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Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) provide insight into the mechanism of human genetic diseases, and majority of functional GWAS mutations target genomic regulatory elements. During erythroid differentiation of human CD34+ cells, we mapped regulatory DNA elements (enhancers and open chromatin regions) by H3K27Ac ChIP-seq and ATAC-seq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and also chose to examine the binding of the BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNA-seq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of erythropoiesis. ChIP-seq for other crucial signaling transcription factors, such as WNT-responsive and TGFb-responsive factors (TCF7L2 and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as "Transcriptional Signaling Centers (TSC)" where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Our bioinformatics-algorithms demonstrated that PU1 and FLI1 binding sites were present in progenitor-specific TSCs whereas KLF1 and NFE2 sites were enriched in TSCs of red blood cells. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor TSC in K562 and HUDEP2 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene and showed defects in erythroid differentiation, demonstrating that TSCs are important to provide optimum gene expression and proper erythroid differentiation. To determine if such TSCs are targeted by GWAS mutations, we have studied 1270 lead and additional 27,799 SNPs in linkage disequilibrium with lead SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). Surprisingly, we observed that, out of the 3831 "functional" SNPs that fall within non-exonic H3K27Ac enhancers, while only 5% (188) of RBC-SNPs target only blood-master-transcription-factor motifs, at least 48% (1821) of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFb signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Additionally, these RBC-trait-SNPs are specifically enriched in GATA+SMAD1 co-bound TSCs and fall within signaling factor binding sites. We validated such SNPs that target SMAD-motifs. The SNP rs9467664 is associated with the MCV-trait near HIST1H4A, a gene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Another MCV-associated SNP rs737092 targets a SMAD motif within an erythroid-specific TSC near RBM38 gene. T-allele, in comparison with C-allele, that retains SMAD1 binding showed more expression in luciferase-based reporter assays specifically under BMP stimulation suggesting that rs737092 compromise BMP-responsiveness. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors under stimulation. Such aberrant signaling events over time could lead to "signalopathies", ultimately resulting in phenotypic variations of RBC traits. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) could provide insight into the mechanism of human genetic diseases. Here we have studied SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). During erythroid differentiation of human CD34+ cells, we mapped enhancers and open chromatin regions by H3K27Ac ChIPseq and ATACseq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNAseq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of differentiation. ATACseq and H3K27Ac patterns demonstrated that GATA+SMAD1 co-occupied regions correlate with open chromatin and super enhancers at every stage, whereas GATA-only regions are associated with genes with low/basal level of expression during differentiation. ChIPseq for other crucial signaling transcription factors, such as cAMP-responsive and TGFb-responsive factors (CREB and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as "signaling centers" where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Surprisingly, we observed that while only 15% of RBC-SNPs target blood-master-transcription-factor motifs, at least 70% of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFβ signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Our bioinformatics-algorithms demonstrated that, in contrast to GATA-only sites, SMAD1+GATA co-bound signaling centers harbor cis -acting motifs and display enriched binding of cell-type specific transcription factors (e.g. PU1 and FLI1 in progenitor vs. KLF1 and NFE2 in differentiated cells). Such distinct identities of signaling centers could serve as codes to distinguish progenitor-specific genes from erythroid-specific genes, and govern their stage-specific expression. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor signaling center in K562 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene. This suggests a signaling factor SMAD1 is important within signaling centers to obtain optimum gene expression. Moreover, a progenitor factor PU1 direct binding of SMAD1 to progenitor-specific signaling centers since with overexpression of PU1 in K562 cells, SMAD1 occupancy was concomitantly increased in selective genomic regions where PU1 binding was increased. More than 80% of the RBC-trait-SNPs are enriched within SMAD1-bound signaling centers. Such SNPs either destroy or create new signaling factor binding sites, e.g. SMAD motifs. We validated one such SNP associated with the MCV-trait near HIST1H4A, agene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors. Such aberrant signaling events over time could lead to "signalopathies", ultimately resulting in phenotypic variations of RBC traits. Disclosures Zon: Fate, Inc.: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Scholar Rock, Inc: Consultancy, Equity Ownership; Stemgent: Consultancy.