Periodic Reporting for period 1 - IRF4 Degradation (Using a novel protein degradation approach to uncover IRF4-regulated genes in plasma cells)
Reporting period: 2019-04-01 to 2021-03-31
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.
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.
As we had encountered protein function issues after tagging IRF4, it was important to confirm that AID-tagged E2A and E2-2 proteins were normal. E2A is essential for early B cell development and E2-2 is required for the development of plasmacytoid dendritic cells, hence we could use these two immune populations as evidence for tagged protein functionality. I performed a thorough characterization of the immune system of these two mouse models and could confirm that E2A-AID and E2-2-AID proteins are functional, especially as evidenced by normal B cell development and pDC populations, respectively.
Since we had the E2A-AID mouse model, and it proved to be normal, we decided to use this to investigate the molecular functions of E2A in pro-B cells. This question is as unanswered as for IRF4 in ASCs. When E2A is lost, B cells do not develop and are instead arrested at a progenitor stage. To this end, I characterized the degradation kinetics of E2A in cultured pro-B cells and ASCs in parallel, as well as E2-2-AID in ASCs.
It is a known issue in the AID/Tir1 field that spontaneous degradation can occur prior to auxin addition, i.e.not induced. In the ASC system, there is substantially more spontaneous degradation than in pro-B cells, and this could affect our interpretation of results. We are currently circumventing these problems by adapting our TIR1 system (to a recently published “TIR2” system). For this reason, we decided to focus on SLAMseq analysis of E2A-depleted pro-B cells.
I successfully performed SLAMseq for E2A-depleted cultured pro-B cells, and a preliminary analysis of the data identified several factors known to be important for B cell development as directly regulated genes of E2A. A more thorough analysis is ongoing, and from these results we will continue to characterize potentially novel genes, verifying their role in pro-B cells using CRISPR/Cas9 gene editing in cultured pro-B cells, and in the mouse when there are conditional alleles available.
Since we had the E2A-AID mouse model, and it proved to be normal, we decided to use this to investigate the molecular functions of E2A in pro-B cells. This question is as unanswered as for IRF4 in ASCs. When E2A is lost, B cells do not develop and are instead arrested at a progenitor stage. To this end, I characterized the degradation kinetics of E2A in cultured pro-B cells and ASCs in parallel, as well as E2-2-AID in ASCs.
It is a known issue in the AID/Tir1 field that spontaneous degradation can occur prior to auxin addition, i.e.not induced. In the ASC system, there is substantially more spontaneous degradation than in pro-B cells, and this could affect our interpretation of results. We are currently circumventing these problems by adapting our TIR1 system (to a recently published “TIR2” system). For this reason, we decided to focus on SLAMseq analysis of E2A-depleted pro-B cells.
I successfully performed SLAMseq for E2A-depleted cultured pro-B cells, and a preliminary analysis of the data identified several factors known to be important for B cell development as directly regulated genes of E2A. A more thorough analysis is ongoing, and from these results we will continue to characterize potentially novel genes, verifying their role in pro-B cells using CRISPR/Cas9 gene editing in cultured pro-B cells, and in the mouse when there are conditional alleles available.
Despite some technical setbacks, we have still been able to study the role of an essential transcription factor (E2A) in early B cell development, therefore shedding light on an aspect of B cell biology that was previously impossible to interrogate. With the use of the AID system, we will determine the directly regulated genes of E2A in pro-B cells, and therefore learn about pathways that are required for this specialised immune compartment to develop. Such knowledge can be applied towards diseases of early B cell developmental dysfunction, such as B cell leukaemias, autoimmunity and immunodeficiency. This study of E2A in pro-B cells has helped us to establish the protocols required to continue our studies of E2A, E2-2 and IRF4 in ASCs.
Aside from immunology, this project has expanded the toolkit available for the AID system. We have gained extensive experience in the degradation system, which is gaining popularity as a method for interrogating protein function. We are, to our knowledge, the only group using this system in vivo (i.e. with mice and not cell lines). It will be a great strength once we have the TIR2 system available as well, paving the way for further projects and collaborations.
Aside from immunology, this project has expanded the toolkit available for the AID system. We have gained extensive experience in the degradation system, which is gaining popularity as a method for interrogating protein function. We are, to our knowledge, the only group using this system in vivo (i.e. with mice and not cell lines). It will be a great strength once we have the TIR2 system available as well, paving the way for further projects and collaborations.