ALBERT

Description: Aberrant erythroid differentiation can give rise to anemias and leukemias. Regulated transcription elongation at erythroid loci is vital for specific differentiation steps during blood development. Transcriptional intermediary factor 1 gamma (TIF1γ), whose gene is mutated in the blood deficient zebrafish moonshine mutant, recruits positive elongation factors to erythroid genes, thus relieving paused Pol II. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a chemical suppressor screen in the bloodless moonshine mutant. Progeny of heterozygous moonshine mutants were treated at 50% epiboly with 4,000 individual chemical compounds with mostly known functional targets. A rescue in blood formation was assessed for by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 22 hours post fertilization (hpf). Using this strategy we have identified peroxisome proliferator-activated receptor alpha (PPARα) agonists, most importantly, Clofibrate to rescue βe3 globin expression at 22 hpf in 70 - 90% of moonshine embryos in a dose-dependent manner. To address whether the rescue by Clofibrate is due to its activation of the PPARα receptor, we either knocked down PPARα using morpholinos or treated zebrafish embryos with the PPARα antagonist GW6471. In both cases we observed a significant reduction in Clofibrate-mediated rescue. To identify the PPARα-interacting proteome in an erythroid progenitor context, human K562 erythroleukemia cells expressing doxycyclin-inducible Flag-PPARα were generated. In these cells, PPARα target genes such as ANGPTL4 and PDK4 are activated starting four hours after doxycyclin addition and this activation is significantly reduced in the presence of the PPARα antagonist GW6471. Large-scale Flag-immunoprecipitation followed by mass spectrometric analysis identified the heterodimerization partner of PPARα, RXR, co-activators (NCOA1, NCOA6) and co-repressors (NCOR2), furthermore 24 subunits of the mediator complex, six subunits of the cohesin (loading) complex, seven RNA polymerase (Pol) II subunits as well as the Cyclin T1 subunit of P-TEFb and both subunits (SUPT5H, SUPT4H1) of DSIF, two proteins with a positive role in transcription elongation. In co-immunoprecipitation experiments using K562 cells, doxycyclin-induced PPARα activation leads to an enhanced interaction of Pol II with both, the Cyclin T1 and CDK9 subunits of P-TEFb. Morpholino-mediated knockdown of the mediator complex subunit med1 prevents Clofibrate-mediated rescue of βe3 globin expression in moonshine embryos. Together these data suggest that PPARα functionally interacts with the mediator complex in hematopoietic progenitors, leading to increased recruitment of the transcription elongation factors to lineage-specific genes. Our studies provide a basic understanding of the processes that regulate transcription elongation in the differentiation of hematopoietic cells, and could lead to novel therapeutic strategies for the treatment of blood diseases and leukemia. Disclosures Zon: FATE Therapeutics, Inc.: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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.