טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYuval Argaman
SubjectIdentifying the Mechanisms Causing Transgene Imprinting in
the Nematode Caenorhabditis elegans
DepartmentDepartment of Biology
Supervisor Professor Lamm Ayelet
Full Thesis textFull thesis text - English Version


Abstract

Genomic imprinting is the expression of a certain allele depending on its parent-of-origin.  Genomic imprinting exists in plants, insects and mammals.  Many imprinted genes are required for normal development and defects in genomic imprinting or imprinted loci result in developmental aberrations.  The main mechanism that was found throughout the phyla to govern the establishment, maintenance and inheritance of imprinting patterns is DNA methylation.  Other epigenetic mechanisms found to influence genomic imprinting are non-coding RNAs (ncRNAs) and chromatin remodeling. 

The nematode Caenorhabditis elegans is widely used as a model organism for studying many biological phenomena.  Strikingly, C. elegans does not have DNA methylation, but has an expanded machinery of the dsRNA-mediated gene silencing known as RNA interference (RNAi), as well as ncRNAs and chromatin remodeling.  RNAi has a central role in genome defense in C. elegans.  It has been reported that a GFP transgene is expressed in a parent-of-origin-dependent manner in C. elegans.  This suggests that transgene imprinting can occur in a DNA methylation-indepdenent manner in C. elegans

We therefore set to determine what epigenetic mechanism(s) that govern transgene imprinting in C. elegans

To find the mechanism, we first established a new protocol to estimate fluorescence expression and tested it on the imprinted transgene.  In addition to parent-of-origin expression, we found that uncrossed homozygote males express the imprinted transgene in higher levels that homozygote hermaphrodites.  This effect was not persistent in animals homozygous to another derivative of the imprinted transgene that was integrated on another chromosome.  We named it “a sex dependent effect”.  We also found that the sex-dependent effect was retained in hermaphrodite and male progeny heterozygote to either a maternally- or paternally-derived imprinted transgene.  This indicates that the transgene imprinting is coupled to the sex of the worm. 

In order to study the molecular basis for transgene imprinting in C. elegans, we chose to analyze the effects of RNAi on transgene imprinting.  We focused on RDE-1 and RRF-1.  RDE-1 is a member of the argonaute protein family and is necessary for RNAi-mediated transgene silencing in C. elegans via primary small RNAs, while RRF-1 is an RNA-dependent RNA Polymerase (RdRP) required for secondary small RNA biogenesis. We found thatthe parent-of-origin-dependent transgene expression was retained on an rde-1(-/-) background. However, the parent-of-origin effect seems to be attenuated in hermaphrodites on an rrf-1(-/-) background.  Interestingly, the sex-dependent effect was persistent in homozygous and heterozygous worms of both sexes and on both mutant backgrounds.  This implies that RDE-1 and RRF-1 are not involved in sex-dependent transgene expression, and that other mechanisms may be involved.   Our results suggest a model in which the parent-of-origin effect is mediated by differential inheritance of secondary small RNAs generated by RRF-1 in the RNAi amplification step.  This step is required for a prolonged, persistent and systemic RNAi response.  Overall, our results are consistent with previous studies showing RNAi-dependent epigenetic memory; and add further knowledge about the nature of the small RNA species involved in this phenomenon.