SUMMARY Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed for enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.GATA1 | cis-regulatory elements | noncoding mutations | Mendelian erythroid disorders W hole-exome sequencing (WES) and targeted sequencing approaches have greatly accelerated our ability to identify causal genetic lesions in both previously implicated and novel genes underlying monogenic disorders (1, 2). In hematology, WES has been extremely useful for identifying unknown genetic etiologies for various disorders, such as those affecting red blood cell (RBC) production, including Diamond-Blackfan anemia and congenital dyserythropoietic anemia (3-5), disorders of RBC structure and function (6, 7), and disorders affecting other aspects of hematologic function (2, 8). Despite this considerable success, however, more than 50% of cases of presumed monogenic diseases are refractory to current WES approaches (9). Although resolving these remaining cases will benefit from improvements in exome capture (10), read alignment (11), and variant annotation methodologies (11), the importance of genetic variation occurring within regulatory elements (REs) outside of the traditionally investigated coding sequences in hematologic and other diseases is being increasingly appreciated (12).Whole-genome sequencing (WGS) approaches are becoming progressively more available and affordable, but separating pathogenic genetic variation from benign or unrelated mutations remains especially difficult outside of protein-coding gen...

Highlights d A FACS-based strategy separates mononucleated and binucleated cardiomyocytes d Mononucleated and binucleated cardiomyocytes are transcriptionally distinct d Binucleation results in the silencing of E2f transcriptional targets d Increased binucleation due to the loss of Ect2 impairs regenerative potential

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Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

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In the original article, Table 1 incorrectly stated that the affected individual in family II has a head circumference of À7.9 SD from the mean. Consistent with the descriptive text, this number should be À9.7 SD. The article has been corrected online. The authors regret the error.

Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume, MCV). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.