Characterization of H3.3 Histone Variant HIS-74 in the Germline of Caenorhabditis elegans Open Access

ElJalby, Mahmoud Montasser (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/3j333293v?locale=en
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Abstract

Histone proteins wrap DNA to form nucleosomes and compact eukaryotic chromosomes. Histones exist as variants that play significant roles in gene regulation and expression. Histone variant H3.3, which is structurally similar to histone H3, is associated with transcription. Three H3.3 histone variants (HIS-71, HIS-72 and HIS-74) have been identified in C. elegans but of these only HIS-71 and HIS-72 have been partially characterized. This study seeks to characterize the role and function of HIS-74, an H3.3 variant that differs from the other H3.3. variants. A GFP-tagged HIS-74 transgene demonstrates germline-specific expression with differences in its stability within the genome between sperm and oocytes. A strain carrying a deletion allele that removes both his-74 and an adjacent gene, ccch-3, exhibits embryonic lethality. RNA interference (RNAi) of the ccch-3 gene alone exhibits no significant lethality, indicating that deletion of the ccch-3 gene is not responsible for the lethal phenotype in the strain. Mapping and outcrossing experiments could not separate the lethality from the deletion, further implicating the loss of his-74 in the lethality. However, a mutation with a premature stop codon in his-74 has no phenotype and we have not yet been able to rescue the mutant strain with a transgene covering the deletion. A his-74-/- knockout line is currently being generated using CRISPR/Cas9 technology. Further studies will seek to better understand the HIS-74 function and interplay with the other two H3.3 histone variants HIS-71 and HIS-72.

Table of Contents

Introduction 1

Results 3

The his-74 Deletion May be Responsible for Embryonic Lethality 3

The Lethality of the his-74 Deletion is Inconclusive 4

HIS-74::GFP Is Strictly Maternally Loaded in the C. elegans Embryo 7

Conclusion 9

Materials and Methods 12

Nematode strains and maintenance 12

RNAi of the ccch-3 gene 13

Obtaining L1 C. elegans 14

Identifying whether the lethal phenotype is due to his-74 gene deletion or a linked mutation 15

DNA Extraction and PCR Amplification 15

References 17

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