Immunochemical Characterization and Transacting Properties of Upstream Stimulatory Factor Isoforms

Abstract

The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was as efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.

The dual role of helix – loop – helix-zipper protein USF in ribosomal RNA gene transcription in vivo

“…It should be noted that expression of USF1 and USF2 separately results in homodimer formation whereas simultaneous expression of the two polypeptides can form heterodimer (Sirito et al, 1994). Because each one of the polypeptides is in considerable excess over the endogenous levels, overexpression of each of the polypeptides individually must lead to higher level of homodimer over the heterodimer population in the cells, while overexpression of both subunits theoretically would result in 1:2:1 (USF1/USF1:USF1/ USF2:USF2/USF2) dimer distribution as been observed in vitro (Sirito et al, 1994;Viollet et al, 1996) or in predominance of heterodimers. Most of USF1 and USF2 are known to exist as heterodimers in vivo while the homodimers are underrepresented (Sirito et al, 1994;Viollet et al, 1996).…”

“…Because each one of the polypeptides is in considerable excess over the endogenous levels, overexpression of each of the polypeptides individually must lead to higher level of homodimer over the heterodimer population in the cells, while overexpression of both subunits theoretically would result in 1:2:1 (USF1/USF1:USF1/ USF2:USF2/USF2) dimer distribution as been observed in vitro (Sirito et al, 1994;Viollet et al, 1996) or in predominance of heterodimers. Most of USF1 and USF2 are known to exist as heterodimers in vivo while the homodimers are underrepresented (Sirito et al, 1994;Viollet et al, 1996). Co-transfection of USF1 and USF2 into cells can, indeed mimic the in vivo pattern and that the ratios of the USF1 and USF2 cDNAs transfected also dictates the dimer composition (Viollet et al, 1996).…”

Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells

“…The role played by USF in the regulation of gene expression has just recently started to be investigated. One of the assumptions is that USF1 and USF2 serve as transcription factors with general housekeeping transcriptional regulatory roles (Gogswell et al, 1995;Luo and Sawadogo, 1996;Reisman and Rotter, 1993;Viollet et al, 1996).…”

“…Most of the USF transcription complexes are composed of USF1-USF2 (Viollet et al, 1996). Surprisingly, the only protein from a HeLa cDNA expression library, besides Jun, that was able to associate with c-Fos, was USF2 (Blannar and Rutter, 1992).…”

DNA binding of USF is required for specific E-box dependent gene activation in vivo

“…The following antibodies were used: rabbit anti-Max antibody (Upstate Biotechnology Inc.); rabbit anti-USF-1 antibody (IgGM), a rabbit anti-USF-2a/2b antibody (IgGG), a rabbit anti-USF-2a-speci®c antibody (IgGO) (Viollet et al, 1996); rabbit anti-Mnt antibody (#6823) (kind gift of Dr RN Eisenman, Seattle, WA, USA); rabbit anti-USF antibody which recognizes all USF-1 and USF-2 proteins (kind gift of Dr M Sawadogo, Houston, TX, USA). Nuclear extracts and electrophoretic mobility shift assays (EMSA) Nuclear extracts were prepared as described before (Gaubatz et al, 1995).…”