Rsl, a Novel Regulator of Sexually Dimorphic Liver Genes

描述(申请人提供)：在非生殖组织和生殖组织中，男性和女性之间存在着许多生理差异。在动物身上得到了很好的研究，但在人类身上只得到了有限的关注的一个例子是肝脏的性别二型性。在啮齿动物中，这种现象是激素、发育和组织特异性基因表达控制的复杂相互作用的范例。几种代谢类固醇和药物或在生殖过程中起作用的蛋白质在啮齿类动物的肝脏中以性别特异的模式表达。诱导是通过对脑下垂体的类固醇作用来引导生长激素分泌的性别特异性。携带变异的RSL(性别限制调节)等位基因的小鼠显示出对男性特有基因表达的额外控制。RSL表型被发现是小鼠性限制蛋白基因SLP的隐性修饰，导致雄性特有的SLP存在于雌性和雄性中。我们已经证明，RSL影响所有男性特有的肝脏基因，包括一些细胞色素P450和参与信息素信号传递的主要尿蛋白(MUP)。RSL的调节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，并导致靶基因在男性和女性中表达增加，我们假设RSL决定了转录抑制。我们之前的建议侧重于通过位置克隆来识别RSL，利用这个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KRAB(Kruppel Association Box)锌功能蛋白(ZFP)，这些蛋白是已知的转录抑制因子。它们的一致性被解释RSL表型的序列变异和BAC转基因小鼠的表型拯救所证实。KRAB-ZFP是第一个被分配给任何KRAB-ZFP的生物学功能，这是值得注意和重要的，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，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，即现在称为Rs11和Rsi2的KRAB-ZFP，它们之间的合作是定量的还是定性的，以及它是如何与激素诱导协同工作的：i)通过完整的RSL等位基因特征来确定RSL的分子效应，并通过单个基因的转基因拯救来验证RSL的同一性；H)表征RSL的多个转录本，并通过基因“敲入”实验，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；4)在分子和生理水平上研究RSL的更广泛的意义，特别是在青春期控制和青春期的控制方面。我们的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.