Altered Translation of GATA1 in Diamond-Blackfan Anemia and the Role of Cyclin D3 in Erythropoiesis
Year: 2015
Type: dissertation
Abstract: Advances in genomic technology have led to the identification of numerous genetic variants that are associated with specific phenotypes. Whole-exome sequencing is often applied to identify rare variants in the context of Mendelian disorders, whereas genome-wide association studies (GWAS) have identified thousands of common variants associated with specific, quantifiable human traits. In both instances, identifying the specific causal variant and subsequently determining its mechanism of action, even on a small scale, remains difficult. In this thesis, we took advantage of the recent identification of the first non-ribosomal protein gene mutation found in a family with Diamond-Blackfan anemia (DBA). We followed up on the molecular etiology of these patients and discovered new insights with respect to the pathophysiology of DBA. Furthermore, we functionally investigated the role of common genetic variation on red blood cell traits, leading to new biological insights of cell cycle regulators in terminal erythroid differentiation. In contrast to the more subtle effects in phenotype due to common genetic variation, rare genetic variants associated with Mendelian disorders are often deleterious and may require a significant amount of clinical attention. DBA is a rare congenital disease and is considered a disorder of the ribosome, as over 50 % of patients have heterozygous loss of function mutations in ribosomal protein genes, resulting in ribosomal haploinsufficiency. Although ribosomal proteins are ubiquitously expressed, the cell-type specific defect in the form of the anemia has puzzled researchers. Recently, in rare cases of DBA, mutations in the essential erythroid transcription factor GATA1 were described, but it remained unclear whether these patients shared a common pathophysiology as those with ribosomal haploinsufficiency. We show that ribosomal haploinsufficiency leads to impaired translation of GATA1 mRNA and connect these two seemingly disparate sets of molecular lesions. We suggest that the amount of functional ribosomes is depleted in DBA, decreasing the overall translation initiation capacity. This is supported by specific inhibition of translation initiation by the small molecule 4EGI-1, phenocopying many in vitro aspects of DBA. We attribute the apparent selective translational defect to a highly structured 5’ untranslated region of the GATA1 mRNA, restricting its efficient translation when the cellular translation initiation potential is impaired. Most importantly, analysis of transcriptional signatures of primary erythroid patient cells with ribosomal protein mutations supports impairment in GATA1 transcriptional activity in DBA, and ectopic GATA1 expression in these cells greatly improved impaired erythropoiesis. These findings have led to new insights into the pathophysiology of DBA and provide not only an explanation for the specificity of the observed defect but suggest potential therapeutic avenues such as GATA1 gene therapy approaches. More generally, this work implicates selectively impaired translation of specific transcripts as a mechanism of human disease. GWASs have been enormously successful at identifying genetic associations but usually provide limited immediate insight to the underlying biology of these associations. By following up on genetic variants associated with red blood cell count and the average volume of a red blood cell, we identified an unexpected role in terminal erythropoiesis for a common cell cycle regulator. The causal variant mapped to a transcription factor binding site bound by erythroid transcription factors. Reporter gene assays showed differential enhancer activity for the associated alleles, suggesting altered transcriptional regulation of the nearby gene, cyclin D3. By following up on cyclin D3, a detailed analysis showed that downregulation of cyclin D3 levels lead to premature cell cycle arrest, without ostensible defects in differentiation. As a result, erythroid progenitor cells underwent fewer cell divisions and remained larger in size, preserving overall hemoglobin levels. Collectively, these data suggest that varying levels of cyclin D3 in terminal erythropoiesis contribute to the natural variation observed in red blood cell parameters in the human population. Together, both studies illustrate how the functional follow up of rare and common human genetic variation leads to new insights and a deeper understanding of the underlying biology that effect basic cellular processes such as cell cycle regulation and translation in physiology and disease.
Author: Ludwig, Leif
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1864616354