The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer

Foamy virus for efficient gene transfer in regeneration studies

Background Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. Results We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. Conclusions FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species

Foamy virus for efficient gene transfer in regeneration studies

Background Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. Results We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. Conclusions FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species

Ibrutinib Displays Atrial-Specific Toxicity in Human Stem Cell-Derived Cardiomyocytes

Summary: Ibrutinib (IB) is an oral Bruton's tyrosine kinase (BTK) inhibitor that has demonstrated benefit in B cell cancers, but is associated with a dramatic increase in atrial fibrillation (AF). We employed cell-specific differentiation protocols and optical mapping to investigate the effects of IB and other tyrosine kinase inhibitors (TKIs) on the voltage and calcium transients of atrial and ventricular human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). IB demonstrated direct cell-specific effects on atrial hPSC-CMs that would be predicted to predispose to AF. Second-generation BTK inhibitors did not have the same effect. Furthermore, IB exposure was associated with differential chamber-specific regulation of a number of regulatory pathways including the receptor tyrosine kinase pathway, which may be implicated in the pathogenesis of AF. Our study is the first to demonstrate cell-type-specific toxicity in hPSC-derived atrial and ventricular cardiomyocytes, which reliably reproduces the clinical cardiotoxicity observed. : The authors employ cell-specific cardiac differentiation protocols, RNA-seq, and optical mapping to demonstrate atrial-specific toxicity of ibrutinib, a first-in-class BTK inhibitor. Other tyrosine kinase inhibitors (TKIs) with the same drug target do not affect atrial electrophysiology. Nilotinib and vandetanib, two TKIs known to be associated with QT prolongation and risk of sudden death, demonstrated ventricular-specific electrophysiologic dysregulation. Keywords: cardiac electrophysiology, tyrosine kinase inhibitors, atrial fibrillation, drug screening, optical mapping, RNA-se

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.ISSN:1757-651

Hierarchical clustering of regeneration, lateral wound and limb bud samples identifies three main phases of limb regeneration.

<p>Samples were clustered using Pearson's correlation as similarity measure (sample key at bottom; numbers represent hours after injury, Prox =  mature sample from the upper arm at time 0 h, all other samples are from the lower arm). From 0–12 hours, at each time point, corresponding amputation and lateral wound samples cluster closest together. Starting at 24 hours, successive amputation samples are more similar to each other than to the corresponding lateral wound time points, indicating a divergence between regeneration and lateral wound gene programs. From 120 hours onward, the amputated samples are most similar to the developing limb bud, indicating that the limb development program has been re-established.</p

Validation of the gene expression profiling using microarray.

<p>A. Validation of gene expression changes by qPCR The expression profiles of seven representative genes- two with a strong signal from the microarray (<i>Psca and DK45 (RV_Am_asm_3322</i>)), two with medium signal (<i>Wnt5a and Wnt5b</i>) and three with a weak signal (<i>Fgf10, Tgm5 and DK35 (ET_Am_asm_6446</i>)) are shown. Time course of gene expression measured by microarray is shown on left, and qPCR on the right. The replicates of the microarray were normalized as described in the Methods section. qPCR data were normalized to the levels of <i>Rpl4</i> (Large ribosomal protein 4), which showed uniform expression levels in all microarray samples. In both, microarray and qPCR data, the normalized expression level at 0 hours is set to 1. The gene profiles obtained by microarray and by qPCR are remarkably similar although the dynamic range is 2–3 fold greater when measured by qPCR. All data points represent the mean of three biological replicates. Error bars show standard deviation. B. Validation of the time-course progression Cell cycle regulator expression reflects blastema formation in the regeneration time course. Top, G1/S-genes, <i>Mcm</i> and <i>Pcna</i> show a second peak at 288 hours in regeneration sample but not in the lateral wound sample. Bottom, G2/M-genes, <i>CyclinB, Plk, Cdc20</i>, remain highly expressed in the regeneration time course whereas they decline by 168 hours in the lateral wound time course. Each line depicts the trace of one representative probe for the gene averaged over three replicates with the error bars representing standard deviation. For each probe the median value of all measurements is set to 1. Solid lines: amputation time course, dotted line: lateral wound time course. See also Figure S1.</p

Gene ontology enrichment on the list of amputation-specific genes selected by two-way ANOVA analysis.

<p>Enrichment was calculated using g:Profiler on the list of 93 human RefSeq protein IDs corresponding to amputation-specific genes selected by two-way ANOVA analysis. 85 IDs from the list of 93 were recognized as unambiguous and were used to calculate the enrichment. Columns represent: #Array: total number of genes present on the array, that is associated to functional term, #List: number of genes in the input list that are associated to functional term, P-value: enrichment P-value, GO term: term name. The vertical alignment of the term name depicts its depth in the GO term hierarchy.</p