Sox2 uses multiple domains to associate with proteins present in Sox2-protein complexes

Abstract

Master regulators, such as Sox2, Oct4 and Nanog, control complex gene networks necessary for the self-renewal and pluripotency of embryonic stem cells (ESC). These master regulators associate with co-activators and co-repressors to precisely control their gene targets. Recent studies using proteomic analysis have identified a large, diverse group of co-activators and co-repressors that associate with master regulators, including Sox2. In this report, we examined the size distribution of nuclear protein complexes containing Sox2 and its associated proteins HDAC1, Sall4 and Lin28. Interestingly, we determined that Sox2 and HDAC1 associate with protein complexes that vary greatly in size; whereas, Lin28 primarily associates with smaller complexes, and Sall4 primarily associates with larger complexes. Additionally, we examined the domains of Sox2 necessary to mediate its association with its partner proteins Sall4, HDAC1 and HDAC2. We determined that Sox2 uses multiple and distinct domains to associate with its partner proteins. We also examined the domains of Sox2 necessary to mediate its self-association, and we determined that Sox2 self-association is mediated through multiple domains. Collectively, these studies provide novel insights into how Sox2 is able to associate with a wide array of nuclear proteins that control gene transcription.

Figure 1. Fractionation of nuclear protein complexes from ESC.

Nuclear proteins were isolated from dox-induced mouse ESC engineered to express Flag-Sox2 from a dox-inducible transgene. Nuclear proteins were then size fractionated using a Superdex™-200 column under non-denaturing conditions. Fractions were concentrated and western blot analyses were conducted using the indicated antibodies: α-Sox2 (top), α-Sall4 (upper-middle), α-HDAC1 (lower-middle), or α-Lin28 (bottom), as described in the Materials and Methods.

Figure 2. Domains of Sox2 used for association with Sall4.

(A) Schematic diagrams of the Flag-Sox2 expression constructs used for domain mapping studies. (B) Mapping domains of Sox2 that mediate its association with Sall4. 293T cells were transiently transfected with an expression construct for Sall4 and the Sox2 constructs shown, and nuclear extracts were prepared 1 day later. Nuclear extracts (input lanes) used for co-immunoprecipitation of Sall4 are presented as western blot analyses, probing for Sall4 (top-left) or the Flag-Sox2 constructs indicated (bottom-left). M2-beads were used to co-immunoprecipitate Flag-Sox2 constructs and their associated proteins. Immunoprecipitate eluates were used in western blot analysis (right panels), and were probed for either Sall4 (top-right) or the Flag-Sox2 constructs (bottom-right). These experiments were repeated, and similar results were observed.

Figure 3. Domains of Sox2 used for association with HDAC1.

(A) Mapping domains of Sox2 that mediate its association with HDAC1. 293T cells were transiently transfected with an expression construct for HDAC1 and the Sox2 constructs shown, and nuclear extracts were prepared 1 day later. Nuclear extracts (input lanes) used for co-immunoprecipitation of HDAC1 are presented as western blot analyses, probing for HDAC1 (top-left) or the Flag-Sox2 constructs indicated (bottom-left). M2-beads were used to co-immunoprecipitate the Flag-Sox2 constructs and their associated proteins. Immunoprecipitate eluates were used for western blots (right panels), and were probed for either HDAC1 (top-right) or the Flag-Sox2 constructs (bottom-right). (B) Co-immunoprecipitation of HDAC1 by Flag-Sox2-HMG or Flag-Sox2-ΔHMG. Experimental design was the same as described for Figure 3A. The experiments described were repeated, and similar results were observed.

Figure 4. Co-immunoprecipitation of endogenous 293T cell HDAC2 by exogenous Flag-Sox2 constructs.

The indicated constructs were transiently transfected into 293T cells, and nuclear proteins were prepared 1 day later. Flag-Sox2 proteins and associated proteins were co-immunoprecipitated from nuclear extracts using M2-beads. Immunoprecipitate eluates were used in western blot analyses, and were probed for either α-HDAC2 (top) or α-Flag (bottom). Protein for the control (mock) lane was from un-transfected 293T cells. This experiment was repeated, and similar results were observed.

Figure 5. Mapping domains necessary for Sox2 to mediate its self-association.

(A) 293T cells were transiently transfected with expression constructs for GFP-Sox2 and the Flag-Sox2 constructs indicated. Nuclear extracts (input lanes) used for co-immunoprecipitation of Flag-Sox2 constructs and associated proteins are presented as western blot analyses (left panels). α-Sox2 was used to visualize GFP-Sox2 (top-left), and α-Flag was used to visualize the Flag-Sox2 constructs indicated. M2-beads were used to co-immunoprecipitate Flag-Sox2 constructs and their associated proteins. Immunoprecipitation eluates were used in western blot analyses (right panels), and probed for GFP-Sox2 (α-Sox2, top-right) or the Flag-Sox2 proteins (α-Flag, bottom-right). (B) Co-immunoprecipitation of GFP-Sox2-ΔHMG by Flag-Sox2-ΔHMG. Experimental design was the same as described for Figure 5A. These experiments were repeated multiple times, and similar results were observed.

Figure 6. Model of Sox2-protein associations.

(A) Sall4 associates with Sox2 primarily through a region C-terminal to the HMG domain of Sox2. The region N-terminal of the HMG domain of Sox2 also mediates the association between Sox2 and Sall4; whereas, the HMG domain of Sox2 may weakly interfere with the association between Sox2 and Sall4, as indicated by the dashed line. (B) HDAC1 primarily associates with Sox2 through Sox2 amino acids 124 to 180. The dashed line indicates interference of association observed between Flag-Sox2 and HDAC1 by the Sox2-HMG domain. (C) Endogenous HDAC2 (from 293T cells) primarily associates with Flag-Sox2 through the HMG domain of Sox2. (D) Flag-Sox2 associates with GFP-Sox2 through multiple domains.