Mechanisms of Chromosome-Wide Gene Regulation in Caenorhabditis Elegans

Abstract

Chromatin organization in the interphase nucleus is dynamic and complex. It impacts gene expression, tissue development, and cell homeostasis. Chromatin dynamics can be established through specific mechanisms that includes those mediated by condensin, a chromatin motor complex. However, our understanding of how condensins and other chromatin modifying mechanisms collaborate to regulate gene expression over an entire chromosome during interphase is incomplete because condensins also function in cell division and any disruption of that role leads to lethality. My dissertation work addresses this gap, by utilizing the microscopic nematode Caenorhabditis elegans and its dosage compensation (DC) machinery as a model for chromosome-wide gene regulation by a chromatin motor complex that cooperates with other chromatin-modifying mechanisms, like the nuclear RNAi machinery. DC is the process that equalizes gene expression of the X chromosomes between hermaphrodites and males, reducing the gene output of the 2 hermaphrodite X chromosomes to equal the single male X. My genetic and biochemical data suggest that the C. elegans DC specific chromatin motor complex, condensin IDC, uses energy to compact the X chromosomes. My data also shows how X-chromosome regulation also requires the nuclear RNAi machinery to compact the X chromosomes. This first part of this dissertation examines how the C. elegans dosage compensation specific chromatin motor complex, condensin IDC, which is not required for cell division, compares to the mitotic condensins that are conserved throughout all eukaryotes. Condensin IDC differs from condensin I in C. elegans by a single subunit, DPY-27. DPY-27 takes the place of SMC-4, a Structural Maintenance of Chromosome (SMC) protein. SMC proteins function in condensin complexes. During mitosis, they are responsible for hydrolyzing ATP for energy to condense DNA. I used sequence alignments, biochemistry, molecular biology, and genetics to understand how this additional SMC protein, DPY-27, compares to other SMC proteins. I demonstrated that DPY-27 is capable of hydrolyzing ATP and this function is required for X-chromosome DC. When this function is mutated in the worms, their X chromosomes lose compaction and fail to gain histone modifications that help reduce their gene expression for DC. The second part of this dissertation examines the role of the nuclear RNAi machinery in X-chromosome DC. The nuclear RNAi machinery is a conserved pathway that can silence gene expression through small interfering RNAs (siRNAs). The siRNAs help guide Argonaute proteins to nascent RNA and shut down transcription of that locus by recruiting histone modifying proteins to silence gene expression. Using genetics and molecular biology, we demonstrated that two Argonaute proteins, HRDE-1 and NRDE-3 have direct roles in DC, and loss of these proteins results in compromised DC and X-chromosome gene regulation. We also demonstrated that these proteins cooperate with the X-signal element SEX-1, which controls the main regulatory switch for hermaphrodite development and DC. Together these findings enlighten us on how condensin IDC and the nuclear RNAi machinery are responsible for regulating gene expression of the entire X chromosomes. While both have been studied previously, this detailed study of DPY-27 as an SMC protein establishes condensin IDC as a genuine chromatin motor complex and sets the stage for future studies to use C. elegans as a model of condensin-mediated gene regulation. Second, the specific role of Argonaute proteins in X-chromosome DC helps expand our understanding of how multiple chromatin-modifying mechanisms work in tandem to regulate gene expression.