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.
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