Alterations in GATA1s Chromatin Occupancy Lead to Hyperproliferative, Cytokine-Independent Megakaryopoiesis

Down syndrome, or Trisomy 21 (T21), is uniquely associated with transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS). Both TAM and ML-DS harbor mutations in the erythro-megakaryocytic transcription factor GATA1 that lead to exclusive expression of GATA1s, a truncated isoform lacking the amino terminus. Consistent with the megakaryocytic phenotype and gene signature of TAM and ML-DS, we found that hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) with T21 and GATA1s display absent erythropoiesis but hyperproliferation of megakaryocytes and cytokine independence. Single-cell RNA sequencing of iPSC-derived hematopoietic progenitor cells (HPCs) revealed that T21 and GATA1s drive early commitment to the megakaryocyte lineage and block maturation, recapitulating a leukemic phenotype.

To investigate the mechanisms underlying these diseases, we combined bulk RNA sequencing, ATAC-seq (assay for transposase-accessible chromatin with sequencing), and CUT&RUN (cleavage under targets and release using nuclease) for GATA1 in T21 HPCs expressing wtGATA1 vs. GATA1s. Multipotent CD41⁺235⁺ HPCs were differentiated and flow-purified from isogenic T21/wtGATA1 and T21/GATA1s iPSCs. RNA sequencing of these HPCs identified 1,292 differentially expressed genes (|log₂FC| >0, adjusted p <0.05). Gene set enrichment analysis (GSEA) revealed enrichment of cell cycle pathways in T21/GATA1s HPCs, including G2/M checkpoint, E2F targets, and MYC targets. Parallel ATAC-seq identified 19,277 differentially accessible regions (adjusted p <0.05) in T21 HPCs expressing wtGATA1 vs. GATA1s. GSEA of differentially expressed genes with corresponding changes in chromatin accessibility again revealed positive enrichment of G2/M checkpoint genes, E2F targets, and MYC targets as well as negative enrichment of heme metabolism, consistent with the cytokine-independent hyperproliferation of megakaryocytes we observed.

To determine whether GATA1s chromatin occupancy drives these transcriptional changes, we performed CUT&RUN for GATA1 in CD41⁺235⁺ HPCs and CD41⁺42b⁺ megakaryocytes differentiated from T21/wtGATA1 and T21/GATA1s iPSCs. In HPCs, GATA1s binding was globally decreased; compared to wtGATA1, 2,340 regions had decreased occupancy whereas only 62 regions had increased binding (false discovery rate ≤0.05). Genes with decreased GATA1s occupancy, chromatin accessibility, and expression in T21/GATA1s HPCs included CCND1 and CDKN1A, both of which inhibit cell cycle progression in G1 phase and play critical roles in the RB pathway. Notably, GATA1 itself plays an important role in this pathway through the GATA1-RB-E2F complex, which represses E2F to control cell proliferation, but GATA1s lacks the motif to form this complex. In stark contrast to the HPCs, GATA1s occupancy was globally increased in T21/GATA1s megakaryocytes (10,299 regions with increased occupancy compared to 279 regions with decreased occupancy; false discovery rate ≤0.05), including at both canonical megakaryocytic and erythroid loci. The essential megakaryocyte gene RUNX1 demonstrated increased chromatin accessibility and GATA1s occupancy, suggesting its role in the megakaryocyte bias and proliferation we observed with GATA1s.

Taken together, our results suggest that GATA1s in T21 HPCs simultaneously promotes cell proliferation and megakaryopoiesis, yielding hyperproliferative, immature megakaryocytes due to absence of GATA1s binding in HPCs and inappropriate persistence of GATA1s at key loci in megakaryocytes. Our findings recapitulate the hallmarks of malignant transformation and offer mechanistic insights into the progression from T21 to TAM and ML-DS.