Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals

During meiosis a competent diploid cell replicates its DNA once and then undergoes two consecutive divisions followed by haploid gamete differentiation. Important aspects of meiotic development that distinguish it from mitotic growth include a highly increased rate of recombination, formation of the synaptonemal complex that aligns the homologous chromosomes, as well as separation of the homologues and sister chromatids during meiosis I and II without an intervening S-phase. Budding yeast is an excellent model organism to study meiosis and gametogenesis and accordingly, to date it belongs to the best studied eukaryotic systems in this context. Knowledge coming from these studies has provided important insights into meiotic development in higher eukaryotes. This was possible because sporulation in yeast and spermatogenesis in higher eukaryotes are analogous developmental pathways that involve conserved genes. For budding yeast a huge amount of data from numerous genome-scale studies on gene expression and deletion phenotypes of meiotic development and sporulation are available. In contrast, mammalian gametogenesis has not been studied on a large-scale until recently. It was unclear if an expression profiling study using germ cells and testicular somatic control cells that underwent lengthy purification procedures would yield interpretable results. We have therefore carried out a pioneering expression profiling study of male germ cells from Rattus norvegicus using Affymetrix U34A and B GeneChips. This work resulted in the first comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis and gametogenesis. We have identified 1268 differentially expressed genes in germ cells at different developmental stages, which were organized into four distinct expression clusters that reflect somatic, mitotic, meiotic and post-meiotic cell types. This included 293 yet uncharacterized transcripts whose expression pattern suggests that they are involved in spermatogenesis and fertility. A group of 121 transcripts were only expressed in meiotic (spermatocytes) and postmeiotic germ cells (round spermatids) but not in dividing germ cells (spermatogonia), \n \nSertoli \ncells or two somatic control tissues (brain \nand skeletal muscle). Functional analysis reveals \nthat most of the known genes in this \ngroup fulfill essential functions during meiosis, \nspermiogenesis (the process of sperm maturation) \nand fertility. Therefore it is highly possible \nthat some of the �30 uncharacterized transcripts \nin this group also contribute to these \nprocesses. A web-accessible database (called \nreXbase, which was later on integrated into \nGermOnline) has been developed for our expression \nprofiling study of mammalian male \nmeiosis, which summarizes annotation information \nand shows a graphical display of expression \nprofiles of every gene covered in our \nstudy. \nIn the budding yeast Saccharomyces cerevisiae \nentry into meiosis and subsequent progression \nthrough sporulation and gametogenesis \nare driven by a highly regulated transcriptional \nprogram activated by signal pathways \nresponding to nutritional and cell-type cues. \nAbf1p, which is a general transcription factor, \nhas previously been demonstrated to participate \nin the induction of numerous mitotic as \nwell as early and middle meiotic genes. In \nthe current study we have addressed the question \nhow Abf1p transcriptionally coordinates \nmitotic growth and meiotic development on a \ngenome-wide level. Because ABF1 is an essential \ngene we used the temperature-sensitive \nallele abf1-1. A phenotypical analysis of mutant \ncells revealed that ABF1 plays an important \nrole in cell separation during mitosis, \nmeiotic development, and spore formation. In \norder to identify genes whose expression depends \non Abf1p in growing and sporulating \ncells we have performed expression profiling \nexperiments using Affymetrix S98 GeneChips \ncomparing wild-type and abf1-1 mutant cells \nat both permissive and restrictive temperature. \nWe have identified 504 genes whose normal expression \ndepends on functional ABF1. By combining \nthe expression profiling data with data \nfrom genome-wide DNA binding assays (ChIPCHIP) \nand in silico predictions of potential \nAbf1p-binding sites in the yeast genome, we \nwere able to define direct target genes. Expression \nof these genes decreases in the absence \nof functional ABF1 and whose promotors are \nbound by Abf1p and/or contain a predicted \nbinding site. \n \nAmong 352 such bona fide direct target genes \nwe found many involved in ribosome biogenesis, \ntranslation, vegetative growth and meiotic \ndevelopement and therefore could account for \nthe observed growth and sporulation defects of \nabf1-1 mutant cells. Furthermore, the fact that \ntwo members of the septin family (CDC3 and \nCDC10 ) were found to be direct target genes \nsuggests a novel role for Abf1p in cytokinesis. \nThis was further substantiated by the observation \nthat chitin localization and septin ring \nformation are perturbed in abf1-1 mutant cells.