Functional Analysis of the Caenorhabditis elegans HP1 Homolog HPL-2 in a Chromatin Context

  • Elizabeth Victoria Miller

Student thesis: Doctoral Thesis

Abstract

The heterochromatin 1 (HP1) family of non-histone chromosomal proteins is evolutionarily conserved and is involved in numerous biological processes, including the stabilization of heterochromatin, a state of compacted DNA along a protein scaffold. HP1 proteins and trimethylated histone H3 on lysine 9 (H3K9me3) are major constituents of heterochromatin and have been characterized extensively in vitro. The binding of HP1 proteins to H3K9 methylation marks plays an essential role in mammalian development and chromatin organization. However, due to their critical function, dissecting the molecular mechanism by which HP1 proteins exert their function in vivo is difficult. C. elegans is a unique model because not only are deletion mutants of the two HP1 homologs, HPL-1 and HPL-2, viable, but also H3K9 methylation is not essential to worm development. Interestingly, HPL-2 is alternatively spliced to generate two HP1 proteins, but in vivo experimentation has vastly ignored the potential contributions of the alternative transcripts to hpl-2 function, thus obfuscating which phenotypes associated with hpl-2 knockdown are due to the loss of one or more of the splicing variants. In this dissertation, I characterized the HPL-2 splicing variants (A and B) on a biochemical level in relation to the canonical human HP1b protein and on a physiological level in splicing variant-specific knockout worms. I show that both recombinant HPL-2A and HPL-2B bind H3K9me3 through their chromodomain (CD). But while HPL-2A acts as a canonical HP1 protein, namely it dimerizes and phase-separates like hHP1b, HPL-2B does not. In contrast to recombinant protein, in extracts both proteins rely on other factors, such as the MBT domain-containing protein LIN-61, for their recruitment to H3K9me3. Although HPL-2A and HPL-2B display distinct characteristics in vitro, both hpl-2a and hpl-2b worms are phenotypically wildtype. In agreement, knockout of either splicing variant leads to upregulated expression of the other one, suggesting a certain level of functional redundancy. Nevertheless, I show that the C-terminal extension of HPL-2B, which is absent in HPL-2A, resembles that of the CEC-4 heterochromatin anchor. I therefore hypothesize that the main functions of HPL-2 are distinct: HPL-2A mediates chromatin compaction and HPL-2B facilitates heterochromatin anchoring to the nuclear periphery.
Date of AwardSep 2020
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorWolfgang Fischle (Supervisor)

Keywords

  • C.elegans
  • HP1
  • HPL-2
  • chromatin
  • LIN-61

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