Rsl, a Novel Regulator of Sexually Dimorphic Liver Genes

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DESCRIPTION (provided by applicant): Numerous physiological differences between males and females exist in nonreproductive as well as reproductive tissues. An example that is well studied in animals but has received limited attention in humans is sexual dimorphism of the liver. In rodents, this phenomenon is a paradigm for the complex interplay of hormonal, developmental and tissue specific control of gene expression. Several proteins that metabolize steroids and drugs or that function in reproduction are expressed in sex-specific patterns in rodent liver. Induction occurs via steroid action on the pituitary to direct sex-specific profiles of growth hormone secretion. Mice carrying variant Rsl (regulator of sex-limitation) alleles reveal an additional control of male-specific gene expression. The rsl phenotype was discovered as a recessive modifier of the mouse sex-limited protein gene, Slp, causing male-specific Slp to be present in females as well as males. We have shown that rsl affects all male-specific liver genes, including some cytochrome P450s and major urinary proteins (MUPs) involved in pheromone signaling. Rsl regulation is independent of androgen or growth hormone control, yet is only evident after puberty. Since genetic variation of Rsl is recessive and leads to increased expression of the target genes, in males as well as females, we hypothesized that Rsl dictates transcriptional repression. Our previous proposal focused on identifying Rsl by positional cloning, using rich genetic variation in this system to gain an inroad to the novel regulation. We show that Rsl encodes a pair of KRAB (Kruppel associated box) zinc f'mger proteins (ZFPs), which are known to be transcriptional repressors. Their identity is confirmed by sequence variations that account for rsl phenotypes, and rescue of the phenotype by BAC transgenesis in mice. Regulation of liver sexual dimorphism is the first biological function to be assigned to any KRAB-ZFP, which is remarkable and important, since ZFPs are the largest class of genes in the human genome, second in mice only to pheromone and odorant receptors, and KRAB-ZFPs are one-third of this class, yet none of their physiological roles are known. We will dissect the mechanism by which these two KRAB-ZFPs, now called Rsll and Rsi2, divide the labor of repressing malespecific liver genes, whether their cooperation is quantitative or qualitative, and how it operates in concert with hormonal induction, in the following four alms: I) Determine molecular effects of Rsl by complete characterization of rsl alleles, and verify Rsl identity by transgenic rescue with individual genes; H) Characterize the multiple transcripts of Rsl, and, with gene "knock-in" experiments, their temporal and spatial expression; HI) Elucidate the molecular mechanism of Rsl regulatory function and its interaction with the hormonal induction pathway; IV) Examine a broader significance of Rsl, at molecular and physiologic levels, particularly in regard to control of, and at, puberty. Our results will be broadly significant to mechanisms that establish and maintain gene silencing.