Tet2 Loss in Hematopoietic Stem Cells Triggers Chromatin Reorganization through DNA Methylation Shifts

Introduction: Loss of function (KO) mutations in TET2 (ten-eleven translocation 2) are the most common epigenetic modifier mutations in Myelodysplastic Syndromes (MDS). TET2 catalyzes conversion of 5-methylcytosine to 5-hydroxymethytosine. Its loss is hence thought to alter the epigenetic landscape in long-term hematopoietic stem cells (LT-HSCs), thereby changing gene expression and leading to improved clonal fitness. Changes to DNA methylation can impact higher-order chromatin organization, with profound effects on transcriptional output (Shen & Laird, Cell 2013). Testing this paradigm in LT-HSCs has been challenging given the large number of cells needed for unbiased studies of chromatin organization.

Methods: To obtain sufficient numbers of purified HSC populations for Hi-C and correlative assays, we optimized an ex-vivo expansion protocol (Wilkinson et al, Nature 2019). This protocol which employs polyvinyl alcohol and a defined cytokine cocktail in serum-free media resulted in a 500-fold expansion of phenotypically homogeneous murine LT-HSCs (CD150+Lin-Sca1+cKit+) over 14 days, generating ~5 million cells per adult mouse. We performed a low-input HiC protocol (tagHiC; Zhang et al, Cell Rep 2020) with 0.5 million ex-vivo expanded WT and Tet2 KO (Ko et al, PNAS 2011) murine LT-HSC cells. TagHiC libraries were sequenced to ~700 million paired-end reads per sample, generating over 300 million valid junction reads, sufficient for a 20-40 kb Hi-C data resolution. Valid read pairs thus determined were analyzed for chromatin compartments: A (or euchromatic) and B (or heterochromatic) and Topologically Associated Domains (TADs) using HiC-Pro and HiC Explorer packages. TAD-separation scores, a measure of the strength of TAD insulation (higher scores reflecting stronger boundaries), were also calculated. Additionally, we compared the methyl-seq data between WT and Tet2 KO conditions (Hon et al., Mol Cell 2014), for determining hypermethylated differentially methylated regions (hyperDMRs) and hypomethylated DMRs (hypoDMRs) in the Tet2 KO state. DMRs were intersected with A/B compartment regions and TAD data to assess differences in A/B compartment shifts and TAD-separation scores between the hypo- and hyper- DMR regions.

Results: Marked A/B compartment shifts and changes in TAD-separation scores were evident in Tet2 KO cells. HypoDMRs in Tet2 KO exhibited 6016 A-to-B shifts and 8627 B-to-A shifts, while hyperDMRs in Tet2 KO showed 1900 A-to-B shifts and 1705 B-to-A shifts. The Fisher’s Exact Test for A-to-B shifts showed an odds ratio of 0.63 (p = 3.53e-36), indicating that hypoDMRs are less likely to shift from A to B compared to hyperDMRs. Conversely, for B-to-A shifts, the odds ratio was 1.60 (p = 3.53e-36), suggesting that hypoDMRs are more likely to shift from B to A than hyperDMRs. The compartment shifts indicate that hyperDMRs are significantly more likely to transition from A-to-B compartments in TET2 KO cells, indicating reduced chromatin activity and gene repression.

Analysis of TAD-separation scores revealed that hyperDMR intersection regions (n=16737) in WT cells had a median score of 0.12, which reduced to 0.06 in mutant cells (t-stat: 24.5, p = 2.01e-131). Conversely, in hypoDMR intersection regions (n=57855), WT cells showed a median score of -0.06, which increased to 0.0 in mutant cells (t-stat: -7.9, p = 1.50e-15). These indicate that hyperDMR-containing TAD regions exhibit reduced separation in TET2 KO, suggesting disruption of TAD boundaries, while hypoDMR-containing TADs showed increased separation, indicating stronger TAD boundaries.

Conclusion: Our ex-vivo expansion strategy enables high-resolution HiC analysis of LT-HSCs. Using this method, we showed that Tet2 loss reorganizes chromatin compartments, making hyperDMRs more inactive and hypoDMRs more active. Our findings strongly suggest that Tet2’s effect on DNA methylation influences higher-order chromatin compartmentalization. Disruption of TAD boundaries compromises gene-regulatory networks, potentially leading to pathogenic enhancer-promoter (EP) interactions. Ongoing work will integrate epigenetic marks (histone), chromatin accessibility (ATAC-seq), and methylation (Ox-BS/BS-seq) to further understand how rewiring of EP interactions across weakened TAD boundaries causes dysregulated gene expression to confer clonal advantage in Tet2 KO.