A Conserved 12-Amino Acid Motif in Sall1 Recruits the Nucleosome Remodeling and Deacetylase Corepressor Complex

Abstract

Sall1 is a multi-zinc finger transcription factor that represses gene expression and regulates organogenesis. In this report, we further characterize the domain of Sall1 necessary for repression. We show that endogenous Sall1 binds to the nucleosome remodeling and deacetylase corepressor complex (NuRD) and confirm the functionality of the Sall1-associating macromolecular complex by showing that the complex possesses HDAC activity. NuRD is involved in global transcriptional repression and regulation of specific developmental processes. The mechanism by which sequence-specific DNA-binding proteins associate with NuRD is not well understood. We have identified a highly conserved 12-amino acid motif in the transcription factor Sall1 that is sufficient for the recruitment of NuRD. Single amino acid substitutions defined the critical amino acid peptide motif as RRKQXK-PXXF. This motif probably exhibits a more general role in regulating gene expression, since other proteins containing this domain, including all Sall family members and an unrelated zinc finger protein Ebfaz, mediate transcriptional repression and associate with NuRD. These results also have important implications for the pathogenesis of TownesBrocks, a syndrome caused by SALL1 mutations.It is well established that changes in chromatin structure are associated with activation and silencing of gene expression. The packaging of DNA in the nucleosome acts to inhibit the accessibility of DNA to transcriptional regulators and the molecular machinery required for gene expression. Gaining accessibility to DNA relies on two major mechanisms that include ATP-dependent chromatin remodeling and multiple types of modifications of nucleosomal histones (1-3). Two well described macromoleclar complexes, NuRD 2 and Sin3 (reviewed in Ref. 4), are instrumental in facilitating these enzymatic activities to establish transcriptional repression.The NuRD complex is distinguished by its ability to exhibit both histone deacetylase and ATP-dependent nucleosome remodeling activity. NuRD has been purified from mammalian and Xenopus cells and is ϳ2 MDa in size (5-9). The mammalian complex is composed of at least eight polypeptides. The histone deacetylase proteins, HDAC1 and HDAC2, and two associated proteins, RbAp46 and RbAp48, are core components that are common to both the NuRD and Sin3 repression complexes. In addition to histone deacetylase activity, the NuRD complex has ATP-dependent nucleosome remodeling activity because of its association with the ATPase, Mi-2. The biochemical functions of the remaining NuRD-specific components, the methyl-CpGbinding protein, MBD3, and MTA1 and MTA2 are not well defined (reviewed in Ref. 4).NuRD is widely conserved across the animal and plant kingdoms and has been shown to play a critical role in regulating gene expression during embryonic development (reviewed in Ref. 4). NuRD has been shown to inhibit Ras signaling during vulval development through effects on both the synMuvA and synMuvB pathways in Caenorhabditis elegans (10 -15). In Dro...

A Phosphomimetic Mutation in the Sall1 Repression Motif Disrupts Recruitment of the Nucleosome Remodeling and Deacetylase Complex and Repression of Gbx2

“…The PCR product was digested with EcoRI (5Ј-end of cDNA) and BlpI (nucleotide 1106) and ligated into pCS2-XsalF that was also EcoRI-BlpI-digested. The GAL4DB and eukaryotic GST expression constructs for N-terminal Sall1-(2-136) or -(2-12) have been described previously (10) and were used as templates to create Sall1 point mutants with the Stratagene QuikChange site-directed mutagenesis kit. The following mutagenic oligonucleotide primers were designed to substitute amino acid 2 found in the N-terminal motif of Sall1 from a serine to an alanine or a glutamic acid in the context of Sall1-(2-136) and Sall1-(2-12): GAL4DB-S2A (5Ј-GGGGGATCCATGGCGCG-GAGGAAGC-3Ј and 5Ј-GCTTCCTCCGCGCCATGGATCC-CCC-3Ј), GAL4DB-S2E (5Ј-GGGGGATCCATGGAGCGGA-GGAAGC-3Ј and 5Ј-GCTTCCTCCGCTCCATGGATCCCC-3Ј) and GST-S2A (5Ј-GCGTGGATCCATGGCGCGGAGGA-AGCA-3Ј and 5Ј-TGCTTCCTCCGCGCCATGGATCCA-CGC-3Ј), GST-S2E (5Ј-CGCGTGGATCCATGGAGCGGAG-GAAGCAAGC-3Ј and 5Ј-TGCTTCCTCCGCGCCATGGAT-CCACGC), or in the context of full-length Sall1-(2-1322) and Flu-S2A (5Ј-CCGCCACCATGGCGCGGAGGAAGC-3Ј and 5Ј-GCTTCCTCCGCGCCATGGTGGCGGT-3Ј).…”

“…Genetic evidence in Drosophila has demonstrated a cell autonomous role for spalt as a transcriptional repressor (7)(8)(9). Similarly, in recent work we revealed that Sall proteins contain an N-terminal repression domain that recruits the nucleosome remodeling and deacetylase (NuRD) 2 complex, revealing a strong correlation between repression and NuRD complex interaction (10). We reported a peptide motif (SRM) that is necessary and sufficient for Sall1-mediated repression and NuRD recruitment (10).…”

“…Similarly, in recent work we revealed that Sall proteins contain an N-terminal repression domain that recruits the nucleosome remodeling and deacetylase (NuRD) 2 complex, revealing a strong correlation between repression and NuRD complex interaction (10). We reported a peptide motif (SRM) that is necessary and sufficient for Sall1-mediated repression and NuRD recruitment (10). The NuRD complex possesses ATP-dependent and histone deacetylase chromatin-remodeling activities and consists of several subunits, including HDAC1, HDAC2, RbAp46, RbAp48, Mi-2␤, MBD3, MTA1, and MTA2 (reviewed in Ref.…”

Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo

“…Two different splice variants have been reported for sem-4/Sall (Fig. 2D), lacking (splice "b") or not (splice "a") a conserved 12-amino acids motif found in the N terminus of SALL proteins that has been involved in the recruitment of the NuRD complex and MTA1 (12,13). Both splice variants, under the control of the egl-5/Hox promoter, were able to rescue the "no PDA" phenotype of the sem-4/SALL null mutant (Fig.…”

“…MTA1 is a transcriptional modulator and appears to have an important role in cellular identity as its expression has been correlated with invasive cancers and metastasis (10) and as loss of MTA1 in ES cells impairs their pluripotency (4). Although SALL4 and MTA1 have also been found to associate with the nucleosome remodelling and histone deacetylase (NuRD) complex (11)(12)(13), most NuRD components did not appear to associate with the NODE complex (4).…”