Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders

Abstract

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.GATA1 | cis-regulatory elements | noncoding mutations | Mendelian erythroid disorders W hole-exome sequencing (WES) and targeted sequencing approaches have greatly accelerated our ability to identify causal genetic lesions in both previously implicated and novel genes underlying monogenic disorders (1, 2). In hematology, WES has been extremely useful for identifying unknown genetic etiologies for various disorders, such as those affecting red blood cell (RBC) production, including Diamond-Blackfan anemia and congenital dyserythropoietic anemia (3-5), disorders of RBC structure and function (6, 7), and disorders affecting other aspects of hematologic function (2, 8). Despite this considerable success, however, more than 50% of cases of presumed monogenic diseases are refractory to current WES approaches (9). Although resolving these remaining cases will benefit from improvements in exome capture (10), read alignment (11), and variant annotation methodologies (11), the importance of genetic variation occurring within regulatory elements (REs) outside of the traditionally investigated coding sequences in hematologic and other diseases is being increasingly appreciated (12).Whole-genome sequencing (WGS) approaches are becoming progressively more available and affordable, but separating pathogenic genetic variation from benign or unrelated mutations remains especially difficult outside of protein-coding gen...

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

“…To identify the gene(s) whose expression is modulated by rs78744187 to influence basophil production, we performed in situ perturbation of the +39-kb enhancer using CRISPR/Cas9-mediated mutagenesis in CD34 + human hematopoietic stem and progenitor cells (HSPCs) (43). We targeted the +39-kb enhancer using two guides that flank the rs78744187 variant (Fig.…”

The genetics of human hematopoiesis and its disruption in disease

“…On one hand, GATA1 bound cis‐regulatory elements (CREs) are frequently impacted by common genetic variation associated with hematopoietic traits, but these variants primarily tune the activity of such CREs with effect sizes below what is required to cause frank disease (Ulirsch et al , ). On the other end of the spectrum, mutations disrupting the GATA1 motif in critical CREs can cause a variety of monogenic blood disorders due to impaired gene expression (Manco et al , ; Campagna et al , ; Kaneko et al , ; Wakabayashi et al , ). Moreover, mutations in GATA1 itself can result in a range of phenotypes that include a complete absence of erythropoiesis, as is the case in DBA, or more subtle defects in red blood cell and platelet production (Crispino & Horwitz, ; Abdulhay et al , ).…”

Concise Review: Epigenetic Regulation of Hematopoiesis: Biological Insights and Therapeutic Applications

“…More recently, CRISPR‐Cas9‐mediated in situ disruption of the GATA1 motifs present in the ALAS2 , UROS , and PKLR regulatory regions reproduced the gene downregulation observed in patients harboring mutation of these binding sites . Interestingly, the disruption of the GATA1 motifs seems to affect the binding of the GATA1‐dependent activation complex .…”

“…Genetic variants mapping to cis ‐regulatory elements can be experimentally validated to unravel the molecular mechanisms underlying the observed phenotypes. They can affect enhancer and promoter activity, for example, by either disrupting or creating de novo binding sites for transcription factors, thus deregulating the expression of the target genes (Figs. ).…”