Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein

Abstract

The far upstream element (FUSE) binding protein (FBP), a singlestranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.DNA conformation ͉ KH-domain ͉ ssDNA sequence specificity

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

“…As the FUSE first melts in response to the torque generated by ongoing transcription (Figure 10, A and B), 5,12,13 FBP sequence selectively engages DNA via its KH motifs. 2,3,15,22 Then FBP loops and stimulates the p89/ XPB/ERCC3 3 0 to 5 0 helicase/translocase of TFIIH at the promoter increasing transcription. 4e6 As transcription intensifies, the melted DNA bubble at FUSE expands upstream, allowing the recruitment of FIR via DNA-protein interactions and protein-protein interaction between a shallow groove in FIR RNA recognition motif 2 and the most NH 2 terminal a-helix of FBP ( Figure 10, A and B).…”

“…Therefore, the Fubp1-floxed allele, deleted of the Neo cassette, was crossed with B6 mice that expressed an actin-Cre transgene that yielded germline deletion of exons 8 through 13, removing entirely KH motifs 2 and 3 and the amino terminal half of KH4, yielding an allele absolutely incapable of expressing a protein that binds properly with single-strand nucleic acids. 2,3,15 This deletion was easily distinguished from the wild-type (WT) allele with the use of the primers and PCR screen in Supplemental Figure S1A and S2, as shown in Supplemental Figure S1B. …”

Overexpression of far upstream element binding proteins: A mechanism regulating proliferation and migration in liver cancer cells

“…24 However, it has recently been demonstrated that the c-myc FUSE significantly varies from the perfect consensus sequence recognized by FBPs, pointing to the existence of additional sites that bind FBPs more strongly. 25 Several segments within the STMN1 promoter indeed are closely related to the proposed optimal FBP binding sequence. The functional relevance of these segments in the FBP-1-driven regulation of stathmin must be confirmed experimentally.…”

The Splicing Factor FUBP1 Is Required for the Efficient Splicing of Oncogene MDM2 Pre-mRNA

“…However, in oligodendrogliomas, where its expression is abolished due to mutations, FUBP1 is considered a tumor suppressor (28 -30). Functionally, FUBP1 is a DNA helicase V (31) and is best known for its role in the transcriptional up-regulation of c-MYC through its binding of single-stranded DNA at the far upstream element (32) via the four tandem K homology motifs in its central domain (33,34). However, it is capable of binding singlestranded RNA and post-transcriptional functions have been described for FUBP1 in mRNA turnover and translation control (35)(36)(37)(38).…”