Biophysical and electrochemical studies of protein-nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail

One-loop f(R) gravity in de Sitter universe

Motivated by the dark energy issue, the one-loop quantization approach for a family of relativistic cosmological theories is discussed in some detail. Specifically, general $f(R)$ gravity at the one-loop level in a de Sitter universe is investigated, extending a similar program developed for the case of pure Einstein gravity. Using generalized zeta regularization, the one-loop effective action is explicitly obtained off-shell, what allows to study in detail the possibility of (de)stabilization of the de Sitter background by quantum effects. The one-loop effective action maybe useful also for the study of constant curvature black hole nucleation rate and it provides the plausible way of resolving the cosmological constant problem.Comment: 25 pages, Latex file. Discussion enlarged, new references added. Version accepted in JCA

A Genome-Wide Analysis of Open Chromatin in Human Epididymis Epithelial Cells Reveals Candidate Regulatory Elements for Genes Coordinating Epididymal Function1

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33 542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium

Identification and Characterization of Cell Type–Specific and Ubiquitous Chromatin Regulatory Structures in the Human Genome

The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type–specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type–specific gene expression as well as cell type–specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

This is the author's accepted manuscript. The final published article is available from the link below. Copyright @ Springer-Verlag 2008.In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing

Detection of regulator genes and eQTLs in gene networks

Genetic differences between individuals associated to quantitative phenotypic traits, including disease states, are usually found in non-coding genomic regions. These genetic variants are often also associated to differences in expression levels of nearby genes (they are "expression quantitative trait loci" or eQTLs for short) and presumably play a gene regulatory role, affecting the status of molecular networks of interacting genes, proteins and metabolites. Computational systems biology approaches to reconstruct causal gene networks from large-scale omics data have therefore become essential to understand the structure of networks controlled by eQTLs together with other regulatory genes, and to generate detailed hypotheses about the molecular mechanisms that lead from genotype to phenotype. Here we review the main analytical methods and softwares to identify eQTLs and their associated genes, to reconstruct co-expression networks and modules, to reconstruct causal Bayesian gene and module networks, and to validate predicted networks in silico.Comment: minor revision with typos corrected; review article; 24 pages, 2 figure

Alterations to chromatin in intestinal macrophages link IL-10 deficiency to inappropriate inflammatory responses

Intestinal macrophages are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters intestinal macrophage programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient mice. In both bone-marrow-derived and intestinal macrophages, we identified chromatin accessibility changes associated with bacterial exposure and IL-10-deficiency. Surprisingly, IL-10-deficient intestinal macrophages adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, if IL-10 protein was added to cells that had previously been IL-10-deficient, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model where IL-10-deficiency leads to chromatin alterations that contribute to a loss of intestinal macrophage tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation

A genome-wide analysis of open chromatin in human tracheal epithelial cells reveals novel candidate regulatory elements for lung function

Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements

The UCSC Genome Browser Database: update 2006

The University of California Santa Cruz Genome Browser Database (GBD) contains sequence and annotation data for the genomes of about a dozen vertebrate species and several major model organisms. Genome annotations typically include assembly data, sequence composition, genes and gene predictions, mRNA and expressed sequence tag evidence, comparative genomics, regulation, expression and variation data. The database is optimized to support fast interactive performance with web tools that provide powerful visualization and querying capabilities for mining the data. The Genome Browser displays a wide variety of annotations at all scales from single nucleotide level up to a full chromosome. The Table Browser provides direct access to the database tables and sequence data, enabling complex queries on genome-wide datasets. The Proteome Browser graphically displays protein properties. The Gene Sorter allows filtering and comparison of genes by several metrics including expression data and several gene properties. BLAT and In Silico PCR search for sequences in entire genomes in seconds. These tools are highly integrated and provide many hyperlinks to other databases and websites. The GBD, browsing tools, downloadable data files and links to documentation and other information can be found at