Elucidating mechanisms that regulate gene expression in red blood cell production is fundamental to understanding cellular differentiation, and to developing new therapies for red cell disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression. Recently, we and others have identified erythroid-specific enhancer elements through genome-scale profiling of chromatin features. Further analysis has uncovered GATA1-interacting coactivators and their combinations as candidate drivers of enhancer function. These studies established a comprehensive catalog of erythroid enhancer elements, yet the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. Given that enhancers are frequently targeted by disease-associated genetic variations, it is imperative to fill this gap in knowledge. The objective of this project is to determine the protein complexes and long-range DNA interactions responsible for enhancer assembly and function in situ during erythropoiesis. The central hypothesis is that erythroid enhancers are assembled by specific combinations of lineage-regulating transcription factors such as GATA1 to recruit transcriptional coactivators and to initiate long-range chromatin interactions for gene transcription. This hypothesis has been formulated on the basis of our preliminary studies of an erythroid-specific super-enhancer comprised of three individual enhancers that regulates the SLC25A37 gene, a mitochondrial iron transporter essential for iron metabolism and heme synthesis, and an innovative approach to identify macromolecules associated with a single genomic locus using the endonuclease-deficient Cas9 (dCas9) and single guide RNA (sgRNA) components of the CRISPR system. Guided by these preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify and characterize SLC25A37 enhancer-associated proteins in situ by dCas9 affinity purification, followed by quantitative proteomic analysis and validation studies. 2) Determine SLC25A37 enhancer long-range DNA interactions using dCas9-mediated chromatin interaction analysis by paired-end sequencing (dCas9-ChIA-PET). By comparing the frequency and location of chromatin interactions between individual SLC25A37 enhancers and their temporal changes during erythropoiesis, these analyses will establish functional links between chromatin looping and enhancer function. 3) Define the functional requirement of 41 erythroid disease-associated, evolutionarily conserved super-enhancers using an engineered dCas9-LSD1 repressor complex in a dCas9 knockin mouse model. Together these studies will not only elucidate the mechanisms for the genetic control of a principal regulator of erythroid iron metabolism, but also provide new tools for in situ analysis of enhancer- regulating components. Such results are expected to advance our understanding of the composition and function of enhancers in coordinating erythroid gene expression. Ultimately, such knowledge has the potential to inform the design of therapeutic strategies to target non-coding regulatory elements in red cell disorders.

Public Health Relevance

The proposed studies are aimed to better understand how non-coding regulatory DNA sequences contribute to the program of gene expression that orchestrates the production of functional red blood cells. Elucidating the molecular mechanisms that regulate these sequences, termed transcriptional enhancers, will lead to an improved understanding of erythroid cell development and differentiation. Given that enhancers are frequently identified as targets of disease or trait-associated genetic variations, these studies may provide new clues for the design of target-based therapeutics for human red cell disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK111430-04
Application #
9754814
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2016-09-19
Project End
2021-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pediatrics
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Vo, Linda T; Kinney, Melissa A; Liu, Xin et al. (2018) Regulation of embryonic haematopoietic multipotency by EZH1. Nature 553:506-510
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
Huang, Jialiang; Li, Kailong; Cai, Wenqing et al. (2018) Dissecting super-enhancer hierarchy based on chromatin interactions. Nat Commun 9:943
Liu, Xin; Zhang, Yuannyu; Chen, Yong et al. (2018) CAPTURE: In Situ Analysis of Chromatin Composition of Endogenous Genomic Loci by Biotinylated dCas9. Curr Protoc Mol Biol 123:e64
Kim, Jiyeon; Hu, Zeping; Cai, Ling et al. (2017) CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. Nature 546:168-172
Wang, Yuan; Zhang, Yuannyu; Ding, Guanqiao et al. (2017) Temporal dynamics of cardiac hypertrophic growth in response to pressure overload. Am J Physiol Heart Circ Physiol 313:H1119-H1129
Ans├│, Elena; Weinberg, Samuel E; Diebold, Lauren P et al. (2017) The mitochondrial respiratory chain is essential for haematopoietic stem cell function. Nat Cell Biol 19:614-625
Liu, Xin; Zhang, Yuannyu; Ni, Min et al. (2017) Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation. Nat Cell Biol 19:626-638
Luc, Sidinh; Huang, Jialiang; McEldoon, Jennifer L et al. (2016) Bcl11a Deficiency Leads to Hematopoietic Stem Cell Defects with an Aging-like Phenotype. Cell Rep 16:3181-3194