The Cell Type–Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia

Abstract

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven-translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human HSPCs. Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of pre-leukemia/clonal hematopoiesis.

Myelodysplastic Syndrome associated TET2 mutations affect NK cell function and genome methylation

“…4b , lower panel, Supplementary Data 3 ), methylation of TYROBP (coding for DAP12) and TNF was predicted to significantly inhibit the NK cell-mediated cytotoxicity pathway as defined by the KEGG database ( p < 0.05, KEGG pathway hsa04650) 25 . Reports suggested variations in methylation by TET2 of the CpG loci according to their position regarding the gene body and the respective regulatory elements 24 , 26 , 27 . We analyzed the percentage of 5hmC in the gene bodies and 10 kb upstream or downstream on the whole genome 26 .…”

“…Reports suggested variations in methylation by TET2 of the CpG loci according to their position regarding the gene body and the respective regulatory elements 24 , 26 , 27 . We analyzed the percentage of 5hmC in the gene bodies and 10 kb upstream or downstream on the whole genome 26 . Then, the mean of methylation has been calculated for genes involved in pathways of interest for NK cell biology.…”

“…From the mechanistic point of view, our results are reminiscent of the work by Wu et al describing the role of the ascorbic acid in promoting KIR locus demethylation through the mobilization of Runx3, TET2, and TET3 22 . Interestingly, it has been recently shown that TET2 KO HSC treated with ascorbic acid could increase the 5hmC levels suggesting a role for TET proteins other than TET2 in this rescuing 26 . Herein, we showed that the addition of ascorbic acid to NK cells allows the restoration of KIR expression.…”

Clonal Hematopoiesis: Role in Hematologic Non-Hematologic

“…TET2 mutations in primary human HSCs/HPCs determine an increased self-renewal activity, increased colony-forming activity in vitro , defective erythroid/megakaryocytic differentiation, and myeloid skewing, associated with a decrease of 5hmC at the level of erythroid-associated gene loci. 253 The study of the methylation profile of CHIP and CCUS subjects provided evidence that TET2 mutations are associated with DNA hypermethylation at enhancer DNA sites: the hypermethylated sites are functionally related to genes involved in leukocyte function and immune response and to ETS-related and C/EBP-related transcription factor motifs. Most TET2-associated hypermethylation sites are shared between CHIP and AML; some AML-specific hypermethylation sites are located at active enhancer DNA elements in HSCs.…”

Viewing AML through a New Lens: Technological Advances in the Study of Epigenetic Regulation

“…The new models used to address these questions in the context of DNA methylation in human cells rely on CRISPR-Cas9 editing using ribonucleoproteins (RNPs) delivered through nucleofection or electroporation. The deletion of TET2 in cultured umbilical cord blood CD34+ cells, a rich source of HSCs, has been used to model the loss-of-function mutations commonly found in CHIP and myeloid malignancies [ 65 , 66 ]. These models show a competitive advantage of TET2 -edited cells, with increased myeloid differentiation and reduced 5hmC levels at the promoters and enhancers of genes regulating erythroid differentiation (e.g., leading to a decreased expression of GATA1 and KLF1 ) ( Figure 1 b).…”