Abstract
Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.
Highlights
Different transcription factors operate together at promoters and enhancers to regulate gene expression
Binding of all four factors was strongly reduced when the PBX homeobox 1 (Pbx1):Prep1 recognition sequence was intact but the noncanonical nuclear transcription factor Y (Nf-y)-binding site mutated (CCTAT to TTTCT in M4). These results suggest that both binding of Pbx1:Prep1 as well as binding of Nf-y to the composite motif DECAext motif is required for genomic loading of specificity protein 2 (Sp2) on the Amd1 promoter
Several lines of evidence revealed that the GC box is not the target sequence of Sp2 in vivo. (i) Sp2 mutants lacking the zinc finger DNA-binding domain bind to GC box-containing promoters as efficiently as WT Sp2. (ii) The GC box is not locally enriched in Sp2 ChIP peak summits. (iii) Mutation of the GC box in the Amd1 promoter does not affect binding of endogenous Sp2
Summary
Sp2, specificity protein 2; MEF, mouse embryonic fibroblast; TALE, 3-amino acid loop extension; Pbx, pre-B-cell leukemia homeobox 1; Prep, Pbx-regulating protein 1; Nf-y, nuclear transcription factor y; GO, Gene Ontology; FRT, Flp recombination target; DAPA, DNA affinity precipitation assay; qPCR, quantitative PCR; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; Btd-box, button head box. Expression of Sp2 mutants in Sp2ko cells revealed that the zinc finger DNA-binding domain is dispensable, and the Sp-box is required for potentiating genomic binding of Pbx1:Prep. Together our results provide further mechanistic insight into the role of Sp2 as an important cofactor and clearly illustrate how the interplay of different transcription factors determines their genomic binding
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