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Uncovering the role of cis genetic elements in antigenic variation of Plasmodium falciparum using the CRISPR-Cas9 genome editing technology

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

The objectives of the project REACT were focused on the use of CRISPR-Cas9 technology to find out the role of cis-genetic elements on the var gene family expression in Plasmodium falciparum. Direct deletion of such elements proved to be challenging, mainly due to the high degree of similarity between different var genes and high AT-richness in intergenic regions characteristic of P. falciparum. This hindered the design of a specific guide for a given var gene. We succeeded generating lines with dCas9 fused to some epigenetic effectors as GCN5 and Sir2A, although no effect was observed in the transcriptional level of the targeted var gene. CRISPR-Cas9 technology was then applied to study other DNA elements suspected to be involved in var gene activation, so-called RUF6 (RNA of unknown function 6), whose global depletion (simultaneous downregulation by means of CRISPR interference -CRISPRi-) led to a complete loss of var gene expression. Additional research, still in progress, points that these elements may be involved in the relocation of internal var gene clusters to the nuclear periphery and/or in 3D nuclear organisation by means of long-range interactions among them.
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.
During the present project important results regarding the var gene expression regulation and heterochromatin biology in P. falciparum have been generated. Among the main scientific results are the validation of the need of RUF6 elements transcription to keep the var expression and the demonstration that they physically interact between them in vivo. Furthermore, some evidences of the mechanism involved in such interaction was also provided, as we showed that 3’ terminal end of RUF6 genes is able to promote self-aggregation in conditions which favour the formation of G-quadruplexes. To reach these achievements, we have pioneered some techniques in P. falciparum as the global knock-down of a whole gene family (fifteen RUF6 members using CRISPRi) or application of capture C, a technique allowing higher resolution to explore the contacts of a particular region in the genome than obtained with other alternatives that map a global genome-wide interaction like Hi-C. We also obtained valuable results about heterochromatin biology, taking as model the ap2-g, including description of fifty-eight potential factors involved in the heterochromatin epigenetics. Our research showed a protein complex binding to the 3’ end of this gene and many identified actors were RNA related proteins, suggesting an involvement of RNA mediated interaction in either the regulation of AP2-G transcription or the heterochromatin boundary formation at the end of the ORF. The latter option is supported by the fact that replacement of the last 765 bp of the ap2-g by gfp gene produces the extension of the heterochromatin ~2 kb downstream compared to its original location at the immediate vicinity of the stop codon.
Our results contributed to the state of the art in the field of antigenic variation of P. falciparum. Before our research, the role of RUF6 was known to be required for mono-allelic expression of var gene family. However, we provided evidence of its essentiality for any var expression. Our results also opened the gate of the involvement of such elements in the tethering of var central clusters to the nuclear periphery, an unexplored topic in P. falciparum. Also, we provide the possible first description of a DNA region which can be considered as boundary element (sequence which stops the heterochromatin propagation) in P. falciparum, located at the 3’ end of ap2-g. This gene has attracted attention from the scientific community in the last years, and the information we offer might be greatly useful for the topic of heterochromatin biology and sexual commitment, where ap2-g plays a pivotal role. The results from this project help understand the functioning of antigenic variation in P. falciparum and the mechanisms to keep the virulent genes repressed. Such information is invaluable to understand the way the parasite evades the host’ immune response and to develop new therapy options. Also, as we used as model a fundamental gene which triggers the sexual commitment, our data can offer interesting basis for further research focused on transmission blocking. In particular, a set of fifty-eight proteins enriched in the terminal part of ap2-g gene has been already uploaded to PRIDE database (dataset identifier PXD017930).