Periodic Reporting for period 1 - REACT (Uncovering the role of cis genetic elements in antigenic variation of Plasmodium falciparum using the CRISPR-Cas9 genome editing technology)
Okres sprawozdawczy: 2018-01-01 do 2019-12-31
In parallel, we studied the heterochromatin biology of the parasite, an epigenetic mechanism responsible for the by-default repression of more than 400 genes in P. falciparum including all the gene families submitted to antigenic variation, as var genes. To do so, we explored a particular gene, the ap2-g, searching for DNA elements adjacent to the limit between euchromatic and heterochromatic domains, the same strategy previously used to find out the cis-genetic DNA elements found in var genes. This gene provided an excellent model for such purpose, as it could be easily targeted for genetic manipulation, is of outstanding relevance for parasite transmission cycle (master regulator for sexual commitment) and it is a single-gene heterochromatin cluster. Specific DNA-protein interaction was detected only in the terminal region of ap2-g by electrophoretic motility shift assay (EMSA), and its replacement using CRISPR-Cas9 tool resulted in the shift of the boundary between chromatin domains up to 2 kb downstream the stop codon, where it is normally located. Proteomic data pointed to involvement of RNA binding proteins in this process. Our results on this topic supposed the first description of a DNA element potentially acting as boundary element and highlighted the possible role of RNA binding proteins in the triggering of sexual development in P. falciparum or establishment of a boundary between chromatin domains.