Nucleosomal structure and functions, characterization of the hamster cardiac myosin heavy chain genes DNase I hypersensitive sites

grantor: University of TorontoIn order to investigate the role of chromatin structure in cardiac gene expression, DNase I hypersensitive sites (DHS) of the hamster cardiac myosin heavy chain genes (MyHC) were studied. Two cardiac-specific DHSs within the 5 kb upstream region of the $\beta$-MyHC gene were identified. One of the DHSs was mapped to $-$2.3 kb ($\beta$-2.3 kb) region and the other to the proximal promoter region. Although these two sites were readily detectable using nuclei from neonatal hamster hearts, the proximal promoter site disappeared when adult hamster heart nuclei were used, and the $\beta$-2.3 kb site decreased in intensity. Furthermore, the disappearance of the $\beta$-promoter DHS in fetal heart chromatin can be induced by injecting thyroid hormone into pregnant hamsters at late gestational stage. Digestion of nuclei from various tissues by micrococcal nuclease revealed that the $\beta$-MyHC gene promoter exists in an array of specifically-positioned nucleosomes only in fetal heart chromatin. The $\beta$-MyHC gene promoter is DNase I hypersensitive within one of the nucleosomal particles. As well, two DHSs, one mapping to $-$1.9 kb ($\alpha$-1.9 kb) region, the other to the proximal promoter region, were detected within the 4 kb upstream region of the cardiac $\alpha$-MyHC gene using adult heart nuclei. It was found that the $\beta$-2.3 kb site is associated with multiple conserved muscle regulatory motifs. In the $\alpha$-1.9 kb site, a conserved GATA motif was identified. The specific interaction between these upstream DHSs and cardiac nuclear proteins was established using gel mobility shift assays and footprinting analysis. Transient transfection CAT (chloramphenicol acetyl-transferase assays) revealed that (1) the $\beta$-2.3 kb site is not a typical enhancer, and (2) the proximal promoter DHS ($-$294 to +97) of the $\beta$-MyHC gene is able to confer tissue-specific expression of the reporter gene; however, (3) co-transfection of GATA-4 with $\beta$-MyHC promoter CAT construct into fibroblasts leads to a marked increase in CAT activity. In conclusion, this study demonstrated that developmental regulation of cardiac MyHC gene expression involves the interplay between chromatin structure and cardiac nuclear factors.Ph.D

Strategies for Overcoming Resistance in Tumours Harboring BRAF Mutations

The development of resistance to previously effective treatments has been a challenge for health care providers and a fear for patients undergoing cancer therapy. This is an unfortunately frequent occurrence for patients undergoing targeted therapy for tumours harboring the activating V600E mutation of the BRAF gene. Since the initial identification of the BRAF mutation in 2002, a series of small molecular inhibitors that target the BRAFV600E have been developed, but intrinsic and acquired resistance to these drugs has presented an ongoing challenge. More recently, improvements in therapy have been achieved by combining the use of BRAF inhibitors with other drugs, such as inhibitors of the downstream effector mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK). Despite improved success in response rates and in delaying resistance using combination therapy, ultimately, the acquisition of resistance remains a concern. Recent research articles have shed light on some of the underlying mechanisms of this resistance and have proposed numerous strategies that might be employed to overcome or avoid resistance to targeted therapies. This review will explore some of the resistance mechanisms, compare what is known in melanoma cancer to colorectal cancer, and discuss strategies under development to manage the development of resistance

S100A10, a novel biomarker in pancreatic ductal adenocarcinoma

Pancreatic cancer is arguably the deadliest cancer type. The efficacy of current therapies is often hindered by the inability to predict patient outcome. As such, the development of tools for early detection and risk prediction is key for improving outcome and quality of life. Here, we introduce the plasminogen receptor S100A10 as a novel predictive biomarker and a driver of pancreatic tumor growth and invasion. We demonstrated that S100A10 mRNA and protein are overexpressed in human pancreatic tumors compared to normal ducts and nonductal stroma. S100A10 mRNA and methylation status were predictive of overall survival and recurrence‐free survival across multiple patient cohorts. S100A10 expression was driven by promoter methylation and the oncogene KRAS. S100A10 knockdown reduced surface plasminogen activation, invasiveness, and in vivo growth of pancreatic cancer cell lines. These findings delineate the clinical and functional contribution of S100A10 as a biomarker in pancreatic cancer

Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein

A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding–impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the β-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans