Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

Abstract

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes. Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes. high mobility group acid-rich β-galactosidase chloramphenicol acetyltransferase cytomegalovirus δ-crystallin enhancer enhanced chemifluorescence electrophoretic gel mobility shift analysis fibroblast growth factor 4 Pit-Oct-Unc transactivation domain The Sox family of transcription factors is comprised of a diverse group of proteins whose pattern of expression is regulated both spatially and temporally (1Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (752) Google Scholar). Sox factors are related by the homology (usually >50%) found within their high mobility group (HMG)1 DNA binding domains. Sox HMG domains have been found to bind to the consensus sequence 5′-(A/T)(A/T)CAA(A/T)G-3′ in the minor groove of DNA (2Harley V.R. Lovell-Badge R. Goodfellow P.N. Nucleic Acids Res. 1994; 22: 1500-1501Crossref PubMed Scopus (333) Google Scholar). As the number of Sox proteins has grown to more than 20 members in multiple species, including both vertebrates and invertebrates, this family has been divided into seven subgroups, the members of which contain high homology within both the HMG domain and flanking regions (1Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (752) Google Scholar). In some cases, subgroup members have similar patterns of expression during development and perhaps redundant functions. For example, the structurally similar group B members Sox-1, -2, and -3 show overlapping expression in the fetal nervous system, and it has been suggested that they perform similar functions (3Collignon J. Sockanathan S. Hacker A. M. S. M. Goodfellow P.N. Lovell-Badge R. Google M. J. Lovell-Badge R. PubMed Google Scholar). Additionally, the group members and Sox-11 show high homology within their HMG domain and and they also have roles in the nervous M. M. Res. Res. PubMed Scopus Google Scholar). The that the HMG DNA binding domains of many members of the Sox family bind a consensus sequence has the of are selective mechanisms in to a specific Sox protein to a gene regulated by an HMG binding of the that a diverse of mechanisms to DNA binding members of transcription factor which also to Sox of DNA binding has been found within multiple members of the in the of proteins for DNA is regulated by autoinhibitory regions flanking the DNA binding domain J. R. S. A. PubMed Scopus Google PubMed Scopus Google Scholar). Interestingly, the and proteins a similar regulatory their are of the ability of proteins to bind DNA J. PubMed Scopus Google Scholar). the autoinhibitory domains of both and HMG proteins repress DNA they can be by by with which their recruitment to be regulated in a PubMed Scopus Google PubMed Scopus Google Google J. PubMed Scopus Google A. J. PubMed Scopus Google Scholar). Although specific of DNA binding has been observed in Sox it is that elements are of specific Sox transcription to the mechanisms through which Sox is have that recruitment with transcription factors at binding the This is by of the elements that expression of the fibroblast growth factor 4 gene in expression a enhancer PubMed Scopus Google J. A. PubMed Scopus Google which an binding for Sox-2 PubMed Scopus Google A. PubMed Scopus Google M. A. J. Scholar). 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