Elucidating the Role of Integrator Complex in Erythropoiesis

PROJECT SUMMARY Erythropoiesis is a finely orchestrated process that involves generating a ~2.5 million red blood cells per second to maintain homeostasis and prevent anemia. During terminal maturation, erythroid precursors upregulate erythroid-specific genes while silencing non-erythroid genes in the setting of a cell that is rapidly dividing and a nucleus that is condensing in preparation for enucleation. Our group has shown that regulation of RNAPII activity is an essential determinant of erythroid cell function. During terminal erythroid maturation, RNA polymerase II (RNAPII) levels decline dramatically, becoming a scarce resource allocated to erythroid genes, such as alpha and beta globin. The mechanism(s) by which RNAPII is removed from genes unnecessary to erythroid differentiation and shunted to produce mRNA essential for red blood cell function is unknown. The Integrator Complex (INT) is a multi-subunit machinery that associates with RNA polymerase II (RNAPII) and functions as a critical transcription regulator. It is a broad negative regulator of promoter-proximally paused RNAPII. Integrator subunit 11 (INTS11) houses the RNA endonuclease domain vital for Integrator to cleave nascent transcripts at all RNAPII loci, which is an important activity for transcriptional repression and the processing of eRNA. Although INT is highly expressed in maturing erythroid cells, little is known about its function in erythropoiesis. Further, the role of promoter-proximal transcriptional termination in regulating erythroid gene expression has been almost completely unexplored. Our preliminary data reveal that disruption of INT impairs the proliferation and maturation of primary human erythroid cells. Our CUT&RUN analyses in primary erythroid cells uncover that INT is present at genes containing stably paused RNAPII that is subsequently lost before enucleation. Finally, we find that INT binds to erythroid enhancers, including the beta-globin LCR, in a manner correlating with increased transcription. Altogether, these data support a hypothesis where INT enforces RNAPII pausing and termination at non-erythroid genes to maintain RNAPII availability while enhancing the transcription of erythroid genes through eRNA processing. To test this hypothesis, we propose two specific aims: Specific Aim1. Determine the function of the Integrator Complex during terminal erythroid maturation. Specific Aim 2. Delineate mechanisms by which Integrator regulates erythroid gene expression.