Super-enhancer mediated regulation of adult β-globin gene expression: the role of eRNA and Integrator

Abstract

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Co-Regulated Genes and Gene Clusters

“…In accordance with this scenario, a number of studies have demonstrated that the same transcription factors are present both on the enhancer and on the promoters of genes controlled by this enhancer [35][36][37]. Due to the higher density of binding sites for transcription factors on enhancers, it is the enhancers that serve as the nucleation centers for the assembly of activator compartments [38][39][40]. The attraction of promoters to the activator compartment is controlled by architectural proteins.…”

Enhancers, phase separation and the RNA polymerase II transfer model

“…When transcribed, they stay present at the gene locus and upon their depletion a reduction in gene expression was observed, as well as an accumulation of PolII at the LCR. [ 3 ] A similar mechanism has been suggested to activate gene expression at promoters. Paraspeckle promoting protein PSPC1 (an RNA binding protein found in paraspeckles, a type of condensate) was shown to bind RNA at sites of transcription initiation, contributing to recruitment of PolII.…”

“…[3,4] When the Integrator endonuclease subunit INTS11 was depleted in murine erythroleukemia (MEL) cells, an accumulation of PolII was observed at the locus control region (LCR) of β-globin and decreased recruitment to the promoter of β-globin, highlighting the importance of Integrator in PolII release and transfer. [3] Similar observations were apparent upon acute, degron-mediated depletion of INTS11 in mouse embryonic stem cells (mESCs), where depletion of INTS11 caused a build-up of PolII at promoters and accumulation of short RNA transcripts, including eRNAs. [4] In this study, transferred PolII was shown to be predominantly unphosphorylated, with the phosphatase subunit of Integrator being transferred along with PolII.…”

Enhancer-Mediated Formation of Nuclear Transcription Initiation Domains

“…Although eRNAs are generally less stable than mRNAs, their average half-life ranges from 5 to 10 min. The relatively short half-life suggests that eRNA function is transient [ 47 , 48 ].…”