Analysis of the promoter region of the interleukin-2 receptor alpha chain (CD25) gene in human B lymphocytes

Allan, Andrew Barr (1999) Analysis of the promoter region of the interleukin-2 receptor alpha chain (CD25) gene in human B lymphocytes. PhD thesis, University of Glasgow.

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In T and B cells, attenuation of an undefined negative regulatory element binding protein (NRE-BP) binding to a negative regulatory element (NRE) removes the suppression of transcriptional activity upon the promoter of a unique 55kDa IL-2 receptor subunit, CD25. This event leads to the expression of CD25 at the cell surface. In human B cells IL-4, a potent activator of CD25 transcription abolishes ISIRE binding activiy via a cyclic adenosine monophosphate (cAMP)/ cAMP-dependent kinase (PKA)-sensitive pathway. Studies of the IL-4-regulated cAMP/PKA pathway showed that the catalytic subunit of PKA (PKAc) did not have a direct effect on NRE-BP in vitro, suggesting that PKAc has an indirect involvement in the loss of NRE binding activity. Studies with the intracellular signalling modulators Wortmannin and Rapamycin revealed that IL-4 does not attenuate NRE binding activity via P1-3 kinase signalling. Electrophoretic mobility shift assay (EMSA) analysis showed that IL-4 does not directly affect NF-kB activity. The attenuating effect of IL-4 on NRE binding activity was also observed for protein binding to the entire NRRI region. Signalling through the IL-13 receptor, which shares the IL-4Ra subunit with IL-4R, failed to attenuate NRRI binding activity suggesting that IL-4 causes a loss of protein binding to NRRI through the signalling generated by the gammac subunit of the IL-4R complex. EMSA studies using consensus and mutant NRRI oligonucleotides showed that NRE is the dominant site for protein binding within NRRI, although the consensus AP-1 site was required for maximum binding. Supershift experiments showed that the transcription factors YY1 and Ets were unlikely to be NRE-BP and that CBP/p300 is unlikely to form a bridging complex over proteins binding to NRRI. Western blot and EMSA experiments characterised the putative AP-1 binding site as a bona fide AP-1 binding site; an antibody against the AP-1-family protein c-fos supershifted one protein species binding to radiolabelled CD25-NRRI. Transient-transfection of P3HR1 B cells with NRRI deletion mutants confirmed that NRE is the dominant suppressive region of NRRI. Lymphoid cells transfected with CD25 STAT6 deletion mutant constructs showed an increase in reporter activity following treatment with phorbol myristate acetate (PMA). This finding is the first demonstration that PRRII is both functional and mitogen-sensitive in B cells. IL-4- sensitivity was assessed in lymphoid cells transfected with STAT6 CD25-CAT reporter constructs. These studies demonstrated that a putative STAT6 site overlapping NRRII was sensitive to IL-4. The remaining sites were insensitive to IL-4, suggesting they are not functional STAT6 sites. This finding suggests that a second IL-4 signalling mechanism, activation of the JAK/STAT pathway, may be involved in the upregulation of CD25 expression. In summary, IL-4 up-regulates the expression of CD25 in human B cells through a cAMP/PKA-sensitive pathway, which attenuates protein binding to NRRI and may also activate the JAK/STAT pathway. NRE binding activity is unaffected by direct action of PKAc, although this kinase is likely to exert an indirect effect. NRE is the dominant protein binding and functional region of NRRI, and supershift experiments characterised the retinoid sensitive region as an AP-1 binding site. The identity of NRE-BP candidates remains unknown and supershift experiments exclude YY1 and Ets as NRE-BP and show that CBP/p300 does not form a bridging complex linking NRE-BP and AP-1. Studies with deletion mutants of putative CD25 STAT6 sites showed for the first time in human B cells, that PRRII is responsive to mitogenic stimulation and identified a STAT6 site close to NRRII as IL-4 sensitive.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Cushley, Dr. William
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-71277
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 02 Nov 2022 09:08
Thesis DOI: 10.5525/gla.thesis.71277

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