Stag2 dependent chromatin remodeling enforces the erythroid-specific Gata1 cistrome Free

The mechanisms regulating erythropoiesis have been extensively characterized, identifying a complex network of intrinsic and extrinsic factors, such as transcription factors (TFs) and cytokines. However, how TFs achieve lineage specificity remains incompletely understood. This is exemplified by GATA1, which is critical for erythroid (Ery) and megakaryocytic (Mk) fate, yet is expressed in a variety of hematopoietic stem and progenitor cells (HSPCs). Emerging evidence suggests that TFs rely on epigenetic context, such as chromatin accessibility, to drive lineage-restricted gene expression. We previously demonstrated that loss of the cohesin subunit STAG2, which is mutated in ~10% of myelodysplastic syndrome (MDS) cases, disrupts chromatin accessibility, TF function, and lineage differentiation. Here, we show that Stag2 dependent chromatin remodeling is a mechanism by which Gata1 achieves erythroid lineage specificity and that this process is disrupted in MDS.

To define how Stag2 enforces Ery identity, we profiled murine erythropoiesis using scATAC-seq and flow cytometry. During early erythropoiesis, we observed delayed Ery fate specification with impaired opening of Ery defining genes such as Gypa and failure to restrict accessibility at alternative lineage genes such as Pf4 and Fli1. Correspondingly, we observed fewer unipotent erythroid progenitors (EryP), greater Mk progenitors, and proportional shifts in terminally committed pools. These chromatin accessibility perturbations persisted even after terminal erythroid commitment. Consistent with this molecular signature, terminal erythroid differentiation (TED), nuclear condensation, and enucleation were adversely impacted by Stag2^∆.

Given the Mk skewing, we aimed to ascertain if this effect was evident at the EryP cell stage, where Mk programs are repressed to facilitate irreversible Ery commitment. In pseudobulked EryPs, GSEA revealed significant negative enrichment of the Ery differentiation program and positive enrichment of Mk programs. This was corroborated by RNA-seq of Stag2^WT and Stag2^∆EryPs. Subsequently, we queried the significant genes from our RNA-seq and observed that Gata1 was amongst the top TFs enriched in down- and upregulated genes. Since these targets representing differential Gata1 engagement were distinct, we suspected this might represent different lineage-specific Gata1 cistromes. Using published ChIP-seq data from Ery and Mk cells, we defined lineage-specific cistromes and found, by RNA-seq and ATAC-seq, negative enrichment of the Gata1-Ery cistrome and positive enrichment of the Gata1-Mk cistrome in Stag2^∆ EryPs. To test whether altered accessibility redirected Gata1 binding, we performed CUT&RUN for Gata1 in EryPs. We observed reduced Gata1 occupancy at Ery targets and increased occupancy at Mk targets. Next, we identified the consensus genes gaining or losing accessibility, Gata1 occupancy, and expression by intersecting all three datasets. Amongst these genes, Ery GO terms were overrepresented amongst the lost genes and Mk GO terms were overrepresented amongst the gained genes.

Given this altered chromatin state and ectopic Gata1 binding, we hypothesized this may functionally alter lineage specification. In both liquid culture and methylcellulose, we observed that Stag2^∆ EryPs have diminished Ery (Ter119⁺CD71⁺) output and augmented Mk (CD41⁺42d⁺) output.

To assess the relevance of these findings in human cells and MDS patients, we knocked down STAG2 in CD34⁺ HSPCs. STAG2-deficient (STAG2^KD) cells had impaired Ery fate specification, terminal commitment, TED, and enucleation, consistent with our murine model. STAG2^KD cells also displayed features of erythrodysplasia, including multilobed nuclei, a hallmark of MDS. Lastly, RNA-seq of STAG2 mutant MDS patients revealed reduced expression of Ery genes, increased expression of Mk genes, and dysregulation of lineage-specific GATA1 cistromes, highlighting the generalizability of our findings.

Overall, we demonstrate that Stag2 loss disrupts the chromatin landscape in EryPs, leading to misallocation of Gata1 binding and a shift in lineage output. Our findings highlight that Gata1 is influenced by cell type specific chromatin accessibility to execute lineage-specific transcriptional programs. More broadly, our data underscore the relevance of coordinated TF-chromatin interactions in lineage specification and introduce TF-cistrome/chromatin mismatch as a novel paradigm in hematologic disease.