Rsl, a Novel Regulator of Sexually Dimorphic Liver Genes

描述（由申请人提供）：男性和女性在非生殖组织和生殖组织中存在许多生理差异。一个在动物身上研究得很好的例子是肝脏的两性异形，但在人类身上却很少受到关注。在啮齿类动物中，这种现象是激素、发育和组织特异性控制基因表达的复杂相互作用的范例。在啮齿动物的肝脏中，一些代谢类固醇和药物的蛋白质或在生殖中起作用的蛋白质以性别特异性的模式表达。诱导发生通过类固醇作用于垂体，以直接生长激素分泌的性别特异性概况。携带变异Rsl（性别限制调节因子）等位基因的小鼠显示出对雄性特异性基因表达的额外控制。rsl表型被发现是小鼠性别限制蛋白基因Slp的隐性修饰子，导致雄性特异性Slp在雌性和雄性中都存在。我们已经证明rsl影响所有男性特异性肝脏基因，包括一些细胞色素p450和参与信息素信号传导的主要尿蛋白（MUPs）。Rsl的调节是独立于雄激素或生长激素的控制，但只有在青春期后才明显。由于Rsl的遗传变异是隐性的，导致靶基因的表达增加，在男性和女性中，我们假设Rsl决定了转录抑制。我们之前的建议侧重于通过定位克隆鉴定Rsl，利用该系统中丰富的遗传变异来获得新的调控途径。我们发现Rsl编码一对已知是转录抑制因子的KRAB （Kruppel associated box）锌蛋白（ZFPs）。它们的身份通过解释rsl表型的序列变异和在小鼠中通过BAC转基因拯救表型得到证实。肝脏两性二态性的调节是任何KRAB-ZFP的第一个生物学功能，这是显着和重要的，因为zfp是人类基因组中最大的一类基因，在小鼠中仅次于信息素和气味受体，而KRAB-ZFP是这一类基因的三分之一，但它们的生理作用尚不清楚。

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.