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Regulation of gene expression by RelA in activated B lymphocytes By Jaimy P. Joy A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland June, 2017 Abstract B cells undergo dramatic alterations in response to antigen recognition through cell surface receptors, which requires active transcription and translation. The temporal expression of transcripts is germane and integral in determining underlying complex biological phenomena. The transcription factor RelA is rapidly expressed in the nucleus in response to BCR crosslinking, and downregulated within hours. However, many putative NF-B target genes expressed by stimulated B cells do not follow the same kinetic pattern as the transcription factor, suggesting complex transcriptional and post-transcriptional regulation. The current work seeks to explore the mechanisms by which genes are regulated by NF-B in primary murine splenic B cells in response to BCR crosslinking. To do this, we examined the kinetics of genome-wide RelA binding, the inducible transcriptome, and RNA stability of induced genes. NF-B target genes, as inferred from RelA ChIP-seq analyses and pharmacological inhibition of IKK-2 inhibitors, were amongst the most highly induced genes in response to BCR crosslinking. Highly induced transcripts had shorter half-lives (t1/2s) than those with less robust induction one hour after stimulation. The majority of inducible genes with fast t1/2s displayed exponential decay, while the majority of inducible genes with slow t1/2s displayed non-exponential decay. As exponential decay is likely to mean a single-step decay mechanism and the 3’ UTRs are known to play a critical role in RNA stability, we looked to the presence of RNABP consensus motifs in genes of various decay patterns. Our analyses revealed that hnRNPA1 binding sites were highly enriched in the 3’ UTRs of genes with fast, ii exponential decay. siRNA mediated knockdown of hnRNPA1 in two mature B cell lines, Bal17 and M12, revealed greater RNA stability of many genes that have fast, exponential decay and are RelA target genes based on inducible RelA binding and IKK- 2 inhibition. These findings suggest a tantalizing model in which mRNA decay is dependent on transcription factors, such as RelA, that inducibly bind to its target genes and induce RNA. Mentor: Ranjan Sen, Ph.D. Reader: Joel Pomerantz, Ph.D. iii Acknowledgements The journey of 1,000 miles begins with a single step and I am grateful to have taken the first step five and a half years ago. It has been an arduous and tumultuous experience, but just as rewarding, and not one I would trade for anything. The science, friendships, and memories will be treasured for years to come. To my friends and family who loved, supported, and encouraged me even when I didn’t deserve it or show my appreciation for it, thank you. This body of work would have been impossible without your unwavering support. You no longer have to ask me “when will you be done?” and I no longer have to silently resent you for it. To my thesis committee members, Joel, Stuart, and Jef, thank you for the consistent years of reliability and helpfulness. Your keen sense of good science and mentorship have been an invaluable part of my scientific growth and maturity. If only all grad students can be so lucky. To the immunology training program, Mark, Joel, Ms. Angela, Ms. Lin, and Lori, thank you for giving me the opportunity to pursue my Ph.D.. You put up with a lot and I hope to make you proud. To the members of the Sen lab, I could not have asked for a better group of people to call my work family. Everyone, both past and present members, has been an incredible resource and support system. If anyone goes through any of my lab notebooks and comes across random wrinkly patches and bleeding ink, assume I was having a bad day. Finally, to Dr. Sen, the person I am lucky to have called my mentor, I (yes, even I) cannot fully express my gratitude due to the limited nature of the English language. I iv came to Hopkins to be trained as a scientist and I leave knowing that to be true. From journal clubs, lab meetings, one-on-one meetings, seminars, to everything in between, I learned to question everything and be critical in my analyses. I could not have asked to train under a better scientist. Somehow, you also managed to be a good and patient person and set the bar for all PIs unrealistically high. To any future graduate student who stumbles onto this thesis, it gets better, but if it doesn’t, Robert Frost said it best: “it [life] goes on”. As this 1,000-mile journey ends, I wish to finish it in the same way I began, by giving thanks to Christ and trusting that the next one will be just as fulfilling. v Table of Contents Abstract ...........................................................................................................................................ii Acknowledgements ...................................................................................................................... iv Table of Figures .......................................................................................................................... viii Chapter I: NF-B .......................................................................................................................... 1 Introduction: NF-B ...................................................................................................................... 1 NF-B and IB family members ............................................................................................. 1 NF-B signaling ........................................................................................................................ 2 Regulation of NF-B ................................................................................................................. 3 NF- binding: specificity and dynamics .................................................................................. 7 Cell and stimulus-specific activation of NF- ...................................................................... 13 Chromatin accessibility and histone modifications ............................................................ 13 TF and cofactor interaction ................................................................................................... 15 NF-B dependent transcription and target genes ................................................................. 17 Existing NF-B target gene databases ............................................................................... 17 Criteria for NF-B target gene identification ....................................................................... 17 Experimental determination of NF-B target genes .......................................................... 18 Monophasic vs oscillatory NF-B activity ........................................................................... 20 Single cell analyses in the pursuit of NF-B target genes ............................................... 21 Kinetics of NF- target genes ............................................................................................ 22 Rate of gene induction ........................................................................................................... 22 Duration of functional promoter-bound complexes ........................................................... 23 Rate of mRNA degradation ................................................................................................... 24 Outstanding questions ............................................................................................................... 26 B cell biology ............................................................................................................................... 28 B cell development and B cell subsets ............................................................................... 28 Epigenetic landscape of B cells ........................................................................................... 28 NF- in B cells .......................................................................................................................... 31 Project Goals............................................................................................................................... 33 Chapter II: BCR-induced NF-B response in B cells ............................................................ 34 Kinetics of transcriptional response to BCR stimulation ................................................... 34 vi Genome-wide inducible RelA binding in anti-IgM stimulated B cells. ............................ 41 Basal state epigenetic and transcriptional status of genes with iRelA binding ............. 52 Stimulus specificity of NF- in B cells ............................................................................... 58 Determination of bona fide RelA target genes by use of pharmacological inhibition of IKK-2 and genetic ablation of RelA. ..................................................................................... 61 Potential RelA dependent distal regulation of CCL3 and CCL4 ..................................... 73 Chapter III: mRNA decay of RelA-dependent genes in BCR-stimulated B cells ............. 77 Introduction .................................................................................................................................. 77 Factors involved in mRNA stability .........................................................................................
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