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Title: Investigating the role of CREB3L2 in terminal B-cell differentiation and B-cell neoplasia
Author: Al-Maskari, Muna Mohamed Salim
ISNI:       0000 0004 6058 4386
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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CREB3L2 (cAMP Response Element Binding protein 3 Like 2) is a bZIP domain containing transcription factor. It is one of several endoplasmic reticulum (ER)-stress transducers that lead to an unfolded protein response (UPR). CREB3L2 is activated by regulated proteolysis via Site-1/Site-2 (S1P/S2P) proteases, due the accumulation of misfolded proteins in the ER. Such regulation is shared with other transcription factors (TFs) such as ATF6 and SREBP. During B-cell differentiation, the UPR is important for plasma cell adaptation to high levels of antibody secretion. However, to date only one UPR regulator has been implicated in plasma cell differentiation, XBP1. The high expression of CREB3L2 in plasma cells suggests that it may also contribute to secretory adaptation and offers the opportunity for therapeutic targeting. Therefore, the aim of this project is to test the involvement of the transcription factor CREB3L2 in plasma cell differentiation through investigating its expression, regulation and function in human B-cell differentiation and B-cell tumours. Evaluation in myeloma and ABC-DLBL cell lines revealed a direct relationship between CREB3L2 activation and UPR induction. In primary cells, CREB3L2 is expressed at the earliest stages of plasma cell differentiation and then becomes strongly induced and activated as the cells transition to the plasma cell state. To investigate the potential role of CREB3L2, S1P and S2P inhibitors were employed to block it's processing. Inhibition of S1P/S2P during plasma cell differentiation led to a profound reduction in plasmablast number linked to a decrease in proliferation and an increase in autophagy. While few cells survive following treatment with the inhibitors, phenotypically the cells appear normal. In contrast, immunoglobulin secretion is severely reduced. Moreover, inhibition of S1P is associated with a unique gene expression profile involving gene signatures related to IRF4 and mTOR function as well as metabolic pathways. To further investigate the contribution of CREB3L2 to the events observed with S1P/S2P inhibitors, siRNA-mediated knockdown and ChIP-seq experiments were performed in primary differentiating B-cells. Although the changes in gene expression after knockdown did not reach statistical significance, genes showing variation have known roles in ER stress responses, autophagy and plasma cell development. Moreover, ChIP-seq of CREB3L2 demonstrated direct binding to several of the differentially expressed genes. The ChIP-seq experiment also included an evaluation of ATF6 and XBP1 binding in the same cells. The results revealed a high degree of overlap for binding, including to loci previously identified in the gene expression analyses. In conclusion, the results show that S1P/S2P control CREB3L2 processing in differentiating B-cells and suggest that CREB3L2 is involved in the early commitment to plasma cell fate, prior to the requirement for XBP1. CREB3L2 is likely to function in combination with other RIP-activated TFs by regulating genes involved in metabolism, necessary for effective transition to high secretory activity. Thus, S1P/S2P-regulated pathways play a pivotal role in the B-cell to plasma cell transition and represent a previously unrecognised component of the regulatory network that turns these cells into efficient antibody "factories".
Supervisor: Tooze, Reuben ; Doody, Gina Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available