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.

FIGURE 1.

Repression of target promoter activities by SALL4 is reversed by cotreatment of 5-azaC and VPA. A 2-kb SALL4-pGL3 promoter construct was cotransfected with SALL4A- or SALL4B-expressing plasmids in HEK293 cells. Luciferase activity assays were performed from lysates of cells that were untreated (a), treated with VPA only (b), treated with 5-azaC only (c), or cotreated (d). For comparison studies, other Sal gene (SALL1 and SALL3) (e) or OCT4 promoter constructs (f) were used to replace SALL4-pGL3, and repressive effects of both promoter activities by SALL4A were assessed after administration of 5-azaC. All numbers refer to -fold changes, and error bars represent S.D. values of three independent experiments. g, a representative Western blotting analysis shows expression of SALL4A-HA and SALL4B-HA proteins in transfected 293 cells. RLU, relative luciferase units.

FIGURE 2.

Interaction of SALL4 proteins with different DNMTs and HDAC1. a, SALL4 isoforms and mutant SALL4 deletions (Δ1–Δ4) are shown schematically. b, SALL4 isoforms and various HA-tagged SALL4 mutants were transfected into 293 cells in 60-mm culture dishes. After 20 h, cells were lysed, and about 100 μl of cell lysates per 5 μg of anti-HA antibody or IgG control were used for Dynabeads protein G immunoprecipitation. Anti-HA immunoprecipitates were analyzed by Western blotting with antibodies against HDAC1 or the indicated DNMT antibodies. Immunoprecipitation of SALL4 isoforms and different mutants from HEK293 nuclear extracts purified different amounts of DNMT (c) and HDAC (d) activities. Error bars, S.D.

FIGURE 3.

SALL4 isoforms co-occupy the same regions of its own promoter as HDAC1 and DNMT proteins. Top, diagram of SALL4 promoter. Bottom, agarose gel analysis of ChIP PCR products. Antibodies against HA (for SALL4A and SALL4B) or the indicated proteins were used to immunoprecipitate DNA fragments from HEK293 cells, and -fold enrichment was compared with input. One SALL4 mutant (Δ2) and mock (H₂O) were used as control. Four pairs of primers spanning the SALL4 promoter region (−1.1 kb to +1 bp upstream of the ATG site) were used for ChIP-PCR analysis. There is a significant enrichment of the P3 product and slight enrichment of the P4 product for the SALL4 isoforms and for HDAC1 and DNMT protein purified fragments.

FIGURE 4.

Forced expression of SALL4 brings about increased DNA cytosine methylation of target genes in various cell types. a, human primary FF cells were transfected with SALL4A- expressing plasmid or vector control. Bisulfite sequencing shows that the SALL4 proximal promoter (100–720 bp upstream of the start codon) is differentially methylated at specific CpGs or GC-rich sites between the two groups of cells. Open circle, no methylation of cytosine; filled circle, methylated cytosine. b, chromatogram of one sequenced clone derived from bisulfite-treated FF cells that were transiently transfected with SALL4-expressing plasmids or pcDNA3 vector control. The arrows indicate differentially methylated sequences between the two cell types; unmethylated C converted to T upon bisulfite treatment. c, MassARRAY profiles showing methylation status of the PTEN promoter in NB4 leukemia cells 6 days after GFP control or SALL4-expressing lentivirus transduction. Percentages of methylation at each CpG site are indicated. The PTEN promoter region analyzed was chromosome 10, 40,427,070–40,427,349. d, 6 days after virus transduction, total RNA was extracted from the indicated group, and PTEN mRNA was measured by qRT-PCR. *, significant difference from the control group, p < 0.01. Error bars, S.E. (n = 3).

FIGURE 5.

a and b, in mouse bone marrow LSK cells, MassARRAY DNA methylation profiles showing percentages of each methylated CpG site within the indicated promoter regions of the SALL4 gene (a) and EBF1 gene (b) after the indicated lentivirus transduction. c, 7 days after virus transduction, total RNA was extracted from the indicated group, and EBF1 mRNA was measured by qRT-PCR. Error bars, S.E. (n = 4). *, p < 0.05.

FIGURE 6.

Down-regulation of SALL4 led to decreased DNA methylation at its own promoter. a, detection of shRNAs interferential efficiency by two SALL4-specific oligonucleotides. Relative reduction of SALL4 mRNA by 7410 and 7412 was 83.3 and 92.9%, respectively. b, DNA methylation status of the endogenous SALL4 promoter (from −965 through −845) was examined by bisulfite genomic sequencing after control shRNA (pRS) or SALL4-specific shRNA treatment. Filled and open circles, methylation and unmethylation, respectively.

FIGURE 7.

Reexpression of SALL4 and PTEN genes by 5-azaC treatment. The NB4 cells were grown and treated with 5-azaC (200 nm; Sigma) for 72 h with medium replacement every 24 h. After 72 h of treatment, genomic DNA, total RNA, and proteins were isolated from the cells for DNA methylation and mRNA analysis. a, DNMT protein expression in the whole-cell lysates from treated and untreated NB4 cells. Identical amounts (50 μg) of protein were separated and subjected to Western blot analysis with antibodies specific for DNMT1. The membranes were reprobed with anti-β-actin antibody to show equal loading of the protein. b, DNA methylation status of the SALL4 and PTEN genes was examined by bisulfite genomic sequencing with or without 5-azaC treatment (3 days). Filled and open circles, methylation and unmethylation, respectively. c, total RNA was extracted from each group, and mRNA for the indicated genes was measured by qRT-PCR. Error bars, S.E. (n = 3). *, significant differences (p < 0.001) from the untreated group.

FIGURE 8.

Hypothetical model. SALL4 selectively recruits epigenetic modulators (DNMT1, DNMT3A, DNMT3B, DNMT3L, MBD2, HDAC1, HDAC2, and most likely others) to specific DNA sequences of its downstream targets for gene suppression, whereas both classes of epigenetic drugs may block this process and reactivate gene expression.