A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation

Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) are responsible for the majority of urinary tract infections(UTI). To cause UTI, UPEC must adhere to epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is primarily mediated by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contribute to UPEC mediated UTI are autotransporter (AT) proteins. AT proteins possess a range of virulence properties such as adherence, aggregation, invasion and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large AIDA-I type AT protein that contributes to biofilm formation and bladder colonization. In this study, we have characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not binding to ECM proteins. Despite variation in upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and function of the UpaH AT protein

Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these—H-NS and Fis—bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques

Transcription factor binding distribution and properties in prokaryotes

The canonical model of transcriptional regulation in prokaryotes restricted binding site locations to promoter regions and suggested that the binding sequences serve as the main determinants of binding. In this dissertation, I challenge these assumptions. As a member of the TB Systems Biology Consortium, I analyzed and validated ChIP-Seq and microarray experiments for over 100 transcription factors (TFs). In order to study the transcriptional functions of predicted binding sites, I integrated binding and expression data and assigned potential regulatory roles to 20% of the binding sites. Stronger binding sites were more often associated with regulation than weaker sites, suggesting a correlation between binding strength and regulatory impact. Seventy-six percent of the sites fell into annotated coding regions and a significant proportion was assigned to regulatory functions. To study the importance of binding sequences, I compared experimental sites with computational motif predictions. Although a conservative binding motif was found for most TFs, only a fraction of the observed motifs appeared bound in the experiment. Some low-affinity binding sites appeared occupied by the corresponding TF while many high-affinity binding sites were not. Interestingly, I found exactly the same nucleotide sequences (up to 15 residues long) bound in one area of the genome but not bound in another area, pointing to DNA accessibility as an important factor for in vivo binding. To investigate the evolutionary conservation of binding-site occupancy, sequence, and transcriptional impact, I analyzed ChIP-Seq and expression experiments for five conserved TFs for two-to-four Mycobacterial relatives. The regulon composition showed significantly less conservation than expected from the overall gene conservation level across Mycobacteria. Despite expectations, sequence conservation did not serve as a good indicator of whether or not a computationally predicted motif was bound experimentally; and in some cases, a fully conserved motif was bound in one relative but not in the other. Conservation of genic binding sites was higher than expected from the random model, adding to the evidence that at least some genic sites are functional. Understanding the evolutionary story of binding sites allowed me to explain unusual site configurations, some of which indicated a role for DNA looping

A Review on Epigenome Editing using CRISPR-based Tools to Rejuvenate Skin Tissues

Genomic activity is controlled by a sophisticated series of cell functions known as the epigenome. The creation of tools capable of directly altering various processes is required to unravel this intricacy. Additionally, by employing tailored DNA-binding platforms connected with effector domains to serve as targeted transcription factors or epigenetic modifiers, it is possible to control the chemical modifiers that regulate the genome's activity. Neoplastic disorders have received the most attention in the study of epigenetics, though the epigenome's significance in a variety of disease processes is now well acknowledged. Researchers are inspired to investigate novel approaches to revert these pathogenic alterations to their normal patterns by considering the fact that the epigenome profile of individuals with aging skin cells or other skin disorders, including atopic dermatitis, differs from that of healthy individuals. Here in this review, we discuss the use of CRISPR/dCas9 as a cutting-edge and flexible tool for fundamental studies on chromatin structure, transcription regulation, and epigenetic landscapes, as well as the potential of this method in these fields. Furthermore, we review on common and recently invented methods to make epigenetic alterations possible in daughter cells after any mitotic differentiations. In the very near future, CRISPR-based epigenomic editing will become a potent tool for comprehending and regulating biological functions

The Discovery And Investigation Of Jig (CG14850): A Previously Uncharacterized Novel Drosophila Melanogaster Protein

Despite Drosophila being one of the most studied model organisms in the world of research, one-third of 14,000 protein-encoding genes that the Drosophila genome possesses code for proteins that are yet uncharacterized. While investigating some of these uncharacterized Drosophila proteins, we identified a scarce protein type–a protein localized in both mitochondria and nucleus. Proteins, dual-localized in mitochondria and nucleus, establish communication between these organelles. This type of dual-localized protein is among the rarest of proteins, which limits us from fully understanding the mito-nuclear communication mechanism. Here, we tell the story of Jig, which we discovered to be one of those extremely rare proteins that localize in mitochondria, bind to the mitochondrial genome, localize in the nucleus, and bind to the nuclear genome. To my knowledge, I am the first to show that this rare protein is part of the CREB pathway, which is one of the most studied proteins for the mito-nuclear communication mechanism. Jig binds to and transports CREB from mitochondria to nucleus throughout the 3rd larval developmental stage. The Jig-CREB pair binds to DNA in both mitochondrial and nuclear genomes to regulate genes that are necessary for Drosophila development. Knocking down Jig causes disruption of CREB localization to the nucleus, changes mitochondrial morphology and membrane potential and arrests Drosophila development. These results are the first to show a Jig-CREB pathway that is necessary to conduct mito-nuclear communication for development

The nucleohistone compartment in relation to sperm HALO formation, DNA damage and DNA sequence analysis

The presence of DNA damage in mature sperm may be associated with poor chromatin structure due to abnormal protamination. Interestingly, however, approximately 5-15% of the DNA in the human sperm nucleus remains bound to histones. In this study, oxidative stress was used to induce DNA damage in mature sperm aimed at investigating the integrity of sperm chromatin structure. The extent of the damage was assessed by acridine orange, alkaline comet and halo assay (HalospermTM). Experiments were designed to probe the relationship between sperm DNA fragmentation as revealed by halo dynamics and the DNA sequences that constitute the nuclear halo structure, and so provide a more robust link between the halo assay as a discriminator of high-quality sperm and paternal genes that may be disrupted in damaged sperm. Differential Density Gradient Centrifugation (DDGC) was used to resolve human spermatozoa into 90% percoll solution (high density) and 45% percoll solution (low- density) fractions. DNA damage was induced by exposure to H2O2 at two different concentrations (100 and 300 μM) for fixed times. Acridine orange, alkaline comet and halo assay were used conventionally to measure the extent of DNA fragmentation in peroxide-treated cells. In a variant of the halo assay aimed at investigating the differences between protamine and histone-bound DNA, human sperm nuclei were treated with either low or high ionic strength salt solutions to generate nuclear halos. Halos produced from control (undamaged) sperm by HalospermTM or by salt extraction were treated in suspension with restriction enzymes to release halo-DNA, which was analysed by Next Generation Sequencing (NGS). Results of acridine orange, alkaline comet and halo assay revealed that pellets of DDGC processed sperm were far more resistant to H2O2 treatment compared with interface sperm. The efficacy of halo formation as an indicator of DNA damage was shown by the high percentage of strong halos generated by HalospermTM and salt extraction methods from sperm isolated from the pelleted sperm compared with interface sperm. Analysis of NGS data of halos generated by HalospermTM and by low or high salt extraction of nuclear proteins suggests that approximately 2000 genes, many of developmental significance are significantly ‘over-represented’ in nuclear halos compared with residual (nucleoid) DNA. Moreover, the data suggests that halo-DNA was originally associated with the histone compartment of sperm chromatin. The nuclear halo can indicate the level of DNA fragmentation in sperm, and the sequence composition of halos suggest that such fragmentation could compromise important paternally-derived DNA sequences that the oocyte may be unable to repair