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Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases.

ABSTRACT: The stem cell protein SALL4 plays a vital role in maintaining stem cell identity and governing stem cell self-renewal through transcriptional repression. To explore SALL4-mediated mechanisms involved in transcriptional repression, we investigated DNA modifications underlying its regulatory activities. By a luciferase activity assay, we found that both histone deacetylase inhibitor valproic acid (VPA) and DNA methylation inhibitor 5-azacytidine (5-azaC) specifically reversed the repression effect of SALL4 on its own as well as other Sal gene promoter activities. Cotreatment of VPA with 5-azaC in cells almost completely blocked this repression effect. Further co-immunoprecipitation assay and enzyme activity analysis demonstrated that SALL4 protein directly interacted with different DNA methyltransferases (DNMTs) and purified DNMT enzymatic activities from nuclear extracts. In addition, SALL4 isoforms co-occupied the same regions of its own promoter as DNMT corepressors, and ectopic overexpression of SALL4 led to increased CpG island promoter methylation of silenced genes in various cell types. These included primary hematopoietic stem/progenitor cells, fibroblasts, and NB4 leukemic cells. In NB4 cells, treatment of cells with 5-azaC also caused decreased amounts of methylated alleles of SALL4 and PTEN and dramatically increased their mRNA expression. Our studies identify a new mechanism by which SALL4 represses gene expression through interaction with DNMTs. Furthermore, DNMTs and histone deacetylase repressors synergistically contribute to the regulatory effects of SALL4. These findings provide new insights into stem cell self-renewal mediated by SALL4 via epigenetic machinery.

SUBMITTER: Yang J

PROVIDER: S-EPMC3265879 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases.

Yang Jianchang J Corsello Tyler R TR Ma Yupo Y

The Journal of biological chemistry 20111129 3

The stem cell protein SALL4 plays a vital role in maintaining stem cell identity and governing stem cell self-renewal through transcriptional repression. To explore SALL4-mediated mechanisms involved in transcriptional repression, we investigated DNA modifications underlying its regulatory activities. By a luciferase activity assay, we found that both histone deacetylase inhibitor valproic acid (VPA) and DNA methylation inhibitor 5-azacytidine (5-azaC) specifically reversed the repression effect ...[more]

PMID: 22128185

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Project description:We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1-/- or DNMT3B-/- human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1-/- cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.

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Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases.

Publications

Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases.

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