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Local and chromosome-wide regulation of gene expression in Drosophila melanogaster
Local and chromosome-wide regulation of gene expression in Drosophila melanogaster
Within animal species, phenotypic variation is striking both between sexes and among individuals of the same sex. Much of this variation can be attributed to diverse gene expression patterns that evolved due to selection pressures specific to the external physical environment or to the internal chromosomal environment. Mechanisms that regulate expression can act on individual genes (local regulation) or on whole chromosomes (chromosome-wide). This dissertation seeks to examine the evolution of local and chromosome-wide expression regulation using the fruit fly Drosophila melanogaster as a model organism. When sex chromosomes determine sex, usually their ploidy differs between the sexes. In the XY system, present in both mammals and Drosophila, males are hemizygous for the X chromosome. This disparity between sexes exposes the X chromosome to unique selective forces that cause it to evolve different gene content and regulatory mechanisms from the autosomes. Notably, D. melanogaster tissue-specific genes, with the exception of ovary-specific genes, are underrepresented on the X chromosome. Furthermore, in the male soma gene expression is equalized between the single X chromosome and the autosomes through a mechanism known as dosage compensation. In contrast, in the male germline the expression of testis-specific genes residing on the X chromosome is suppressed through a mechanism known as X suppression. The main aim of this thesis is to investigate these aspects of gene regulation on the X chromosome in D. melanogaster. Chapter 1 and part of Chapter 2 focus on the phenomenon of X suppression. In Chapter 1, using testis- and other tissue-specific constructs, I survey autosomal and X-linked reporter gene expression in whole flies and carcasses with the tissue of interest removed. First, by reanalyzing the expression of the testis-specific reporter genes for which X suppression was initially described, I confirm the occurrence of X suppression in testis. Second, I show that X suppression is not a general property of tissue-specific genes, and that the X chromosome is neither a restrictive nor an unrestrictive environment for the expression of genes expressed specifically in the accessory gland (analogous to the mammalian prostate gland), ovary, or Malpighian tubule (analogous to the mammalian kidney). Moreover, I show that X-linkage has no impact on the tissue-specificity of gene expression. These findings suggest that the observed genomic distribution of tissue-specific genes is not the consequence of a chromosome-wide regulatory mechanism. This is the first study to functionally investigate the effect of X-linkage on the expression of tissue-specific genes, other than those specific to the testis. Chapter 2 investigates further the extent of X suppression. Using a ubiquitously-expressed reporter gene with an exogenous promoter in order to exclude any sex- or tissue-specific effects, I survey the expression of X-linked and autosomal reporter genes in testes and male somatic tissues. As expected, X suppression is absent in somatic tissues, which indicates that X suppression exclusively affects testis-expressed genes. Surprisingly, I find that the exogenous reporter gene, which has a basal level of expression in testis, shows no sign of X suppression in the male germline. This demonstrates that the expression level of a gene, together with its sex- and tissue-specificity, can be a major factor that influences the extent of X suppression. Thus, the present work makes a valuable contribution to the characterization of this newly-discovered regulatory mechanism. In Chapter 2, I also perform a pilot study regarding the effect the dosage compensation, which is mediated via the dosage compensation complex (DCC), on the chromosomal distribution of sex-biased genes in various tissues. I use X-linked insertions of the above mentioned ubiquitously-expressed reporter gene and correlate its expression in testis and male somatic tissues (heads and carcasses) with the proximity to different DCC binding sites. I find that the expression level of the X-linked reporter genes is not correlated with their distance to a binding site of DCC components, with the exception of maleless protein (MLE), for which there was a positive correlation between expression level and MLE distance in somatic tissues. Based on my findings, I provide recommendations that will serve as a foundation for a future study of this topic. Chapter 3 examines a candidate gene for local regulatory adaptation. The Metallothionein A (MtnA) gene exhibits expression variation in brains of natural populations of D. melanogaster. By collecting flies and analyzing the deletion frequency in an additional population (Cyprus), I provide further evidence that this expression variation is associated with a 49-bp deletion in the MtnA 3’ untranslated region (UTR), which is present at intermediate frequency in derived populations of the species. These results, supported with population genetic analysis, suggest that the deletion allele has been a target of local adaptation. By performing hydrogen peroxide tolerance assays, I show that the deletion is associated with increased oxidative stress tolerance, which suggests that the deletion (and increased MtnA expression) is an adaption to oxidative stress.
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Argyridou, Eliza
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Argyridou, Eliza (2018): Local and chromosome-wide regulation of gene expression in Drosophila melanogaster. Dissertation, LMU München: Faculty of Biology
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Abstract

Within animal species, phenotypic variation is striking both between sexes and among individuals of the same sex. Much of this variation can be attributed to diverse gene expression patterns that evolved due to selection pressures specific to the external physical environment or to the internal chromosomal environment. Mechanisms that regulate expression can act on individual genes (local regulation) or on whole chromosomes (chromosome-wide). This dissertation seeks to examine the evolution of local and chromosome-wide expression regulation using the fruit fly Drosophila melanogaster as a model organism. When sex chromosomes determine sex, usually their ploidy differs between the sexes. In the XY system, present in both mammals and Drosophila, males are hemizygous for the X chromosome. This disparity between sexes exposes the X chromosome to unique selective forces that cause it to evolve different gene content and regulatory mechanisms from the autosomes. Notably, D. melanogaster tissue-specific genes, with the exception of ovary-specific genes, are underrepresented on the X chromosome. Furthermore, in the male soma gene expression is equalized between the single X chromosome and the autosomes through a mechanism known as dosage compensation. In contrast, in the male germline the expression of testis-specific genes residing on the X chromosome is suppressed through a mechanism known as X suppression. The main aim of this thesis is to investigate these aspects of gene regulation on the X chromosome in D. melanogaster. Chapter 1 and part of Chapter 2 focus on the phenomenon of X suppression. In Chapter 1, using testis- and other tissue-specific constructs, I survey autosomal and X-linked reporter gene expression in whole flies and carcasses with the tissue of interest removed. First, by reanalyzing the expression of the testis-specific reporter genes for which X suppression was initially described, I confirm the occurrence of X suppression in testis. Second, I show that X suppression is not a general property of tissue-specific genes, and that the X chromosome is neither a restrictive nor an unrestrictive environment for the expression of genes expressed specifically in the accessory gland (analogous to the mammalian prostate gland), ovary, or Malpighian tubule (analogous to the mammalian kidney). Moreover, I show that X-linkage has no impact on the tissue-specificity of gene expression. These findings suggest that the observed genomic distribution of tissue-specific genes is not the consequence of a chromosome-wide regulatory mechanism. This is the first study to functionally investigate the effect of X-linkage on the expression of tissue-specific genes, other than those specific to the testis. Chapter 2 investigates further the extent of X suppression. Using a ubiquitously-expressed reporter gene with an exogenous promoter in order to exclude any sex- or tissue-specific effects, I survey the expression of X-linked and autosomal reporter genes in testes and male somatic tissues. As expected, X suppression is absent in somatic tissues, which indicates that X suppression exclusively affects testis-expressed genes. Surprisingly, I find that the exogenous reporter gene, which has a basal level of expression in testis, shows no sign of X suppression in the male germline. This demonstrates that the expression level of a gene, together with its sex- and tissue-specificity, can be a major factor that influences the extent of X suppression. Thus, the present work makes a valuable contribution to the characterization of this newly-discovered regulatory mechanism. In Chapter 2, I also perform a pilot study regarding the effect the dosage compensation, which is mediated via the dosage compensation complex (DCC), on the chromosomal distribution of sex-biased genes in various tissues. I use X-linked insertions of the above mentioned ubiquitously-expressed reporter gene and correlate its expression in testis and male somatic tissues (heads and carcasses) with the proximity to different DCC binding sites. I find that the expression level of the X-linked reporter genes is not correlated with their distance to a binding site of DCC components, with the exception of maleless protein (MLE), for which there was a positive correlation between expression level and MLE distance in somatic tissues. Based on my findings, I provide recommendations that will serve as a foundation for a future study of this topic. Chapter 3 examines a candidate gene for local regulatory adaptation. The Metallothionein A (MtnA) gene exhibits expression variation in brains of natural populations of D. melanogaster. By collecting flies and analyzing the deletion frequency in an additional population (Cyprus), I provide further evidence that this expression variation is associated with a 49-bp deletion in the MtnA 3’ untranslated region (UTR), which is present at intermediate frequency in derived populations of the species. These results, supported with population genetic analysis, suggest that the deletion allele has been a target of local adaptation. By performing hydrogen peroxide tolerance assays, I show that the deletion is associated with increased oxidative stress tolerance, which suggests that the deletion (and increased MtnA expression) is an adaption to oxidative stress.