Regulation of gene expression

Regulation of gene expression (or gene regulation) includes the processes that cells and viruses use to turn the information on genes 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. The first discovered example of a gene regulation system was 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:

• chromatin domains
• Transcription
• Post-transcriptional modification
• RNA transport
• Translation
• mRNA degradation
• Post-translational modifications

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 which are directed by DNA methylation, ncRNA or DNA binding protein.

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 carcinogenesis.

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 These 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.

Posttranscriptional 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 which in turn is regulated and may have an affinity for certain sequences.

• Capping changes the five prime end of the mRNA to a three prime end by 5'-5' linkage, which protects the mRNA from 5' exonuclease, which degrades foreign RNA. The cap also helps in ribosomal binding.
• Splicing removes the introns, noncoding regions that are transcribed into RNA, in order to make the mRNA able to create proteins. Cells do this by spliceosome's binding on either side of an intron, looping the intron into a circle and then cleaving it off. The two ends of the exons are then joined together.
• Addition of poly(A) tail otherwise known as poly-adenylation. Junk RNA is added to the 3' end, and acts as a buffer to the 3' exonuclease in order to increase the half life of mRNA.

In both prokaryotes and eukaryotes a large number of RNA binding protein 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 a early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV)

Circuitry

Up-regulation and down-regulation

Up-regulation is a process which 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. Conversely 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 overly stimulated 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 as how they respond:

• 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 in 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 in which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.

Developmental biology

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

• The collinearity 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.
• Somatogenesis is the creation of segmatation (somites) form a uniform tissue (PSM) sequentially from anterior to posterior, this is a achieved in amniotes possibly by means of two opposing gradients, Retinoic acid in the anterior (wavefront) and an oscillating gradient in the posterior (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.

Theoretical circuits

• Repressor/Inducer: a 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

Generally, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determined 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, it is 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.
• Epigenetic 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 only be compare 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.