Regulation of gene expression

''Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation.''


 * For vocabulary, see Glossary of gene expression terms

Regulation of gene expression (or gene regulation) includes the processes that cells and viruses use to regulate the way that the information in genes is turned into gene products. Although a functional gene product may be an RNA or a protein, the majority of known mechanisms regulate protein coding genes. Any step of the gene's expression may be modulated, from DNA-RNA transcription to the post-translational modification of a protein.

Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. Although in 1951, Barbara Mclintok showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon, discovered by Jacques Monod, in which protein involved in lactose metabolism are expressed by E. coli only in the presence of lactose and absence of glucose.

Furthermore, gene regulation drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms where the different types of cells may possess different gene expression profiles though they all possess the same genome sequence.

Regulated stages of gene expression
Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilised point is Transcription Initiation:


 * Chromatin domains
 * Transcription
 * Post-transcriptional modification
 * RNA transport
 * Translation
 * mRNA degradation

Modification of DNA
In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin structure, which can be altered as a result of histone modifications directed by DNA methylation, ncRNA, or DNA-binding protein. Hence these modifications may up or down regulate the expression of gene. Certain of these modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.

Chemical
Methylation of DNA is a common method of gene silencing. DNA is typically methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG islands" when densely clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment, whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods developed to quantify SNPs, such as Pyrosequencing (Biotage) or MassArray (Sequenom), measuring the relative amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis.

Structural
Transcription of DNA is dictated by its structure. In general, the density of its packing is indicative of the frequency of transcription. Octameric protein complexes called nucleosomes are responsible for the amount of supercoiling of DNA, and these complexes can be temporarily modified by processes such as phosphorylation or more permanently modified by processes such as methylation. Such modifications are considered to be responsible for more or less permanent changes in gene expression levels.

Histone acetylation is also an important process in transcription. Histone acetyltransferase enzymes (HATs) such as CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often, DNA methylation and histone deacetylation work together in gene silencing. The combination of the two seems to be a signal for DNA to be packed more densely, lowering gene expression.

Regulation of transcription
Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by at least five mechanisms:


 * Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e., sigma factors used in prokaryotic transcription).
 * Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene.
 * General transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA.
 * Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
 * Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex. Enhancers are much more common in eukaryote than prokaryotes, where only a few examples exist (to date).

Post-transcriptional regulation
After the DNA is transcribed and mRNA is formed, there must be some sort of regulation on how much the mRNA is translated into proteins. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript. These processes occur in eukaryotes but not in prokaryotes. This modulation is a result of a protein or transcript that, in turn, is regulated and may have an affinity for certain sequences.

Regulation of translation
The translation of mRNA can also be controlled by a number of mechanisms, mostly at the level of initiation. Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist, which often are directed to their target sequence by the secondary structure of the transcript, which may change depending on certain conditions, such as temperature or presence of a ligand (aptamer). Some transcripts act as ribozymes and self-regulate their expression.

Examples of gene regulation

 * Enzyme induction is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme.
 * The induction of heat shock proteins in the fruit fly Drosophila melanogaster.
 * The Lac operon is an interesting example of how gene expression can be regulated.
 * Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV).

Developmental biology
A large number of studied regulatory systems come from developmental biology. Examples include:


 * The colinearity of the Hox gene cluster with their nested antero-posterior patterning
 * It has been speculated that pattern generation of the hand (digits - interdigits) The gradient of Sonic hedgehog (secreted inducing factor) from the zone of polarizing activity in the limb, which creates a gradient of active Gli3, which activates Gremlin, which inhibits BMPs also secreted in the limb, resulting in the formation of an alternating pattern of activity as a result of this reaction-diffusion system.
 * Somitogenesis is the creation of segments (somites) from a uniform tissue (Pre-somitic Mesoderm, PSM). They are formed sequentially from anterior to posterior. This is achieved in amniotes possibly by means of two opposing gradients, Retinoic acid in the anterior (wavefront) and Wnt and Fgf in the posterior, coupled to an oscillating pattern (segmentation clock) composed of FGF + Notch and Wnt in antiphase.
 * Sex determination in the soma of a Drosophila requires the sensing of the ratio of autosomal genes to sex chromosome-encoded genes, which results in the production of sexless splicing factor in females, resulting in the female isoform of doublesex.

Up-regulation and down-regulation
Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell), which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. On the converse, down-regulation is a process resulting in decreased gene and corresponding protein expression.


 * Up-regulation occurs, for example, when a cell is deficient in some kind of receptor. In this case, more receptor protein is synthesized and transported to the membrane of the cell and, thus, the sensitivity of the cell is brought back to normal, reestablishing homeostasis.


 * Down-regulation occurs, for example, when a cell is overstimulated by a neurotransmitter, hormone, or drug for a prolonged period of time, and the expression of the receptor protein is decreased in order to protect the cell (see also tachyphylaxis).

Inducible vs. repressible systems
Gene Regulation can be summarized by the reponse of the respective system:


 * Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.


 * Repressible systems - A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to "repress expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.

Theoretical circuits

 * Repressor/Inducer: an activation of a sensor results in the change of expression of a gene
 * negative feedback: the gene product downregulates its own production directly or indirectly, which can result in
 * keeping transcript levels constant/proportional to a factor
 * inhibition of run-away reactions when coupled with a positive feedback loop
 * creating an oscillator by taking advantage in the time delay of transcription and translation, given that the mRNA and protein half-life is shorter
 * positive feedback: the gene product upregulates its own production directly or indirectly, which can result in
 * signal amplification
 * bistable switches when two genes inhibit each other and have both positive feedback
 * pattern generation

Methods
In general, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determine which genes changed and by how much they did. These are, however, not informative of where the regulation has occurred and may actually mask conflicting regulatory processess (see post-transcriptional regulation), but it is still the most commonly analysed (QPCR and DNA microarray).

When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:


 * The chromatin conformation of the region can be determined by ChIP-chip analysis by pulling down RNA Polymerase II, Histone 3 modifications, Trithorax-group protein, Polycomb-group protein, or any other DNA-binding element to which a good antibody is available.
 * Epistatic interactions can be investigated by synthetic genetic array analysis
 * Due to post-transcriptional regulation, transcription rates and total RNA levels differ significantly. To measure the transcription rates nuclear run-on assays can be done and newer high-throughput methods are being developed, using thiol labelling instead of radioactivity.
 * Only 5% of the RNA polymerised in the nucleus actually exists, and not only introns, abortive products, and non-sense transcripts are degradated. Therefore, the differences in nuclear and cytoplasmic levels can be see by separating the two fractions by gentle lysis.
 * Alternative splicing can be analysed with a splicing array or with a tiling array (see DNA microarray).
 * All in vivo RNA is complexed as RNPs. The quantity of transcripts bound to specific protein can be also analysed by RIP-Chip. For example, DCP2 will give an indication of sequestered protein; ribosome-bound gives and indication of transcripts active in transcription (although it should be noted that a more dated method, called polysome fractionation, is still popular in some labs)
 * Protein levels can be analysed by Mass spectrometry, which can be compared only to QPCR data, as microarray data is relative and not absolute.
 * RNA and protein degradation rates are measured by means of transcription inhibitors (actinomycin D or α-amanitin) or translation inhibitors (Cycloheximide), respectively.