Investigating the Role of spr-5 and met-2 in Exogenous Gene Expression Public

Abstract

Both spr-5 and met-2 are implicated in the epigenetic silencing of endogenous, germline-specific genes in Caenorhabditis elegans (C. elegans) by their demethylase and methyltransferase activity, respectively. To elucidate the role that these genes serve in silencing of exogenous genes in the C. elegans, we asked if gfp (green fluorescent protein) transgenes escape germline silencing in spr-5 (suppressor of presenilin) and spr-5;met-2 (histone methyl transferase-like) mutants. Surprisingly, we found that when we crossed an extrachromosomal array containing a let-858::gfp transgene into a homozygous spr-5;met-2 mutant background not only was there no expression of this transgene in the germline, the sterile double mutant progeny had dramatically reduced expression of LET-858::GFP in somatic cells, suggesting somatic silencing of the transgene construct. This result indicates that the extrachromosomal array was silenced somatically as well as in the germline. Furthermore, out of the approximately 230 spr-5 mutants injected with a gfp::cdk-1 (cyclin dependent kinase) transgene, only five C. elegans lines were produced that inheritably expressed the transgene construct somatically, further implicating SPR-5 in active somatic expression. These findings implicate spr-5 and met-2 in an indirect role in maintaining transgenes in an active state in the soma. These findings may also indicate that spr-5 and met-2 play a role in maintaining the distinction between soma and germline cell identity.

Table of Contents

Background. 1

Epigenetic Identity and Transcriptional Gene Regulation. 1

piRNA Mediated Epigenetic Regulation. 3

met-2 in Epigenetic Germline Regulation. 5

spr-5 in Epigenetic Germline Regulation. 6

spr-5 and met-2 in Epigenetic Exogenous Germline Regulation. 8

Green Fluorescent Protein Marker. 10

C. elegans as a Model Organism. 10

Results. 12

Crossing a let-858::gfp Transgene into spr-5;met-2 Double Mutants. 12

The Biolistically Integrated h2a::gfp Transgene Maps to Chromosome III. 17

The Role of spr-5 in Epigenetic Regulation of Single Copy Integrated Transgenes. 18

Generation of spr-5 mutant mosSCI Injection Strain. 19

mosSCI Single Copy Insertion in spr-5 mutants. 20

Discussion. 26

Apparent Silencing of let-858::gfp in Homozygous spr-5;met-2 Mutants. 26

Lack of Formation of Arrays in spr-5 mosSCI Injections. 28

spr-5;met-2 Mutants Silence Non-Self DNA Somatically. 29

Models. 30

Future Work. 34

Experimental Procedures. 37

Sources. 40

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Biology, General Biology, Cell
Mot-clé	spr-5 methylation met-2 regulation epigenetics H3K4 exogenous Caenorhabditis elegans methyltransferase H3K9 silencing demethylase
Committee Chair / Thesis Advisor	Katz, David J, Emory University
Committee Members	McGill, Tracy, Emory University Kelly, William G, Emory University Spell, Rachelle, Emory University

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