Investigations into the mechanism of DNA binding and transcriptional regulation by the NF-κB subunit RelA

The NF-κB family of dimeric transcription factors function as master regulators of inflammation and the innate immune response. Upon pathway activation, cytoplasmic NF-κB dimers translocate to the nucleus and bind DNA response elements, known as κB DNAs or κB sites, at the promoters or enhancers of pro-inflammatory genes to activate transcription. Like other transcription factors, NF-κB binding affinity to κB DNA is sequence-dependent, however in vitro binding affinity is not a determinant of transcriptional output in the cell. Further, recent reports suggest that nuclear cofactors influence NF-κB DNA binding to κB DNA. This thesis explores what factors contribute to DNA binding by the NF-κB subunit RelA. Chapter 1 introduces gene regulation by NF-κB and outlines current gaps in our understanding of DNA binding by NF-κB. Chapter 2 explores how the central nucleotide of κB DNA modulates RelA DNA binding affinity. Our results show that the central nucleotide of κB DNA dramatically influences RelA:κB DNA complex stability through transient and dynamic interactions not observed in crystal structures. Chapter 3 investigates how locally distributed low affinity κB sites contribute to DNA binding by RelA. Our results show that low affinity binding sites impact RelA DNA binding kinetics and overall affinity, and tandemly organized low affinity κB sites can synergistically activate RelA-dependent transcription. Additionally, DNA-dependent cofactors can associate with RelA on κB DNA to collectively increase promoter occupancy and activate transcription. Chapter 4 explores how nuclear cofactors contribute to RelA DNA binding affinity. Our results show that nuclear cofactors enhance RelA DNA binding in vitro and we identify NME1 as a κB site-specific nuclear cofactor for RelA

PABP1 Drives the Selective Translation of Influenza A Virus mRNA

Influenza A virus (IAV) is a human-infecting pathogen with a history of causing seasonal epidemics and on several occasions worldwide pandemics. Infection by IAV causes a dramatic decrease in host mRNA translation, whereas viral mRNAs are efficiently translated. The IAV mRNAs have a highly conserved 5'-untranslated region (5'UTR) that is rich in adenosine residues. We show that the human polyadenylate binding protein 1 (PABP1) binds to the 5'UTR of the viral mRNAs. The interaction of PABP1 with the viral 5'UTR makes the translation of viral mRNAs more resistant to canonical cap-dependent translation inhibition than model mRNAs. Additionally, PABP1 bound to the viral 5'UTR can recruit eIF4G in an eIF4E-independent manner. These results indicate that PABP1 bound to the viral 5'UTR may promote eIF4E-independent translation initiation

Gut microbial DNA and immune checkpoint gene Vsig4/CRIg are key antagonistic players in healthy aging and age-associated development of hypertension and diabetes

AIMS: Aging is associated with the development of insulin resistance and hypertension which may stem from inflammation induced by accumulation of toxic bacterial DNA crossing the gut barrier. The aim of this study was to identify factors counter-regulating these processes. Taking advantage of the Chromogranin A (CgA) knockout (CgA-KO) mouse as a model for healthy aging, we have identified Vsig4 (V-set and immunoglobulin domain containing 4) as the critical checkpoint gene in offsetting age-associated hypertension and diabetes. METHODS AND RESULTS: The CgA-KO mice display two opposite aging phenotypes: hypertension but heightened insulin sensitivity at young age, whereas the blood pressure normalizes at older age and insulin sensitivity further improves. In comparison, aging WT mice gradually lost glucose tolerance and insulin sensitivity and developed hypertension. The gut barrier, compromised in aging WT mice, was preserved in CgA KO mice leading to major 35-fold protection against bacterial DNA-induced inflammation. Similarly, RNA sequencing showed increased expression of the Vsig4 gene (which removes bacterial DNA) in the liver of 2-yr-old CgA-KO mice, which may account for the very low accumulation of microbial DNA in the heart. The reversal of hypertension in aging CgA-KO mice likely stems from (i) low accumulation of microbial DNA, (ii) decreased spillover of norepinephrine in the heart and kidneys, and (iii) reduced inflammation. CONCLUSION: We conclude that healthy aging relies on protection from bacterial DNA and the consequent low inflammation afforded by CgA-KO. Vsig4 also plays a crucial role in "healthy aging" by counteracting age-associated insulin resistance and hypertension

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration <i>in vitro</i>. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex <i>in vivo</i>. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation