a. Department of Medical Genetics, School of Basic Medical Sciences, Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China;
b. Cyrus Tang Medical Institute, National Clinical Research Center for Hematologic Diseases, State Key Laboratory of Radiation Medicine and Protection, Collaborative Innovation Center of Hematology, Suzhou Medical College, Soochow University, Suzhou, Jiangsu 215123, China;
c. Department of Blood Transfusion, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215123, China
Funds:
Ryo Kurita and Yukio Nakamura (Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Japan) provided HUDEP-2 cells. We thank the members of the Xu laboratory for insightful discussions and comments. This research was supported by the National Natural Science Foundation of China (82170119), the Key Program for Basic Research in Jiangsu Province (BK20243008), the High-Level Personnel Project of Jiangsu Province (JSSCTD202353), the Interdisciplinary Basic Frontier Innovation Program of Suzhou Medical College of Soochow University (YXY2302018), the Cyrus Tang Foundation (CTJC25002), the Pediatric Hematology &
Oncology Key Laboratory of Higher Education Institutions in Jiangsu Province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Collaborative Innovation Center of Hematology (all to P.X.).
The regulation of α-like globin genes, particularly the embryonic ζ-globin gene (HBZ), remains incompletely understood. To identify transcriptional regulators of HBZ, we establish a GFP reporter system based on the HBZ-P2A-GFP allele in erythroid cell lines and conduct a CRISPR/Cas9 screen targeting 1639 transcription factors. This screen identifies SKI as a potent HBZ repressor. Functional validation shows that SKI loss increases HBZ expression without impairing erythropoiesis, whereas SKI overexpression suppresses HBZ. Tet-on-inducible SKI overexpression and auxin-inducible SKI degradation indicate that SKI rapidly represses HBZ transcription. Transcriptome profiling further reveals that SKI deletion activates HBZ while minimally affecting other erythroid genes. Mechanistically, genome-wide occupancy analyses show that SKI binds the distal enhancers HS-10 and HS-40, with partial co-occupancy by BCL11A. Despite this overlap, dual knockout of SKI and BCL11A synergistically increases HBZ expression, as does base editing of the SKI-binding site within HS-10. We also identify a naturally occurring variant (chr16:193207G>A) within this enhancer in α-thalassemia patients with elevated ζ-globin levels. Together, these findings establish SKI as a direct, BCL11A-independent transcriptional repressor of ζ-globin. This work advances our understanding of globin gene regulation and suggests targeted ζ-globin reactivation as a potential therapeutic strategy for α-thalassemia.
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