Project description:To elucidate the function of Airn isoforms in the heart, we conducted loss-of-function experiments in murine cardiomoycyte cell line HL-1.
Project description:To elucidate the function of Airn isoforms in the heart, we conducted RNA immunoprecipitation experiment followed by microarray (RIP-chip) in murine cardiomoycyte cell line HL-1.
Project description:HNF4α is a nuclear receptor regulating the transcription of genes involved mainly in development, cell differentiation and metabolism. Opposite functions for the two classes of P1 and P2 isoforms of HNF4α have recently been highlighted. These classes include 12 variants of HNF4α that can be expressed by the use of two promoters and by alternative splicing. Until now, the characterization of this transcription factor has ignored this diversity and has remained confined to the study of a fraction of the isoforms. We therefore wanted to clarify the situation by specifically characterizing the transcriptional functions of the 12 isoforms of HNF4α. We have generated for this purpose stable lines expressing each isoform of HNF4α in HCT 116 cells. We analyzed the whole transcriptome associated with each isoform by sequencing RNA, as well as their proteome by a BioID approach coupled to quantitative mass spectrometry. We noted major differences in the transcriptional function of the 12 isoforms. The α4, α5 and α6 isoforms have been characterized for the first time, and show a greatly reduced transcriptional potential. We have shown that these isoforms are unable to recognize the consensus response element of HNF4α. The α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity. Several transcription factors and coregulators have been identified as potential specific partners for certain HFH4α isoforms. The IRF-2BP2 co-repressor interacts specifically with isoforms which include the long form of the F domain of HNF4α. This specific interaction could explain the large number of genes modulated negatively by α1 and α2 compared to α3. The analysis integrating the vast amount of transcriptomic and proteomic data allows the identification of transcriptional regulatory mechanisms specific to certain isoforms, demonstrating the importance of considering all isoforms which can have diverse functions.
Project description:Heart disease remains the leading cause of death globally. Although reperfusion following myocardial ischemia can prevent death by restoring nutrient flow, ischemia/reperfusion injury can cause significant heart damage. The mechanisms that drive ischemia/reperfusion injury are not well understood; currently, few methods can predict the state of the cardiac muscle cell and its metabolic conditions during ischemia. Here, we explored the energetic sustainability of cardiomyocytes, using a model for cellular metabolism to predict the levels of ATP following hypoxia. We modeled glycolytic metabolism with a system of coupled ordinary differential equations describing the individual metabolic reactions within the cardiomyocyte over time. Reduced oxygen levels and ATP consumption rates were simulated to characterize metabolite responses to ischemia. By tracking biochemical species within the cell, our model enables prediction of the cell’s condition up to the moment of reperfusion. The simulations revealed a distinct transition between energetically sustainable and unsustainable ATP concentrations for various energetic demands. Our model illustrates how even low oxygen concentrations allow the cell to perform essential functions. We found that the oxygen level required for a sustainable level of ATP increases roughly linearly with the ATP consumption rate. An extracellular O2 concentration of ~0.007 mM could supply basic energy needs in non-beating cardiomyocytes, suggesting that increased collateral circulation may provide an important source of oxygen to sustain the cardiomyocyte during extended ischemia. Our model provides a time-dependent framework for studying various intervention strategies to change the outcome of reperfusion.
Project description:Due to alternative processing pathway and post-transcriptional modifications, precursor miRNAs maturate into various sequence isoforms (isomiRs). These sequence variations may result in the changes of the miRNA seed site, target genes, involvement in signaling pathways and thus function. It is important to mention that knowledge about the targets of isomiR is still poor. To date, isomiR research has only been performed in melanoma, breast, and gastric cancer, but there are no experimental studies conducted in colorectal cancer. Here, we aimed to evaluate the putative targets and functional role in vitro of miR-1246 and its two 5’ isoforms (ISO-miR-1246_a and ISO-miR-1246_G). To our best knowledge, this is the first study showing the important role of 5’isoforms of miR-1246 in colorectal carcinogenesis, while acting on different targetomes and being involved in distinct signaling pathways.