Functional interaction of the retinoblastoma and Ini1/Snf5 tumor suppressors in cell growth and pituitary tumorigenesis

The Ini1 subunit of the SWI/SNF chromatin remodeling complex suppresses formation of malignant rhabdoid tumors in humans and mice. Transduction of Ini1 into Ini1-deficient tumor-derived cell lines has indicated that Ini1 arrests cell growth, controls chromosomal ploidy, and suppresses tumorigenesis by regulating components of the retinoblastoma (Rb) signaling pathway. Furthermore, conditional inactivation of Ini1 in mouse fibroblasts alters the expression of various Rb-E2F-regulated genes, indicating that endogenous Ini1 levels may control Rb signaling in cells. We have reported previously that loss of one allele of Ini1 in mouse fibroblasts results only in a 15% to 20% reduction in total Ini1 mRNA levels due to transcriptional compensation by the remaining Ini1 allele. Here, we examine the effects of Ini1 haploinsufficiency on cell growth and immortalization in mouse embryonic fibroblasts. In addition, we examine pituitary tumorigenesis in Rb-Ini1 compound heterozygous mice. Our results reveal that heterozygosity for Ini1 up-regulates cell growth and immortalization and that exogenous Ini1 down-regulates the growth of primary cells in a Rb-dependent manner. Furthermore, loss of Ini1 is redundant with loss of Rb function in the formation of pituitary tumors in Rb heterozygous mice and leads to the formation of large, atypical Rb(+/-) tumor cells lacking adrenocorticotropic hormone expression. These results confirm in vivo the relationship between Rb and Ini1 in tumor suppression and indicate that Ini1 plays a role in maintaining the morphologic and functional differentiation of corticotrophic cells

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19Arf and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19Arf-p53 signaling, and induces senescence in primary cells

Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site

Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence

MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age-related changes in gene expression in the senescent failing heart. Age-related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age-related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging-associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin-1 (TSP-1). We employed aged mice with a failure-resistant (C57Bl6) and failure-prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age-associated heart failure. In aging-associated heart failure, we linked an aging-induced increase in the ECM proteins CTGF and TSP-1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR-17–92 cluster. Failure-resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR-18/19 changes the levels of ECM proteins CTGF and TSP-1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte-derived miR-18/19 during cardiac aging, in the fine-tuning of cardiac ECM protein levels. During aging, decreased miR-18/19 and increased CTGF and TSP-1 levels identify the failure-prone heart

Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARγ binding sites in mouse adipocytes and PPARγ upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARγ-targets. By integration of four separate datasets of genome-wide PPARγ binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARγ binding within 50 kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated ≥2-fold during adipogenesis and for six of these miRNA loci the PPARγ binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPARγ-dependence of four of these miRNA loci was demonstrated by PPARγ knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPARγ ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPARγ binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPARγ target miRNA genes, showed the PPARγ dependence of four of these genes and demonstrated three as direct PPARγ target genes in mouse adipogenesis

Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4‐ Cre ‐mediated knockout of the tuberous sclerosis complex 1 gene

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96661/1/expphysiol.2012.069674.pd

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function

Smooth Muscle miRNAs Are Critical for Post-Natal Regulation of Blood Pressure and Vascular Function

Phenotypic modulation of smooth muscle cells (SMCs) plays a key role in vascular disease, including atherosclerosis. Several transcription factors have been suggested to regulate phenotypic modulation of SMCs but the decisive mechanisms remain unknown. Recent reports suggest that specific microRNAs (miRNAs) are involved in SMC differentiation and vascular disease but the global role of miRNAs in postnatal vascular SMC has not been elucidated. Thus, the objective of this study was to identify the role of Dicer-dependent miRNAs for blood pressure regulation and vascular SMC contractile function and differentiation in vivo. Tamoxifen-inducible and SMC specific deletion of Dicer was achieved by Cre-Lox recombination. Deletion of Dicer resulted in a global loss of miRNAs in aortic SMC. Furthermore, Dicer-deficient mice exhibited a dramatic reduction in blood pressure due to significant loss of vascular contractile function and SMC contractile differentiation as well as vascular remodeling. Several of these results are consistent with our previous observations in SM-Dicer deficient embryos. Therefore, miRNAs are essential for maintaining blood pressure and contractile function in resistance vessels. Although the phenotype of miR-143/145 deficient mice resembles the loss of Dicer, the phenotypes of SM-Dicer KO mice were far more severe suggesting that additional miRNAs are involved in maintaining postnatal SMC differentiation

Supplementary data for article: Selaković, Ž.; Tran, J. P.; Kota, K. P.; Lazić, M.; Retterer, C.; Besh, R.; Panchal, R. G.; Soloveva, V.; Sean, V. A.; Jay, W. B.; et al. Second Generation of Diazachrysenes: Protection of Ebola Virus Infected Mice and Mechanism of Action. European Journal of Medicinal Chemistry 2019, 162, 32–50. https://doi.org/10.1016/j.ejmech.2018.10.061

Supplementary material for: [https://www.sciencedirect.com/science/article/pii/S0223523418309383?via%3Dihub]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/360]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2966

Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype

Background: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer.Methods: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription-PCR.Results: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours.Conclusion: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer