IRF4 Degradation · Using a novel protein degradation approach to uncover IRF4-regulated genes in plasma cells

Using a novel protein degradation approach to uncover IRF4-regulated genes in plasma cells

Antibody-secreting cells (ASCs) are an essential facet of the adaptive immune response. Their specialised role is to produce and secrete antibodies, which are important for neutralizing pathogens and providing long-lasting immunity to recurring infections. Defects in ASC development or function are associated with immunodeficiency, autoimmune disease, and cancer in the form of multiple myeloma. Despite the importance of ASCs in immunity and disease, little is known about how the development and function of these cells is regulated at the molecular level. ASCs develop from B cells that have been activated during an immune response. A striking feature of ASC differentiation is that the B cell undergoes a complete change in its gene expression programme. These changes are mediated by specific transcription factor proteins. IRF4 is an essential transcription factor for ASC development, and ASCs do not survive if the Irf4 gene is deleted. We sought to discover which genes are directly regulated by IRF4 in ASCs by depleting the IRF4 protein, rather than the gene, using an inducible degradation strategy (the auxin-inducible degradation, or AID, system). The advantage of this approach is depletion of IRF4 in the cell of interest, and thereby we could study gene expression changes that precede the loss of ASCs. The AID system requires tagging the protein of interest with a so-called degron (auxin inducible degron, or AID). Unfortunately, we learned that tagging IRF4 with an AID tag at the C-terminus of the protein disrupted the normal function of IRF4. We decided to proceed by moving the AID tag to the N-terminus of the protein. In the meantime, we were simultaneously generating AID-tagged versions of other proteins essential for ASC development, namely E2A and E2-2. Therefore, the project changed focus to these different transcription factors in order to develop the protocols for auxin-induced degradation and SLAMseq in ASCs. In addition, E2A is required for the development of the B cell lineage (i.e. ASC precursors) at a much earlier developmental stage, the pro-B cell. Pro-B cells are easier to work with in culture than ASCs, and so we could use E2A-AID degradation and SLAMseq analysis in pro-B cells as proof-of-concept for our ASC studies. In doing so, we would also learn about the genes and pathways, regulated by E2A, which are essential for the development of the B cell lineage at the pro-B cell stage.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз