Enhancers are non-coding DNA elements near genes that positively regulate gene expression. Because enhancers often act in a cell type specific manner, they confer biological specificity on gene regulation. Common genetic variation within enhancers appears to be a prevalent means of trait association. Super-enhancers are a recently described set of enhancers highly enriched for regulatory elements of cell lineage-defining genes and genetic variants related to lineage-specific disease susceptibility. However the structural determinants of super-enhancer function remain poorly understood. Enhancers are typically defined by their correlated biochemical features or ectopic potential in reporter assays. Nonetheless the gold-standard test of enhancer activity is loss-of-function mutation to discern the requirement for regulatory sequences in their natural chromosomal environment. Our group has developed a Cas9-mediated in situ saturating mutagenesis technique that allows for high-resolution, high-throughput functional analysis of enhancers in the native genomic setting. The assay involves design and synthesis of guide RNAs saturating a region of interest followed by pooled lentiviral CRISPR screening. This method when coupled with genomic target deep sequencing allows for an evaluation nearing nucleotide resolution of the functional sequences critical for enhancer function. In this proposal, we apply this technique to systematically perturb twenty erythroid-specific super-enhancers of key erythroid genes. We extend the Cas9 mutagenesis strategy by making use of a variant Cas9 to allow for increased genome editing resolution. In addition, we take a variant-informed guide design approach to improve mutagenesis even in the face of natural genomic variation. We utilize bioinformatic methods to identify essential sequences, predict interacting transcription factors, and refine models to estimate enhancer activity. We use biochemical techniques to determine the occupancy of transcription factors and chromatin regulators at required sequences. We focus on defining and testing super-enhancer looping interactions in which the key transcription factors GATA1 and TAL1 participate. Finally we employ prospective reverse genetics to validate both cis-acting sequences and trans-acting factors necessary for super-enhancer function. These studies will help determine emergent cooperative properties of clustered components within super-enhancers. We expect these experiments to inform a further understanding of crucial aspects of erythropoiesis as well as fundamental mechanisms of gene regulation.
More than 98% of the human genome does not encode for genes, though the majority of common genetic variants associated with disease susceptibility are found in these non-coding sequences. Disease- associated variants are highly enriched in enhancers, the switches that determine in which cell types genes are active. This project is designed to apply cutting-edge genome editing technologies and hypothesis-driven biochemical methods to discover the mechanisms whereby highly active enhancers known as super- enhancers determine red blood cell identity.
|Esrick, Erica B; Bauer, Daniel E (2018) Genetic therapies for sickle cell disease. Semin Hematol 55:76-86|
|Yien, Yvette Y; Shi, Jiahai; Chen, Caiyong et al. (2018) FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity. J Biol Chem 293:19797-19811|
|Wattrus, Samuel J; Zon, Leonard I (2018) Stem cell safe harbor: the hematopoietic stem cell niche in zebrafish. Blood Adv 2:3063-3069|
|Uenishi, Gene I; Jung, Ho Sun; Kumar, Akhilesh et al. (2018) NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells. Nat Commun 9:1828|
|Yu, Shan-He; Zhu, Kang-Yong; Zhang, Fan et al. (2018) The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression. Biochim Biophys Acta Gene Regul Mech 1861:106-116|
|Parada-Kusz, Margarita; Penaranda, Cristina; Hagedorn, Elliott J et al. (2018) Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies. Dis Model Mech 11:|
|Rost, Megan S; Shestopalov, Ilya; Liu, Yang et al. (2018) Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish. Blood Adv 2:3418-3427|
|Lahvic, Jamie L; Ammerman, Michelle; Li, Pulin et al. (2018) Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proc Natl Acad Sci U S A 115:9252-9257|
|Liu, Nan; Hargreaves, Victoria V; Zhu, Qian et al. (2018) Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell 173:430-442.e17|
|Whitman, Jared C; Paw, Barry H; Chung, Jacky (2018) The role of ClpX in erythropoietic protoporphyria. Hematol Transfus Cell Ther 40:182-188|
Showing the most recent 10 out of 215 publications