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Distinct requirement of Runx complexes for TCR? enhancer activation at distinct developmental stages.

ABSTRACT: A TCR? enhancer, known as the E? enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of E? during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the E? function. A single mutation at one of the two Runx binding motifs within the E? severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCR? expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated E? activity, demonstrating that Runx complex binding is essential to initiate E? activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCR? expression, whereas E? itself is continuously required for TCR? expression. These findings imply that Runx complexes are essential for E? activation at the initiation phase, but are not necessary for maintaining E? activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for E? activity are differentially regulated, depending on the developmental stage and cellular activation status.

SUBMITTER: Seo W

PROVIDER: S-EPMC5288706 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Distinct requirement of Runx complexes for TCRβ enhancer activation at distinct developmental stages.

Seo Wooseok W Muroi Sawako S Akiyama Kaori K Taniuchi Ichiro I

Scientific reports 20170202

A TCRβ enhancer, known as the Eβ enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of Eβ during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the Eβ function. A single mutation at one of the two Runx binding motifs within the Eβ severely impaired Tcrb activation at the initiation phase in immature thymocytes. Howe ...[more]

PMID: 28150718

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Project description:BackgroundDynamic activation and inactivation of gene regulatory DNA produce the expression changes that drive the differentiation of cellular lineages. Identifying regulatory regions active during developmental transitions is necessary to understand how the genome specifies complex developmental programs and how these processes are disrupted in disease. Gene regulatory dynamics are mediated by many factors, including the binding of transcription factors (TFs) and the methylation and acetylation of DNA and histones. Genome-wide maps of TF binding and DNA and histone modifications have been generated for many cellular contexts; however, given the diversity and complexity of animal development, these data cover only a small fraction of the cellular and developmental contexts of interest. Thus, there is a need for methods that use existing epigenetic and functional genomics data to analyze the thousands of contexts that remain uncharacterized.ResultsTo investigate the utility of histone modification data in the analysis of cellular contexts without such data, I evaluated how well genome-wide H3K27ac and H3K4me1 data collected in different developmental stages, tissues, and species were able to predict experimentally validated heart enhancers active at embryonic day 11.5 (E11.5) in mouse. Using a machine-learning approach to integrate the data from different contexts, I found that E11.5 heart enhancers can often be predicted accurately from data from other contexts, and I quantified the contribution of each data source to the predictions. The utility of each dataset correlated with nearness in developmental time and tissue to the target context: data from late developmental stages and adult heart tissues were most informative for predicting E11.5 enhancers, while marks from stem cells and early developmental stages were less informative. Predictions based on data collected in non-heart tissues and in human hearts were better than random, but worse than using data from mouse hearts.ConclusionsThe ability of these algorithms to accurately predict developmental enhancers based on data from related, but distinct, cellular contexts suggests that combining computational models with epigenetic data sampled from relevant contexts may be sufficient to enable functional characterization of many cellular contexts of interest.

Project description:Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.

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Distinct requirement of Runx complexes for TCR? enhancer activation at distinct developmental stages.

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Distinct requirement of Runx complexes for TCRβ enhancer activation at distinct developmental stages.

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