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Chicago Ridge, IL, United States

Browne J.A.,Lurie Childrens Research Center | Yang R.,Lurie Childrens Research Center | Eggener S.E.,Northwestern University | Leir S.-H.,Lurie Childrens Research Center | Harris A.,Lurie Childrens Research Center
Molecular and Cellular Endocrinology | Year: 2016

The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1β transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment. © 2016 Elsevier Ireland Ltd.

Gillen A.E.,Lurie Childrens Research Center | Gillen A.E.,Northwestern University | Lucas C.A.,Northwestern University | Haussecker P.L.,Weatherall Institute of Molecular Medicine | And 3 more authors.
Chromosoma | Year: 2013

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use. © 2013 Springer-Verlag Berlin Heidelberg.

Clementz A.G.,Lurie Childrens Research Center | Clementz A.G.,Northwestern University | Harris A.,Lurie Childrens Research Center | Harris A.,Northwestern University
Molecular Cancer Research | Year: 2013

The extracellular matrix (ECM) is a critical component of stroma-to-cell interactions that subsequently activate intracellular signaling cascades, many of which are associated with tumor invasion and metastasis. The ECM contains a wide range of proteins with multiple functions, including cytokines, cleaved cell-surface receptors, secreted epithelial cell proteins, and structural scaffolding. Fibrillar collagens, abundant in the normal ECM, surround cellular structures and provide structural integrity. However during the initial stages of invasive cancers, the ECM is among the first compartments to be compromised. Also present in the normal ECM is the nonfibrillar collagen XV, which is seen in the basement membrane zone but is lost prior to tumor metastasis in several organs. In contrast, the tumor microenvironment often exhibits increased synthesis of fibrillar collagen I and collagen IV, which are associated with fibrosis. The unique localization of collagen XV and its disappearance prior to tumor invasion suggests a fundamental role in maintaining basement membrane integrity and preventing the migration of tumor cells across this barrier. This review examines the structure of collagen XV, its functional domains, and its involvement in cell-surface receptor-mediated signaling pathways, thus providing further insight into its critical role in the suppression of malignancy. © 2013 American Association for Cancer Research.

Gosalia N.,Lurie Childrens Research Center | Gosalia N.,Northwestern University | Harris A.,Lurie Childrens Research Center | Harris A.,Northwestern University
Genes | Year: 2015

The contribution of chromatin dynamics to the regulation of human disease-associated loci such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been the focus of intensive experimentation for many years. Recent technological advances in the analysis of transcriptional mechanisms across the entire human genome have greatly facilitated these studies. In this review we describe the complex machinery of tissue-specific regulation of CFTR expression, and put earlier observations in context by incorporating them into datasets generated by the most recent genomics methods. Though the gene promoter is required for CFTR expression, cell-type specific regulatory elements are located elsewhere in the gene and in flanking intergenic regions. Probably within its own topological domain established by the architectural proteins CTCF and cohesin, the CFTR locus utilizes chromatin dynamics to remodel nucleosomes, recruit cell-selective transcription factors, and activate intronic enhancers. These cis-acting elements are then brought to the gene promoter by chromatin looping mechanisms, which establish long-range interactions across the locus. Despite its complexity, the CFTR locus provides a paradigm for elucidating the critical role of chromatin dynamics in the transcription of individual human genes. © 2015 by the authors; licensee MDPI, Basel, Switzerland.

Clementz A.G.,Lurie Childrens Research Center | Clementz A.G.,Northwestern University | Mutolo M.J.,Lurie Childrens Research Center | Mutolo M.J.,Northwestern University | And 9 more authors.
PLoS ONE | Year: 2013

Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone. © 2013 Clementz et al.

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