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.
| Identifer | oai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/665316 |
| Date | 09 1900 |
| Creators | Miller, Elizabeth Victoria |
| Contributors | Fischle, Wolfgang, Biological and Environmental Sciences and Engineering (BESE) Division, Orlando, Valerio, Mahfouz, Magdy M., Jensen, Christian Froekjaer, Becker, Peter |
| Source Sets | King Abdullah University of Science and Technology |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | 2021-09-24, At the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation will become available to the public after the expiration of the embargo on 2021-09-24. |
Page generated in 0.0021 seconds