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Two Inputs into C. elegans Dosage Compensation: Chromosome Confromation and the miRNA-specific Argonaute ALG-2

  • Author(s): Crane, Emily Elizabeth
  • Advisor(s): Meyer, Barbara J
  • et al.
Abstract

Dosage compensation equalizes X-linked gene expression between males (1X) and hermaphrodites (2X) of C. elegans through a protein complex (DCC) that is targeted to the X chromosomes of only XX embryos. The DCC includes five proteins with homology to condensin, a conserved complex essential for the compaction, resolution, and segregation of mitotic and meiotic chromosomes. Similarity between the DCC and condensin suggests that the DCC may achieve chromosome-wide gene repression through changes in X-chromosome structure. The DCC binds X using two classes of sites. rex (recruitment elements on X) sites recruit the DCC in an autonomous, DNA sequence-dependent manner using a conserved motif enriched on X. dox (dependent on X) sites are more prevalent than rex sites, but unlike rex sites are not sequence dependent and are found preferentially in promoters of expressed genes. They cannot recruit the DCC when removed from X.

In Chapter 2, I describe work done to test the theory that the DCC changes the chromosome conformation of the X. I designed and implemented an assay that uses Fluorescence in situ Hybridization (FISH) to assess the sub-cellular location of strong DCC binding sites in embryonic nuclei. I found that five pair-wise combinations of rex sites separated on X by 1 to 5.4 megabases of DNA colocalized in three dimensions at a frequency higher in XX embryos (and XO embryos manipulated to load the DCC on their single X), than in wildtype XO embryos in which the DCC is not bound to X. I have successfully performed, and am in the process of analyzing, 5C experiments (chromosome conformation capture carbon copy) to identify DCC-specific interactions on large regions of the genome. When rex sites colocalize, they frequently do so at the nuclear periphery, coincident with nuclear pore complexes (NPCs). This result suggests that NPCs provide a scaffold for DCC binding and X-chromosome restructuring. Through immunoprecipitation reactions, I showed a biochemical association between DCC components and NPC components. Depletion of individual NPC components disrupts DCC binding to X. Additionally, the DCC colocalizes with NPCs in specific NPC mutants that aggregate the NPC, further demonstrating the importance of the NPC in DCC function. The NPC may act as a scaffold to restructure the X chromosome through DCC binding.

Beyond my work on the mechanism of long-range gene repression in dosage compensation, I have also investigated the role of miRNA pathway members in dosage compensation, which I describe in Chapter 3. Many organisms use non-coding RNAs to achieve X chromosome dosage compensation. I have found that ALG-2, one of the two argonautes that specifically processes miRNAs, functions in C. elegans dosage compensation. RNAi to alg-2 in a weak DCC mutant severely disrupts DCC localization to X and X-chromosome gene repression. Surprisingly, mutations in the second C. elegans miRNA specific argonaute, alg-1, do not disrupt DCC localization or function, suggesting a specific and novel role for alg-2.

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