The ETS Transcription Factor ELF4 Suppresses the Inflammatory Response
Date of Award
Doctor of Philosophy (PhD)
The study of human genetics offers information into how genes and signaling pathways regulate human health. Sequencing and comparative analysis of human genomes allows for the identification of functional genetic elements, and when paired with reverse genetic tools can provide compelling evidence to link a specific genotype to a phenotype. ¬We report the discovery of three unrelated male patients with complete loss-of-function genetic variants in the transcription factor ELF4, an X-linked gene belonging to the ETS family of transcription factors. Patients with inflammatory bowel disorder-like symptoms were subject to whole exome sequencing, leading to the discovery of deficiency in ELF4, X-linked (DEX). Analysis of additional, rare variants in DEX patient genomes shows they do not share additional rare variants. Functional testing of relatively rare ELF4 genetic variants present in healthy human databases shows normal activity in vitro. ELF4 has been evolving under mild negative selection in the human genome. Characterization of the immune system in DEX patients and newly generated mouse models demonstrates ELF4 is not required for development or survival, but rather prevents excessive inflammatory responses. ELISA shows LPS-stimulated PBMCs from patients produce elevated cytokines, including Il-1, Il-6, and IL-12p40. Cytokine/chemokine profiling of serum from patients shows elevated C-reactive protein and CXCL1. RNA sequencing on macrophages reveals a major inflammatory transcriptional network regulated by ELF4, including S100a8, Lcn2, Mefv, Ptges, Trem1, Cxcl2, Cxcl3, Cx3cr1 and others. Further analysis on RNA sequencing on macrophages identifies a subset of interferon stimulated genes (ISGs) promoted by ELF4, including some prominent anti-inflammatory ISGs Il1rn, Il10, and Il27. Key inflammatory genes were recapitulated using THP1 monocyte-like cell line with a deletion in ELF4 using CRISPR/Cas9 technology (e.g. PTGES, TREM1). W205S knock-in mice, generated with the missense variant identified in DEX patients, show quantitatively similar dysregulation of Il1rn, Il10, S100a8, and Trem1, compared to WT mice Re-analysis ChIP sequencing data for Elf4 in mouse BMDMs reveals that Elf4 likely regulates many ISGs in part through direct regulation. Luciferase assays confirm ELF4 can act directly on some of the regulatory sequences of genes discovered by RNA sequencing, including IL1RN, PTGES, and OAS1, but not IL10, IL27, or TREM1. In addition to characterizing disease in male patients and mice with hemizygous ELF4 deficiency, I sought to characterize any contribution in heterozygous females. Because ELF4 is not an escape gene (i.e. it does not escape inactivation of one X-chromosome), a cloning method for evaluating X-inactivation and ELF4 allele usage was developed. PBMCs from the mother of B.1 (W251S ELF4 allele) show a striking 75% usage of the S251 variant allele and only 25% usage of the W251 reference allele. Similarly, in purified CD4+ or CD8+ T cells sorted from heterozygous W250S mice, I measure >65% usage of the S250 variant allele. Together, the data provided here support a role for ELF4 in suppressing the inflammatory response.
Tyler, Paul, "The ETS Transcription Factor ELF4 Suppresses the Inflammatory Response" (2021). Yale Graduate School of Arts and Sciences Dissertations. 433.