Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity

ABSTRACT: Upon perturbations by RAS and by shBMAL, differential effects on the circadian phenotype were observed in wild type and Ink4a/Arf knockout MEFs which could be reproduced by modelling simulations and correlated with opposing cell cycle fate decisions.

ORGANISM(S): Mus musculus

SUBMITTER: Angela Relógio

PROVIDER: E-MTAB-5943 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity.

El-Athman Rukeia R Genov Nikolai N NN Mazuch Jeannine J Zhang Kaiyang K Yu Yong Y Fuhr Luise L Abreu Mónica M Li Yin Y Wallach Thomas T Kramer Achim A Schmitt Clemens A CA Relógio Angela A

PLoS biology 20171207 12

The mammalian circadian clock and the cell cycle are two major biological oscillators whose coupling influences cell fate decisions. In the present study, we use a model-driven experimental approach to investigate the interplay between clock and cell cycle components and the dysregulatory effects of RAS on this coupled system. In particular, we focus on the Ink4a/Arf locus as one of the bridging clock-cell cycle elements. Upon perturbations by the rat sarcoma viral oncogene (RAS), differential e ...[more]

PMID: 29216180

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Project description:Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.

Dataset Information

The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity

Publications

The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity.

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