Dataset Information

Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases.

ABSTRACT:

Background

Ectopic expression of a defined set of transcription factors allows the reprogramming of mammalian somatic cells to pluripotency. Despite continuous progress in primate and rodent reprogramming, limited attention has been paid to cell reprogramming in domestic and companion species. Previous studies attempting to reprogram canine cells have mostly assessed a small number of presumptive canine induced pluripotent stem cell (iPSC) lines for generic pluripotency attributes. However, why canine cell reprogramming remains extremely inefficient is poorly understood.

Methods

To better characterize the initial steps of pluripotency induction in canine somatic cells, we optimized an experimental system where canine fetal fibroblasts (cFFs) are transduced with the Yamanaka reprogramming factors by Sendai virus vectors. We use quantitative PCR arrays to measure the expression of 80 target genes at various stages of canine cell reprogramming. We ask how cFF reprogramming is influenced by small molecules affecting the epigenomic modification 5-hydroxymethylcytosine, specifically L-ascorbic acid and retinoic acid (AA/RA).

Results

We found that the expression and catalytic output of a class of 2-oxoglutarate-dependent (2-OG) hydroxylases, known as ten-eleven translocation (TET) enzymes, can be modulated in canine cells treated with AA/RA. We further show that AA/RA treatment induces TET1 expression and facilitates early canine reprogramming, evidenced by upregulation of epithelial and pluripotency markers. Using a chemical inhibitor of 2-OG hydroxylases, we demonstrate that 2-OG hydroxylase activity regulates the expression of a subset of genes involved in mesenchymal-to-epithelial transition (MET) and pluripotency in early canine reprogramming. We identify a set of transcription factors depleted in maturing reprogramming intermediates compared to pluripotent canine embryonic stem cells.

Conclusions

Our findings highlight 2-OG hydroxylases have evolutionarily conserved and divergent functions regulating the early reprogramming of canine somatic cells and show reprogramming conditions can be rationally optimized for the generation of maturing canine iPSC.

SUBMITTER: Tobias IC

PROVIDER: S-EPMC7725121 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases.

Tobias Ian C IC Kao Mian-Mian C MC Parmentier Thomas T Hunter Hailey H LaMarre Jonathan J Betts Dean H DH

Stem cell research & therapy 20201209 1

<h4>Background</h4>Ectopic expression of a defined set of transcription factors allows the reprogramming of mammalian somatic cells to pluripotency. Despite continuous progress in primate and rodent reprogramming, limited attention has been paid to cell reprogramming in domestic and companion species. Previous studies attempting to reprogram canine cells have mostly assessed a small number of presumptive canine induced pluripotent stem cell (iPSC) lines for generic pluripotency attributes. Howev ...[more]

PMID: 33298190

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Project description:Inhibition of lysyl hydroxylase and prolyl 3-hydroxylase was studied with 23 selected aromatic and aliphatic structural analogues of 2-oxoglutarate and the results were compared with those previously reported for prolyl 4-hydroxylase. All the compounds inhibited the hydroxylases competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fe2+ and the peptide substrate. The inhibition patterns for the three collagen hydroxylases were basically similar, but certain differences in detail emerged. One systematic difference was that lysyl hydroxylase had a higher Ki for almost all the compounds than had the two prolyl hydroxylases. Another interesting difference was that pyridine-2,4-dicarboxylate was the most potent inhibitor of lysyl hydroxylase and prolyl 3-hydroxylase, with Ki values of 50 microM and 3 microM respectively, whereas pyridine-2,5-dicarboxylate was the most potent inhibitor of prolyl 4-hydroxylase. These and other data suggest that the three collagen hydroxylases have similar but not identical 2-oxoglutarate-binding sites. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate and their corresponding benzene derivatives were also found to inhibit 2-oxoglutarate dehydrogenase, but with this enzyme, unlike the collagen hydroxylases, no distinct difference in the Ki values was found between the corresponding pyridine and benzene derivatives. This demonstrates the importance of the metal ion for the binding of various compounds at the 2-oxoglutarate-binding site of the collagen hydroxylases. 2-Oxoadipate was shown to replace 2-oxoglutarate in the lysyl hydroxylase and 2-oxoglutarate dehydrogenase reactions, as has previously been reported for prolyl 4-hydroxylase, whereas no other 2-oxo acid tested had any co-substrate activity. The 2-oxoglutarate-binding site of these enzymes is thus flexible to a certain degree, as it can accommodate molecules of different shapes and volumes. On the basis of the present data pyridine-2,5-dicarboxylate seems to be a quite specific inhibitor of prolyl 4-hydroxylase, the Ki for 2-oxoglutarate dehydrogenase being about 4000-fold higher.

Project description:Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in infection that was reduced at later time points. A similar expression pattern was observed in the parasites. Our analyses provide specific insights into the interplay between human macrophages and Leishmania parasites and constitute an important general resource for the study of how pathogens evade host defenses and modulate the functions of the cell to survive intracellularly.

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Dataset Information

Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases.

Background

Methods

Results

Conclusions

Publications

Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases.

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