Flipping the Hemoglobin Switch and Discovering Regulators Involved in Fetal Hemoglobin Reactivation

The fetal to adult hemoglobin switch is a developmental process by which fetal hemoglobin becomes silenced after birth and replaced by adult hemoglobin. Diseases caused by defective or missing adult hemoglobin, such as Sickle Cell Disease or β-Thalassemia, can be ameliorated by reactivating fetal hemoglobin. We discovered that knockdown or knockout of β-globin, a subunit of adult hemoglobin, led to robust upregulation of γ-globin, a subunit of fetal hemoglobin. This phenomenon suggested that red blood cells have an inherent ability to upregulate fetal hemoglobin in the event that adult hemoglobin is lacking.We developed multiple gene-editing tools in an immortalized erythroid cell model to investigate the molecular mechanisms behind the increase in fetal hemoglobin. Time-course transcriptomics identified ATF4, a transcription factor, as a causal regulator of this response. Further analysis also converged upon downregulation of MYB and BCL11A, known repressors of γ-globin, described in detail in chapter 2. Further work in chapter 3 explores other possible fetal hemoglobin regulators as discovered by CRISPRi arrayed mediated knockdown experiments. This work furthers our understanding of fundamental mechanisms of gene regulation and how cellular and molecular events influence red blood cell differentiation

Flipping the Hemoglobin Switch and Discovering Regulators Involved in Fetal Hemoglobin Reactivation

The fetal to adult hemoglobin switch is a developmental process by which fetal hemoglobin becomes silenced after birth and replaced by adult hemoglobin. Diseases caused by defective or missing adult hemoglobin, such as Sickle Cell Disease or β-Thalassemia, can be ameliorated by reactivating fetal hemoglobin. We discovered that knockdown or knockout of β-globin, a subunit of adult hemoglobin, led to robust upregulation of γ-globin, a subunit of fetal hemoglobin. This phenomenon suggested that red blood cells have an inherent ability to upregulate fetal hemoglobin in the event that adult hemoglobin is lacking.We developed multiple gene-editing tools in an immortalized erythroid cell model to investigate the molecular mechanisms behind the increase in fetal hemoglobin. Time-course transcriptomics identified ATF4, a transcription factor, as a causal regulator of this response. Further analysis also converged upon downregulation of MYB and BCL11A, known repressors of γ-globin, described in detail in chapter 2. Further work in chapter 3 explores other possible fetal hemoglobin regulators as discovered by CRISPRi arrayed mediated knockdown experiments. This work furthers our understanding of fundamental mechanisms of gene regulation and how cellular and molecular events influence red blood cell differentiation

Engineering of the endogenous HBD promoter increases HbA2

The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.ISSN:2050-084

ATF4 Regulates MYB to Increase γ-Globin in Response to Loss of β-Globin

© 2020 The Authors β-Hemoglobinopathies can trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult β-globin. We find that decreased β-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon β-globin knockout decreases the levels of MYB and BCL11A. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could inform strategies to treat hemoglobinopathies.ISSN:2666-3864ISSN:2211-124

Discovery of stimulation-responsive immune enhancers with CRISPR activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs