Bacteria in various habitats are subject to continuously changing environmental conditions, such as nutrient deprivation, heat and cold stress, UV radiation, oxidative stress, dessication, acid stress, nitrosative stress, cell envelope stress, heavy metal exposure, osmotic stress, and others. In order to survive, they have to respond to these conditions by adapting their physiology through sometimes drastic changes in gene expression. In addition they may adapt by changing their morphology, forming biofilms, fruiting bodies or spores, filaments, Viable But Not Culturable (VBNC) cells or moving away from stress compounds via chemotaxis. Changes in gene expression constitute the main component of the bacterial response to stress and environmental changes, and involve a myriad of different mechanisms, including (alternative) sigma factors, bi- or tri-component regulatory systems, small non-coding RNA’s, chaperones, CHRIS-Cas systems, DNA repair, toxin-antitoxin systems, the stringent response, efflux pumps, alarmones, and modulation of the cell envelope or membranes, to name a few. Many regulatory elements are conserved in different bacteria; however there are endless variations on the theme and novel elements of gene regulation in bacteria inhabiting particular environments are constantly being discovered. Especially in (pathogenic) bacteria colonizing the human body a plethora of bacterial responses to innate stresses such as pH, reactive nitrogen and oxygen species and antibiotic stress are being described. An attempt is made to not only cover model systems but give a broad overview of the stress-responsive regulatory systems in a variety of bacteria, including medically important bacteria, where elucidation of certain aspects of these systems could lead to treatment strategies of the pathogens. Many of the regulatory systems being uncovered are specific, but there is also considerable “cross-talk” between different circuits. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria is a comprehensive two-volume work bringing together both review and original research articles on key topics in stress and environmental control of gene expression in bacteria. Volume One contains key overview chapters, as well as content on one/two/three component regulatory systems and stress responses, sigma factors and stress responses, small non-coding RNAs and stress responses, toxin-antitoxin systems and stress responses, stringent response to stress, responses to UV irradiation, SOS and double stranded systems repair systems and stress, adaptation to both oxidative and osmotic stress, and desiccation tolerance and drought stress. Volume Two covers heat shock responses, chaperonins and stress, cold shock responses, adaptation to acid stress, nitrosative stress, and envelope stress, as well as iron homeostasis, metal resistance, quorum sensing, chemotaxis and biofilm formation, and viable but not culturable (VBNC) cells. Covering the full breadth of current stress and environmental control of gene expression studies and expanding it towards future advances in the field, these two volumes are a one-stop reference for (non) medical molecular geneticists interested in gene regulation under stress.

Gene regulation underlies all functional outputs of a cell. Measuring changes in gene expression during biological processes is now routine; however, understanding how complex gene regulatory responses affect cellular functions remains a challenge. To address this problem, I developed network-based strategies to interrogate publicly available gene expression data to better understand how Caulobacter crescentus survives stress. My approach identified GsrN, a conserved small RNA that is directly activated by the general stress sigma factor. I found that GsrN functions as a potent post-transcriptional regulator of survival across distinct conditions including osmotic and oxidative stress. To understand GsrN's role in stress adaptation, I developed a forward biochemical approach to identify the molecular partners of GsrN. This methodology identified the leader of katG mRNA, the sole catalase/peroxidase gene in Caulobacter crescentus, as a regulatory target of GsrN. I further demonstrated that GsrN base pairs to the leader of katG mRNA and activates expression of KatG. In summary, my dissertation outlines new computational and experimental approaches that led to new understanding of the regulation of Caulobacter crescentus stress physiology.

Malfunctioning cellular stress responses have been linked to a host of diseases, such as diabetes, neuronal degenerative diseases, and cancer. Cancer cells are faced with a variety of stress conditions resulting from the tumor microenvironment and their highly proliferative state. microRNAs (miRNAs) are endogenous small non-coding RNAs that contribute to post-transcriptional regulation of gene expression by binding to target sites in the 3' UTR of cognate mRNA, leading to translational inhibition and/or mRNA degradation. miRNA dysregulation leads to aberrant genetic expression and is associated with cellular stress related diseases, including cancer. Activating transcription factor 5 (ATF5) is a widely expressed transcription factor that modulates survival, proliferation, and differentiation and plays a role in homeostasis and cellular stress response. In unstressed conditions, ATF5 expression is suppressed and has a short half-life. Conversely, ATF5 is upregulated by diverse stress conditions and leads to increased cell survival. ATF5 is overexpressed in a wide range of cancers. Furthermore, silencing ATF5 or interfering with its activity leads to selective apoptosis of cancer cells but not non-transformed cells, and decreased tumor growth in vivo, making it a promising target for cancer therapy. Regulation of ATF5 is not fully understood.The purpose of this dissertation was to better understand the regulation of ATF5 in the stress phenotype of cancer cells and investigate the potential regulation of ATF5 by miRNA under cellular stress conditions in cancer cells. We focused on three different microRNA predicted by in silico analysis to target the ATF5 3' UTR: miR-129-5p, mir-433-3p, and miR-520b, and their ability to suppress ATF5 expression, with an added interest in probing for an enhanced combinatorial activity. Although initial findings indicated all three miRNA could interact with ATF5 3' UTR, functional studies revealed a role for miR-520b alone in modulating ATF5 expression under multiple stress conditions in cancer cells. The implications of these findings will be thoroughly discussed. This is the first study to investigate miRNA regulation of ATF5, a stress-response transcription factor, under cellular stress conditions, and we present the first evidence of the mediation of ATF5 expression by miRNA under the stress phenotype of cancer cells.

Changes in Eukaryotic Gene Expression in Response to Environmental Stress focuses on various aspects of eukaryotic cell's response to heat stress (shock) and other stress stimuli. This book is organized into two major sections, encompassing 17 chapters that reflect the emphasis on research utilizing Drosophila, a variety of animal systems, and plants. This book first provides a brief introduction to the organization, sequences, and induction of heat shock proteins and related genes. It then describes the control of transcription during heat shock from the standpoint of molecular biology and evolutionary variations of the mechanisms in organisms with diverse metabolic needs. It goes on to discuss the issue of coordinate and noncoordinate responses of heat shock genes. It presents a model for post-transcriptional regulation on certain aspects of coordinate and noncoordinate regulations. Chapters 6-12 discuss heat shock proteins and genes and the effects of stress on gene expression of sea urchin, avian, and mammalian cells. The second part of the book focuses on the physiological role of heat shock proteins and genes in plants and fungi. It includes a discussion on experimental problems encountered during studies of the mechanisms of inhibition of photosynthesis by unfavorable environmental conditions. The changes in transcription and translation of specific mRNAs in the developing embryo during heat shock at various temperatures are described. The concluding chapters deal with heat shock response in plants, particularly the response in soybeans and maize, covering both physiological and molecular analyses. Research scientists, clinicians, and agriculturists will greatly benefit from the information presented in this book.

Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation factors and rapidly modulate mRNA translation at both the initiation and the elongation stages. Here, I discover that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected mRNA fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and co-translational folding. My results further support that trapped chaperone under stress may prevent the release of elongation factors from ribosomes. My study reveals that translating ribosomes fine-tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a co-translational stress response to maintain the intracellular protein homeostasis. Correspondingly, repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. Understanding the selective translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. Here, I develop an approach (global translation initiation sequencing, GTI-seq) to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. With single nucleotide resolution, I show an unprecedented view of alternative translation initiation in mammalian cells. Furthermore, I uncover a robust translational reprogramming of protein catabolic process, in particular the proteasome system, in response to starvation. This regulatory mode of TIS selection indicates that the scope of selective translation under stress conditions is much broader than anticipated. Collectively, my studies have revealed unprecedented proteome complexity and flexibility through stress-induced translational reprogramming, including ribosome pausing during elongation and wide-spread alternative translation initiation. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases.

HuR is a ubiquitously expressed RNA-binding protein that affects the post-transcriptional life of thousands of cellular mRNAs by regulating transcript stability and translation. HuR can post-transcriptionally regulate gene expression and modulate cellular responses to stress, differentiation, proliferation, apoptosis, senescence, inflammation, and the immune response. It is an important mediator of survival during cellular stress, but when inappropriately expressed, can promote oncogenic transformation. Not surprisingly, the expression of HuR itself is tightly regulated at multiple transcriptional and post-transcriptional levels. Previous studies demonstrated the existence of two alternate HuR transcripts that differ in their 5prime untranslated regions and have markedly different translatabilities. These forms were also found to be reciprocally expressed following cellular stress in kidney proximal tubule cell lines, and the shorter, more readily translatable variant was shown to be regulated by Smad 1/5/8 pathway and bone morphogenetic protein-7 (BMP-7) signaling. In this study, the factors that promote transcription of the longer alternate form were identified. NF-kappaB was shown to be important for expression of the long HuR mRNA, as was a newly identified region with potential for binding the Sp/KLF families of transcription factors. Sp1-like factors were not found to regulate HuR expression, revealing a possible KLF family member activity in regulation of the long isoform. Further analysis revealed that at least seven potential KLF family members are expressed in LLC-PK1 cells. One of these, Kruppel-like factor 8 (KLF8), had previously been shown to have key roles in cell proliferation, survival, and invasiveness, like HuR. Chromatin immunoprecipitation confirmed binding of KLF8 to the putative Sp/KLF binding site under both normal and stressed conditions in LLC-PK1 cells, and overexpression of KLF8 increased HuR mRNA levels. Further, cellular stress induced by thapsigargin treatment of cultured proximal tubule cells or ischemic injury of native rat kidney resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. NF-kappaB protein levels transiently increased during a recovery phase similar to long form HuR mRNA, suggesting it might be one of the predominant transcription factors in regulating the long variant during recovery phase following injury or stress. These results demonstrate that KLF family members, including KLF8, can participate in regulating expression of alternate forms of HuR mRNA along with NF-kappaB and Smad 1/5/8 under different cellular contexts. The differential expression of HuR mRNAs with alternate 5prime untranslated regions and translatabilities provides a mechanism for the stress-responsive regulation of HuR expression by two distinct pathways that include Smad 1/5/8 or KLF8 and NF-kappaB.

The Second Edinburgh International Workshop was held in September, 1984 and took as its topic the coordinated regulation of gene expression. The intention of this series of workshops is to promote exchange of ideas and data between scientists and clinicians whose interests span molecular and cell biology, development and differentiation, oncology, and genetic and developmental pathologies. It is hoped that such interdisciplinary discussions may give rise to fruitful insights. The meetings are structured to give ample time for discussion after each formal presentation and culminate in a session of general discussion which is reported at the end of the volume of proceedings. We are very grateful to the participants, all of whom participated in the discussion and whose contributions were essential to the success of the meeting. Novel ideas were often put forward and explored thoroughly from different angles. We normally expect to achieve quite rapid publication of the proceedings of the meeting and are grateful to authors who produced typescripts of their contributions expeditiously, but, as may sometimes be the case with multi-author works, some contributors had difficulty in meeting schedules for submitting manuscripts or corrections of the text of the discussion, and in one case we have been unable to publish any record of the contribution. Our committment to the publication of the discussion, allowing participants to make corrections to the transcript of the session, such as insertion of references and clarification of oral contributions, has also imposed some delay.

An overview of the current systems biology-based knowledge and the experimental approaches for deciphering the biological basis of cancer.

The cell is a complex biological network that is capable of transitioning to a wide variety of states. Enumerating, defining, and understanding the mechanisms behind cellular states are important problems of Systems Biology. This document contains insights gleaned from the study of three systems wide problems: transcription regulation by NF-kB, oxidative stress in response to reactive oxidative species, and gene expression changes caused by creation of lentiviral mediated cancer models. A consideration by literature review is provided of the historical problem formulations for studying mechanisms of NF-kB target gene regulation. Previous formulations of regulation are useful as frameworks for experimental design of future experiments when considered without bias towards prior assumptions. A description of the construction of a network bridging the multitude of cell responses to hydrogen peroxide is provided along with failed attempts to validate that network. Potential regulation by heme in response to oxidative stress reveals an ever tighter relationship between ROS, metabolism, and cell death. Application of molecular signatures defined from human primary cancers is used for determining the suitability of mouse cancer models generated from lentiviral constructs for the study of human primary cancers. Mouse tumors generated artificially display a surprising degree of concordance with primary cancers. The ability of high throughput technologies to query nearly the entire state of the cell can lead to undesirable complexity. Application of simplifying assumptions derived from the consideration of the biological fundamental problem as opposed from technical limitations allows a reduction of in complexity that elucidates areas for future study.