Genetic Regulation Of Spermatogenesis

利用基因表达序列分析技术(SAGE)建立了小鼠雄性生殖细胞的表达数据库。计算分析导致了特定阶段的通路和启动子模块的确定，以及与精子发生的不同阶段相关的生物网络的构建。此外，这些分析还导致了大量具有阶段特异性选择性剪接变体的基因的鉴定。已有研究表明，选择性剪接是精子发生过程中一种重要的遗传过程。已知许多基因经历了选择性剪接，这赋予了变异体新的活性。然而，对于剪接机制的阶段特异性和变异体的表达还没有系统的研究。我们已经开始对一些基因的新的阶段特异性变体进行表征，包括热休克蛋白4(Hspa4)、H3组蛋白、家族3B(H3F3B)和泛素蛋白连接酶E3A(Ube3a)。我们将以Hspa4为模型，研究选择性剪接在基因功能的阶段特异性调控中的作用及其对剪接变异体生物活性的影响。Hspa4已被证明是在氧化应激下被诱导的，氧化应激对精子的存活和正常功能以及男性生育能力至关重要。我们证实在A型精原细胞、粗线期精母细胞和圆形精细胞中存在三种不同的Hspa4转录本。进一步的生化和功能研究正在进行中，以确定其调控机制和生殖细胞中异构体的生物学功能。 对生殖细胞SAGE数据库的分析也显示出显著的反义转录本的存在。我们特别感兴趣的是来自假基因的反义转录本的存在。在我们鉴定的19个带有反义转录本的基因中，有4个(Uba52.Ch10、Calm2和UBB)在不同的染色体上都有来自其假基因的反义转录本。显然，这些假基因来自各自亲本基因的逆转录，并转座到活跃转录基因的内含子中：Uba52假基因位于Cbx1的内含子中；Calm2假基因存在于Prkar2b的内含子中；Ch10假基因存在于SP3的内含子中；Ubb假基因位于Catper2的内含子中。更有趣的是，假基因的方向与其宿主基因的方向是反平行的。因此，假基因的反义转录本将作为各自宿主基因的加工内含子产生。这增加了两个反平行转录单位通过杂交正义-反义转录本相互作用的可能性。随后的实验证实了反平行基因Uba52-Cbx1、Ch10-Sp3和Calm2-Prkar2b的天然双链RNA的存在。UBB-Catper2的双链RNA的存在尚未得到证实。我们将以Uba52和Cbx1为模型来研究反平行基因对之间的关系。Uba52的功能基因位于8号染色体上，假基因位于11号染色体上，嵌入Cbx1的第一内含子。Uba52、Cbx1和Uba52假基因的正义和反义转录本在小鼠肾细胞系CRL-6436中表达，将作为本研究的模型。 在生殖细胞表达分析的基础上，我们克隆了一个新的mArd1(停滞缺陷1)同源基因，我们将其命名为mArd2，该基因具有睾丸特异性，并在粗线期精母细胞中表达上调。已知mArd1蛋白与辅助蛋白亚基mNat1相互作用，构成功能性N-乙酰转移酶。早期在酵母中的研究已经确定了ARD1从细胞周期调节到DNA修复和重组的不同作用。我们发现mArd2的转录本在雄性减数分裂生殖细胞中优先表达，而编码蛋白的表达被推迟。通过体外蛋白质下拉实验和N-乙酰转移酶活性测定，我们证明了Ard2和Ard1一样，可以与Nat1相互作用并显示N-乙酰转移酶活性。我们的数据表明Ard1和Ard2在功能上是同源的。因此，Ard2的表达可能是从减数分裂开始的X连锁Ard1缺失的代偿机制。已证明Ard1的一种异构体可以乙酰化Hif-1的？？氨基。我们现在正在测试Ard2是否会在生殖细胞中表现出这种活性，即底物特异性改变。我们还在研究启动子元件和DNA甲基化对精子发生过程中Ard2转录激活的影响。 我们根据雄性生殖细胞的表达谱确定的另一个基因是mLin28，它是一个异时性基因，其产物调节线虫的发育时间。LIN28蛋白被认为是调节细胞增殖和分化的决定因素。对林28的生化特性研究较少。我们观察到当生殖细胞分化时，mLin28转录本的转录起始点的不同使用。我们确定了特定的启动子元件和模块，这些元件和模块已知可以引起转录激活效应。MLin28转录本的3！|末端是异质性的；我们分离了显示阶段特异性表达模式的转录本的备选3！|末端。为了更好地了解Lin28的表达调控，我们正在分析该基因的启动子活性。我们还建立了稳定表达HA标记版本的Lin28的P19细胞系，用于鉴定可能与Lin28相互作用并调节其产生的蛋白质和RNA分子。针对Lin28的siRNA转录敲除实验正在进行中，以研究Lin28蛋白的体外功能。

We established an expression database of mouse male germ cells through the use of Serial Analysis of Gene Expression (SAGE). Computational analyses had led to the identification of stage-specific pathways and promoter modules and the construction of biological networks associated with different stages of spermatogenesis. In addition, these analyses led to the identification of a large number of genes with stage-specific alternative spliced variants. It has been suggested that alternative splicing is a prominent genetic process occurring during spermatogenesis. A number of genes have been known to undergo alternative splicing which confers novel activities to the variants. However, there is no systematic study on the stage-specificity of the splicing mechanism and the expression of the variants. We have initiated the characterization of novel stage-specific variants of a number of genes including heat shock protein 4 (Hspa4), H3 histone, family 3B (H3f3b) and ubiquitin protein ligase E3A (Ube3a). We will use Hspa4 as a model to investigate the role of alternative splicing in stage-specific regulation of gene function and its impact on the biological activity of the splice variants. Hspa4 has been shown to be induced in response to oxidative stress, which is critical for the survival and normal functioning of spermatozoa and male fertility. We confirmed the presence of three distinctive transcripts of Hspa4 in type A spermatogonia, pachytene spermatocytes and round spermatids. Further biochemical and functional studies are underway to characterize its regulatory mechanisms and the biological functions of the isoforms in germ cells. Analysis of the germ cell SAGE database also revealed the prominent presence of antisense transcripts. We are particularly intrigued by the presence of antisense transcripts derived from pseudogenes. Among the 19 genes with antisense transcripts that we identified, four (Uba52. Ch10, Calm2 and Ubb) had antisense transcripts derived from their pseudogenes on different chromosomes. Apparently these pseudogenes were derived from reverse transcripts of the respective parent genes and transposed to the intron of actively transcribed genes: Uba52 pseudogene resides in the intron of Cbx1; Calm2 pseudogene is present in the intron of Prkar2b; Ch10 pseudogene is contained in the intron of Sp3; and Ubb pseudogene is located in the intron of Catsper2. More interestingly, the orientation of the pseudogenes is anti-parallel to that of their host genes. Thus, the antisense transcripts of the pseudogenes will be produced as processed introns of their respective host genes. This raises the possibility that the two anti-parallel transcription units interact through hybridization of the sense-antisense transcripts. Subsequent experiments confirmed the presence of native double-stranded RNA of the anti-parallel genes, namely, Uba52-Cbx1, Ch10-Sp3, and Calm2-Prkar2b. Presence of double-stranded RNA of Ubb-Catsper2 has not been confirmed yet. We will examine the relationship between the anti-parallel gene pairs using Uba52 and Cbx1 as a model. The functional gene of Uba52 is on chromosome 8 while its pseudogene is on chromosome 11 embedded in the first intron of Cbx1. Uba52, Cbx1, and the sense and antisense transcript of the Uba52 pseudogene are expressed in mouse kidney cell line CRL-6436, which will be used as a model for the study. Based on expression analysis of the germ cells we had cloned a novel mArd1 (Arrest Defective 1) homolog, which we named mArd2 that demonstrated testis-specificity and elevated expression in pachytene spermatocytes. The mArd1 protein is known to interact with an auxiliary protein subunit mNAT1 to constitute a functional N-acetyltransferase. Earlier studies in yeasts had identified a diverse role for ARD1 from cell cycle regulation to DNA repair and recombination. We showed that the transcript of mArd2 is preferentially expressed in male meiotic germ cells while the expression of the encoded protein was delayed. By performing in vitro protein pull-down assays and N-acetyltransferase activity assay, we demonstrated that Ard2, like Ard1, could interact with Nat1 and display N-acetyltransferase activity. Our data imply that Ard1 and Ard2 are functionally homologous. The expression of Ard2 may therefore be responsible for the compensation of the loss of X-linked Ard1 starting from meiosis. An isoform of Ard1 has been shown to acetylate the ??-amino group of Hif-1??. We are now testing if Ard2 would display such activity, i.e. altered substrate specificity, in germ cells. We are also examining the effect of promoter elements and DNA methylation on transcriptional activation of Ard2 during spermatogenesis. Another gene we identified based on expression profiling of male germ cells is mLin28, which is a heterochronic gene whose product regulates developmental timing in C. elegans. Lin28 protein had been suggested to regulate the decision between cellular proliferation and differentiation. The biochemical properties of Lin28 are not well studied. We observed differential use of transcription start sites of mLin28 transcripts as germ cells differentiate. We identified specific promoter elements and modules which were known to elicit transcriptional activation effects. The 3!| end of mLin28 transcripts was heterogeneous; we had isolated alternative 3!| ends of the transcripts which displayed stage-specific expression patterns. To better understand the regulation of Lin28 expression, we are analyzing promoter activity of this gene. We also generated a P19 cell line stably expressing a HA-tagged version of Lin28 for the identification of proteins and RNA molecules that may interact with Lin28 and regulate its production. Transcript knockdown experiments with siRNA against Lin28 are in progress to study the function of Lin28 protein in vitro.