The X chromosome as a unique environment for gene expression in Drosophila melanogaster

Belyi, Aleksei (2021): The X chromosome as a unique environment for gene expression in Drosophila melanogaster. Dissertation, LMU München: Fakultät für Biologie

Abstract

The evolution of genetic sex determination in many organisms has led to the development of heteromorphic sex chromosomes. Such chromosomes often acquire specific regulatory mechanisms and gene content due to the absence of recombination between them. In D. melanogaster, two major chromosomal regulatory mechanisms, X chromosome-specific dosage compensation and X suppression in the male germline, create a unique environment for gene expression in different sexes and across tissues. In the male soma, X-linked genes are dosage compensated by having their expression up-regulated, a process mediated by the binding of the dosage compensation complex (DCC). Previous studies of X-linked gene expression found a negative correlation between a gene’s male-to-female expression ratio and its distance to the nearest DCC binding site in somatic tissues, including head and brain, which suggests that dosage compensation influences sex-biased gene expression. However, a limitation of the previous studies was that they focused on endogenous X-linked genes and, thus, could not disentangle the effects of chromosomal position from those of gene-specific regulation. In the first part of my thesis, we addressed this limitation by examining the expression of a CMV-lacZ reporter gene construct consisting of the Escherichia coli lacZ gene under the control of the minimal human cytomegalovirus (CMV) promoter inserted at many locations spanning the X chromosome. By doing so, we were able to test the effect of a reporter gene’s proximity to a DCC binding site on its expression in males, females and the male-to-female expression ratio across different tissues. We observed a negative correlation between the male-to-female expression ratio of the reporter gene and its distance to the nearest DCC binding site in somatic tissues but not in gonads. A reporter gene’s location relative to a DCC binding site had a greater influence on its expression than the local regulatory elements of neighbouring endogenous genes, suggesting that intra-chromosomal variation in the strength of dosage compensation is a major determinant of sex-biased gene expression. Average levels of sex-biased expression did not differ between head and brain, but there was greater positional effect variation in the brain, which may explain the observed excess of endogenous sex-biased genes located on the X chromosome in this tissue. In the male germline, however, there is no DCC-mediated dosage compensation, and the expression of X-linked genes is suppressed through a mechanism analogous to the meiotic sex chromosome inactivation that occurs in mammals. In contrast to the latter, in Drosophila the X suppression is not complete and its extent is correlated with the gene’s expression level in testes. As the genetic and molecular mechanisms behind this X suppression are unknown, in the second part of my dissertation, we used a forward genetic screen to discover the genes responsible for the X suppression. For this, we performed chemical mutagenesis on males with an X-linked copy of the lacZ reporter gene controlled by the ocnus promoter, which has testis-specific expression and previously had shown a strong effect of X suppression. With this approach, we detected two mutant lines that we named INvolved in X Suppression (INXS1 and INXS2), which showed a strong increase of reporter gene expression relative to the control line. Analysis of endogenous X-linked gene expression confirmed a general increase in expression in both mutants. Also, males of both INXS mutants were sterile and displayed complete sperm immobility. Taken together, these phenotypes indicate suggest that there is partial or complete relaxation of X suppression in the male germline. We found four top candidate genes on the X (INXS1: CG13003; INXS2: CG1314) and third chromosomes (INXS1 and INXS2: CG31525; INXS1: CG42654), with all genes except CG13003 having high testis-specific expression. The genes CG13003, CG31525 and CG42654 showed a partial association with increased reporter gene expression in INXS1, whereas, in INXS2, both CG1314 and CG31525 showed a complete association. However, with a preliminary functional analysis of the CG13003, CG1314 and CG31525 genes using targeted RNA interference (RNAi) knockdown in different germline cell types and whole body, we were not able to find direct evidence for their role in the X suppression. This study generated the first mutant lines with partial or complete disruption of X suppression and identified several promising targets for further functional testing.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Drosophila, Sex chromosomes, Dosage compensation, Gene regulation, Sex-biased expression, Reporter gene, The X chromosome supression
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. September 2021
• 1. Bericht­erstatter:in: Parsch, John