Analysis of the Transcriptional Program Governing Meiosis and Gametogenesis in Yeast
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Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals Inauguraldissertation zur Erlangung der W¨urde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakult¨at der Universit¨atBasel von Ulrich Schlecht Aus Tokyo (Japan) Basel, 2006 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakult¨at auf Antrag von Prof. Michael Primig, Prof. Peter Philippsen und Prof. Urs Meyer. Basel, den 10.07.2006 Prof. Hans-Jakob Wirz Dekan 2 ’Everything in its right place’ - Radiohead 3 Table of contents Table of contents Abstract 6 General introduction 8 Meiosis and gametogenesis . 8 Meiotic development in mammals . 8 Transcriptional control of meiosis in budding yeast . 8 Knowledge databases . 10 Goals of this work . 11 Chapter 1: Expression profiling of Mammalian Male Meiosis and Gametogen- esis Identifies Novel Candidate Genes for Roles in the Regulation of Fertility 12 Abstract 12 Introduction 12 Results 13 Experimental design . 13 Identification of differentially expressed transcripts in somatic versus germ cells . 14 Identification of transcripts represented by probe sets through similarity search . 17 Clustering gene expression profiles in somatic and germ cells . 18 Somatic tissue profiling of germ cell genes . 21 Array data validation by Q-PCR ................................ 21 Online data-access and visualization . 22 Discussion 24 Mining mammalian meiosis . 24 Experimental design and data analysis . 24 Germ cell expression and function . 25 Materials and Methods 26 Acknowledgements 28 Chapter 2: Genome-wide Analysis of ABF1 -target Genes During Mitotic Growth and Meiotic Development 29 Abstract 29 Introduction 29 Results 33 Experimental design . 33 Phenotypes of abf1-1 mutant strains . 35 Expression profiling of abf1-1 mutant cells (mitosis) . 38 4 Table of contents Genes differentially expressed in YPD . 40 Genes differentially expressed in YPA . 45 Expression profiling of abf1-1 mutant cells (meiosis) . 48 Genome-wide identification of Abf1p-bound loci . 51 In silico prediction of ABF1 -target sites . 57 Combining the three data sets . 57 Data mining of ABF1 -target genes . 60 Genes essential for vegetative growth . 60 Genes induced and repressed in acetate . 62 Genes involved in meiotic development . 62 Abf1p and ARS functionality . 67 Abf1p and cytokinesis . 67 Discussion 69 Experimental strategy . 69 Abf1p and DNA replication . 72 Abf1p acts via interaction with different regulatory complexes . 73 ABF1 -target genes and phenotypes of abf1-1 ......................... 75 Materials and Methods 77 Acknowledgements 79 General conclusion and outlook 80 References 82 List of abbreviations 96 Acknowledgements 96 Curriculum vitae 97 Erkl¨arung 99 5 Introduction Abstract toli cells or two somatic control tissues (brain and skeletal muscle). Functional analysis re- During meiosis a competent diploid cell repli- veals that most of the known genes in this cates its DNA once and then undergoes two group fulfill essential functions during meiosis, consecutive divisions followed by haploid ga- spermiogenesis (the process of sperm matura- mete differentiation. Important aspects of mei- tion) and fertility. Therefore it is highly possi- otic development that distinguish it from mi- ble that some of the ∼30 uncharacterized tran- totic growth include a highly increased rate scripts in this group also contribute to these of recombination, formation of the synaptone- processes. A web-accessible database (called mal complex that aligns the homologous chro- reXbase, which was later on integrated into mosomes, as well as separation of the homo- GermOnline) has been developed for our ex- logues and sister chromatids during meiosis I pression profiling study of mammalian male and II without an intervening S-phase. Bud- meiosis, which summarizes annotation infor- ding yeast is an excellent model organism to mation and shows a graphical display of ex- study meiosis and gametogenesis and accord- pression profiles of every gene covered in our ingly, to date it belongs to the best studied study. eukaryotic systems in this context. Knowl- In the budding yeast Saccharomyces cere- edge coming from these studies has provided visiae entry into meiosis and subsequent pro- important insights into meiotic development in gression through sporulation and gametogene- higher eukaryotes. This was possible because sis are driven by a highly regulated transcrip- sporulation in yeast and spermatogenesis in tional program activated by signal pathways higher eukaryotes are analogous developmental responding to nutritional and cell-type cues. pathways that involve conserved genes. Abf1p, which is a general transcription factor, For budding yeast a huge amount of data has previously been demonstrated to partici- from numerous genome-scale studies on gene pate in the induction of numerous mitotic as expression and deletion phenotypes of meiotic well as early and middle meiotic genes. In development and sporulation are available. In the current study we have addressed the ques- contrast, mammalian gametogenesis has not tion how Abf1p transcriptionally coordinates been studied on a large-scale until recently. It mitotic growth and meiotic development on a was unclear if an expression profiling study us- genome-wide level. Because ABF1 is an es- ing germ cells and testicular somatic control sential gene we used the temperature-sensitive cells that underwent lengthy purification pro- allele abf1-1. A phenotypical analysis of mu- cedures would yield interpretable results. We tant cells revealed that ABF1 plays an im- have therefore carried out a pioneering expres- portant role in cell separation during mitosis, sion profiling study of male germ cells from meiotic development, and spore formation. In Rattus norvegicus using Affymetrix U34A and order to identify genes whose expression de- B GeneChips. This work resulted in the first pends on Abf1p in growing and sporulating comprehensive large-scale expression profiling cells we have performed expression profiling analysis of mammalian male germ cells under- experiments using Affymetrix S98 GeneChips going mitotic growth, meiosis and gametoge- comparing wild-type and abf1-1 mutant cells nesis. We have identified 1268 differentially at both permissive and restrictive temperature. expressed genes in germ cells at different de- We have identified 504 genes whose normal ex- velopmental stages, which were organized into pression depends on functional ABF1. By com- four distinct expression clusters that reflect so- bining the expression profiling data with data matic, mitotic, meiotic and post-meiotic cell from genome-wide DNA binding assays (ChIP- types. This included 293 yet uncharacterized CHIP) and in silico predictions of potential transcripts whose expression pattern suggests Abf1p-binding sites in the yeast genome, we that they are involved in spermatogenesis and were able to define direct target genes. Ex- fertility. A group of 121 transcripts were only pression of these genes decreases in the absence expressed in meiotic (spermatocytes) and post- of functional ABF1 and whose promotors are meiotic germ cells (round spermatids) but not bound by Abf1p and/or contain a predicted in dividing germ cells (spermatogonia), Ser- binding site. 6 Introduction Among 352 such bona fide direct target genes we found many involved in ribosome biogene- sis, translation, vegetative growth and meiotic developement and therefore could account for the observed growth and sporulation defects of abf1-1 mutant cells. Furthermore, the fact that two members of the septin family (CDC3 and CDC10 ) were found to be direct target genes suggests a novel role for Abf1p in cytokinesis. This was further substantiated by the obser- vation that chitin localization and septin ring formation are perturbed in abf1-1 mutant cells. 7 Introduction General introduction et al., 2001) mammalian gametogenesis has not been studied on a genomic scale until recently. Meiosis and gametogenesis The identification of meiosis- and germ-cell- specific transcripts in multicellular organisms Meiosis is the developmental pathway by which is a complex task because gonads contain dif- sexually reproducing diploid organisms gener- ferent types of cells, only some of which are ate haploid germ cells. It plays an impor- germ cells. However, it is possible to obtain in- tant role in promoting genetic diversity, remov- formative expression data using microarrays as ing recessive lethal mutations from populations recently shown by different studies using var- and maintaining constant chromosome num- ious mammalian model systems (Pang et al., bers from one generation to the next. Meio- 2003; Schultz et al., 2003; Schlecht et al., 2004). sis can be regarded as a specialized form of These large-scale expression analyses of mam- the mitotic cell cycle with three major differ- malian male reproductive tissue were based on ences. First, following pre-meiotic DNA syn- profiling the process of spermatogenesis using thesis, high levels of recombination occur be- total testis samples or germ cell populations tween homologous chromosomes during mei- purified from animals at different stages of sex- otic prophase. Second, sister-centromere co- ual maturity (Schultz et al., 2003; Almstrup hesion and coorientation are coupled to sepa- et al., 2004; Ellis et al., 2004; Shima et al., ration and independent assortment of parental 2004; Schlecht et al., 2004). Understanding chromosomes. Third, two consecutive nuclear the genetic program controlling the mitotic and divisions