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Full-Text Articles in Cell Biology

Optimization Of Transcription Factor Binding Map Accuracy Utilizing Knockout-Mouse Models, Wolfgang Krebs, Susanne V. Schmidt, Alon Goren, Dominic De Nardo, Larisa Labzin, Anton Bovier, Thomas Ulas, Heidi Theis, Michael Kraut, Eicke Latz, Marc Beyer, Joachim L. Schultze Dec 2014

Optimization Of Transcription Factor Binding Map Accuracy Utilizing Knockout-Mouse Models, Wolfgang Krebs, Susanne V. Schmidt, Alon Goren, Dominic De Nardo, Larisa Labzin, Anton Bovier, Thomas Ulas, Heidi Theis, Michael Kraut, Eicke Latz, Marc Beyer, Joachim L. Schultze

Open Access Articles

Genome-wide assessment of protein-DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify 'hyper ChIPable ...


Two Ways To Fold The Genome During The Cell Cycle: Insights Obtained With Chromosome Conformation Capture, Job Dekker Nov 2014

Two Ways To Fold The Genome During The Cell Cycle: Insights Obtained With Chromosome Conformation Capture, Job Dekker

Open Access Articles

Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of chromosome conformation during prophase leading to the formation of mitotic chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing chromosome conformation capture combined with classical models of chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic chromosomes. One striking observation is that ...


Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo Sep 2014

Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo

Katherine A. Fitzgerald

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis ...


Exosome-Mediated Delivery Of Functionally Active Mirna-155 Inhibitor To Macrophages, Fatemeh Momen-Heravi, Shashi Bala, Terence Bukong, Gyongyi Szabo Sep 2014

Exosome-Mediated Delivery Of Functionally Active Mirna-155 Inhibitor To Macrophages, Fatemeh Momen-Heravi, Shashi Bala, Terence Bukong, Gyongyi Szabo

Gyongyi Szabo

Exosomes, membranous nanovesicles, naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis. Here, we showed that B cell-derived exosomes can function as vehicles to deliver exogenous miRNA-155 mimic or inhibitor into hepatocytes or macrophages, respectively. Stimulation of B cells significantly increased exosome production. Unlike in parental cells, baseline level of miRNA-155 was very low in exosomes derived from stimulated B cells. Exosomes loaded with a miRNA-155 mimic significantly increased miRNA-155 levels in primary mouse hepatocytes and the liver of miRNA-155 knockout mice. Treatment of RAW macrophages with miRNA-155 inhibitor loaded exosomes resulted in statistically ...


Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo Sep 2014

Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo

Gyongyi Szabo

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis ...


A System For Genome-Wide Histone Variant Dynamics In Es Cells Reveals Dynamic Macroh2a2 Replacement At Promoters, Ozlem Yildirim, Jui-Hung Hung, Ryan J. Cedeno, Zhiping Weng, Christopher J. Lengner, Oliver J. Rando Aug 2014

A System For Genome-Wide Histone Variant Dynamics In Es Cells Reveals Dynamic Macroh2a2 Replacement At Promoters, Ozlem Yildirim, Jui-Hung Hung, Ryan J. Cedeno, Zhiping Weng, Christopher J. Lengner, Oliver J. Rando

Open Access Articles

Dynamic exchange of a subset of nucleosomes in vivo plays important roles in epigenetic inheritance of chromatin states, chromatin insulator function, chromosome folding, and the maintenance of the pluripotent state of embryonic stem cells. Here, we extend a pulse-chase strategy for carrying out genome-wide measurements of histone dynamics to several histone variants in murine embryonic stem cells and somatic tissues, recapitulating expected characteristics of the well characterized H3.3 histone variant. We extended this system to the less-studied MacroH2A2 variant, commonly described as a "repressive" histone variant whose accumulation in chromatin is thought to fix the epigenetic state of differentiated ...


Hsa-Mir-30c Promotes The Invasive Phenotype Of Metastatic Breast Cancer Cells By Targeting Nov/Ccn3, Jason R. Dobson, Hanna Taipaleenmaki, Yu-Jie Hu, Deli Hong, Andre J. Van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian, Jitesh Pratap Aug 2014

Hsa-Mir-30c Promotes The Invasive Phenotype Of Metastatic Breast Cancer Cells By Targeting Nov/Ccn3, Jason R. Dobson, Hanna Taipaleenmaki, Yu-Jie Hu, Deli Hong, Andre J. Van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian, Jitesh Pratap

Open Access Articles

BACKGROUND: For treatment and prevention of metastatic disease, one of the premier challenges is the identification of pathways and proteins to target for clinical intervention. Micro RNAs (miRNAs) are short, non-coding RNAs, which regulate cellular activities by either mRNA degradation or translational inhibition. Our studies focused on the invasive properties of hsa-mir30c based on its high expression in MDA-MB-231 metastatic cells and our bioinformatic analysis of the Cancer Genome Atlas that identified aberrant hsa-mir-30c to be associated with poor survival.

METHODS: Contributions of hsa-mir-30c to breast cancer cell invasion were examined by Matrigel invasion transwell assays following modulation of hsa-mir-30c ...


Genomic Occupancy Of Runx2 With Global Expression Profiling Identifies A Novel Dimension To Control Of Osteoblastogenesis, Hai Wu, Troy W. Whitfield, Jonathan A.R. Gordon, Jason R. Dobson, Phillip W.L. Tai, Andre J. Van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian Mar 2014

Genomic Occupancy Of Runx2 With Global Expression Profiling Identifies A Novel Dimension To Control Of Osteoblastogenesis, Hai Wu, Troy W. Whitfield, Jonathan A.R. Gordon, Jason R. Dobson, Phillip W.L. Tai, Andre J. Van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian

Open Access Articles

BACKGROUND: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed.

RESULTS: By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation--proliferation, matrix deposition and mineralization--we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we ...


Rnase-Mediated Protein Footprint Sequencing Reveals Protein-Binding Sites Throughout The Human Transcriptome, Ian M. Silverman, Fan Li, Anissa Alexander, Loyal Goff, Cole Trapnell, John L. Rinn, Brian D. Gregory Jan 2014

Rnase-Mediated Protein Footprint Sequencing Reveals Protein-Binding Sites Throughout The Human Transcriptome, Ian M. Silverman, Fan Li, Anissa Alexander, Loyal Goff, Cole Trapnell, John L. Rinn, Brian D. Gregory

Departmental Papers (Biology)

Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP–RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.