Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Language

English (en)

Date of Award

8-29-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Sarah C.R. Elgin

Abstract

Heterochromatin is classically defined as densely staining regions of the genome; these domains are typically late replicating and show little recombination. Correct assembly of heterochromatin is critical for chromosome stability. Assembly begins with histone deacetylation and H3 lysine 9 di- and trimethylation: H3K9me2/3); the methylated H3 is typically bound by Heterochromatin Protein 1a: HP1a). Heterochromatin predominates at pericentric and telomeric domains --regions abundant in transposable elements: TEs) and satellite repeats. Transcription of these TEs has been found to generate a platform for assembly of heterochromatin through RNAi in S. pombe and A. thaliana, and may play a critical role in Drosophila melanogaster. However, the precise role of RNAi in heterochromatin assembly for a metazoan system such as flies remains unclear. However, 1360, a DNA transposable element in D. melanogaster, has been found to be sufficient to promote heterochromatin assembly in a repeat-rich region, as shown by a variegating phenotype of a hsp70-white reporter. RNAi components and heterochromatin factors such as HP1a were both implicated in this 1360-sensitive variegation, a form of position effect variegation: PEV).

Here, I sought to determine the extent and mechanism of TE-sensitive PEV. A collection of 1360-sensitive landing pad insertion lines containing the hsp70-w reporter was generated. This tool allows for the repeated sampling of altered 1360 constructs in a variety of chromatin contexts, a useful a platform to study the attributes of 1360-sensitive variegation as well as PEV generally. We found 1360-sensitive PEV to extend to sites outside of annotated heterochromatin, although most sensitive sites lie within or proximal to heterochromatic masses. I used biochemical approaches to show that 1360-sensitive PEV corresponds to HP1a accumulation over the hsp70-w promoter region, confirming that the silencing is due to heterochromatin assembly. The deletion of sites within the 1360 element with homology to the PIWI-interacting RNAs: piRNAs) in 1360 suppressed PEV, as did dominant mutations in PIWI domain proteins. Similar results were obtained using Invader4, a retrotransposon, in the same landing pad site. The results support a mechanism that uses piRNAs for transposon-sensitive HP1a-silencing, likely early in development, with persistent effects observed in the adult somatic tissue of the eye.

To determine if the sequence determinants required for 1360-sensitive silencing in a euchromatic region: as seen above) also operate in a repetitious sequence environment, where interspersed signals may operate cooperatively, I investigated a 1360-sensitive site in the piRNA generating locus 42AB. We find that mutations in piwi, along with many prototypical Su(var) mutations, result in weak suppression of variegation at this site, while an ago2 mutation enhances variegation. Tests of various fragments of the TEs do not reveal a strong dependency on piRNA matching sequences, contrary to the euchromatic site driven to a heterochromatic form by the added TE. These findings indicate that suppression of PEV by mutations in the genes for RNAi components occurs in a limited number of heterochromatic domains, predominantly those near gene clusters - sites typically found at the border between euchromatin and heterochromatin. Thus chromosomal context appears to be an important determinant for RNAi-dependent 1360-sensitive PEV. This finding helps to reconcile reports of inconsistent PEV effects from mutations in RNAi components that have been carried out using reporters in different domains. Collectively, these results indicate the TEs can act as sequence determinants of heterochromatin assembly at a subset of genomic sites using an RNAi-mediated targeting mechanism.

Comments

Permanent URL: http://dx.doi.org/10.7936/K75B00GH

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