Garp/LRRC32 defines the erythroid-megakaryocyte fate decision and enables β-globin program activation
Bibliographic record
Abstract
Abstract Erythroid commitment from multipotent progenitors is a tightly regulated process, yet the molecular drivers during the earliest stages, prior to erythropoietin (Epo) responsiveness, remain incompletely understood. GARP (Glycoprotein A Repetitions Predominant, encoded by LRRC32) is a membrane protein best known for binding to latent TGF-β on regulatory T cells; its role in hematopoiesis has never been investigated before. Here, we uncover an essential function of GARP in erythroid commitment preventing megakaryocytic skewing and regulating globin transcription. Ubiquitous Lrrc32 deletion (Lrrc32null) led to fatal anemia in mice, characterized by complete loss of Ter119+ erythroid cells in the bone marrow (BM), markedly elevated plasma Epo, and thrombocytosis. Anemia was fully rescued by transplantation of wild-type BM, suggesting a cell-intrinsic defect. Genetic ablation of TLR9 or type I interferon receptor failed to rescue the phenotype, excluding a role for inflammatory signaling in Lrrc32null-mediated anemia. In contrast, erythroid-specific deletion using Epo receptor (EpoR)-Cre mice did not cause anemia, indicating a requirement for GARP prior to EpoR expression. Moreover, we observed GARP expression in early erythroid progenitors, with highest levels preceding CD71 upregulation and declining thereafter, defining a narrow temporal window of GARP expression. High-dimensional flow cytometry in the BM of Lrrc32nullmice showed expanded hematopoietic stem and progenitor populations upstream of BFU-E, suggesting a lineage-specific block before this stage. In vitro differentiation of CD117+Lrrc32nullcells recapitulated this arrest, with failure of erythroid differentiation and reduced cell survival. Bulk RNA-seq and proteomics of Lrrc32nullCD117+ cells cultured in the presence of erythroid differentiation media revealed transcriptional de-differentiation with suppression of erythroid programs. Co-differential expression analysis showed that Lrrc32nullprogenitors lose erythroid identity and acquire signatures associated with other myeloid lineages, consistent with a rewiring of lineage potential. Electron microscopy showed excessive intracellular vacuolization indicating a non-apoptotic failure of maturation, likely reflecting megakaryocytic bias. Single-cell CITE-seq profiling confirmed a dramatic change of the erythroid trajectory, while megakaryocytic lineages remained intact. These findings suggest that GARP is required to resolve the erythroid-megakaryocyte bifurcation at the MEP level. We also performed CRISPR-mediated Lrrc32 deletion in zebrafish embryos, which caused early lethality, reduced Gata1 expression, and impaired hemoglobinization (benzidine staining) 5 days post fertilization. Globin gene analysis showed significantly reduced embryonic β-globin expression, while α-globin levels were elevated, indicating dysregulated globin gene expression. In a human erythroleukemia cell line (HEL), GARP deletion increased γ-globin expression, mimicking a fetal-like globin program. This occurred despite upregulation of the canonical γ-globin repressors BCL11A and MYB, along with HBS1L, NR2F2, and NR2C1, suggesting a dysregulated transcriptional network of the globin program in the absence of GARP. Mechanistically, confocal imaging of murine BM and nuclear fractionation of HEL cells revealed intracellular and nuclear localization of GARP, indicating roles beyond surface TGF-β presentation. Given that β-globin activation requires full engagement of the erythroid transcriptional network, its suppression in GARP-deficient cells may reflect a failure to establish erythroid chromatin architecture. Thus, loss of β-globin expression likely marks incomplete commitment rather than impaired maturation. In conclusion, this study uncovers a previously unrecognized role for GARP in erythroid lineage commitment. It acts at a critical bifurcation point to suppress megakaryocytic skewing and enable β-globin program activation. Its loss causes cell-autonomous erythroid failure before CFU-E formation, leading to fatal anemia. Mechanistically, GARP may act both at the membrane to regulate TGF-β availability and within the nucleus to stabilize erythroid chromatin structure. These findings define a novel regulatory axis connecting TGF-β signaling, chromatin priming, and globin activation in hematopoietic lineage fate determination, with broad implications for understanding anemia, hematopoietic development, and lineage commitment biology.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".