25 a Zebrafish Model of Myelodysplastic Syndrome Produced Through Tet2 Genomic Editing

Abstract

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. T ET2 belongs to the TET (ten-eleven translocation) family of methylcytosine oxidases, which require 2-oxoglutarate, oxygen, and Fe(II) for their activity. TET2, like TET1 and TET3, modifies the methylation status of the genome, regulating the transcription of specific genes by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5caC). Each of the last 3 products is recognized and excised by thymine DNA glycosylase (TDG), completing the removal of the 5-methyl group and regenerating unmodified cytosine (1). Hydroxylation of 5mC by the TET enzymes, returning cytosine to its unmethylated state, has been shown to be crucial to many aspects of embryonic development, including embryonic stem cell (ESC) renewal, epigenetic programming of zygotic cells, and meiosis of primordial germ cells (PGCs) (reviewed in references 2 and 3).A variety of alterations, including deletions and missense, nonsense, and frameshift mutations, inactivate the TET2 enzyme in different types of human myeloid malignancies, such as myelodysplastic syndromes (MDS) (25 to 35%) (4-7), myeloproliferative neoplasms (MPN) (2 to 20%) (8, 9), de novo acute myeloid leukemia (AML) (12 to 17%) (10-14), secondary AML (24 to 32%) (11,12), and chronic myelomonocytic leukemia (CMML) (50 to 60%) (5). In these diseases, TET2 gene alterations lead to a marked reduction in global levels of 5hmC (15). TET2 mutations have also been identified in the hematopoietic cells of otherwise healthy adults over 50 years of age who have "clonal skewing" of their bone marrow cells (16), indicating that TET2 mutations may represent one of the first mutations leading to clonal expansion and the eventual development of myeloid malignancies.The role of TET2 mutations in myeloid malignancies has been studied in a number of mouse models (17-20). The hematopoietic stem cells (HSCs) in these models have low 5hmC content and exhibit increased self-renewal ability and a competitive advantage over wild-type HSCs for repopulating hematopoietic lineages. Tet2 knockout mice are viable and fertile and appear to develop normally. However, as they age, Tet2-deficient mice are prone to develop myeloid malignancies, predominantly CMML, with 20 to 30% developing disease after 8 months of age, clearly suggesting that additional genetic lesions are needed to initiate myeloid malignancy.Thus, the essential role of TET2 in maintaining the normal growt...

DNA methylation and hydroxymethylation in stem cells

“…It has been found that the status of 5hmC and demethylation in the intermediate CpG promoters (ICPs) of those lineage‐specific genes, particularly at specific regions or CpG sites of these ICPs, significantly affects erythropoiesis . In contrast, a subsequent similar study, based on zinc finger nuclease technology, revealed neither change in Cmyb and CD41 expression nor defect in embryonic erythropoiesis . The homozygous Tet2 m / m zebrafish lines showed decreased levels of 5hmC specifically in haematopoietic cells of the kidney marrow rather than other cell types, which indicates the unique function of TET2 in haematopoietic cells that cannot be compensated for by other TETs .…”

Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis

“…Recently, the first zebrafish models of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) were reported. 59,60 Gjini and colleagues studied zebrafish with homozygously targeted tet2 mutation often found in MDS. The authors used zinc finger nuclease technology to establish a stable zebrafish line with loss-of-function mutations in the tet2 gene and characterized a phenocopy of the human MDS condition in these fish after 24 months, with cytopenias and dysplastic cell populations in the kidney marrow.…”

“…The authors used zinc finger nuclease technology to establish a stable zebrafish line with loss-of-function mutations in the tet2 gene and characterized a phenocopy of the human MDS condition in these fish after 24 months, with cytopenias and dysplastic cell populations in the kidney marrow. 59 Tet2 mutations alone in human patients often result in clinically benign clonal hematopoietic proliferation, and require secondary and sometimes tertiary mutations for progressing into clinical apparent MDS/AML. Given the length of time the tet2-mutated state required to have apparent manifestation, it is possible that secondary mutations could have been acquired, similar to the pathophysiology of MDS in humans.…”

Application of genome editing technologies to the study and treatment of hematological disease

“…The MDS comprise a group of clonal HSC malignancies characterized by ineffective hematopoiesis leading to peripheral blood cytopenias, and a progressive increase in bone marrow blasts leading to the development of AML (Delhommeau et al, 2009;Fragoso and Barata, 2014;Greenberg et al, 2002;Heaney and Golde, 1999;Jhanwar, 2015). Gjini et al used ZFN genome editing technology to disrupt the Tet2 catalytic domain in zebrafish (Gjini et al, 2015). Homozygous tet2 mutant zebrafish showed normal embryonic and larval hematopoiesis, but developed progressive clonal MDS as they aged, with myeloid and erythroid dysplasia.…”

“…Homozygous tet2 mutant zebrafish showed normal embryonic and larval hematopoiesis, but developed progressive clonal MDS as they aged, with myeloid and erythroid dysplasia. As homozygous tet2 mutant zebrafish are viable and fertile, this mutant line provides an ideal model for phenotype-based small molecule drug screens to identify compounds with specific activity against tet2 mutant cells (Gjini et al, 2015).…”