Epigenetic regulation of germ cell identity

Project leader : Valérie Robert

People involved : Cécile Bedet, Steve Garvis, Marion Herbette, Marine Mercier

Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types following fertilization. Studies in a range of organisms suggest that dynamic changes in chromatin organization are essential for these processes.

The highly conserved Set1/MLL family of histone methyltransferases deposit H3K4 di- and tri-methylation (H3K4me2 and -me3) at the promoter of transcriptionally active genes. We have found that the C. elegans SET-2/SET1  methyltransferase is responsible for H3K4me2 and -me3 in the distal germline (Figure 1) (Xiao et al., 2011 ; Herbette et al., 2020 ).

Figure 1 : (A) Schematic representation of a C.elegans germline showing germ cells at different steps of proliferation

(mitotic zone) and differentiation (meiosis). (B) H3K4me3 immunostaining of germline nuclei at different steps of pro-

liferation/differentiation in wild-type and set-2 mutant animals (from Herbette et al., 2020).

set-2 mutant animals show increased genome instability, which may be related to a role for SET-2 in germline chromatin organization (Herbette et al., 2017; Herbette et al., 2020). In addition, at the stressful temperature of 25°C, mutant animals show a transgenerational loss of fertility that is reversible and correlates with loss of germ cell identity (Figure 2) (Xiao et al., 2011; Robert et al., 2014).

Figure 2 : (A) Temperature-sensitive and reversible transgenerational loss of fertility in set-2 mutant animals (data from Benedicte

Billard de Saint Laumer. (B and C) At 25°C, pachytene germ cells lose their typical "fried egg" shape (B, picture taken after 3 to 4

generations  at 25°C) and germlines  express somatic  markers (C, here a  pan-neuronal  marker after 3 to 4 generations at 25°C)

(from Robert et al., 2014).

We want to understand how H3K4 methylation, alone or in combination with other epigenetic factors, influence germ cell identity. Using transcriptome profiling of set-2 mutants across generations combined with novel bioinformatics tools, we have identified a set of genes (contributors) whose misregulation contributes to transgenerational loss of fertility in the absence of SET-2/SET1 dependent H3K4 methylation (Robert et al., 2020); collaboration with the groups of Susan Strome (UCSC) and Gaël Yvert (LBMC); Figure 3).

Figure 3: Identification of genes whose deregulation contributes to  transgenerational loss of germ cell identity in  the absence of

SET-2 at 25°C. (A) Experimental design. (B) Within-Class analysis on paired samples (dissected germlines) identifies a specific set

of genes that defines a path to sterility. (C) Distribution of genes whose misregulation contributes to loss of fertility. (from Robert

et al., 2020).

This analysis shows that loss of fertility and germline identity results from downregulation of genes encoding germline functions and upregulation of transcription factors, X-linked genes and TGF-ß pathway components (Figure 4).

Figure 4: A model for H3K4 methylation contribution to the maintenance of germline identity in C. elegans. Current experiments

are investigating molecular mechanisms involved in this control.

Using ChIP-seq, we are currently characterizing how activating and repressive histone marks are deregulated at contributor genes during the transgenerational loss of germline identity in set-2 mutants. We are also investigating additional mechanisms that may underly loss of germline identity in set-2 mutant, including changes in the population of small RNAs (collaboration with the Cecere lab, Institut Pasteur).

Relevant lab publications :

Herbette, M., Mercier, M. G., Michal, F., Cluet, D., Burny, C., Yvert, G., et al. (2017). The C. elegans SET-2/SET1 histone H3 Lys4 (H3K4) methyltransferase preserves genome stability in the germline. DNA Repair 57, 139–150. doi:10.1016/j.dnarep.2017.07.007.

Herbette, M., Robert, V., Bailly, A., Gely, L., Feil, R., Lleres, D., et al. (2020). A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization. Cells. doi:10.3390/cells9092049.

Robert, V. J., Knutson, A. K., Rechtsteiner, A., Garvis, S., Yvert, G., Strome, S., et al. (2020). Caenorhabditis elegans SET1/COMPASS Maintains Germline Identity by Preventing Transcriptional Deregulation Across Generations. Front. Cell Dev. Biol. 8. doi:10.3389/fcell.2020.561791.

Robert, V. J., Mercier, M. G., Bedet, C., Janczarski, S., Merlet, J., Garvis, S., et al. (2014). The SET-2/SET1 histone H3K4 methyltransferase maintains pluripotency in the Caenorhabditis elegans germline. Cell reports 9, 443–450. doi:10.1016/j.celrep.2014.09.018.

Xiao, Y., Bedet, C., Robert, V. J. P., Simonet, T., Dunkelbarger, S., Rakotomalala, C., et al. (2011). Caenorhabditis elegans chromatin-associated proteins SET-2 and ASH-2 are differentially required for histone H3 Lys 4 methylation in embryos and adult germ cells. Proceedings of the National Academy of Sciences of the United States, 8305