Hematopoiesis is regulated by transcription factors that activate lineage-specific programs. Transcription initiates, and pauses; the pause is then released, a process guided by cdk9, a kinase that phosphorylates Pol II. TIF1? is a chromatin factor that is mutated in the bloodless moonshine zebrafish mutant: we have demonstrated that a mouse knockout of TIF1? has decreased marrow erythropoiesis, no B cells, and expanded myelopoiesis. Using chemical genetics in the zebrafish, we found that clofibrate and leflunomide, activators of the nuclear receptors PPAR? and SXR, rescue blood development in the moonshine mutant. We now propose to examine the genetic program that establishes this rescue, including an evaluation of gene expression in the rescued mutants. We will purify the PPAR? and SXR complexes by affinity tagging and mass spectroscopy, and use zebrafish genetics to establish the downstream pathways that affect the rescue. A prior genetic screen for moonshine demonstrated rescue with mutations in the PAF1 complex or in spt5, both of which regulate transcription elongation. The paf1 zebrafish mutant is rescued by mating to a cdk9 mutant, which suggests that some transcripts hyper-elongate in the paf1 mutant. A recent large-scale knockdown screen for chromatin factors that are required for erythropoiesis during zebrafish development revealed that antisense morpholinos to 6 chromatin factors reduce red blood cell development, and knockdown of 9 chromatin factors increases erythropoiesis: we will assess whether these chromatin factor genes can be rescued by mutations in PAF1, spt5, or cdk9. We will also determine whether activators of specific nuclear hormone pathways can rescue the chromatin factor mutants. Our study will provide a comprehensive analysis of chromatin factors and how they regulate transcription elongation, and will offer the clinical opportunity to modify these pathways for the treatment of blood diseases such as sickle cell anemia and thalassemia. 1
Blood genes are turned on and off by factors that induce DNA to open or close. Gene expression involves an initiation event, followed by a pause ? the pause must then be released, like the brake on a car. This release process is disrupted in many blood diseases. We propose to study the pause release mechanism with a method that uses zebrafish genetics. Specific drugs added to the water also that release the brake: we will evaluate how these drugs work. Other factors open (or unravel) the DNA: we will evaluate zebrafish with mutations in these factors to understand such interactions. Our studies should lead to new strategies for treating patients with a variety of blood diseases. A surprising result of our current work is that transcriptional pausing can be regulated by drug treatments, and manipulation of pausing by compounds could open a new avenue for therapies. This proposal will define new mechanisms of gene expression in blood cells, and this information will be used to define drugs that activate specific blood genes. A mission of NHLBI is to treat diseases such as sickle cell anemia and thalassemia. The drugs could be active in the treatment of these diseases. To date, little is known about transcription pausing in the heart and lung. This platform and technology could form the basis for an understanding of transcription in these organs.
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| Yamauchi, Takuji; Masuda, Takeshi; Canver, Matthew C et al. (2018) Genome-wide CRISPR-Cas9 Screen Identifies Leukemia-Specific Dependence on a Pre-mRNA Metabolic Pathway Regulated by DCPS. Cancer Cell 33:386-400.e5 |
| Gehrke, Jason M; Cervantes, Oliver; Clement, M Kendell et al. (2018) An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol 36:977-982 |
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