Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells

Abstract

Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls.

Fig. 1.

Sall4 is a major regulator in mouse ES cells. (A) Sall4 bound to promoters that over-represent a broad classification of GO annotations. These included various potential regulatory and developmental annotations. Analysis was done with DAVID, and the x axis represents the gene number. (B) Further classification of developmentally important genes over-represented in the Sall4 binding pool. For the organ development annotation, the over-representation was insignificant (P < 0.074) but notable. The x axis represents the gene number. P values are inset following each bar and were calculated by using Fisher's Exact Test based on over-representation in comparison to the genome. (C) Sall4 binds promoter regions belonging to a variety of pathways that have definitive roles in development, suggesting that Sall4 may control a wide variety of developmental processes. Listed genes are only representative of the Sall4-bound genes in each pathway. P values for this analysis are not presented because of the low number of genes within each pathway. Classification was done by using Ingenuity Pathway Analysis (www.ingenuity.com).

Fig. 2.

Coimmunoprecipitation and co-occupancy of Sall4, Oct4, and Nanog. (A) Transient transfection of W4 ES cells with a Sall4-HA construct exhibited protein expression detected by both anti-HA and anti-Sall4 antibodies (the latter data not shown) in the cell extract (left). Oct4 and Nanog are detected by using respective antibodies in the whole ES cell extract (input). Immunoprecipitation of Sall4-HA with an anti-HA antibody revealed both Oct4 and Nanog bands, whereas immunoprecipitation with an IgG antibody detected neither protein. (B) Venn diagram showing the overlapping target genes of Sall4, Oct4, and Nanog as determined by ChIP-Chip and ChIP-PET experiments. These complexes may function in the regulation of stem cell pluripotency.

Fig. 3.

Decreased expression of iPS genes in Sall4^+/− ES cells. Ectopic expression of 4 key transcription factors, Oct4, Sox2, c-Myc, and Klf4, produces iPS cells. (A) Sall4 binds to promoter regions of Oct4, Sox2, c-Myc, and Klf4 as shown by ChIP-PCR. (B) Following adenovirus induced removal of 1 Sall4 allele, expression of all 4 transcription factors is decreased as measured by Q-RT-PCR. The Sall4/Gapdh ratio in control cells was set at 1. The values are the mean of triplicate reactions, and the bars indicate SD.

Fig. 4.

The role of Sall4 in H3K27 methylation regulation (A) Sall4 binds to 422 GAHM that are methylated at H3K27. Some of the GAHMs are also bound by PRC1 (Rnf2, Phc1) and PRC2 (Suz12, Eed). One hundred sixty-four of these genes are associated with Sall4, PRC1, and PRC2 (inner orange circle) with 69 PRC2-bound genes and 29 PRC1-bound genes also bound by Sall4 (outer orange circle). However, Sall4 binds 164 of GAHM-H3K27 that do not bind the polycomb group proteins (outer gray circle). (B) Two hundred fifty-eight genes are bound by one or more polycomb group protein(s) and Sall4. Of these, 81 have developmental functions that display significantly over-represented (P < 0.001) binding to genes associated with various developmental processes (orange). Binding of Sall4 to GAHM-H3K27, but neither PRC1 nor PRC2 occurs for 164 genes, with 23 genes having developmental processes that are notable but not statistically significant (P < 0.07; white). This suggests that two or more mechanisms may interact to regulate cell fate through histone methylation H3K27. The x axis represents the gene number. These GO annotations are not mutually exclusive, and P values were determined by using Fisher's Exact Test.

Fig. 5.

Sall4 target genes are associated with bivalent domains. Venn diagram displaying the GAHMs bound by Sall4 within HCNEs. Notably, the majority of Sall4-bound genes within characterized HCNEs are marked by bivalent histone methylation domains including a cluster of homeobox genes (see Table S3).