The Integrator Complex Is Essential for Erythropoiesis

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.11-11
• Main Authors: Lv, Xiurui, Getman, Michael, Rahman, Nabil, Murphy, Kristin, Wagner, Eric J, Steiner, Laurie A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-190077

Erythropoiesis is a finely orchestrated process that involves the generation of 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 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 (Murphy Blood 2021). 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 are unknown. The Integrator Complex (INT) is a modular and multi-subunit machinery that binds to paused RNAPII and promotes promoter-proximal termination. INT is highly expressed in maturing erythroid cells, although little is known about the function of INT during erythropoiesis. We hypothesize that INT facilitates promoter-proximal termination at non-erythroid genes to maintain RNAPII availability during terminal erythroid maturation. The balance between kinase and phosphatase activity is a key regulator of RNAPII activity. Phosphorylation of the c-terminal domain of RNAPII by pTEFb facilitates RNAPII pause release, and promotes active transcription. Alternatively, subunit 8 of INT (INTS8) can dephosphorylate the c-terminal domain of RNAPII through association with protein phosphatase 2a (PP2A) (Huang Mol Cell 2020), enforcing RNAPII pausing and antagonizing pTEFb activity. Integrator subunit 11 then cleaves the nascent RNA causing promoter-proximal transcriptional termination (Wu PNAS 2017). To gain insights into the function of INT in erythropoiesis, we utilized the RNP method of Crispr/Cas 9 genome editing (Gundry Cell Reports 2016) to delete INTS8 from CD34+ HSPCs, and then subjected them to erythroid differentiation and colony forming assays. Disruption of INTS8 significantly impaired erythroid proliferation and viability. It also delayed erythroid maturation, evident by delayed GPA expression, less mature morphology, and lower rates of benzidine positivity. Moreover, INTS8 disruption led to a decline in erythroid colony-forming ability and decreased expression of alpha and beta globin. Tog