Periodic Reporting for period 1 - BICE (Behavioural Individuality in C. elegans)
Periodo di rendicontazione: 2020-09-01 al 2022-08-31
Epigenetic marks such as histone methylation help to define how the DNA code is read. Alterations in this process can result in altered gene expression, a loss of cellular identity and cancer. To preserve expression patterns, epigenetic information must be correctly maintained. Thus, during the copying of the DNA strands that occurs before cell division, epigenetic marks must be transmitted to the new DNA strands. There are indications from cellular models that the machinery which duplicates DNA also participates in the transmission of epigenetic information. However, the replisome, the ensemble of proteins that participate in DNA replication, has not been characterised in a higher organism such as the roundworm C. elegans. In this project, we probed whether mutations in C. elegans replication components alter the transmission of epigenetic information. First, we used a fluorescent reporter assay to characterise interactions between replisome components in collaboration with another lab. Secondly, we tested the replisome mutants for fertility defects over generations, a phenotype that is associated with alterations in epigenetic transmission. Both approaches yielded novel insights into the function of the replisome, such as new genetic interactions that have not been reported to date. In addition, we found that combined loss of two non-essential factors increases gene expression, in regions that are normally repressed. We have indications that this increased expression in the double mutant is due to a loss of repressive histone marks. Furthermore, the double mutant exhibits a loss of fertility over generation which is reversible, and which can be repressed by downregulation of a histone demethylase. Our work describes for the first time the interplay of these two DNA replication factors and its effects on the transmission of epigenetic information.
We screened various replication mutants for defects in the transmission of epigenetic marks using two assays. First, we measured the expression of a normally repressed fluorescent transgene in the mutant backgrounds. In this way, we determined various previously unknown genetic interactions between replication factors. Some of this data is in line with previous publications from lower organisms like yeast. However, other results indicate that the function of several components of the C. elegans replisome differs from the function of their yeast counterparts. These data will be published in cooperation with another lab in a joint manuscript.
Secondly, we found that the combined loss of two non-essential replication factors leads to a loss of fertility over generations. The double mutant also exhibits a reduction in repressive histone marks, and increased expression in regions that are normally regulated by those repressive marks. The reduced fertility is reversible and can be suppressed by downregulation of a histone demethylase. In summary, we have indications that the replication factors are important for the faithful transmission of repressive histone marks. The manuscript describing these results is currently in preparation.
Secondly, we found that the combined loss of two non-essential replication factors leads to a loss of fertility over generations. The double mutant also exhibits a reduction in repressive histone marks, and increased expression in regions that are normally regulated by those repressive marks. The reduced fertility is reversible and can be suppressed by downregulation of a histone demethylase. In summary, we have indications that the replication factors are important for the faithful transmission of repressive histone marks. The manuscript describing these results is currently in preparation.
For the first time, we characterised the role of replication components in the transmission of epigenetic information in a higher eukaryotic organism. The faithful transmission of epigenetic marks during cell division is necessary to prevent the loss of cell identity, which can result in cancer.