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Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

ABSTRACT: BACKGROUND:The 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation. RESULTS:We used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC. CONCLUSION:The present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease.

SUBMITTER: Cakouros D

PROVIDER: S-EPMC6317244 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

Cakouros Dimitrios D Hemming Sarah S Gronthos Kahlia K Liu Renjing R Zannettino Andrew A Shi Songtao S Gronthos Stan S

Epigenetics & chromatin 20190103 1

<h4>Background</h4>The 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation.<h4>Results</h4>We used siRNA knoc ...[more]

PMID: 30606231

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Project description:The TET dioxygenases erase mediate DNA dedemethylation in pre-implantation embryos and in primordial germ cells, yet limited studies address their contribution to the global gain of DNA methylation following implantation. Here, we show that Tet1 is expressed and non-redundantly contributes to 5-hydroxymethylctyosine (5hmC) non-redundantly in the pre-gastrulation mouse epiblast. Ablation of Tet1 in primed epiblast cells results in widespread loss of 5hmC associated with gain of 5-methylcytosine at CpG islands and promoters. Moreover, Tet1 is expressed, albeit at lower levels, in the extra-embryonic ectoderm. Tet1-deficiency in the pre-streak mouse embryos causes dysregulation of early lineage regulators in the epiblast and increased expression of metabolic genes in the extra-embryonic ectoderm. Our studies reveal a distinct role of Tet1 in regulating the methylome landscape of the post-implantation mammalian epiblast and a hitherto unknown gene repressive effect in the extra-embryonic lineage, providing insights into the early developmental origins of epigenetic-based basis of imprinting and developmental disorders. Lysates of E6.25 epiblast (E) and extra-embryonic (ExE) tissues were pooled, based on retrospective genotyping, from at least 6 embryos (collected from 2-3 litters). Four pooled biological replicates were collected for each tissue compartment and each genotype, i.e. wild type (wt), Tet1gt/wt (Zwt), Tet1gt/gt(ZZ); in 3 of 4 pooled replicates, the E and ExE samples were obtained from the same litters and given the same numbering. One ExE sample of ZZ was lost. RNA was purified using the RNeasy micro Kit (Qiagen) to obtain 1-10 ng of RNA per pool. RNA quality was assessed using a Bioanalyzer 6000 Pico chip and only samples with RNA integrity number (RIN) > 8.0 were processed further. First-strand cDNA synthesis and amplification was performed by using the SMARTer Ultra Low InputÂ RNA amplification kit v3 (Clontech). Libraries were prepared using NEBNext Ultra DNA library prep for Illumina and sequenced on NextSeq 500 in high-output to generate 75-bp single-end reads.Â Â Low quality ends and adapter sequences wereÂ trimmed off from the Illumina reads withÂ FastX 0.0.13 andÂ cutadaptÂ 1.7.1. Using FastX andÂ ShortRead 1.16.3, we filtered subsequently small readsÂ (length < 35 bp), polyA-reads (>90% of the bases equal A),Â ambiguous reads (containing N) and lowÂ quality reads (>50% of theÂ bases < Q25).

Dataset Information

Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

Publications

Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

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