Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing. Disclosures No relevant conflicts of interest to declare.

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6–GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory condition, which is contributed by heme-activated pro-inflammatory macrophages. While previous studies addressed heme ability to stimulate macrophage inflammatory skewing through TLR4/ROS signaling, how heme alters cell functional properties remains unexplored. Macrophage-mediated immune cell recruitment and apoptotic cell (AC) clearance are relevant in the context of SCD, where tissue damage, cell apoptosis and inflammation occur due to vasoocclusive episodes, hypoxia and ischemic injury. Here we show that heme strongly alters macrophage functional response to AC damage by exacerbating immune cell recruitment and impairing cell efferocytic capacity. In SCD, heme-driven excessive leukocyte influx and defective efferocytosis contribute to exacerbated tissue damage and sustained inflammation. Mechanistically, these events depend on heme-mediated activation of TLR4 signaling and suppression of the transcription factor PPARg and its coactivator PGC1a. These changes reduce efferocytic receptor expression and promote mitochondrial remodeling, resulting in a coordinated functional and metabolic reprogramming of macrophages. Overall, this results in limited AC engulfment, impaired metabolic shift to mitochondrial fatty acid b-oxidation and ultimately reduced secretion of the anti-inflammatory cytokines IL-4 and IL-10, with consequent inhibition of continual efferocytosis, resolution of inflammation and tissue repair. We further demonstrate that impaired phagocytic capacity is recapitulated by macrophage exposure to sickle patients'plasma and improved by hemopexin-mediated heme scavenging, PPARg agonists or IL-4 exposure through functional and metabolic macrophage rewiring. Our data indicate that therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PGC1a/PPARg modulation significantly ameliorate tissue damage associated with SCD pathophysiology.

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This article has been corrected: During the assembly of Figure 2B, the same image was inadvertently used for both H1299 (pEGFP-C3) and H1299 (pEGFP-4.1N) groups at 0h time point. The proper Figure 2 B specific for H1299 (pEGFP-C3) and H1299 (pEGFP-4.1N) groups are shown below. The authors declare that these corrections do not change the results or conclusions of this paper.