Phenotypic and molecular consequences of coding and noncoding TET2 perturbations in the human hematopoietic system

Abstract

The hematopoietic or blood system comprises trillions of specialized cells critical to sustaining life, all derived from the same multipotent progenitor known as the hematopoietic stem cell (HSC). One central question in the study of hematopoiesis is how the HSC pool is maintained and faithfully differentiates into all components of the blood. HSCs rely on epigenetic modifications to regulate the balance of self-renewal and differentiation during blood production. As such, mutations in epigenetic regulatory proteins are overrepresented in blood diseases, including cancer. This thesis examines the function of one of these critical epigenetic factors, known as ten-eleven translocation methylcytosine dioxygenase 2 (TET2). TET2-inactivating mutations contribute to epigenetic changes and disrupt gene regulation, but the phenotypes and molecular mechanisms underlying their contribution to blood malignancies in humans remain limited.

For this reason, we generated a human primary cell model of TET2 deficiency using CRISPR-Cas9 genome editing in CD34+ hematopoietic stem and progenitor cells (HSPCs). We show that TET2 inactivity leads to an increased frequency in human HSPCs and a clonogenic bias to monocytic progenitors using immunophenotyping and colony-forming unit assays respectively. We then demonstrate that these phenotypes are associated with accessible chromatin changes affecting predominately cell morphogenesis and immune-related genes using the assay for transposable-accessible chromatin with sequencing (ATAC-seq). Bioinformatic analyses revealed that these regions were specifically enriched for binding motifs of CTCF and ETS family factors, a set of CpG-rich sequences that are demethylated during hematopoiesis for proper gene regulation. Similar molecular alterations were observed in humans harboring naturally occurring germline TET2 variants using a peripheral leukocyte DNA methylation array. These variants were also associated with an increased risk of developing clonal hematopoiesis (a pre-malignant HSC state characterized by increased mutant cell fitness). Lastly, we demonstrate that disruptions of a TET2 enhancer resemble phenotypes from TET2 coding sequence mutations in human HSCs. This suggests an additional mechanism for disease predisposition. Collectively, these results affirm that TET2 regulates the balance between progenitor self-renewal and differentiation in a coordinated fashion with various transcription factors and chromatin regulators.

Next, we extended our investigation to red blood cell progenitors (erythroid) using the human umbilical cord CD34 cell-derived HUDEP-2 cell system. We demonstrate that loss of TET2 impairs their differentiation and impacts the expression of critical erythroid genes using immunophenotyping, morphological assessments, and gene expression profiling. These genes notably include HBA1 and HBB (adult globin subunits), ALAS2 (heme synthesis factor), GYPA (glycoprotein), and SCL/TAL1 (a master transcription factor). The phenotypic consequences of TET2 disruption were not attributable to changes in apoptosis, cell cycling, or ATP-dependent cell viability, indicating an additional mechanism that requires further investigation. Together, these results prove that TET2 is required for normal erythropoiesis and hemoglobin production.

In conclusion, our research has led to a better understanding of the function of TET2 during human hematopoiesis through leveraging recent advances in computational and experimental approaches. We hope this dissertation will guide future investigations of TET2 and have a positive impact on blood cancer research.