Male-specific transcription initiation of the C4-Slp gene in mouse liver follows activation of STAT5

Abstract

The mouse genes encoding the constitutively expressed complement component C4 and its closely related isoform C4-Slp (sex-limited protein), which is expressed only in male animals of several strains, provide a unique model to study sequence elements and trans-acting factors responsible for androgen responsiveness. Our previous studies have shown that hormonal induction of C4-Slp is mediated by a sex-specific pattern of growth hormone secretion. Promoter analyses in vitro have led to contradictory conclusions concerning the significance of C4-Slp-specific sequences in the 5' flanking region. Mutant mice carrying the H-2(aw18) haplotype, which is characterized by a large deletion in the S region covering the C4 and 21-OHase A genes, permit the direct in vivo analysis of C4-Slp transcription, unhindered by the presence of C4. Run-on analysis of transcription in liver nuclei of males and females of this strain demonstrated a 100-fold higher transcriptional activity in males, essentially determined at the transcription initiation level. The androgen dependence of transcription initiation was confirmed by run-on analysis of testosterone-treated females, where transcriptional activity started after 6 days of androgen treatment and reached male levels after 20 days. Conversely, the growth hormone-regulated activity of transcription factor STAT5 was already detected in liver nuclei after 48 hr of androgen treatment. Furthermore, we demonstrate that activated STAT5 recognizes in vitro two upstream gamma interferon-activated sequence (GAS) elements of the C4-Slp gene, centered at positions -1969 and -1831. We postulate that binding of STAT5 to these C4-Slp-specific GAS elements plays a crucial role in the chromatin remodelings that lead to transcriptional competence of the C4-Slp gene in the liver.

Figure 1

RNase protection analysis of liver steady-state RNAs. (A) Liver RNA (30 μg) from B10^aw18 mice (males, lanes 1 and 2, or females, lanes 3 and 4) or BALB/c female mice (lane 6) were hybridized to the transcription start-site probe (spliced and unspliced protected fragments of 121 nt and 234 nt, respectively, are shown schematically). Lanes: 1, 3, and 6, nuclear RNAs; 2 and 4, total RNA; 5, tRNA, negative control. (B) C4-Slp expression in B10^aw18 or BALB/c females injected for 20 days with 700 μg of testosterone propionate (+T, lanes 2 and 4). A positive control is in lane 3 (B10^aw18 male). Lanes 1 and 5 are untreated controls. Thirty micrograms of total RNA was hybridized to the distal C4/C4-Slp probe (244 nt; described in ref. 2), which distinguishes C4 (100 nt) and C4-Slp (191 nt).

Figure 2

Run-on analysis of newly synthesized transcripts. The sense or antisense oligonucleotides used as probes in the run-on assays are depicted on a schematic C4-Slp gene. Numbers represent the exons from which oligonucleotides have been designed. Two control probes (promoter and actin) are also shown schematically. The probes were immobilized on filters and hybridized with labeled nascent nuclear RNA from B10^aw18 males or females, or females injected for 10 days with 700 μg of testosterone propionate. Autoradiograms are shown after 72 hr of exposure. The scatter plot summarizes different run-on experiments, each performed on a pool of nuclei from three B10^aw18livers and analyzed quantitatively by using the PhosphorImager. The ratio exon 1/act of each experiment is reported on a logarithmic scale. Long (30 min, •) or short (10 min, ○) labeling times were used, and 6d, 10d, and 20d represent the days of testosterone treatment.

Figure 3

Elongation run-on analysis along C4 and C4-Slp genes. Probes used are identical to those described in Fig. 2. Labeled nuclear RNA was extracted from BALB/c males (30 min or 10 min) or females (30-min labeling). Autoradiograms after 72 hr of exposure are shown. The scatter plots represent exon 1/actin and exon 2/actin ratios, respectively, as obtained from several experiments performed on BALB/c males and females or on B10^aw18 and BALB/c males. Long (30 min, •) or short (10 min, ○) labeling times were used.

Figure 4

Time course of liver STAT5 activation upon testosterone treatment. (A) Mobility shifts using liver nuclear extracts from males or from females treated with testosterone for various times. Arrows mark the position of the shifted band in experiments with different times of gel migration. The probe was an oligonucleotide carrying the STAT5 consensus sequence (see Materials and Methods). Exposure times were 15 hr and 48 hr for male and female extracts, respectively. (B) Early detection of activated STAT5 in liver nuclear extracts of androgen-treated females (2 or 4 days). The probe was the same as in A. An arrow points to a weak but significant shifted band observed after 2 days of androgen treatment. Exposure time was 4 days.

Figure 5

Selective STAT5 binding to upstream GAS sites of C4-Slp. C4-Slp GAS sequences and the corresponding C4 control sequences are shown along the top of the figure, and the most conserved nucleotides of the consensus sequence for STAT5 binding are underlined. Numbers indicate the position of the first T in the C4-Slp sequence. Retardation gel shift assays were performed with probes encompassing GAS 1 and GAS 2 sequences (described in Materials and Methods) and liver nuclear extracts from male (m) or female (f) B10^aw18 mice. Competition was performed with 50-fold excess of unlabeled probe in the binding reaction. An arrow indicates the position of the protein–probe complex. Signals of various intensities at the top of each lane correspond to the origin of gel migration. Exposure was for 2 days.