Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma María Berdascoa, Santiago Roperoa,b, Fernando Setiena, Mario F. Fragaa,c, Pablo Lapunzinad,Re´ gine Lossone, Miguel Alaminosf, Nai-Kong Cheungg, Nazneen Rahmanh, and Manel Estellera,i,1 aCancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, 08907 L’Hospitalet, Spain; bDepartment of Biochemistry and Molecular Biology, School of Medicine, University of Alcala, 28801 Madrid, Spain; cCancer Epigenetics Unit, Instituto Universitario de Oncología del Principado de Asturias, Oviedo University-Hospital Universitario Central de Asturias, 33004 Oviedo, Spain; dInstituto de Gene´tica Me´dica y Molecular, Hospital Universitario La Paz, 28050 Madrid, Spain; eInstitut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire and Institut Clinique de la Souris, Illkirch, Strasbourg 67404, France; fDepartment of Histology, Granada University and Hospital Clinico Foundation, 18071 Granada, Spain; gDepartment of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; hSection of Cancer Genetics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom; and iInstitucio Catalana de Recerca i Estudis Avanc¸ats, 08010 Barcelona, Spain Edited by Stanley M. Gartler, University of Washington, Seattle, WA, and approved October 27, 2009 (received for review June 19, 2009) Sotos syndrome is an autosomal dominant condition characterized protein–protein interaction (8). NSD1 has histone methyltrans- by overgrowth resulting in tall stature and macrocephaly, together ferase activity, demonstrated by the use of a recombinant protein- with an increased risk of tumorigenesis. The disease is caused by containing SET domain of NSD1 that has the ability to methylate loss-of-function mutations and deletions of the nuclear receptor the histone lysine residues H3-K36 and H4-K20 (9) and in the SET domain containing protein-1 (NSD1) gene, which encodes a context of leukemia cells within the fusion protein NUP98-NSD1 to histone methyltransferase involved in chromatin regulation. How- methylate H3-K36 in association with gene activation (10). There ever, despite its causal role in Sotos syndrome and the typical is growing evidence that deregulation of SET domain-containing accelerated growth of these patients, little is known about the proteins, such as NSD1, has an important role in cellular transfor- putative contribution of NSD1 to human sporadic malignancies. mation (11), with the leukemia-translocated H3-K4 and H3-K36 Here, we report that NSD1 function is abrogated in human neu- histone methyltransferases hDOT1L and MLL, respectively, being roblastoma and glioma cells by transcriptional silencing associated further examples (12, 13). The case for NSD1 is even more with CpG island-promoter hypermethylation. We also demonstrate interesting because its genetic disruption in Sotos syndrome is that the epigenetic inactivation of NSD1 in transformed cells leads associated with an elevated risk of cancer (1–4), as occurs with to the specifically diminished methylation of the histone lysine other childhood overgrowth conditions such as Beckwith- residues H4-K20 and H3-K36. The described phenotype is also Wiedemann (14). Because patients with NSD1 germ-line genetic observed in Sotos syndrome patients with NSD1 genetic disrup- disruption have an increased risk of developing malignancy before tion. Expression microarray data from NSD1-depleted cells, fol- adulthood, including neuroblastoma, Wilms tumors, and hemato- lowed by ChIP analysis, revealed that the oncogene MEIS1 is one logical malignancies, a tumor-suppressor function for NSD1 might of the main NSD1 targets in neuroblastoma. Furthermore, we show be proposed. This putative role is also supported by the presence of that the restoration of NSD1 expression induces tumor suppressor- genomic rearrangements involving NSD1 in leukemias (10, 15) and like features, such as reduced colony formation density and inhi- breast cancer cells (16). However, somatic mutations of NSD1 have bition of cellular growth. Screening a large collection of different not been described in sporadic neoplasms. Transcriptional inacti- tumor types revealed that NSD1 CpG island hypermethylation was vation by cytosine/phosphate/guanine (CpG) island promoter hy- a common event in neuroblastomas and gliomas. Most impor- permethylation is an alternative mechanism for the inactivation of tantly, NSD1 hypermethylation was a predictor of poor outcome in high-risk neuroblastoma. These findings highlight the importance tumor suppressor genes (17–19). Similar scenarios to that outlined of NSD1 epigenetic inactivation in neuroblastoma and glioma that for NSD1 have been described for familial tumor-suppressor genes, leads to a disrupted histone methylation landscape and might have such as hMLH1 and BRCA1, which are very rarely mutated in a translational value as a prognostic marker. sporadic tumors, but undergo epigenetic inactivation in noninher- ited neoplasms (17–19). cancer ͉ DNA methylation ͉ epigenetics ͉ histone ͉ overgrowth Here, we demonstrate that NSD1 undergoes CpG island pro- moter methylation-associated gene silencing in human neuroblas- toma and glioma cells. NSD1 epigenetic inactivation is associated otos syndrome is an autosomal dominant condition character- with global diminished levels of trimethylated histone lysine resi- ized by physical overgrowth during the first years of life, a S dues H4-K20 and H3-K36, and NSD1 protein absence in the 5Ј end distinctive facial appearance, and learning disability (1, 2) with an regulatory of its target genes, such as the oncogene MEIS1. increased incidence of malignant neoplasms (1–4). The distinctive Furthermore, the reintroduction of NSD1 reduces colony forma- head shape and size has led to Sotos syndrome sometimes being tion and cell growth, supporting a tumor-suppressor role. Most called cerebral gigantism (1, 2). Mutations in the nuclear receptor SET [su(var)3–9, enhancer-of-zeste, trithorax] domain containing protein-1 (NSD1) gene are found in patients exhibiting the clinical Author contributions: M.B., S.R., F.S., M.F.F., P.L., N.R., and M.E. designed research; M.B., symptoms of Sotos syndrome (1, 2, 5). There are occasional S.R., F.S., and M.F.F. performed research; P.L., R.L., M.A., N.-K.C., and N.R. contributed new individuals with NSD1 defects that overlap clinically with Sotos reagents/analytic tools; M.B., S.R., F.S., M.F.F., M.A., and M.E. analyzed data; and M.B. and syndrome and other conditions such as Weaver syndrome (6, 7). M.E. wrote the paper. The vast majority of NSD1 mutational mechanisms, including The authors declare no conflict of interest. truncating, missense, and splice-site mutations and deletions, result This article is a PNAS Direct Submission. in loss of function of the NSD1 protein (1, 2). Freely available online through the PNAS open access option. The NSD1 protein contains a SET domain and other functional 1To whom correspondence should be addressed. E-mail: [email protected]. domains, including plant homeodomain and proline-tryptophan- This article contains supporting information online at www.pnas.org/cgi/content/full/ tryptophan-proline domains, both of which are involved in a 0906831106/DCSupplemental. 21830–21835 ͉ PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906831106 Downloaded by guest on September 28, 2021 important, the presence of aberrant methylation at the NSD1 determine whether they recapitulate the diminished levels of these promoter predicts worse survival in high-risk neuroblastoma marks observed in NSD1 epigenetically silenced cancer cells. The patients. assessment of seven Sotos syndrome samples carrying different inactivating genetic disruptions of NSD1 showed that these patients Results indeed had significantly decreased trimethylated H3-K36 and H4- NSD1 Promoter CpG Island Hypermethylation Leads to Gene Inacti- K20 (Fig. 2C and Fig. S1) compared with lymphoblastoid cell lines vation. NSD1 is a gene candidate for hypermethylation-associated from healthy donors, again supporting a central role for NSD1 in inactivation in human cancer because a 5Ј-CpG island is located the formation of the aforementioned trimethylated lysine residues. around the transcription start site (Fig. 1A). To analyze the DNA -methylation status of the promoter-associated CpG island, we Loss of NSD1 Recruitment to 5؅-Regulatory Regions of Growth screened 72 human cancer cell lines from 12 different cell malig- Promoting Genes in Hypermethylated Cancer Cells: The Example of the nancy types (Table S1), using bisulfite genomic sequencing and MEIS1 Oncogene in Neuroblastoma. The divalent specificity to meth- methylation-specific PCR targeted to the area surrounding the ylate H4-K20 and H3-K36 in vitro (9) is currently thought to be transcription start site. NSD1 CpG island promoter hypermethyl- unique to NSD1, and our findings indicate that there is also double ation was found in three cancer cell lines: LAI-5S (neuroblastoma), substrate specificity in vivo. H4-K20 methylation appears to be LAN-I (neuroblastoma), and U373-MG (glioblastoma) (Fig. 1 A associated with transcriptional silencing (23, 24) and H3-K36 and B). All normal tissues analyzed, including lymphocytes and methylation has been found primarily in active genes throughout brain, were completely unmethylated at the NSD1 promoter (Fig. the gene body (25, 26), but it also might also