of the these abnormalities can lead to cancer (10–12). DNA replication machinery, and is required for normal replication In this study we examined the function of L3MBTL1 in mam- fork progression, suggesting that L3MBTL1 causes DNA damage, malian cells and found that L3MBTL1 interacts with several at least in part, by perturbing DNA replication. An activated DNA components of the DNA replication machinery. Depletion of CELL BIOLOGY damage response and genomic instability are common features in L3MBTL1 in cells was sufficient to trigger the DDR and pro- tumorigenesis and a consequence of overexpression of many on- mote genomic instability. Thus, L3MBTL1 is essential for main- cogenes. We propose that the loss of L3MBTL1 contributes to the − taining DNA replication, providing a mechanism for its role as development of 20q hematopoietic malignancies by inducing rep- a putative tumor suppressor protein. licative stress, DNA damage, and genomic instability. Results H4K20me1/2 binding protein | chromatin reader Depletion of L3MBTL1 Inhibits Cell Proliferation and Causes G2/M Arrest. To assess the role of L3MBTL1 in cell cycle regulation, we he l3mbtl1 gene is located on the long arm of chromosome 20q, depleted L3MBTL1 mRNA and protein in U2OS cells using Twithin a region on 20q12 commonly deleted in several myeloid lentiviral vectors that expressed several shRNAs directed against malignancies, including myeloproliferative neoplasms, myelodys- its ORF. Down-regulation of L3MBTL1 mRNA and protein was plastic syndromes, and acute myeloid leukemia (1). It has been achieved efficiently with three different shRNAs (≥90% knock- proposed that the 20q12 locus contains one or more tumor sup- down; Fig. 1 A and B). The depleted cells were monitored for pressors, which when lost contribute to the development of these S phase entry by using BrdU incorporation, and we detected disorders. L3MBTL1 is a human homolog of the Drosophila a marked decrease of S phase cells and the accumulation of cells in polycomb group (PcG) protein L(3)MBT. Homozygous mutations the G2/M phase (Fig. 1C and Fig. S1A). This effect was also ob- served in MRC5 normal diploid fibroblasts, Cal51, T98G, and of the Drosophila l3mbt gene cause malignant transformation of – the adult optic neuroblasts and ganglion mother cells in the larval K562 cells following L3MBTL1 knockdown (Fig. S1 B D), dem- onstrating that it is not cell-type specific; it occurred in non- brain (2). Somatic, focal deletions of other human L3MBTL family fi members, the l3mbtl2 and l3mbtl3 genes, have recently been found cancerous cells as well as hematopoietic K562 cells. These ndings indicate that these cell cycle effects are relevant to the myeloid in human medulloblastoma (3). These findings suggest that compartment. L3MBTL1 is a candidate tumor suppressor gene in myeloid ma- lignancies associated with 20q12 deletions. Depletion of L3MBTL1 Generates DNA Breaks. Cell cycle alterations The L3MBTL1 protein contains three MBT repeats, which as- triggered by L3MBTL1 depletion suggest that cells may be ex- sume a three-bladed propeller-like architecture, as well as a Zn periencing replicative stress, leading to DNA damage. To verify fi nger and an SPM dimerization domain (4, 5). We previously this hypothesis, we examined two DNA damage markers, the demonstrated that L3MBTL1 functions as an HDAC-independent production of the phosphorylated histone H2A.x (γH2A.x), transcriptional repressor (6) that binds preferentially to mono- and which localizes to sites of double-stranded DNA breaks (DSBs) dimethylated lysines of histones via the second of its three MBT (13), and the distribution of 53BP1, which is recruited to DNA repeats (7, 8). The three MBT domains of L3MBTL1 are sufficient within minutes following DNA damage by binding to H4K20me2 to compact nucleosomal arrays. This compaction requires that the and possibly other modified histones (14). We observed an in- nucleosome contain a mono- or dimethylated lysine 26 on histone crease in both γH2A.x and 53BP1 foci following L3MBTL1 de- H1b or lysine 20 on histone H4 (H4K20) (7). L3MBTL1 binds to chromatin most prominently during the S phase of the cell cycle, concomitant with the appearance of the monomethylated H4K20 Author contributions: N.G. designed research; N.G., F.P., A.F., F.V., and S.M. performed (H4K20me1) mark (8), suggesting that the biological function of research; N.G. analyzed data; and N.G., J.H., and S.D.N. wrote the paper. L3MBTL1 may be related to DNA replication. The authors declare no conflict of interest. The accurate duplication of DNA during replication is es- 1To whom correspondence should be addressed. E-mail: [email protected]. sential for maintaining genomic stability, as uncorrected errors This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. made during this process can lead to DNA breaks, which gen- 1073/pnas.1017092108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1017092108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Fig. 1. L3MBTL1 depletion inhibits cell proliferation and causes G2/M arrest. U2OS cells were infected with control shRNA or one of several different lentiviral shRNAs target- ing L3MBTL1. Levels of L3MBTL1 mRNA (A) and protein (B) were measured by quantitative RT-PCR and immunoblotting 48 h after infection. L3MBTL1 mRNA levels detected were adjusted for loading discrepancies using Hprt mRNA as the loading standard, and the levels of mRNA detected were plotted as a percent of the L3MBTL1 observed in U2OS cells infected with control shRNA. To quantitate depletion of L3MBTL1, the relative protein levels of L3MBTL1 were ad- justed using the α-tubulin loading control and quantified relative to the protein level present in the control sample (set as 1). (C) U2OS cells were infected with control shRNA or with L3MBTL1 shRNA #3. After 48 h, cells were incubated with BrdU, stained with BrdU-APC antibody and propidium iodide (PI), and analyzed by flow cytometry. The distribution of BrdU (y axis) and PI (x axis) is plotted. pletion with all three shRNAs in U2OS (Fig. 2 A and B) and in cells from progressing into mitosis. To explore this pathway, we MRC5 fibroblasts (Fig. S2A). first examined the phosphorylation of the ATM kinase, one hall- We also measured the formation of DNA strand breaks in mark of the DDR pathway (16), and detected increased phospho- L3MBTL1-depleted cells using the comet assay, which visualizes ATM foci that overlapped with 53BP1 foci in L3MBTL1-depleted damaged DNA on a single-cell level (15). Comet tails were de- cells (Fig. 3A). We also examined whether the downstream com- tected in depleted U2OS cells (Fig. 2C and Fig. S2B), MRC5 and ponents of the ATR/ATM-triggered DDR pathway were activated Cal51 cells (Fig. 2D), indicating DNA strand breakage. We treated following L3MBTL1 depletion using immunoblotting to monitor MRC5 fibroblasts with varying doses of gamma irradiation to es- changes in phosphorylation of the key components of this system, tablish a range of tail moments (Fig. 2D) and determined that including Chk1 and Chk2. As shown in Fig. 3B, increased levels of depletion of L3MBTL1 caused extensive DNA damage, similar to phospho-Ser317-Chk1, phospho-Ser345-Chk1, and phospho- that induced by 5 Gy of irradiation. Thr68-Chk2 were detected in cells at 24, 48, and 72 h postinfection, similar to that observed in cells irradiated with 10 Gy. Loss of L3MBTL1 Activates the DDR and Affects H4K20 Methylation Depletion of L3MBTL1 altered the activities as well as the Status. DNA damage triggers a checkpoint response that prevents levels of a number of proteins associated with checkpoint regu- Fig. 2. Depletion of L3MBTL1 generates DNA breaks. (A) U2OS cells infected with one of several shRNAs against L3MBTL1 or with control shRNA were stained with antibodies against 53BP1 and γH2A.x. (B) DNA damage foci were quan- titated in control and L3MBTL1 knockdown cells based on whether they contained more or less than five foci per cell. (C) The tail moment was calculated and plotted from three in- dependent comet assays of U2OS cells treated with control shRNA or L3MBTL1 shRNA U2OS cells at 24 and 48 h after in- fection. (D) Calculated tail moments from comet assays of control and L3MBTL1-depleted MRC5 and Cal51 cells 48 h af- ter infection are plotted.
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