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Epigenetic modifications of splicing factor genes in myelodysplastic syndromes and acute myeloid leukemia.

Wong JJ, Lau KA, Pinello N, Rasko JE - Cancer Sci. (2014)

Bottom Line: The only evidence of epigenetic effects was hypermethylation of the YBX3 promoter in U937 cells in conjunction with an enrichment of histone marks associated with gene silencing.Hypermethylation of the ZRSR2 promoter was also detected in 7/173 (4%) cases but was not associated with decreased mRNA expression (P = 0.1204).We conclude that DNA hypermethylation does not frequently silence splicing factors in MDS and AML.

View Article: PubMed Central - PubMed

Affiliation: Gene and Stem Cell Therapy Program, Centenary Institute, Camperdown, New South Wales, Australia; Sydney Medical School, University of Sydney, Camperdown, New South Wales, Australia.

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Silencing of YBX3 in U937 cells is associated with promoter DNA hypermethylation and altered histone modifications. (a) YBX3 promoter showing individual CpGs (vertical black line) and their distance from the transcriptional start site (+1). Recognition site for BstU1 used in combined bisulfite restriction analysis (COBRA) assays for YBX3 is shown on the map. (b) Gel electrophoretogram of COBRA showing PCR amplicons of the YBX3 promoter following bisulfite conversion and digestion with BstU1 of genomic DNA from leukemic cell lines and controls. (c) Clonal bisulfite sequencing confirming hypermethylation at the YBX3 promoter in U937 cells. Each row represents a single cloned PCR amplicon aligned to the map shown in (a). Black and white circles denote methylated and unmethylated CpGs, respectively. (d) Reversal of DNA hypermethylation in 5-Aza-2′deoxycytidine (5-AZA)-treated U937 cells as detected by clonal bisulfite sequencing. (e) Quantitative RT-PCR showing increased expression of YBX3 following treatment of U937 cells with 5-AZA. (f) Fold enrichment of YBX3 promoter sequence bound to H3K4me3 (activation mark) and (g) H3K27me3 (silencing mark) in HL-60 and U937 cells normalized to IgG control. +ve Con, positive control, completely methylated human genomic DNA; N1, N2, N3, negative controls, peripheral blood DNA from three healthy individuals.
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fig02: Silencing of YBX3 in U937 cells is associated with promoter DNA hypermethylation and altered histone modifications. (a) YBX3 promoter showing individual CpGs (vertical black line) and their distance from the transcriptional start site (+1). Recognition site for BstU1 used in combined bisulfite restriction analysis (COBRA) assays for YBX3 is shown on the map. (b) Gel electrophoretogram of COBRA showing PCR amplicons of the YBX3 promoter following bisulfite conversion and digestion with BstU1 of genomic DNA from leukemic cell lines and controls. (c) Clonal bisulfite sequencing confirming hypermethylation at the YBX3 promoter in U937 cells. Each row represents a single cloned PCR amplicon aligned to the map shown in (a). Black and white circles denote methylated and unmethylated CpGs, respectively. (d) Reversal of DNA hypermethylation in 5-Aza-2′deoxycytidine (5-AZA)-treated U937 cells as detected by clonal bisulfite sequencing. (e) Quantitative RT-PCR showing increased expression of YBX3 following treatment of U937 cells with 5-AZA. (f) Fold enrichment of YBX3 promoter sequence bound to H3K4me3 (activation mark) and (g) H3K27me3 (silencing mark) in HL-60 and U937 cells normalized to IgG control. +ve Con, positive control, completely methylated human genomic DNA; N1, N2, N3, negative controls, peripheral blood DNA from three healthy individuals.

Mentions: Our COBRA analysis indicated that methylation was absent at the promoter regions of SF3B1, SRSF2, U2AF1, ZRSR2, SF3A1, HNRNPR, MATR3, and ZFR in cell lines (Fig. 1). The YBX3 promoter was hypermethylated only in the monocytic leukemia cell line U937 (Fig. 2a–c). Hypermethylation of YBX3 in U937 cells was associated with its reduced mRNA expression. Inhibition of DNA methylation using the DNA methyltransferase inhibitor 5-Aza-2′deoxycytidine increased the expression of YBX3 following reversal of CpG methylation (Fig. 2d,e), indicating that promoter DNA hypermethylation was associated with the regulation of its expression. DNA methylation also occurred in conjunction with altered histone modifications at the promoter of this gene. We found >100-fold enrichment of the histone mark associated with active transcription, H3K4me3, at the YBX3 promoter in YBX3-expressing HL-60 cells (Fig. 2f). Enrichment of H3K4me3 was not present at the YBX3 promoter in U937 cells, which lacks YBX3 expression (Fig. 2f). The silencing mark, H3K27me3 was enriched twofold at the promoter of YBX3 in U937 but not HL-60 cells (Fig. 2g). Thus, appropriate histone modifications occur concomitantly with promoter DNA methylation to induce silencing of YBX3 in U937 cells.


Epigenetic modifications of splicing factor genes in myelodysplastic syndromes and acute myeloid leukemia.

Wong JJ, Lau KA, Pinello N, Rasko JE - Cancer Sci. (2014)

Silencing of YBX3 in U937 cells is associated with promoter DNA hypermethylation and altered histone modifications. (a) YBX3 promoter showing individual CpGs (vertical black line) and their distance from the transcriptional start site (+1). Recognition site for BstU1 used in combined bisulfite restriction analysis (COBRA) assays for YBX3 is shown on the map. (b) Gel electrophoretogram of COBRA showing PCR amplicons of the YBX3 promoter following bisulfite conversion and digestion with BstU1 of genomic DNA from leukemic cell lines and controls. (c) Clonal bisulfite sequencing confirming hypermethylation at the YBX3 promoter in U937 cells. Each row represents a single cloned PCR amplicon aligned to the map shown in (a). Black and white circles denote methylated and unmethylated CpGs, respectively. (d) Reversal of DNA hypermethylation in 5-Aza-2′deoxycytidine (5-AZA)-treated U937 cells as detected by clonal bisulfite sequencing. (e) Quantitative RT-PCR showing increased expression of YBX3 following treatment of U937 cells with 5-AZA. (f) Fold enrichment of YBX3 promoter sequence bound to H3K4me3 (activation mark) and (g) H3K27me3 (silencing mark) in HL-60 and U937 cells normalized to IgG control. +ve Con, positive control, completely methylated human genomic DNA; N1, N2, N3, negative controls, peripheral blood DNA from three healthy individuals.
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fig02: Silencing of YBX3 in U937 cells is associated with promoter DNA hypermethylation and altered histone modifications. (a) YBX3 promoter showing individual CpGs (vertical black line) and their distance from the transcriptional start site (+1). Recognition site for BstU1 used in combined bisulfite restriction analysis (COBRA) assays for YBX3 is shown on the map. (b) Gel electrophoretogram of COBRA showing PCR amplicons of the YBX3 promoter following bisulfite conversion and digestion with BstU1 of genomic DNA from leukemic cell lines and controls. (c) Clonal bisulfite sequencing confirming hypermethylation at the YBX3 promoter in U937 cells. Each row represents a single cloned PCR amplicon aligned to the map shown in (a). Black and white circles denote methylated and unmethylated CpGs, respectively. (d) Reversal of DNA hypermethylation in 5-Aza-2′deoxycytidine (5-AZA)-treated U937 cells as detected by clonal bisulfite sequencing. (e) Quantitative RT-PCR showing increased expression of YBX3 following treatment of U937 cells with 5-AZA. (f) Fold enrichment of YBX3 promoter sequence bound to H3K4me3 (activation mark) and (g) H3K27me3 (silencing mark) in HL-60 and U937 cells normalized to IgG control. +ve Con, positive control, completely methylated human genomic DNA; N1, N2, N3, negative controls, peripheral blood DNA from three healthy individuals.
Mentions: Our COBRA analysis indicated that methylation was absent at the promoter regions of SF3B1, SRSF2, U2AF1, ZRSR2, SF3A1, HNRNPR, MATR3, and ZFR in cell lines (Fig. 1). The YBX3 promoter was hypermethylated only in the monocytic leukemia cell line U937 (Fig. 2a–c). Hypermethylation of YBX3 in U937 cells was associated with its reduced mRNA expression. Inhibition of DNA methylation using the DNA methyltransferase inhibitor 5-Aza-2′deoxycytidine increased the expression of YBX3 following reversal of CpG methylation (Fig. 2d,e), indicating that promoter DNA hypermethylation was associated with the regulation of its expression. DNA methylation also occurred in conjunction with altered histone modifications at the promoter of this gene. We found >100-fold enrichment of the histone mark associated with active transcription, H3K4me3, at the YBX3 promoter in YBX3-expressing HL-60 cells (Fig. 2f). Enrichment of H3K4me3 was not present at the YBX3 promoter in U937 cells, which lacks YBX3 expression (Fig. 2f). The silencing mark, H3K27me3 was enriched twofold at the promoter of YBX3 in U937 but not HL-60 cells (Fig. 2g). Thus, appropriate histone modifications occur concomitantly with promoter DNA methylation to induce silencing of YBX3 in U937 cells.

Bottom Line: The only evidence of epigenetic effects was hypermethylation of the YBX3 promoter in U937 cells in conjunction with an enrichment of histone marks associated with gene silencing.Hypermethylation of the ZRSR2 promoter was also detected in 7/173 (4%) cases but was not associated with decreased mRNA expression (P = 0.1204).We conclude that DNA hypermethylation does not frequently silence splicing factors in MDS and AML.

View Article: PubMed Central - PubMed

Affiliation: Gene and Stem Cell Therapy Program, Centenary Institute, Camperdown, New South Wales, Australia; Sydney Medical School, University of Sydney, Camperdown, New South Wales, Australia.

Show MeSH
Related in: MedlinePlus