(L5103) Studying the immune response genes expression regulation of Drosophila melanogaster

Anton Musabirov (Lomonosov Moscow State University, Lomonosov Moscow State University)| Y.A. Menchits (Lomonosov Moscow State University)| A.V. Guz (Lomonosov Moscow State University)| L.A. Lebedeva (Lomonosov Moscow State University)| Z.M. Kachaev (Lomonosov Moscow State University)| Y.V. Shidlovsky (Lomonosov Moscow State University)

The main control of the synthesis of the antimicrobial peptides in the cells of Drosophila melanogaster is carried out by two signalling pathways IMD and Toll, which include the classic transcription factors of the NF-kB family (Dif, Dorsal and Relish). The Toll pathway is activated in response to gram-positive bacteria and fungi, and the IMD pathway is activated in response to gram-negative bacteria.

In addition to transcription factors, the transcriptional apparatus of the cells includes the co-activators of transcription, such as the SAYP protein, which was described earlier in our laboratory. Co-activators do not bind directly to DNA, but assemble different parts of the transcriptional complex, which includes chromatin-remodelling complex, the specific transcription factors and the general transcription factors, which bind to RNA Pol II. SAYP homologues were found in the genomes of all multicellular animals. The protein mediates various functions of ontogenesis.

We found that knockdown of SAYP leads to a decrease in expression of the specific genes of the IMD and Toll immune response pathways. In addition, the proteomic analysis of proteins associated with SAYP revealed sequences of another transcription factor, which is called DEAF-1. This protein is also involved in the immune response and ontogenesis. These prerequisites determined the task for studying the interactions of SAYP, Dif, Dorsal, Relish and DEAF-1 factors and their influence on the genes expression activation.

We have obtained antibodies to the described factors. The most optimal epitopes (presumably exposed and accessible for recognition) were selected; the corresponding fragments of the cDNA genes were inserted into the expression vector and the conditions of expression in E.coli cells were matched. Recombinant proteins were produced and purified by metal-chelate affinity chromatography using Ni-Sepharose. The presence of these proteins in the fractions was confirmed by MALDI. The proteins have been used to immunize rabbits. The antibodies have been affinity purified from sera. The specificity of the antibodies was verified by Western blot analysis of the Drosophila embryonic nuclear extract.

We have developed a model system for activating the IMD and Toll pathways. Escherichia coli and Micrococcus luteus cells were grown, washed and resuspended in water. The suspensions were inactivated and added to the S2 D. melanogaster cell cultures. Using qPCR, we have observed the activation of the immune response genes.

Using the model system, we checked the recruitment of two model genes (Cecropin A1, Metchnikowin) to the promoters of the studied transcription co-activator. Chromatin immunoprecipitation has shown the binding of studied co-factor to gene promoters upon their activation.

Immunoprecipitation using the obtained antibodies to DEAF-1 and SAYP has shown the co-precipitation of DEAF-1 protein with SAYP. This indicates that these proteins may jointly participate in the regulation of the immune response genes expression. We are currently looking for the individual domains of these proteins that mediate their interactions.