Mechanisms of Pol II Recruitment to the Human Beta-Globin Locus Control Region Super-Enhancer

Abstract

The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.