Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers

Abstract

We have isolated human cDNA clones for USF2, a new member of the upstream stimulatory factor (USF) family of transcription factors. Analysis of these clones revealed the existence of highly conserved elements in the C terminal region of all USF proteins. These include the basic region, helix-loop-helix (HLH) motif, and, in the case of the human proteins, the C-terminal leucine repeat (LR). In addition, a highly conserved USF-specific domain is located immediately upstream of the basic region. Using in vitro translated proteins, we found that all members of the USF family bound DNA as dimers. The N-terminal portion of USF, including the USF-specific domain, was entirely dispensable for dimer formation and DNA-binding. However, deletion mutants of USF2 lacking the LR were deficient in DNA-binding activity. Interestingly, each of the USF proteins could form functional heterodimers with the other family members, including the sea urchin USF, which does not have a LR motif. This indicates that the conserved LR in human USF is not required for dimer formation, and influences only indirectly DNA-binding.

Figure 1

Nucleotide sequence of human USF2 cDNA clones and deduced amino acid sequence. Shown is the entire sequence of clone hUSF2-C that was isolated from a human Namalwa cell cDNA library. Positions of the 5′ end of two other USF2 clones (hUSF2-C1 and hUSF2-A2), isolated from a HeLa cell cDNA library, are indicated (see Materials and Methods). The presumptive polyadenylation signal is underlined.

Figure 2

Conserved domains in the C-terminal region of the various USF proteins. The amino acid sequence of the C-terminal region of USF2 was aligned with the same region in human USF1 (amino acids 157–310; Gregor et al., 1990), Xenopus laevis USF (amino acids 150–303; Kaulen et al, 1991), and sea urchin USF (amino acids 148–265; Kozlowski et al., 1991). Location of the various conserved domains is indicated above the sequences. Unchanged residues are outlined in gray.

Figure 3

Domains of USF2 required for DNA-binding and dimer formation. A. Schematic of the various USF proteins. Shown are the USF proteins encoded by the pBS/ET-USF2, pBS/ET-USF2a, pBS/ET-USF2b, and PBS/ET-USF2c constructs (see Materials and Methods), which contain respectively amino acids 15–234, 103–234, 15–184, and 15–195/221–234 of hUSF2-C. The human USF1 (amino acids 1–310) and sea urchin USF (amino acids 1–265) were encoded by clones dI2 (Gregor et al., 1990) and 436–1 (Kozlowski et al., 1991), respectively. B. SDS gel analysis of in vitro translation products. Aliquots of reticulocyte lysates programmed with RNA derived from the various USF2 subclones, as indicated above each lane, were analyzed for the presence of translation products by SDS-PAGE. In lanes 5, 8, and 9, two different proteins were cotranslated. C. Localization of the minimum region of USF2 required for DNA-binding. The in vitro translated proteins shown in B were analyzed for DNA-binding activity by EMSA, using as a probe an oligonucleotide containing a consensus USF binding site (see Materials and Methods). The migrations of the different USF2 dimers are indicated at left. Longer exposure of the gel failed to detect any binding activity in the case of the USF2 LR mutants b and c (lanes 5 and 6). D. Formation of USF2 homodimers. USF2, or the N-terminal truncated USF2a mutant, were either translated separately (lanes 1, 2, 4, and 5) or cotranslated (lane 3), and the resulting DNA-binding species analyzed by EMSA. In lane 4, the individual proteins were simply mixed during the binding reaction. In lane 5, the two proteins were mixed, then heated for 7 minutes at 65°C prior to incubation with the oligonucleotide probe. The arrow at left indicates the migration of endogenous rabbit reticulocyte USF. The two arrows at right indicate the migration of heterodimers between rabbit USF and the human USF2 and USF2a proteins. E. Formation of USF heterodimers. The ability of USF2 to heterodimerize with human USF1 (lane 3) as well as with the sea urchin USF (lane 6) was investigated by cotranslating these proteins with the N-terminally truncated USF2a and analyzing the resulting DNA binding species by EMSA. Control reactions (lanes 1, 2, 3, and 4) were used to determine the migration of the individual homodimers, as indicated.

Figure 4

Southern and Northern analysis of human USF2. A. In each lane, 5 μg of HeLa DNA, digested by various restriction endonucleases as indicated, were resolved on a 0.8% agarose gel along with a ³²P-labeled Hind III digest of λ DNA as size markers (lane M). After transfer to nitrocellulose, the blot was probed with the BamH I fragment corresponding to nucleotides 33 to 510 of hUSF2-C. Hybridization of this probe was observed with single genomic DNA fragments of 9.6 kb, 17 kb, 2 kb, 23 kb, 25 kb, and 1.7 kb after digestion with BamH I, EcoR I, Pst I, Kpn I, Bgl I, and Pvu II, respectively (lanes 2–5, and 7–8). Hybridization was also observed with 15.5- and 3.3-kb Hind III fragments (lane 1) and 13.6- and 6.5-kb Hinc II fragments (lane 6). The size of the DNA markers (in kb) is indicated at left. B. 2 μg of either total (lane 1) or poly(A)-selected HeLa RNA (lane 2) were separated on 1.2% agarose-formaldehyde gel and transferred to nylon membrane. The radioactive probe was the same as in A. The size (in kb) and migration of RNA markers run on the same gel is indicated at left.

Figure 5

Presence of USF2 in purified preparation of HeLa USE The various protein samples were separated by SDS gel electrophoresis and transferred to nitrocellulose. One portion of the filter was stained with Fast Green (A). The rest of the filter was treated with immune rabbit serum to USF2, and specific binding of the antibodies was visualized with alkaline phosphatase-conjugated goat antibodies to rabbit IgGs (B). Lanes 1: proteins from 3 μl of an IPTG-induced culture of E. coli cells transformed with plasmid _PUR-hUSF2 (see Materials and Methods). Lanes 2: 340 ng of purified His₆-delUSF protein (see Materials and Methods). Lanes 3: 160 ng of purified HeLa USF (Mono S fraction). The two arrows at left indicate the respective migration of the β-galactosidase-USF2 and polyhistidine-USF1 fusion proteins. The open arrow at right indicates the migration of the 44- and 43-kDa polypeptides characteristic of purified HeLa USF preparations (Sawadogo et al., 1988).