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Poulin Lab

Research

Developmental Epigenetics

From Signalling To Transcription

High throughput RNAi screens in C. elegans ¦ Chromatin modifications and the embryo ¦ Chromatin modifications and the vulva

H3K4me3 is not detected on the X chromosome. Anti-H3K4me3 is red, DAPI is blue, and green is anti-H3K27me2.
H3K4me3 is not detected on the X chromosome. Anti-H3K4me3 is red, DAPI is blue, and green is anti-H3K27me2.

High throughput RNAi screens in C. elegans

Performing RNAi by feeding in C. elegans was an important step that made loss of function analysis truly high throughput. However, the bottleneck of the RNAi feeding procedure remains the phenotypic analysis, which is still at best semi-quantitative. We have developed a Food Consumption (FC) assay that is based on how much bacteria is eaten by the worms as function of the time, which is an indication of fitness. The experiments are performed in 96-well format in liquid, and the food concentration is followed using a plate reader. We are currently using the FC assay to screen for genetic interactions between chromatin factors and cancer signalling pathways, to map genetic variation effect, and to test small compounds.

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Chromatin modifications and the embryo

One of the most fascinating process of early embryogenesis is the production of cells with two fundamentally different programmes: the somatic programme, which produces cells that are destined to die, and the germline programme responsible for immortality of the species. Hence, the germline blastomeres face a dilemma at each cell division: to retain pluripotency or to enable differentiation.

A two-cell stage embryo photograph using Nomarsky microscopy and DAPI staining.
A two-cell stage embryo photograph using Nomarsky microscopy and DAPI staining.

Since the germline blastomeres generate both somatic cells and germ cells, these specialised cells must “remember and distinguish” between these two programmes. To understand the basic principles by which the germline blastomeres retain pluripotency, and yet erase/suppress the germline programme could have far reaching implications. For example stem cells have to retain their pluripotency, and ensure that the differentiation is not enabled, and importantly following differentiation pluripotency must be suppressed or the cell could become a cancer stem cell. Hence, our work could impact on our basic understanding of pluripotency versus differentiation. We are currently investigating the role that chromatin modifications, especially methylation, play in this process.

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Chromatin modifications and the vulva

The C. elegans vulva system has been instrumental to identify and characterise components of multiple core signalling pathways, notably of the conserved EGFR/RAS/RAF/MAPK signalling pathway. It has also shed light on the molecular mechanisms involved in attenuation of RAS signalling. Chromatin remodelling complexes have been shown to play an important role in the process of attenuation, notably via regulation of EGF expression and regulation of negative or positive modulators of the RAS signalling cascade.

In my lab, we have performed large-scale and targeted RNAi screens in a divers sensitised genetic backgrounds (gap-1 or sumo mutants) to reveal attenuators of the RAS signalling. This functional strategy has identified ~100 candidates, amongst these, we are currently investigating the C. elegans MLL complex and the bromodomain containing protein BET-1.

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