The COUP-TFs compose a family of functionally related transcription factors

Abstract

The chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are members of the steroid/thyroid hormone receptor superfamily and function in transcriptional regulation of a wide variety of genes. The COUP-TFs purified from HeLa nuclear extract by COUP-affinity chromatography are composed of multiple M(r) forms. The Low M(r) COUP-TFs (43,000, 44,000, 46,000, and 47,000 M(r)) produce a relatively fast migrating complex (C1) with DNA in electrophoresis mobility shift assays, while the high M(r) forms (66,000, 68,000, 72,000, and 74,000 M(r)) produce a slower migrating (C2) complex. The high M(r) COUP-TFs were purified by gel filtration chromatography and independently formed the C2 DNA complex, probably acting as dimers. The high M(r) forms are indistinguishable from the low M(r) COUP-TFs in DNA binding and in enhancement of in vitro transcription from the ovalbumin promoter. The finding of multiple COUP-TF forms led us to clone a second low M(r) COUP-TF, "COUP-TF2." The COUP-TF2 sequence has very strong homology with COUP-TF1. The N-termini of COUP-TF1 and COUP-TF2 are least similar, but both contain glutamine-rich and proline-rich motifs, putative activation domains.

Figure 1

Purified COUP-TFs are composed of multiple M_r forms and produce complexes with COUP DNA of two distinct mobilities. Left panel: Electrophoretic mobility shift (EMSA) analysis of COUP-TFs. Approximately 30 ng of COUP-TFs, purified from HeLa nuclear extract by COUP affinity chromatography, bound a radiolabeled fragment of the ovalbumin promoter (−269 to −44). The two COUP-TF specific complexes, Cl and C2, are visible on the autoradiograph of the native 5% polyacrylamide gel, as is the free probe seen below. Right panel: Sodium dodecyl sulfate-PAGE analysis of purified COUP-TFs. Three hundred ng of purified COUP-TFs (above) were separated and silver-stained on a 10% polyacrylamide gel. The protein bands observed fall into three classes: low M_r COUP-TFs, responsible for the Cl complex in the left panel; high M_r COUP-TFs, responsible for the C2 complex; and hERRl, a relatively weak COUP-binding protein.

Figure 2

Dimers of high and low M_r COUP-TFs independently form C2 and Cl complexes, respectively. High and low M_r COUP-TFs were separated by gel filtration chromatography, and even-numbered fractions were analyzed by EMSA. The high M_r COUP-TFs formed the C2 complex on DNA, while the low M_r COUP-TFs formed Cl, as indicated. Elution of M_r standards from the column indicated that the native forms of high and low M_r COUP-TFs are approximately 130,000 and 90,000 M_r, respectively.

Figure 3

Individual forms of the high M_r COUP-TFs bind COUP specifically. Individual bands of the high M_r COUP-TFs were electroeluted from a preparative SDS-polyacrylamide gel, renatured, and tested in an EMSA. The results from only one high M_r COUP-TF are shown because all four were equivalent. The C2 complex (with brackets at right) was efficiently reduced by the addition of 10-fold and 50-fold molar excesses of unlabeled COUP oligonucleotide (COUP), but not by a 50-fold molar excess of mutant COUP oligonucleotide (mCOUP).

Figure 4

The high and low M_r COUP-TFs have identical DNase 1 footprints over the ovalbumin COUP element. Purified high and low M_r COUP-TFs were bound to the end-labeled −269 to −44 fragment of the ovalbumin promoter. During DNase 1 treatment, they protected identical areas on the coding strand of the ovalbumin promoter over the COUP element (−90 to −70; −70 is toward the bottom of the figure), as seen on this autoradiograph of a sequencing gel. The DNase 1 digestion of the probe in the absence of COUP-TFs is shown at left, for comparison.

Figure 5

The high M_r COUP-TFs are activators of transcription from the ovalbumin promoter. The purified high M_r COUP-TFs activated transcription of a 377-base G-free cassette under the control of the ovalbumin promoter (−219 to −1). Each assay included fractions of HeLa nuclear extract containing RNA polymerase, its cofactors, and S300-II. Also added were nucleotides (including ³²P.UTP), buffers, and the internal control template pAdML₂₀₀, with the adenovirus major late promoter directing the formation of a 200-base G-less transcript. Specific transcripts from ovalbumin (OV) and adenovirus major late (AdML) promoters are seen at appropriate positions on this autoradiograph of a sequencing gel. The specificity of the COUP-TF enhancement of transcription was tested with 50-fold molar excesses of COUP oligonucleotide (COUP) or progesterone response element-containing oligonucleotides (PRE).

Figure 6

A second low M_r COUP-TF, COUP-TF2, has been cloned and sequenced. In the upper panel, the sequencing strategy is shown for the COUP-TF2 sequence. Sites for generating restriction fragments are pictured, along with the ATG translation start site and the DNA binding domain (boxed). Arrows denote sequencing information obtained, and circles indicate where specific oligonucleotide primers were used. A bar showing the relative size of 100 bases is at the lower right for reference. Shown below is the 2216-base sequence of a COUP-TF2 cDNA, identified by screening a lambda gt10 HeLa cell cDNA library with a COUP-TF1 probe. The inferred amino acid sequence is shown under the base sequence. The sequences are numbered at left for convenience. The DNA-binding domain, determined by homology to COUP-TF1, is boxed.

Figure 7

The COUP-TF2 protein is highly homologous to COUP-TF1. The amino acid sequence of COUP-TF2 is aligned with that of COUP-TF1 to show the high level of positions with identical residues (indicated by a dash in the COUP-TF1 sequence). Spacing to optimize the alignment is designated by dots. The three conserved regions of the steroid/thyroid hormone receptor superfamily are boxed, with the most N-terminal being the DNA binding domain (region I), followed in order by regions II and III. Amino acid residues are numbered at right for convenience.