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9 Identification of a Novel Gene Regulatory Element in Human Erythroid Progenitor Cells

Program: Oral and Poster Abstracts
Type: Oral
Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Red Cells: Emerging Gene Regulatory Mechanisms in Erythroid Differentiation
Hematology Disease Topics & Pathways:
Research, Translational Research, CHIP, genomics, bioinformatics, hematopoiesis, Biological Processes, molecular biology, Technology and Procedures, gene editing
Saturday, December 9, 2023: 10:00 AM

Vincent P Schulz, PhD1*, Kimberly Lezon-Geyda, PhD1*, Peiying Shan, MD2*, Julien Papoin, MS3*, Mohandas Narla, DSc4,5, Laurie A. Steiner, MD6, Lionel Blanc, PhD3, James Palis, MD7 and Patrick G Gallagher, MD8,9

1Department of Pediatrics, Yale University School of Medicine, New Haven, CT
2Dept of Pediatrics, Yale University School of Medicine, New Haven, CT
3Feinstein Institutes for Medical Research, Northwell, Manhasset, NY
4Research Laboratory of Red Cell Physiology, New York Blood Center, New York
5LFKRI, New York Blood Center Enterprises, New York, NY
6Department of Pediatrics, University of Rochester, Rochester, NY
7University of Rochester Medical Center, Rochester, NY
8Yale University School of Medicine, New Haven, CT
9Dept of Pediatrics, Ohio State University, Nationwide Childrens Hospital, Columbus, OH

Early erythropoiesis involves commitment of multi-lineage progenitors to erythroid progenitor cells and differentiation into erythroid burst-forming unit (BFU-E) cells, then erythroid colony-forming unit (CFU-E) cells. In contrast to terminal erythroid differentiation, regulation of gene expression in early erythropoiesis is poorly defined. To address this knowledge gap, we identified active enhancers in erythroid progenitor cells linked to known disease genes and genes associated with erythrocyte traits in genome wide association studies. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from mobilized peripheral blood were cultured towards the erythroid lineage. From these cultures, FACS-based methods were utilized to purify four stages of erythroid progenitor cells, termed EP1 to EP4, and five populations of terminally differentiated erythroblasts, from proerythroblasts to orthochromatic erythroblasts. ChIP-seq was performed using antibodies to H3K4me1, H3K27Ac, H3K27me3, and H3K9me2/3. Chromatin accessibility was determined by ATAC-seq. Patterns of gene expression were determined by RNA-seq. Active enhancers were identified using the ModHMM algorithm. Numerous erythroid progenitor cell-specific active enhancers were identified near genes linked to known disease genes and GWAS-associated erythrocyte traits. rs162375, a single nucleotide polymorphism (SNP) linked to mean corpuscular hemoglobin, was in a 475bp Long Terminal Repeat Retrotransposon (LTR) element upstream of the RRP1B gene. The LTR enhancer near the RRP1B gene is a member of the LTR15 family. Transposable elements are a source of novelty in primate gene regulation when they acquire binding sites for critical tissue-specific transcription factors, and then these tissue-specific regulatory regions direct tissue-specific expression of nearby genes. The SNP-containing LTR15 erythroid progenitor-specific enhancer upstream of RRP1B contains 2 conserved GATA motifs and a conserved RUNX motif. The enhancer region had a peak of open chromatin determined by ATAC-seq, and H3K27 acetylation and strong GATA1 binding determined by ChIP-seq. These findings were present only in erythroid progenitor cells, not in earlier hematopoietic cells, terminally differentiating erythroid cells, or other non-erythroid cells. CRISPR-based gene editing was utilized to delete the LTR15 enhancer in CD34+ HSPCs cultured towards the erythroid lineage into erythroid progenitor cells. mRNA expression of RRP1B was decreased to 70.2% of wild type. Luciferase-based reporter gene studies demonstrated the enhancer increased expression of a minimal promoter 3.2-fold over background. Mutation of the GATA and RUNX sites individually or together decreased reporter gene activity to background levels. Bioinformatic analyses identified 291 additional erythroid progenitor cell specific LTR15 element active enhancers, with most containing two GATA motifs and a RUNX motif. The majority of the novel enhancer regions were also marked by progenitor cell-specific ATAC peaks and strong GATA1 binding in ChIP-seq. GATA1 and RUNX, both present in early erythropoiesis, are required to activate the regulatory elements in erythroid progenitor cells. Decreasing levels of RUNX in terminal erythroid differentiation parallels loss of enhancer activity. Since LTR15 first appeared in Catarrhine, old-world monkeys, these data indicate this novel regulatory element has recently evolved to contribute to regulation of gene expression in early erythropoiesis in primates.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH