Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma

Abstract

Sotos syndrome is an autosomal dominant condition characterized by overgrowth resulting in tall stature and macrocephaly, together with an increased risk of tumorigenesis. The disease is caused by loss-of-function mutations and deletions of the nuclear receptor SET domain containing protein-1 (NSD1) gene, which encodes a histone methyltransferase involved in chromatin regulation. However, despite its causal role in Sotos syndrome and the typical accelerated growth of these patients, little is known about the putative contribution of NSD1 to human sporadic malignancies. Here, we report that NSD1 function is abrogated in human neuroblastoma and glioma cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also demonstrate that the epigenetic inactivation of NSD1 in transformed cells leads to the specifically diminished methylation of the histone lysine residues H4-K20 and H3-K36. The described phenotype is also observed in Sotos syndrome patients with NSD1 genetic disruption. Expression microarray data from NSD1-depleted cells, followed by ChIP analysis, revealed that the oncogene MEIS1 is one of the main NSD1 targets in neuroblastoma. Furthermore, we show that the restoration of NSD1 expression induces tumor suppressor-like features, such as reduced colony formation density and inhibition of cellular growth. Screening a large collection of different tumor types revealed that NSD1 CpG island hypermethylation was a common event in neuroblastomas and gliomas. Most importantly, NSD1 hypermethylation was a predictor of poor outcome in high-risk neuroblastoma. These findings highlight the importance of NSD1 epigenetic inactivation in neuroblastoma and glioma that leads to a disrupted histone methylation landscape and might have a translational value as a prognostic marker.

Fig. 1.

Analysis of NSD1 CpG island promoter methylation status and gene function in human cancer cell lines. (A) Schematic depiction of the NSD1 CpG island around the transcription start site (long black arrow). CpG dinucleotides are represented as short vertical lines. Location of bisulfite genomic sequencing and methylation-specific PCR primers are indicated as black and white arrows, respectively. Results of bisulfite genomic sequencing of 10 individual clones in two regions of the NSD1 CpG island are shown. Presence of a methylated or unmethylated cytosine is indicated by a black or white square, respectively. NL, normal lymphocytes; NB, normal brain. (B) Methylation-specific PCR for the NSD1 gene in cancer cell lines. The presence of a PCR band under lane M indicates methylated genes, while the presence under lane U indicates unmethylated genes. In vitro methylated DNA (IVD) is used as positive control. (C) RT-PCR analysis of NSD1 expression. Treatment with the demethylating agent (ADC+) reactivates NSD1 gene expression in the NSD1-hypermethylated cancer cell lines LAI-5S, LAN-1, and U373-MG. (D) Western blot analysis of NSD1 expression. The NSD1-hypermethylated cell lines LAI-5S and U373-MG do not express the NSD1 protein, in comparison with the unmethylated SK-N-JD and SK-N-BE(2)C cells. Treatment with the demethylating agent (ADC+) reactivates NSD1 gene expression. (E) Immunofluorescence analysis of NSD1 expression. The NSD1 CpG island methylated cell line LAI-5S does not stain for the NSD1 protein, in comparison with the unmethylated SK-N-JD cells. [Magnifications: 40× (LAI-5S) and 20× (SK-N-JD).]

Fig. 2.

Histone modifications and histone modifiers according to NSD1 status. (A) Western blot and immunofluorescence analysis of 3Me-K20-H4 and 3Me-K36-H3 in neuroblastoma cells. The NSD1-hypermethylated cell lines LAI-5S and LAN-1 show reduced levels of the 3Me-K20-H4 and 3Me-K36-H3 marks, in comparison with the unmethylated SK-N-BE(2)C and SK-N-JD cells. (Magnification: 40×.) (B) Western blot analysis of the histone modifiers SET2 and SMYD2 (methylation of H3-K36) and Pr-SET7/8 and SUV4–20h1,h2 (methylation of H4-K20) show similar expression with independence of the NSD1 epigenetic inactivation status. (C) Western blot analysis of 3Me-K20-H4 and 3Me-K36-H3 in Sotos syndrome samples. The lymphoblastoid cell lines from seven Sotos syndrome patients (R604X to OGS661) show reduced levels of the 3Me-K20-H4 and 3Me-K36-H3 marks, in comparison with unmethylated SK-N-JD neuroblastoma cells or lymphoblastoid cell lines from healthy donors (CCL256.1 and GMO8729).

Fig. 3.

Loss of NSD1 recruitment to the 5′ end of the MEIS1 oncogene in neuroblastoma. (A) (Left) NSD1 depletion by RNA interference in SK-N-JD cells is associated with reduced levels of 3Me-K20-H4 and 3Me-K36-H3. (Right) NSD1 expression upon RNA interference monitored by RT-PCR in CpG island unmethylated SK-N-JD cells. (B) Quantitative RT-PCR shows the high up-regulation of the MEIS1 transcript upon NSD1 depletion. (C) Determination by conventional and quantitative ChIP of NSD1 occupancy at the 5′ end promoter region of the MEIS1 gene. The presence of the NSD1 protein is evident in unmethylated neuroblastoma cells (SK-N-JD) and lymphoblastoid cells derived from healthy donors (GMO8729), whereas it is absent or extremely diminished in neuroblastoma cells with NSD1 epigenetic inactivation (LAI-5S) and lymphoblastoid cells from a Sotos syndrome patient (OGS55). (D) Quantitative RT-PCR and Western blot analysis shows the high up-regulation of the MEIS1 transcript in neuroblastoma cells with NSD1 CpG island hypermethylation (LAI-5S and LAN-1) compared with NSD1 unmethylated cells (SK-N-JD and SK-N-BE (2)C) (Left) and in Sotos syndrome lymphoblastoid cells (OGS55 to R2017W) compared with lymphoblastoid cells from healthy donors (CCL256.1 and GMO8729) (Right). (E) Quantitative ChIP for histone modification marks at the 5′ end of the MEIS1 gene. NSD1 epigenetic silencing (LAI-5S) and genetic loss (OGS55) both are associated with a depletion in repressive marks (3Me-K20-H4, 3Me-K9-H3 and 3Me-K27-H3) and an enrichment in active marks (3Me-K4-H3) compared with the NSD1 unmethylated SK-N-JD cells.

Fig. 4.

Tumor suppressor-like properties of NSD1. (A) Colony formation assay. (Left) NSD1 expression monitored by RT-PCR and Western blot analysis in empty vector and NSD1-transfected LAI-5S cells and densitometric quantification of the colony formation density. (Right) Examples of the colony focus assay after 2-week selection with G418 and methylene blue staining. (B) A decrease of cell viability over time, determined by the MTT assay, upon NSD1 transfection is observed. (C) Effect of NSD1 depletion on cell growth in the NSD1 unmethylated SK-N-JD neuroblastoma cells. (Left) An increase of cell viability over time, determined by the MTT assay, upon NSD1 depletion is observed. (Right) Optical image of the effect of NSD1 reduction on the in vitro growth of SK-N-JD cells at 48 h. (Magnification: 10×.)

Fig. 5.

NSD1 CpG island hypermethylation in primary human malignancies. (A) Analysis of NSD1 methylation by methylation-specific PCR. The presence of a PCR band under lane M indicates methylated genes, whereas the presence under lane U indicates unmethylated genes. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) are used as negative and positive control for unmethylated and methylated genes, respectively. NSD1 hypermethylation is observed in primary neuroblastomas (N1-N6) and gliomas (G1-G6), but it was absent in other tumor types (example, colorectal cancer, CRC1-CRC6). (B) Kaplan–Meier analysis of NSD1 promoter hypermethylation in neuroblastoma patients. NSD1 promoter hypermethylation was significantly associated with lower overall survival (P = 0.048) and progression-free survival (P = 0.026).