ALBERT

Description: Understanding the cell-autonomous as well as niche contributions governing erythropoiesis is critical for directed differentiation approaches of hematopoietic stem cells into differentiated red blood cells (RBCs) to treat blood disorders such as anemias and leukemias. Transcriptional intermediary factor 1 gamma (TIF1γ) is essential for erythropoiesis from zebrafish to mammals. Zebrafish moonshine mutant embryos defective for tif1γ do not make red blood cells (RBCs) due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. Loss of factors involved in transcription elongation control, PAF1 and spt5, rescues the moonshine RBC defect. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a high-content chemical suppressor screen in the bloodless moonshine mutant using 3,500 compounds. Among the suppressors, we identified leflunomide, an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme for de novo pyrimidine synthesis. Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar both rescue the formation of primitive erythroid cells in 61% (38/62) and 68% (50/74) of moonshine embryos, respectively. Blastula transplant experiments revealed that tif1γ, in addition to its cell-autonomous role, plays a role in the hematopoietic niche for RBC development. Through in-vivo metabolomics analyses we have identified nucleotide metabolism as the most significantly altered process in moonshine mutants, including elevated levels of uridine monophosphate and low levels of nicotinamide adenine dinucleotide (NAD+). Low NAD+ levels are accompanied by a reduced oxygen consumption rate in tif1γ-depleted embryos by Seahorse analysis. In support, genome-wide transcriptome analysis coupled with chromatin immunoprecipitation studies revealed genes encoding coenzyme Q (CoQ) metabolic enzymes as direct TIF1γ targets. DHODH is the only enzyme of the pyrimidine de novo synthesis pathway located on the inner mitochondrial membrane and its activity is coupled to that of the electron transport chain (ETC). Rotenone, a potent ETC complex I inhibitor reverses the rescue of the blood defect by DHODH inhibition in moonshine embryos. Since DHODH function is linked to mitochondrial oxidative phosphorylation via CoQ activity, we asked whether alterations in mitochondrial metabolism might be causal for the RBC defect in moonshine mutants. Indeed, treatment with the CoQ analog decylubiquinone results in rescue of βe3 globin expression in 26% (33/126) of moonshine embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases and consequences for erythroid differentiation protocols. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Description: 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.

Description: Background: EphrinA1-Fc abolishes acute I/R injury and attenuates nonreperfused cardiac injury 4 days after permanent occlusion in mice. The goal of this study was to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of coronary artery ligation, to determine the degree to which early salvage effects translate to reduced adverse remodeling after 4 weeks of nonreperfused myocardial infarction (MI) in wild-type B6 and EphA2-R-M (EphA2 receptor null) mice. Methods: At 4 weeks post-MI, echocardiography, histologic and immunohistochemical analyses of B6 mouse hearts were performed. Primary mouse cardiac fibroblasts (FBs) isolated from B6 mice cultured in the presence of low and high dose ephrinA1-Fc, both with and without pro-fibrotic TGF-β stimulation and Western blots, were probed for relative expression of remodeling proteins MMP-2, MMP-9 and TIMP-1, in addition to DDR2 and (p)SMAD2/3/totalSMAD2/3. Results: EphrinA1-Fc preserved a significant degree of contractile function, decreased adverse left ventricular remodeling, attenuated excessive compensatory hypertrophy, and decreased interstitial fibrosis in wild-type (WT) B6 mouse hearts. In contrast, most of these parameters were poorer in ephrinA1-Fc-treated EphA2-R-M mice. Of note, fibrosis was proportionately decreased, implying that other EphA receptor(s) are more important in regulating the pro-fibrotic response. Primary FBs showed disparate alteration of MMP-2, MMP-9 and TIMP-1, as well as DDR2 and p-SMAD2/3/totalSMAD2/3, which indicates that matrix remodeling and cardiac fibrosis in the injured heart are influenced by ephrinA1-Fc. Conclusion: This study demonstrates the capacity of a single administration of ephrinA1-Fc at the onset of injury to attenuate long-term nonreperfused post-MI ventricular remodeling that results in progressive heart failure, and the important role of EphA2 in mitigating the deleterious effects.

Description: 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.