Rsl, a Novel Regulator of Sexually Dimorphic Liver Genes

描述（由申请方提供）：雄性和雌性之间在非生殖和生殖组织中存在许多生理差异。一个在动物中得到充分研究但在人类中受到有限关注的例子是肝脏的两性异形。在啮齿动物中，这种现象是基因表达的激素、发育和组织特异性控制之间复杂相互作用的典范。代谢类固醇和药物或生殖功能的几种蛋白质在啮齿动物肝脏中以性别特异性模式表达。诱导通过类固醇作用于垂体，以指导生长激素分泌的性别特异性特征而发生。 携带变体Rsl（性别限制调节因子）等位基因的小鼠揭示了对雄性特异性基因表达的额外控制。rsl表型被发现作为小鼠性别限制蛋白基因Slp的隐性修饰物，导致雄性特异性Slp存在于雌性以及雄性中。我们已经表明，rsl影响所有男性特定的肝脏基因，包括一些细胞色素P450和主要的尿蛋白（MUP）参与信息素信号。Rsl的调节不受雄激素或生长激素的控制，但只在青春期后才明显。由于Rsl的遗传变异是隐性的并且导致靶基因在雄性以及雌性中的表达增加，我们假设Rsl支配转录抑制。我们之前的建议集中在通过定位克隆来鉴定Rsl，利用该系统中丰富的遗传变异来获得对新调控的进展。 我们发现Rsl编码一对KRAB（Kruppel associated box）锌指蛋白（ZFP），这是已知的转录抑制因子。它们的同一性通过解释rsl表型的序列变异和通过小鼠中的BAC转基因拯救表型来证实。肝脏性二态性的调节是第一个被分配给任何KRAB-ZFP的生物学功能，这是显着的和重要的，因为ZFP是人类基因组中最大的基因类别，在小鼠中仅次于信息素和气味受体，KRAB-ZFP是这类基因的三分之一，但它们的生理作用尚不清楚。 我们将在以下四个方面剖析这两个KRAB-ZFP（现在称为Rsl 1和Rsl 2）分工抑制雄性特异性肝基因的机制，无论它们的合作是定量的还是定性的，以及它如何与激素诱导协同作用：I）通过rsl等位基因的完整表征来确定Rsl的分子效应，并通过用单个基因的转基因拯救来验证Rsl身份; H）表征Rsl的多个转录物，并且用基因“敲入”实验表征它们的时间和空间表达; HI）阐明Rsl调节功能的分子机制及其与激素诱导途径的相互作用; IV）在分子和生理水平上，特别是关于青春期的控制和青春期时，检查Rsl的更广泛的意义。我们的研究结果将对建立和维持基因沉默的机制具有广泛的意义。

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.