Defense Date

8-29-2019

Graduation Date

Fall 12-20-2019

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Philip E. Auron

Committee Member

Michael I. Jensen Seaman

Committee Member

Kyle W. Selcer

Committee Member

Deborah L. Galson

Keywords

Interleukin 1 beta (IL-1β), Spi1/PU.1, Gene Transcription, T cell receptor activation, CCAAT-enhancer-binding protein β (C/EBPβ), Protein–Protein Molecular Docking, Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB), THP-1 Monocytic cell line, Toll-like Receptor 4 (TLR4) activation, Inflammation

Abstract

Cytokines are key regulators of the inflammatory response and play an important role in facilitating intercellular communication between various immune cell types. Interleukin‑1β (IL‑1β) is a potent pro-inflammatory cytokine that is required for robust initiation of innate immune response and subsequent development of adaptive immunity. IL-1β is first synthesized as an inactive cytoplasmic, non‑glycosylated, precursor molecule (proIL‑1β) by monocytes and macrophages in response to invading pathogenic microbes. The activation of caspase‑1 by inflammasomes cleaves proIL-1β into mature biologically active IL-1β that is released from cells via a non-classical, endoplasmic reticulum‑independent secretory pathway directly from the cytoplasm via Gasdermin D membrane pores. IL-1β binds to its heterodimeric receptor (IL‑1R1·IL1RAP) on various immune cells, resulting in an inflammatory response that in short transient bursts can be therapeutic. However, increased and sustained expression of IL-1β is known to be associated with many autoinflammatory diseases. Therefore, understanding the molecular mechanisms regulating human IL1B gene transcription is important for discovering potential regulatory control points in this immunologically important gene, as well as for designing more effective and safer anti‑IL‑1β therapies than those currently available. Most of our present understanding of IL1B gene regulation is from activated monocytes, but not much is known about its regulation in lymphoid CD4 T cells. Previous studies from our lab have shown that the Spi-1/PU.1 (Spi1) transcription factor is absolutely necessary for IL1B gene transcription in monocytes. Using mRNA expression and Chromatin Immunoprecipitation (ChIP), we have shown that the IL1B gene in TCR‑activated CD4 T cells is transcribed from a low-activity bivalent H3K4me3+/H3K27me3+ promoter in a Spi1‑independent manner. Our lab has previously reported that IL1B gene transcription is dependent upon long‑range chromatin looping mediated by a mutual DNA‑binding domain interaction between two transcription factors, Spi1 at the promoter and C/EBPβ at a distal enhancer. Using in vitro interaction and computational analysis, we have generated a predicted molecular structure of the C/EBPβ:Spi1 protein‑protein interaction and demonstrated that C‑terminal tail residues beyond the C/EBPβ leucine zipper are critical for mediating its interaction with the DNA‑binding domain of Spi1. We have also reported that a small molecule can effectively bind to a pocket on Spi1 disrupting its interaction with C/EBPβ, thereby significantly reducing IL1B gene transcription in TLR4-activated THP‑1 monocytes. Using mRNA kinetic expression profiling, relatively‑specific transcription factor inhibition and ChIP, we have identified a differential involvement of NF‑κB and C/EBPβ transcription factors in TLR4‑dependent IL1B gene transcription in monocytes. We have also demonstrated that serine phosphorylation of Spi1 plays an important role in facilitating IL1B gene expression.

Language

English

Additional Citations

1. Pulugulla, S.H., Packard, T.A., Galloway, N.L.K., Grimmett, Z.W., Doitsh, G., Adamik, J., Galson, D.L., Greene, W.C., and Auron, P.E. (2018). Distinct mechanisms regulate IL1B gene transcription in lymphoid CD4 T cells and monocytes. Cytokine 111, 373-381.

2. Pulugulla, S.H., Workman, R., Rutter, N.W., Yang, Z., Adamik, J., Lupish, B., Macar, D.A., El Abdouni, S., Esposito, E.X., Galson, D.L., Camacho, C.J., Madura, J.D., and Auron, P.E. (2018). A combined computational and experimental approach reveals the structure of a C/EBPβ-Spi1 interaction required for IL1B gene transcription. J Biol Chem 293, 19942-19956.

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