Restoration of genome-wide 5-hydroxymethylation by azacitidine and ascorbate in TET2-deficient human pre-leukemic HSPCs
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA817283 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18361300_1.fastq.gz | Fastqsanger.gz | |||
| SRR18361300_2.fastq.gz | Fastqsanger.gz | |||
| SRR18361400_1.fastq.gz | Fastqsanger.gz | |||
| SRR18361400_2.fastq.gz | Fastqsanger.gz | |||
| SRR18361500_1.fastq.gz | Fastqsanger.gz |
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Project description: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 serial replating and competitive advantage, defective erythroid/megakaryocytic differentiation, and in vivo myeloid skewing coupled with reduction of 5hmC at erythroid regulators. Azacitidine and ascorbate restored 5hmC abundance and slowed/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 treat pre-leukemia/clonal hematopoiesis and prevent leukemogenesis.
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Project description:Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA.
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Project description:Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA.
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Project description:Ascorbate (vitamin C) is an important antioxidant in leaves with multiple functions in photosynthesis and its levels are tightly regulated by light quality. The majority of work to date has focussed on regulation of ascorbate levels via biosynthesis. However, the pathway of ascorbate degradation, which is localised in the apoplast may additionally play a significant role. The degradative pathway begins with ascorbate oxidation by the enzyme ascorbate oxidase (AO) and hence we have examined the impact of manipulation of ascorbate oxidase activity on the content of ascorbate and the acclimation of photosynthesis to changing light levels. Leaf ascorbate content was highly light dependent in Nicotiana tobaccum being double in high compared with low light conditions. In contrast ascorbate oxidase activity was not altered by changing light levels. Transgenic tobacco lines with reduced or enhanced AO activity exhibited no morphological phenotype and photosynthesis under high light was similarly impaired irrespective of the measured AO activity. However further investigation between AO activity and leaf acclimation to changing light intensity revealed a suite of metabolic and transcriptome changes indicating a role for the apoplastic ascorbate pool in chloroplast to nucleus communication. In all genotypes, transcripts associated with ascorbate synthesis and recycling were less abundant following transition from high to low light. However transcripts associated with glutathione recycling, light harvesting, reaction centre function and the Calvin cycle were all increased in abundance. Following the transition from high to low light increases in leaf threonate, an ascorbate degradation product, were proportional to AO activity. A number of chloroplast synthesised amino acids were significantly increased both during high light and following transfer back to LL for 12 h. Similarly, several primary carbohydrates and TCA intermediates were significantly enhanced following high light treatment. In addition to light effects, AO activity had a significant impact on ï¢-alanine, aspartate and threonine. Intriguingly, a large number of fatty acids were reduced in antisense AO and wild-type plants immediately after high light stress however the content of these compounds were significantly increased in overexpressing sense lines. We conclude that AO mediated turnover plays an important role in adjusting cellular ascorbate content to photosynthetic need in a fluctuating light environment and that light mediated signals rapidly adjust that the apoplastic ascorbate pool.
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Project description:Ascorbate is the most abundant water-soluble antioxidant in plants, and it is an essential molecule for normal plant development. It is present in all green plants, with very different concentrations in different plant species. While ascorbate accumulation is a trait of nutritional, and therefore, agronomical interest, the impact of different concentrations over cellular homeostasis remains elusive. In order to shed light over this question, we leveraged Arabidopsis lines with very low ascorbate (vtc2 mutant with 20% of WT ascorbate levels), and low ascorbate concentration (vtc4 mutant with 65% of WT levels), and we generated a line that accumulates 165% of WT levels (vtc2/OE-VTC2). An 80% reduction of ascorbate increased the expression of genes implicated in defense against pathogens, but repressed genes associated with abiotic stress responses. Unexpectedly, lines with increased (165% of WT) and decreased (65% of WT) ascorbate levels shared 85% of induced transcription factors and the GO terms associated with their transcriptional programs. Among the group of genes whose expression is positively correlated with ascorbate content, we identified TAA1/WEI8, a gene encoding a tryptophan aminotransferase that catalyzes the first step of auxin biosynthesis. Using a combination of genetic and pharmacological approaches in fluorescent and histochemical reporter lines for auxin biosynthesis and signaling activity, we revealed that TAA1- and TAA1-RELATED2 (TAR2)-mediated auxin biosynthesis is necessary for plants to cope with increased ascorbate concentration in a light-dependent manner, revealing a new layer of complexity in the regulatory landscape of redox homeostasis.
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Project description:Clinical response to azacitidine in MDS is associated with distinct DNA methylation changes in HSPCs
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Project description:Cytopenia in at least one of the hematopoietic lineages (RBCs, WBCs or platelets) is a hallmark feature of MDS, indicating the lack of ability to support HSPC proliferation and the retention of their multilineage potential in the bone marrow. Here, wel investigate MDS-MSCs (with and without 5-azacitidine pre-treatment) as feeder layers for healthy CD34+ HSPCs. We performed RNA sequencing of co-cultured CD34+ HSPCs to compare their transcriptomic signatures after exposure to treated vs untreated MDS-MSCs.
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Project description:Clinical response to azacitidine in MDS is associated with distinct DNA methylation changes in HSPCs [Bisulfite-Seq]
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Project description:Clinical response to azacitidine in MDS is associated with distinct DNA methylation changes in HSPCs [RNA-Seq]
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Project description:Ascorbate activates CD30 expression and causes widespread specific demethylation of the epigenome of serum free cultured hESC. The genetic and epigenetic integrity of human Embryonic Stem cells (hESCs) is critical to their future applications in research and medicine. hESC cultured in serum free media can accumulate point mutations, aneuploidy and progressive epigenetic changes over prolonged culture in vitro. We have identified ascorbate as the only molecule in the very widely used Knock-out serum replacement medium that is sufficient to induce expression of CD30, a biomarker for aneuploidy in hESCs. In fact, we show that hESC cultured in the presence of ascorbate for 20 passages not only display demethylation of the CD30 locus, but exhibit widespread and remarkably specific and consistent demethylation of 1,847 genes in both HES2 and HES3 cells. The specific ascorbate induced demethylation changes in the hESC epigenome, of which 86% are shared between the two lines, identify a subset of genes in hESC, including CD30, that are sensitive to serum free culture medium induced epigenetic changes. Experiment. HES2 cells were maintained on 20% Fetal calf serum on mouse embryonic fibroblast feeder layers with mechanical dissection. Using HES2 after 99 mechanical passages (P99), hESC were grown for 17-20 (+17 - +20) passages in the presence of 20% Knock-Out Serum Replacement (Invitrogen) either with (+ASC) or without (-ASC) Ascorbate with enzymatic culturing.
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