The Analysis of Regulatory DNA: Current Developments, Knowledge and Applications Uncovering Gene Regulation

This introductory review aims at summarizing the vast and varied field about how transcript abundance is controlled in eukaryotes. The field of transcriptional regulation is a very exciting one right now. Many new discoveries are turning many concepts we have had about eukaryotic transcription sideways, if not occasionally, upside-down. Furthermore, important topics will be pointed out that are explored in more detail as their own chapters of this eBook. The regulation of transcription is a multi-step process that can be and is regulated at every level, from the access of DNA, to the recruitment and process of transcription, on to the regulation of RNA stability, function and form. The complete introductory review encompasses the first two chapters of this book. The first part of the review focuses on what is currently known and studied with respect to new paradigms for the mechanism of transcription and the protein components that modulate it. The second half of the review covers the canonical core promoter and varied types of promoter classes that are now being revealed through genomic studies.

Transcription is mediated through a variety of protein complexes that are not only responsible for the actual mechanism of transcription, but also are regulatory, in that their interactions at promoters can be altered by modifications, additional proteins, or homolog/paralog substitution. One important aspect of promoter recognition is mediated via proteins which can recognize and bind to DNA. Research in this area has led to the identification and characterization of core promoter elements that function to stimulate transcription in vitro and in vivo. One of the greatest revolutions in eukaryotic gene regulation is the discoveries that promoters, including their cores, are not as simply explained by naming some of their elements that compose them. This chapter looks at which core elements have been discovered, identified and characterized and places them in the context of the recent genomic scale studies that are being currently made. Furthermore, some insights into different known promoter classes concerning their architecture, regulation and classification are addressed. Finally, we discuss and present data about a core promoter that sits downstream of the transcription start site in plants, a likely case of intron-mediated enhancement. Many, if not all, of these new discoveries are raising the questions about how gene regulation is managed in vivo under continually changing conditions.

The term epigenetics defines heritable changes of gene expression that are not driven by the primary nucleotide sequence of a gene, but rather by the reorganisation of chromatin structures. In eukaryotes, chromatin allows the compaction of DNA into the nucleus via its basic structural subunit, the nucleosome, in which DNA is wrapped around histone octamers. This structure is timely and spatially remodeled to fit the needs of DNA replication and chromosome segregation during cell division processes, RNA transcription and DNA repair. In this chapter, we describe the two major mechanisms of epigenetic regulation: DNA methylation and covalent modifications of nucleosomal histone proteins, their crosstalk, and how their combinations switch on and off gene expression.

microRNAs (miRNAs) are a group of ∼22-nucleotide endogenous non-coding RNAs identified in both animal and plant genomes. miRNAs play an important regulatory role in almost all fundamental processes and are involved in cellular alterations and diverse disease progression. miRNA can regulate a target gene at both the transcriptional level and the post-transcriptional level. In miRNA directed transcriptional gene silencing, miRNAs can induce heterochromatin formation via binding to the promoter regions of their target genes. In post-transcriptional gene regulation, miRNAs regulate gene expression via attenuating protein translation or promoting mRNA degradation. Since the discovery of this small regulator, bioinformatics methods have become standard techniques to elucidate hierarchical functions of miRNAs in gene regulatory networks. In this chapter, we briefly compare the characteristics for metazoans and plant miRNAs in terms of genomic features, miRNA biogenesis and miRNA target recognition. We then focus on the chromatin effects raised by nuclear localized miRNAs. We also discuss computational tools used for analyzing the miRNA’s macro role in gene networking. Available web-based resources for miRNAs are summarized.

Phylogenetic footprinting is an evolutionary tool used for the identification of functional elements in selected genomes. This method relies on the search for similarities among ancestrally related sequences and has been widely used to identify cis-regulatory elements in non-coding genomic regions. Conserved non-coding elements (CNEs) have been shown to be crucial for controlling gene transcription in vertebrate genomes. Here we present practical guidelines for discovering new cis-regulatory elements using phylogenetic footprinting and discuss pros and cons of different approaches for the functional characterization of these elements. In addition, we present two case studies to provide practical examples of how this powerful technique is used. The first case study illustrates the in silico identification of the Myostatin gene promoter conserved in vertebrates and its biological validation. The second case study describes the search for cis-regulatory elements of the Raldh2 gene and demonstrates the conserved biological function of an intronic CNE responsible for modulating the gene activity in the embryonic dorsal spinal cord in vertebrates. The comparison of these two case studies highlights the similarities and differences regarding bioinformatic strategies and validation methods for the identification and functional characterization of promoters and distal cis-regulatory elements (enhancers/silencers).

The analyses of molecular mechanisms that control eukaryote gene expression throughout growth and development is an important issue in understanding expression changes during environmental stress, pathogen response or cancer. Information flow within a cell is governed by DNA-binding proteins such as transcription factors and their binding to DNA-motifs, but database searches revealed that qualitative data for most DNA-protein interaction is lacking. In addition, routine methods for the serial analysis of DNA-protein interaction are still missing. Here, we review the pros and cons of a wide range of laboratory methods for the functional analyses of DNA-protein interaction, their prerequisites, benefits and pit-falls. Some of these are standard in vitro or in vivo methods, such as the electrophoretic mobility shift assay (EMSA), the systematic evolution of ligands by exponential enrichment (SELEX) or the chromatin Immunoprecipitation (ChIP) to name only a few of them. However, only the yeast-one-hybrid assay, the protein-on-DNA microarrays and the DNA-protein-Interaction-ELISA (DPI-ELISA) are applicable for high-throughput proteomics/interactomics analyses of DNA-binding proteins, which would significantly improve our understanding of signaling processes at subcellular level and, in addition, allow us to predict improved gene regulatory networks from systems biology data.

Promoters, regions of DNA located upstream of genes, are the first line of control of gene expression. Cloning wild type promoter constructs upstream of reporter genes allow researchers to use them as reporters for in vitro and in vivo studies of transcriptional regulation and gene expression. Synthetic promoters are also designed in order to gain control of spatial (tissue-specific expression) and/or temporal (chemically or environmentally inducible) expression of a gene of interest. Elaboration of genetically controlled systems also leads to more complex tightly dynamic networks of synthetic promoters. In the present review, we highlight the potential applications of reporter, inducible and synthetic promoters as tools to study gene transcription and elaborate in vitro and in vivo biological systems with an outstanding impact on gene transfer and gene therapy. In the future, engineered promoters will provide the basis for highly sophisticated genetic manipulations in biological processing, biopharmaceutical applications, gene therapy and tissue engineering applications for in vivo models.

Bioinformatics and especially sequence analysis has come a long way. Nevertheless, finding cis elements is still difficult, as one is usually confronted with several problems simultaneously, hindering the efficient identification of cis elements. Here, we describe the general structure of a core promoter including its known elements and then move on to finding regulatory cis regulatory elements that might drive the expression of a gene. We review several commonly used tools and briefly touch on the techniques these use and conclude by showing that next generation sequencing techniques might provide enough additional information to make reliable cis element identification easier in the future.

The specific binding of a transcription factor to its DNA target site – the cis-element – located in the gene promoter, is considered the pivotal event in gene transcriptional regulation. However, frequently, transcriptional regulation is not mediated by single binding events but by the cooperative binding of several transcription factors such that individual cis-elements function jointly as composite elements. Furthermore, promoter regions generally do not harbor only one, but several and different cis-elements resulting in complex interactions and divers expression patterns. Here, we review primarily bioinformatic approaches to capture this regulatory complexity and look at different algorithmic strategies and associated available software solutions to identify composite cis-elements. Furthermore, efforts to describe the resulting complexity of gene regulation and the relation to whole genome architectural properties are discussed. Finally, approaches and available web-based information resources to utilize information on transcription factor – target gene binding events to infer complex gene regulatory networks are presented.

Transcription factors are proteins that bind to short sequence motifs on DNA typically called cisregulatory elements. These cis-regulatory elements are characterized by their linear base pair sequence as well as specific features of their three-dimensional structure. These structural features play an important role in the recognition and binding of proteins to DNA.

Various computational approaches have been used to model protein-DNA interaction interfaces at an atomic level. We describe their most promising scope of applications and discuss their assets and drawbacks. Structure-based computational methods require three-dimensional protein-DNA complexes gained either by X-ray crystallography or by in silico modeling. Docking approaches, molecular dynamics, and Monte Carlo simulations are promising techniques to model transcription factor-DNA complexes in silico. Both experimentally determined and ab initio designed protein-DNA complexes can be analyzed by statistical methods. We describe the differences of several statistical potentials and how they were obtained. Position weight matrices obtained from structure-based approaches can then be used to scan efficiently and more accurately genome-wide for transcription factor binding sites.

In a case study on WRKY-DNA complexes we present a computational modeling technique for the ab initio design of a specific transcription factor-DNA complex. This procedure is generally applicable to similar problems. The resulting three-dimensional interaction interface provides the basis for studying specific side chain and base interactions. Moreover, the results give hints towards varying specificity and function of different representatives of the WRKY protein family. This study provides valuable insights into the interplay between transcription factors and DNA in three dimensions and opens up new perspectives for their design.