Narla Mohandas

Thrombotic events are the leading cause of death in myeloproliferative neoplasms (MPNs) driven by the JAK2 Val ⁶¹⁷ →Phe (JAK2V617F) mutation. We investigated how JAK2V617F alters hemostasis and thrombosis in humans and in lineage-specific knockin mice to test whether red blood cell (RBC) mechanics contribute directly to clot pathology. Jak2 ^F/+ Vav-Cre knockin mice form structurally aberrant clots characterized by extended clot formation times and unstable hemostatic plug formation. Whole-blood clot contraction in vitro was impaired in humans and mice carrying the JAK2V617F mutation, even when Jak2 ^V617F erythrocytes were resuspended in normal plasma with normal platelets. Thus, in addition to increasing the hematocrit, the JAK2V617F mutation imparted intrinsic changes in erythrocytes critical for mediating abnormal clot formation. Biophysical analysis showed that Jak2 ^V617F erythrocytes had stiffened membranes, making them less compressible during clot contraction. Proteomic and lipidomic analysis of Jak2 ^V617F erythrocytes revealed the loss of key membrane and cytoskeleton proteins, a sparse spectrin cytoskeletal network, increased membrane lipid content, and deep plasma membrane invaginations that were consistent with biaxial cytoskeletal stretching and altered lipid packing. These JAK2V617F-driven changes in erythropoiesis generated intrinsically rigid erythrocytes that impaired clot contraction and produced larger and more occlusive, yet mechanically unstable thrombi. This mechanism may explain the contradictory coexistence of thrombosis and hemorrhage in MPNs and why both complications improve when blood counts are lowered, suggesting that targeting erythrocyte mechanics may provide previously unidentified therapeutic opportunities.

Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 proteins) and mapped the oxygen-dependent interactome. These datasets reveal an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N-terminus. We identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident with increased Band 3-deoxyhemoglobin contacts. This reversible assembly functions as an oxygen-sensitive switch coordinating redox and glycolytic remodeling. Humanized mice lacking Band 3 N-terminal segments exhibit impaired oxygen-dependent regulation of BLVRB binding to band 3, impaired hypoxic activation of glycolysis, reduced 2,3-bisphosphoglycerate synthesis, and diminished exercise tolerance, demonstrating physiological relevance. Population-scale cis-pQTLs for SLC4A1 and BLVRB suggest functions beyond canonical heme catabolism. Mechanistically, biochemical analyses in vitro suggest that hemoglobin β (HBB), Band 3, and BLVRB can undergo S-nitrosation and may participate in trans-nitrosation reactions with the glycolytic enzyme GAPDH, whose modification at C152 inhibits enzymatic activity in vitro. Collectively, these findings define a Band 3-BLVRB axis that integrates oxygen-dependent protein interactions with thiol-based redox chemistry, providing a framework for understanding how an anucleate cell achieves metabolic adaptability through reversible protein-protein interactions and post-translational modification. These findings suggest that perturbation of the Band 3-BLVRB axis may influence oxygen delivery and metabolic flexibility during hypoxic stress, with potential relevance to high-altitude adaptation, exercise physiology, and cardiopulmonary disease.

The transcription factor GATA1 has pleiotropic hematopoietic functions, particularly in erythroid and megakaryocytic ontogeny. While mechanistic investigations have uncovered many facets of GATA1 biology, how GATA1 co-regulates divergent cell fates remains incompletely characterized. We previously described that loss of Stag2, a member of the cohesin complex and a recurrent mutational target in myelodysplastic syndrome (MDS) and myeloid leukemia of Down Syndrome, results in altered chromatin accessibility, transcription factor function, and cell differentiation. Hence, we hypothesized that chromatin accessibility determines GATA1 cistrome specificity and lineage fate decisions. To understand the connection between chromatin accessibility and GATA1, we comprehensively studied erythropoiesis in Stag2∆ mice. Defects in Stag2-deficient hematopoiesis included reduced numbers of erythroid progenitors (EryPs), impaired terminal erythroid differentiation, increased number of MkPs, and increased megakaryocytes. RNA- and ATAC-sequencing of EryPs revealed altered patterns of Gata1 target gene expression with altered accessibility in conjunction with loss of expression of erythroid targets and gain of megakaryocyte targets. Despite unchanged Gata1 expression, Gata1 occupancy was reprogrammed from erythroid to megakaryocyte targets with Fli1 motifs enriched at Stag2∆ Gata1 binding sites. Functionally, we observed that Stag2-deficient EryPs have diminished erythroid output and augmented megakaryocyte output in orthogonal differentiation assays, which was partially reversed with Fli1 knockdown. Human models and primary MDS patients recapitulated the essential phenotypic and molecular features of our in vivo murine MDS model. Collectively, this study establishes chromatin accessibility as a determinant of transcription factor binding specificity, revealing an accessibility-driven Gata1 retargeting mechanism underlying MDS dyserythropoiesis.

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using in vivo models, we uncover divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1, which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

Erythropoiesis, the process by which hematopoietic stem cells differentiate into mature red blood cells, is tightly regulated by a complex network of transcriptional and epigenetic mechanisms. While helicase-like transcription factor (HLTF) is known for its roles in transcriptional regulation, chromatin remodeling, and genome stability, its function in erythroid development has remained unexplored. Here, we identify HLTF as a novel regulator of GATA1 and erythropoiesis. HLTF directly binds the GATA1 promoter, enhancing its transcription. In vitro and in vivo assays demonstrated that HLTF promotes erythroid proliferation, survival, and terminal differentiation. HLTF knockout impairs erythropoiesis, effects which are partially rescued by GATA1 overexpression. Multi-omics analyses (RNA-seq, ATAC-seq, and CUT&Tag) reveal that HLTF maintains chromatin accessibility and activates erythroid gene networks. HLTF interacts with GATA1, co-occupies erythroid regulatory regions, and facilitates GATA1 genomic binding. Notably, GATA1 also transcriptionally activates HLTF, forming a positive feedback loop. In erythroid disorders, HLTF is up-regulated in polycythemia vera (PV) and down-regulated in myelodysplastic syndromes (MDS). Knockout of HLTF in PV patient-derived cells suppressed erythroid hyperplasia, reduced chromatin accessibility, and impaired GATA1 binding. Together, these findings reveal HLTF as a transcriptional regulator of GATA1 and a pivotal modulator of erythropoiesis, providing new insights into erythroid lineage control and erythroid-related diseases.

Erythropoiesis is precisely regulated by multilayered networks. It is crucial for maintaining steady-state hemoglobin levels and ensuring effective oxygen transport. Alternative polyadenylation (APA) is a post-transcriptional regulatory mechanism generating multiple mRNA isoforms from a single gene based on specific 3′ untranslated region sequences. While APA plays a vital role in various cellular processes, the underlying mechanism in erythropoiesis remains largely unexplored. In this study, we employed an integrative approach, combining bioinformatics analyses and experimental validations, to systematically investigate the role of APA in erythropoiesis. We mapped the APA landscape during erythroid differentiation and identified significant APA shifts essential for the differentiation of erythroid cells from burst-forming unit erythroid (BFU–E) to colony-forming unit erythroid (CFU–E). Notably, our findings highlighted polyadenylate-binding protein cytoplasmic 1 (PABPC1) as the primary regulator of APA during these stages. Functional analyses have revealed that knockdown of PABPC1 disrupts erythroid progenitor cell proliferation and differentiation. These results implicate an essential role of PABPC1 in modulating cell fate through APA regulation. Furthermore, we found that decreased PABPC1 levels increased the usage of the proximal polyadenylation sites in the TSC22 domain family member 1 (TSC22D1) gene. This shift led to elevated expression of TSC22D1, uncovering a novel mechanism by which APA influences erythroid progenitor expansion and differentiation. Our findings provide novel insights into APA regulation in early erythropoiesis and suggest potential therapeutic strategies for diseases associated with erythropoietic disorders.

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using novel in vivo models, we uncover strikingly divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1 , which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

Malaria elimination faces challenges from drug resistance, stemming from mutations within the parasite’s genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature’s approach, and implementing methodologies to render indispensable host proteins inactive, holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain, which shares consensus sequence with the zinc endopeptidase family, possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor, BEL-A, we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally, thiorphan, a metallo-endopeptidase inhibitor, which specifically inhibits Kell activity, inhibited Plasmodium infection at nanomolar concentrations. Interestingly, individuals in malaria-endemic regions exhibit low Kell expression and activity, indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril, demonstrated potent antimalarial activity in vivo, highlighting Kell’s protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic.