Gene wiki reviewSALL4, the missing link between stem cells, development and cancer
Introduction
SALL4, a member of the spalt-like (SALL) gene family (SALL1 to SALL4), was originally cloned based on its DNA sequence homology to the homeotic gene in Drosophila, spalt (sal) (Kohlhase et al., 1996; Frei et al., 1988; Kuhnlein et al., 1994). SALL4 plays an essential role in maintaining the pluripotent and self-renewal properties of embryonic stem cells (ESCs). After birth, SALL4 expression is down-regulated and absent in most adult tissues. However, SALL4 is re-expressed in various cancers. This review summarizes our current knowledge about the structure and expression patterns of SALL4, its role in stem and cancer cells, and the existing understanding of its molecular functions.
In humans, SALL4 is located in 20q13.2, consisting of four coding exons and 3162 bp of coding sequence (SALL4A; AY172738) (Kohlhase et al., 2002a, Deloukas et al., 2001). GenBank sequences AY170621 (SALL4B mRNA, 1851 bp) and AY170622 (SALL4C mRNA, 831 bp) suggest that two alternative splicing products exist in addition to the full length SALL4A mRNA. The B mRNA consists of exon 1, 1020 bp of 5′ end of exons 2, 3 and 4, whereas in the C mRNA, exon 2 is spliced out (Kohlhase et al., 2005) (Fig. 1). Another SALL4 transcript containing different exons 1a and 1b instead of the original exon 1 identified by 5′ RACE has also been reported (Bohm et al., 2006).
Like the protein encoded by sal, SALL4 belongs to a group of transcription factors characterized by multiple cys(2)his(2) (C2H2)-type zinc finger domains distributed over the entire protein. Human SALL4 contains a single C2H2 zinc finger near the N-terminus, two C2H2 zinc finger clusters in the middle portion, and one at the C-terminus of the protein (de Celis and Barrio, 2009) (Fig. 1). SALL4A has all four zinc finger clusters while SALL4B lacks the zinc finger clusters ZF2 and ZF3. The most C-terminal ZF4 cluster of Sall4 is both necessary and sufficient for its localization to the heterochromatin (Sakaki-Yumoto et al., 2006). In addition to zinc fingers, SALL4 also contains a glutamine (Q)-rich region that is highly conserved in all invertebrate and vertebrate SALL family members. This domain was shown to be necessary for interactions between Spalt family proteins (Sweetman et al., 2003). Furthermore, SALL4 A and B isoforms are able to form homodimers and heterodimers (Rao et al., 2010), possibly through their Q-rich regions. SALL4 is a nuclear protein and its subcellular localization is mediated through at least one conserved nuclear localization signal (NLS) at amino acids (AA) 64–67. A single mutation that changes lysine 64 into arginine (K64 into R64) is sufficient to disrupt its subcellular distribution and compromises its function in vivo (Wu et al., 2014).
Post-translational modifications of SALL4 such as phosphorylation (Zoumaro-Djayoon et al., 2011), ubiquitination (Wilson et al., 2012) and sumoylation (Yang et al., 2012a) have been reported. The functions of these post-translational modifications are largely unknown. One study reported that lysine residues 156, 316, 374, and 401 were essential for SALL4B sumoylation, which seemed to affect the protein stability, its interaction with Oct4, and its transactivation function (Yang et al., 2012a). The role of ubiquitination of SALL4 is still unknown.
In murine development, Sall4 protein expression is first observed at the two-cell stage due to maternal contribution, and later in some cells of the 8- to 16-cell-stage mouse embryo after zygotic transcription has initiated (Elling et al., 2006). In late blastocysts, the Sall4 RNA and Sall4 protein become enriched in the inner cell mass (ICM) and the trophectoderm. Finally, by E11.5, Sall4 expression is observed in the midbrain, the rostral edge of the forebrain, maxillary arch, genital tubercle, limb buds, tail, and left ventricular myocardium (Kohlhase et al., 2002b, Koshiba-Takeuchi et al., 2006). In adult mice, Sall4 expression is mostly restricted to germ cells, wherein it is highly expressed in undifferentiated spermatogonia and oocytes in primordial, primary, and secondary follicles (Eildermann et al., 2012, Miettinen et al., 2014, Cao et al., 2009a). Similarly, expression of SALL4 in adult human tissue is restricted to the testis and ovary (Kohlhase et al., 2002a) (Fig. 2). One exception to this expression pattern is human CD34 + hematopoietic stem/progenitor cells (HSPCs) (Gao et al., 2013a).
SALL4 has isoform-specific gene expression pattern in the testis, ESCs, and fetal liver cells. Sall4a is expressed in postnatal day 7 (PND7) testis, while Sall4b is expressed from PND0 onward (Gassei and Orwig, 2013). Sall4a is more abundant than Sall4b in undifferentiated mouse ESCs, and neither isoform is found upon induction of ESC differentiation (Rao et al., 2010). Furthermore, the A isoform, and not B, is detected in fetal livers, while several human hepatocellular carcinoma (HCC) cell lines express both SALL4A and SALL4B (Oikawa et al., 2009).
Very little is known about the expression regulation of SALL4 and its isoforms. Several studies have focused on the promoter region of this gene (Bohm et al., 2006, Bard et al., 2009, Yang et al., 2010). While one study reported on SALL4's intronic enhancer (Wu et al., 2006), its distal regulatory element(s) remains uninvestigated. In breast cancer, SALL4 was proposed to be a downstream target gene of STAT3 (Bard et al., 2009). During intestinal metaplasia of the gastric epithelial cells, SALL4 was identified as a direct target of caudal-related homeobox 1 (CDX1) protein (Fujii et al., 2012). In murine ESC, several reports have demonstrated that Sall4 protein participates in an interconnected autoregulatory circuit with Oct4, Sox2, and Nanog, wherein each of the four factors can regulate its own expression as well as that of others (Yang et al., 2008a; Zhang et al., 2006; Lim et al., 2008). Within this circuit, one study reported that Sall4 can negatively self-regulate and antagonize Oct4's activation function to balance its own expression level (Yang et al., 2010). Further, a TCF/LEF consensus sequence was reported in the SALL4 promoter region and SALL4 expression could be activated by LEF1 or TCF4E, indicating that SALL4 is a target of the canonical WNT signaling (Bohm et al., 2006). Posttranscriptional regulation of SALL4 has also been observed. Specifically in glioma, one study reported that miR-107 can bind the 3′UTR of SALL4 mRNA and modulate its expression. In a metastatic breast cancer model (Lin et al., 2015), SALL4 was found to be a target of miR-33b. In murine ESC, miR-294 and let-7c were reported to have opposing effects on Sall4 expression (Melton et al., 2010).
Epigenetic regulation of SALL4 has also been observed. Specifically, the DNA methylation status of SALL4 seems to correlate with its expression. In fact, SALL4 is hypomethylated and highly expressed in induced pluripotent stem (iPS) cells and ESCs, comparing to differentiated cells (Fig. 3) (Nishino et al., 2010, Amabile et al., 2015). While one study focused on the methylation status of SALL4 promoter region (Yang et al., 2012b), the major CpG island of SALL4 is located at the exon 1/intron 1 region as reported by other SALL4 methylation studies (Lin et al., 2013, Ueno et al., 2014). Future experiments are thus needed to explore the effects of upstream regulator(s) on SALL4 DNA methylation status and how this epigenetic status affects cell fate.
Section snippets
Function of SALL4 in stem cells and development
Sall4 has been shown to be involved in the proper development of the ICM and Sall4-null mice do not survive beyond embryonic day E6.5 (Elling et al., 2006; Sakaki-Yumoto et al., 2006; Zhang et al., 2006). Although Sall4-null blastocysts have no defects in lineage commitment of the ICM or trophectoderm in vivo, Sall4-null ICM cells show defected proliferation in vitro (Sakaki-Yumoto et al., 2006). Down-regulation of Sall4 in ESCs via shRNA leads to reduced Pou5f1 expression and increased Cdx2
SALL4 in cancers
Analyses of its expression and epigenetic status have shown that SALL4 is de-regulated and aberrantly expressed in various cancers (review in Zhang et al., 2015a) such as leukemia (Ma et al., 2006), germ cell tumors (Mei et al., 2009, Cao et al., 2011a), hepatocellular carcinoma (HCC) (Oikawa et al., 2009, Yong et al., 2013a), gastric cancer (Miettinen et al., 2014, Zhang et al., 2014, Ushiku et al., 2010, Ikeda et al., 2012, Osada et al., 2014, Sugai et al., 2010), colorectal carcinoma (
Molecular dissection of SALL4 functions in cells
To understand the mechanism(s) underlying SALL4 function(s) in various cells, efforts have been made to search for both SALL4 protein interaction partners and its downstream targets. The PTEN/AKT pathway is the best-understood example that integrates our current understanding of these two aspects of SALL4's molecular functions. Based on chromatin immunoprecipitation followed by microarray (ChIP-chip) data in normal human CD34 + cells, leukemic NB4 cells, and human ESCs, PTEN was identified to be
Concluding remarks
The fate of a specific cell type is governed by genetic and epigenetic factors, and can be redirected or reprogrammed through modifications of the key factors involved. Identifying and understanding the key players in cell fate determination will help us direct a cell type-specific gene expression profile, which is ultimately responsible for cellular identity and function. The knowledge thus gained can help us direct cellular differentiation for the purpose of tissue regeneration and repair, or
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgments
This review and the corresponding Gene Wiki article are written as part of the Gene Wiki Review series — a series resulting from a collaboration between the journal GENE and the Gene Wiki Initiative. The Gene Wiki Initiative is supported by the National Institutes of Health (GM089820). Additional support for Gene Wiki Reviews is provided by Elsevier, the publisher of GENE. This work is also supported in part through NIH grant P01HL095489 and R03CA184531, research funds from Leukemia and
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Both authors contributed equally to the work.