Generally,
in progression to cancer, hundreds of genes are silenced or activated.
Although silencing of some genes in cancers occurs by mutation, a large
proportion of carcinogenic gene silencing is a result of altered DNA
methylation. DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes.
Altered expressions of microRNAs also silence or activate many genes in progression to cancer (see microRNAs in cancer). Altered microRNA expression occurs through hyper/hypo-methylation of CpG sites in CpG islands in promoters controlling transcription of the microRNAs.
Silencing of DNA repair genes through methylation of CpG islands
in their promoters appears to be especially important in progression to
cancer (see methylation of DNA repair genes in cancer).
Genes may also have distant promoters (distal promoters) and
these frequently contain CpG islands as well. An example is the
promoter of the DNA repair gene ERCC1, where the CpG island-containing promoter is located about 5,400 nucleotides upstream of the coding region of the ERCC1 gene. CpG islands also occur frequently in promoters for functional noncoding RNAs such as microRNAs.
Transcription silencing due to methylation of CpG islands
In humans, DNA methylation occurs at the 5′ position of the pyrimidine ring of the cytosine residues within CpG sites to form 5-methylcytosines. The presence of multiple methylated CpG sites in CpG islands of promoters causes stable inhibition (silencing) of genes.
Silencing of transcription of a gene may be initiated by other
mechanisms, but this is often followed by methylation of CpG sites in
the promoter CpG island to cause the stable silencing of the gene.
Transcription silencing/activation in cancers
In cancers, loss of expression of genes
occurs about 10 times more frequently by transcription silencing
(caused by promoter hypermethylation of CpG islands) than by mutations.
As Vogelstein et al. point out, in a colorectal cancer there are
usually about 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations.
In contrast, in colon tumors compared to adjacent normal-appearing
colonic mucosa, there are about 600 to 800 heavily methylated CpG
islands in promoters of genes in the tumors while these CpG islands are
not methylated in the adjacent mucosa.
Using gene set enrichment analysis, 569 out of 938 gene sets
were hypermethylated and 369 were hypomethylated in cancers.
Hypomethylation of CpG islands in promoters results in increased
transcription of the genes or gene sets affected.
One study
listed 147 specific genes with colon cancer-associated hypermethylated
promoters and 27 with hypomethylated promoters, along with the frequency
with which these hyper/hypo-methylations were found in colon cancers.
At least 10 of those genes had hypermethylated promoters in nearly 100%
of colon cancers. They also indicated 11 microRNAs
whose promoters were hypermethylated in colon cancers at frequencies
between 50% and 100% of cancers. MicroRNAs (miRNAs) are small
endogenous RNAs that pair with sequences in messenger RNAs to direct post-transcriptional repression.
On average, each microRNA represses or inhibits transcriptional
expression of several hundred target genes. Thus microRNAs with
hypermethylated promoters may be allowing enhanced transcription of
hundreds to thousands of genes in a cancer.
Transcription inhibition and activation by nuclear microRNAs
For more than 20 years, microRNAs have been known to act in the cytoplasm to degrade transcriptional expression of specific target gene messenger RNAs (see microRNA history). However, recently, Gagnon et al.
showed that as many as 75% of microRNAs may be shuttled back into the
nucleus of cells. Some nuclear microRNAs have been shown to mediate
transcriptional gene activation or transcriptional gene inhibition.
DNA repair genes with hyper/hypo-methylated promoters in cancers
DNA repair genes are frequently repressed in cancers due to hypermethylation of CpG islands within their promoters. In head and neck squamous cell carcinomas at least 15 DNA repair genes have frequently hypermethylated promoters; these genes are XRCC1, MLH3, PMS1, RAD51B, XRCC3, RAD54B, BRCA1, SHFM1, GEN1, FANCE, FAAP20, SPRTN, SETMAR, HUS1, and PER1.
About seventeen types of cancer are frequently deficient in one or
more DNA repair genes due to hypermethylation of their promoters. As summarized in one review article, promoter hypermethylation of the DNA repair gene MGMT
occurs in 93% of bladder cancers, 88% of stomach cancers, 74% of
thyroid cancers, 40%-90% of colorectal cancers and 50% of brain cancers. Promoter hypermethylation of LIG4 occurs in 82% of colorectal cancers. This review article also indicates promoter hypermethylation of NEIL1 occurs in 62% of head and neck cancers and in 42% of non-small-cell lung cancers; promoter hypermetylation of ATM occurs in 47% of non-small-cell lung cancers; promoter hypermethylation of MLH1 occurs in 48% of squamous cell carcinomas; and promoter hypermethylation of FANCB occurs in 46% of head and neck cancers.
On the other hand, the promoters of two genes, PARP1 and FEN1, were hypomethylated and these genes were over-expressed in numerous cancers. PARP1 and FEN1 are essential genes in the error-prone and mutagenic DNA repair pathway microhomology-mediated end joining. If this pathway is over-expressed, the excess mutations it causes can lead to cancer. PARP1 is over-expressed in tyrosine kinase-activated leukemias, in neuroblastoma, in testicular and other germ cell tumors, and in Ewing's sarcoma, FEN1 is over-expressed in the majority of cancers of the breast, prostate, stomach, neuroblastomas, pancreatic, and lung.
DNA damage appears to be the primary underlying cause of cancer. If accurate DNA repair is deficient, DNA damages tend to accumulate. Such excess DNA damage can increase mutational errors during DNA replication due to error-prone translesion synthesis. Excess DNA damage can also increase epigenetic alterations due to errors during DNA repair. Such mutations and epigenetic alterations can give rise to cancer (see malignant neoplasms). Thus, CpG island hyper/hypo-methylation in the promoters of DNA repair genes are likely central to progression to cancer.
In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, i.e. observable trait. The genetic information stored in DNA
represents the genotype, whereas the phenotype results from the
"interpretation" of that information. Such phenotypes are often
expressed by the synthesis of proteins that control the organism's
structure and development, or that act as enzymes catalyzing specific metabolic pathways.
The
process of transcription is carried out by RNA polymerase (RNAP), which
uses DNA (black) as a template and produces RNA (blue).
The production of a RNA copy from a DNA strand is called transcription, and is performed by RNA polymerases, which add one ribonucleotide at a time to a growing RNA strand as per the complementarity law of the nucleotide bases. This RNA is complementary to the template 3′ → 5′ DNA strand, with the exception that thymines (T) are replaced with uracils (U) in the RNA.
In prokaryotes, transcription is carried out by a single type of RNA polymerase, which needs to bind a DNA sequence called a Pribnow box with the help of the sigma factor
protein (σ factor) to start transcription. In eukaryotes, transcription
is performed in the nucleus by three types of RNA polymerases, each of
which needs a special DNA sequence called the promoter and a set of DNA-binding proteins—transcription factors—to initiate the process (see regulation of transcription below). RNA polymerase I is responsible for transcription of ribosomal RNA (rRNA) genes. RNA polymerase II (Pol II) transcribes all protein-coding genes but also some non-coding RNAs (e.g., snRNAs, snoRNAs or long non-coding RNAs). RNA polymerase III transcribes 5S rRNA, transfer RNA (tRNA) genes, and some small non-coding RNAs (e.g., 7SK). Transcription ends when the polymerase encounters a sequence called the terminator.
mRNA processing
While transcription of prokaryotic protein-coding genes creates messenger RNA (mRNA) that is ready for translation into protein, transcription of eukaryotic genes leaves a primary transcript of RNA (pre-RNA),
which first has to undergo a series of modifications to become a mature
RNA. Types and steps involved in the maturation processes vary between
coding and non-coding preRNAs; i.e. even though preRNA molecules for both mRNA and tRNA undergo splicing, the steps and machinery involved are different. The processing of non-coding RNA is described below (non-coring RNA maturation).
The processing of premRNA include 5′ capping, which is set of enzymatic reactions that add 7-methylguanosine (m7G) to the 5′ end of pre-mRNA and thus protect the RNA from degradation by exonucleases. The m7G cap is then bound by cap binding complex heterodimer (CBC20/CBC80), which aids in mRNA export to cytoplasm and also protect the RNA from decapping.
Another modification is 3′ cleavage and polyadenylation.
They occur if polyadenylation signal sequence (5′- AAUAAA-3′) is present
in pre-mRNA, which is usually between protein-coding sequence and
terminator. The pre-mRNA is first cleaved and then a series of ~200
adenines (A) are added to form poly(A) tail, which protects the RNA from
degradation. The poly(A) tail is bound by multiple poly(A)-binding proteins (PABPs)
necessary for mRNA export and translation re-initiation. In the inverse
process of deadenylation, poly(A) tails are shortened by the CCR4-Not 3′-5′ exonuclease, which often leads to full transcript decay.
Illustration
of exons and introns in pre-mRNA and the formation of mature mRNA by
splicing. The UTRs (in green) are non-coding parts of exons at the ends
of the mRNA.
A very important modification of eukaryotic pre-mRNA is RNA splicing. The majority of eukaryotic pre-mRNAs consist of alternating segments called exons and introns. During the process of splicing, an RNA-protein catalytical complex known as spliceosome
catalyzes two transesterification reactions, which remove an intron and
release it in form of lariat structure, and then splice neighbouring
exons together. In certain cases, some introns or exons can be either
removed or retained in mature mRNA. This so-called alternative splicing
creates series of different transcripts originating from a single gene.
Because these transcripts can be potentially translated into different
proteins, splicing extends the complexity of eukaryotic gene expression
and the size of a species proteome.
Extensive RNA processing may be an evolutionary advantage
made possible by the nucleus of eukaryotes. In prokaryotes,
transcription and translation happen together, whilst in eukaryotes, the
nuclear membrane separates the two processes, giving time for RNA processing to occur.
Non-coding RNA maturation
In most organisms non-coding genes (ncRNA)
are transcribed as precursors that undergo further processing. In the
case of ribosomal RNAs (rRNA), they are often transcribed as a pre-rRNA
that contains one or more rRNAs. The pre-rRNA is cleaved and modified
(2′-O-methylation and pseudouridine
formation) at specific sites by approximately 150 different small
nucleolus-restricted RNA species, called snoRNAs. SnoRNAs associate with
proteins, forming snoRNPs. While snoRNA part basepair with the target
RNA and thus position the modification at a precise site, the protein
part performs the catalytical reaction. In eukaryotes, in particular a
snoRNP called RNase, MRP cleaves the 45S pre-rRNA into the 28S, 5.8S,
and 18S rRNAs. The rRNA and RNA processing factors form large aggregates
called the nucleolus.
In the case of transfer RNA (tRNA), for example, the 5′ sequence is removed by RNase P, whereas the 3′ end is removed by the tRNase Z enzyme and the non-templated 3′ CCA tail is added by a nucleotidyl transferase. In the case of micro RNA (miRNA),
miRNAs are first transcribed as primary transcripts or pri-miRNA with a
cap and poly-A tail and processed to short, 70-nucleotide stem-loop
structures known as pre-miRNA in the cell nucleus by the enzymes Drosha and Pasha. After being exported, it is then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC), composed of the Argonaute protein.
Even snRNAs and snoRNAs themselves undergo series of modification
before they become part of functional RNP complex. This is done either
in the nucleoplasm or in the specialized compartments called Cajal bodies. Their bases are methylated or pseudouridinilated by a group of small Cajal body-specific RNAs (scaRNAs), which are structurally similar to snoRNAs.
RNA export
In eukaryotes most mature RNA must be exported to the cytoplasm from the nucleus. While some RNAs function in the nucleus, many RNAs are transported through the nuclear pores and into the cytosol.
Export of RNAs requires association with specific proteins known as
exportins. Specific exportin molecules are responsible for the export of
a given RNA type. mRNA transport also requires the correct association
with Exon Junction Complex
(EJC), which ensures that correct processing of the mRNA is completed
before export. In some cases RNAs are additionally transported to a
specific part of the cytoplasm, such as a synapse; they are then towed by motor proteins that bind through linker proteins to specific sequences (called "zipcodes") on the RNA.
Translation
For some RNA (non-coding RNA) the mature RNA is the final gene product.
In the case of messenger RNA (mRNA) the RNA is an information carrier
coding for the synthesis of one or more proteins. mRNA carrying a single
protein sequence (common in eukaryotes) is monocistronic whilst mRNA carrying multiple protein sequences (common in prokaryotes) is known as polycistronic.
During
the translation, tRNA charged with amino acid enters the ribosome and
aligns with the correct mRNA triplet. Ribosome then adds amino acid to
growing protein chain.
Every mRNA consists of three parts: a 5′ untranslated region (5′UTR),
a protein-coding region or open reading frame (ORF), and a 3′
untranslated region (3′UTR). The coding region carries information for
protein synthesis encoded by the genetic code to form triplets. Each triplet of nucleotides of the coding region is called a codon
and corresponds to a binding site complementary to an anticodon triplet
in transfer RNA. Transfer RNAs with the same anticodon sequence always
carry an identical type of amino acid. Amino acids are then chained together by the ribosome
according to the order of triplets in the coding region. The ribosome
helps transfer RNA to bind to messenger RNA and takes the amino acid
from each transfer RNA and makes a structure-less protein out of it. Each mRNA molecule is translated into many protein molecules, on average ~2800 in mammals.
In prokaryotes translation generally occurs at the point of
transcription (co-transcriptionally), often using a messenger RNA that
is still in the process of being created. In eukaryotes translation can
occur in a variety of regions of the cell depending on where the protein
being written is supposed to be. Major locations are the cytoplasm for soluble cytoplasmic proteins and the membrane of the endoplasmic reticulum for proteins that are for export from the cell or insertion into a cell membrane.
Proteins that are supposed to be expressed at the endoplasmic reticulum
are recognised part-way through the translation process. This is
governed by the signal recognition particle—a protein that binds to the ribosome and directs it to the endoplasmic reticulum when it finds a signal peptide on the growing (nascent) amino acid chain.
Folding
Protein before (left) and after (right) folding
Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA into a linear chain of amino acids. This polypeptide lacks any developed three-dimensional structure (the left hand side of the neighboring figure). The polypeptide then folds into its characteristic and functional three-dimensional structure from a random coil.
Amino acids interact with each other to produce a well-defined
three-dimensional structure, the folded protein (the right hand side of
the figure) known as the native state. The resulting three-dimensional structure is determined by the amino acid sequence (Anfinsen's dogma).
The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded. Failure to fold into the intended shape usually produces inactive proteins with different properties including toxic prions. Several neurodegenerative and other diseases are believed to result from the accumulation of misfolded proteins. Many allergies are caused by the folding of the proteins, for the immune system does not produce antibodies for certain protein structures.
Enzymes called chaperones assist the newly formed protein to attain (fold into) the 3-dimensional structure it needs to function. Similarly, RNA chaperones help RNAs attain their functional shapes. Assisting protein folding is one of the main roles of the endoplasmic reticulum in eukaryotes.
Translocation
Secretory
proteins of eukaryotes or prokaryotes must be translocated to enter the
secretory pathway. Newly synthesized proteins are directed to the
eukaryotic Sec61 or prokaryotic SecYEG translocation channel by signal peptides. The efficiency of protein secretion in eukaryotes is very dependent on the signal peptide which has been used.
Protein transport
Many proteins are destined for other parts of the cell than the cytosol and a wide range of signalling sequences or (signal peptides)
are used to direct proteins to where they are supposed to be. In
prokaryotes this is normally a simple process due to limited
compartmentalisation of the cell. However, in eukaryotes there is a
great variety of different targeting processes to ensure the protein
arrives at the correct organelle.
Not all proteins remain within the cell and many are exported, for example, digestive enzymes, hormones and extracellular matrix
proteins. In eukaryotes the export pathway is well developed and the
main mechanism for the export of these proteins is translocation to the
endoplasmic reticulum, followed by transport via the Golgi apparatus.
Regulation of gene expression
The patchy colours of a tortoiseshell cat are the result of different levels of expression of pigmentation genes in different areas of the skin.
Regulation of gene expression refers to the control of the amount and
timing of appearance of the functional product of a gene. Control of
expression is vital to allow a cell to produce the gene products it
needs when it needs them; in turn, this gives cells the flexibility to
adapt to a variable environment, external signals, damage to the cell,
and other stimuli. More generally, gene regulation gives the cell
control over all structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism.
Numerous terms are used to describe types of genes depending on how they are regulated; these include:
A constitutive gene is a gene that is transcribed continually as opposed to a facultative gene, which is only transcribed when needed.
A housekeeping gene
is a gene that is required to maintain basic cellular function and so
is typically expressed in all cell types of an organism. Examples
include actin, GAPDH and ubiquitin.
Some housekeeping genes are transcribed at a relatively constant rate
and these genes can be used as a reference point in experiments to
measure the expression rates of other genes.
A facultative gene is a gene only transcribed when needed as opposed to a constitutive gene.
An inducible gene is a gene whose expression is either responsive to environmental change or dependent on the position in the cell cycle.
Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification
of a protein. The stability of the final gene product, whether it is
RNA or protein, also contributes to the expression level of the gene—an
unstable product results in a low expression level. In general gene
expression is regulated through changes in the number and type of interactions between molecules that collectively influence transcription of DNA and translation of RNA.
Some simple examples of where gene expression is important are:
Cyclin expression levels control progression through the eukaryotic cell cycle.
Transcriptional regulation
When
lactose is present in a prokaryote, it acts as an inducer and
inactivates the repressor so that the genes for lactose metabolism can
be transcribed.
Regulation of transcription can be broken down into three main routes
of influence; genetic (direct interaction of a control factor with the
gene), modulation interaction of a control factor with the transcription
machinery and epigenetic (non-sequence changes in DNA structure that
influence transcription).
The lambda repressor transcription factor (green) binds as a dimer to major groove of DNA target (red and blue) and disables initiation of transcription.
Direct interaction with DNA is the simplest and the most direct
method by which a protein changes transcription levels. Genes often have
several protein binding sites around the coding region with the
specific function of regulating transcription. There are many classes of
regulatory DNA binding sites known as enhancers, insulators and silencers. The mechanisms for regulating transcription are very varied, from blocking key binding sites on the DNA for RNA polymerase to acting as an activator and promoting transcription by assisting RNA polymerase binding.
The activity of transcription factors is further modulated by
intracellular signals causing protein post-translational modification
including phosphorylated, acetylated, or glycosylated.
These changes influence a transcription factor's ability to bind,
directly or indirectly, to promoter DNA, to recruit RNA polymerase, or
to favor elongation of a newly synthesized RNA molecule.
The nuclear membrane in eukaryotes allows further regulation of
transcription factors by the duration of their presence in the nucleus,
which is regulated by reversible changes in their structure and by
binding of other proteins. Environmental stimuli or endocrine signals may cause modification of regulatory proteins eliciting cascades of intracellular signals, which result in regulation of gene expression.
More recently it has become apparent that there is a significant
influence of non-DNA-sequence specific effects on transcription. These
effects are referred to as epigenetic
and involve the higher order structure of DNA, non-sequence specific
DNA binding proteins and chemical modification of DNA. In general
epigenetic effects alter the accessibility of DNA to proteins and so
modulate transcription.
In eukaryotes, DNA is organized in form of nucleosomes. Note how the DNA (blue and green) is tightly wrapped around the protein core made of histoneoctamer (ribbon coils), restricting access to the DNA. From PDB: 1KX5.
In eukaryotes the structure of chromatin, controlled by the histone code, regulates access to DNA with significant impacts on the expression of genes in euchromatin and heterochromatin areas.
Enhancers, transcription factors, Mediator complex and DNA loops in mammalian transcription
Regulation of transcription in mammals. An active enhancer regulatory region is enabled to interact with the promoter region of its target gene by formation of a chromosome loop. This can initiate messenger RNA (mRNA) synthesis by RNA polymerase II (RNAP II) bound to the promoter at the transcription start site
of the gene. The loop is stabilized by one architectural protein
anchored to the enhancer and one anchored to the promoter and these
proteins are joined together to form a dimer (red zigzags). Specific
regulatory transcription factors
bind to DNA sequence motifs on the enhancer. General transcription
factors bind to the promoter. When a transcription factor is activated
by a signal (here indicated as phosphorylation
shown by a small red star on a transcription factor on the enhancer)
the enhancer is activated and can now activate its target promoter. The
active enhancer is transcribed on each strand of DNA in opposite
directions by bound RNAP IIs. Mediator (a complex consisting of about
26 proteins in an interacting structure) communicates regulatory signals
from the enhancer DNA-bound transcription factors to the promoter.
Enhancers
are regions of the genome that are major gene-regulatory elements.
Enhancers control cell-type-specific gene expression programs, most
often by looping through long distances to come in physical proximity
with the promoters of their target genes.
Multiple enhancers, each often at tens or hundred of thousands of
nucleotides distant from their target genes, loop to their target gene
promoters and coordinate with each other to control expression of their
common target gene.
The schematic illustration at the left shows an enhancer looping
around to come into close physical proximity with the promoter of a
target gene. The loop is stabilized by a dimer of a connector protein
(e.g. dimer of CTCF or YY1),
with one member of the dimer anchored to its binding motif on the
enhancer and the other member anchored to its binding motif on the
promoter (represented by the red zigzags in the illustration). Several cell function specific transcription factors (there are about 1,600 transcription factors in a human cell) generally bind to specific motifs on an enhancer
and a small combination of these enhancer-bound transcription factors,
when brought close to a promoter by a DNA loop, govern level of
transcription of the target gene. Mediator (a complex usually
consisting of about 26 proteins in an interacting structure)
communicates regulatory signals from enhancer DNA-bound transcription
factors directly to the RNA polymerase II (pol II) enzyme bound to the
promoter.
Enhancers, when active, are generally transcribed from both
strands of DNA with RNA polymerases acting in two different directions,
producing two eRNAs as illustrated in the Figure.
An inactive enhancer may be bound by an inactive transcription factor.
Phosphorylation of the transcription factor may activate it and that
activated transcription factor may then activate the enhancer to which
it is bound (see small red star representing phosphorylation of
transcription factor bound to enhancer in the illustration). An activated enhancer begins transcription of its RNA before activating transcription of messenger RNA from its target gene.
DNA methylation and demethylation in transcriptional regulation
DNA methylation is the addition of a methyl group to the DNA that happens at cytosine.
The image shows a cytosine single ring base and a methyl group added on
to the 5 carbon. In mammals, DNA methylation occurs almost exclusively
at a cytosine that is followed by a guanine.
DNA methylation is a widespread mechanism for epigenetic influence on gene expression and is seen in bacteria and eukaryotes
and has roles in heritable transcription silencing and transcription
regulation. Methylation most often occurs on a cytosine (see Figure).
Methylation of cytosine primarily occurs in dinucleotide sequences where
a cytosine is followed by a guanine, a CpG site. The number of CpG sites in the human genome is about 28 million. Depending on the type of cell, about 70% of the CpG sites have a methylated cytosine.
Methylation of cytosine in DNA has a major role in regulating
gene expression. Methylation of CpGs in a promoter region of a gene
usually represses gene transcription while methylation of CpGs in the body of a gene increases expression. TET enzymes play a central role in demethylation of methylated cytosines. Demethylation of CpGs in a gene promoter by TET enzyme activity increases transcription of the gene.
Transcriptional regulation in learning and memory
The identified areas of the human brain are involved in memory formation.
In a rat, contextual fear conditioning (CFC) is a painful learning experience. Just one episode of CFC can result in a life-long fearful memory. After an episode of CFC, cytosine methylation is altered in the promoter regions of about 9.17% of all genes in the hippocampus neuron DNA of a rat. The hippocampus
is where new memories are initially stored. After CFC about 500 genes
have increased transcription (often due to demethylation of CpG sites in
a promoter region) and about 1,000 genes have decreased transcription
(often due to newly formed 5-methylcytosine at CpG sites in a promoter
region). The pattern of induced and repressed genes within neurons
appears to provide a molecular basis for forming the first transient
memory of this training event in the hippocampus of the rat brain.
In particular, the brain-derived neurotrophic factor gene (BDNF) is known as a "learning gene." After CFC there was upregulation of BDNF
gene expression, related to decreased CpG methylation of certain
internal promoters of the gene, and this was correlated with learning.
Transcriptional regulation in cancer
The majority of gene promoters contain a CpG island with numerous CpG sites. When many of a gene's promoter CpG sites are methylated the gene becomes silenced. Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations.
However, transcriptional silencing may be of more importance than
mutation in causing progression to cancer. For example, in colorectal
cancers about 600 to 800 genes are transcriptionally silenced by CpG
island methylation. Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs. In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-expressed microRNA-182 than by hypermethylation of the BRCA1 promoter (see Low expression of BRCA1 in breast and ovarian cancers).
Post-transcriptional regulation
In eukaryotes, where export of RNA is required before translation is
possible, nuclear export is thought to provide additional control over
gene expression. All transport in and out of the nucleus is via the nuclear pore and transport is controlled by a wide range of importin and exportin proteins.
Expression of a gene coding for a protein is only possible if the
messenger RNA carrying the code survives long enough to be translated.
In a typical cell, an RNA molecule is only stable if specifically
protected from degradation. RNA degradation has particular importance in
regulation of expression in eukaryotic cells where mRNA has to travel
significant distances before being translated. In eukaryotes, RNA is
stabilised by certain post-transcriptional modifications, particularly
the 5′ cap and poly-adenylated tail.
Intentional degradation of mRNA is used not just as a defence
mechanism from foreign RNA (normally from viruses) but also as a route
of mRNA destabilisation. If an mRNA molecule has a complementary sequence to a small interfering RNA then it is targeted for destruction via the RNA interference pathway.
Three prime untranslated regions and microRNAs
Three prime untranslated regions (3′UTRs) of messenger RNAs
(mRNAs) often contain regulatory sequences that post-transcriptionally
influence gene expression. Such 3′-UTRs often contain both binding sites
for microRNAs
(miRNAs) as well as for regulatory proteins. By binding to specific
sites within the 3′-UTR, miRNAs can decrease gene expression of various
mRNAs by either inhibiting translation or directly causing degradation
of the transcript. The 3′-UTR also may have silencer regions that bind
repressor proteins that inhibit the expression of a mRNA.
The 3′-UTR often contains microRNA response elements (MREs).
MREs are sequences to which miRNAs bind. These are prevalent motifs
within 3′-UTRs. Among all regulatory motifs within the 3′-UTRs (e.g.
including silencer regions), MREs make up about half of the motifs.
As of 2014, the miRBase web site, an archive of miRNAsequences
and annotations, listed 28,645 entries in 233 biologic species. Of
these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were
predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). Friedman et al.
estimate that >45,000 miRNA target sites within human mRNA 3′UTRs
are conserved above background levels, and >60% of human
protein-coding genes have been under selective pressure to maintain
pairing to miRNAs.
Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs.
Other experiments show that a single miRNA may repress the production
of hundreds of proteins, but that this repression often is relatively
mild (less than 2-fold).
The effects of miRNA dysregulation of gene expression seem to be important in cancer. For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.
The effects of miRNA dysregulation of gene expression also seem
to be important in neuropsychiatric disorders, such as schizophrenia,
bipolar disorder, major depression, Parkinson's disease, Alzheimer's
disease and autism spectrum disorders.
Translational regulation
Neomycin
is an example of a small molecule that reduces expression of all
protein genes inevitably leading to cell death; it thus acts as an antibiotic.
Direct regulation of translation is less prevalent than control of
transcription or mRNA stability but is occasionally used. Inhibition of
protein translation is a major target for toxins and antibiotics, so they can kill a cell by overriding its normal gene expression control. Protein synthesis inhibitors include the antibiotic neomycin and the toxin ricin.
Post-translational modifications
Post-translational modifications (PTMs) are covalent
modifications to proteins. Like RNA splicing, they help to
significantly diversify the proteome. These modifications are usually
catalyzed by enzymes. Additionally, processes like covalent additions to
amino acid side chain residues can often be reversed by other enzymes.
However, some, like the proteolytic cleavage of the protein backbone, are irreversible.
PTMs play many important roles in the cell. For example, phosphorylation is primarily involved in activating and deactivating proteins and in signaling pathways.
PTMs are involved in transcriptional regulation: an important function
of acetylation and methylation is histone tail modification, which
alters how accessible DNA is for transcription. They can also be seen in the immune system, where glycosylation plays a key role. One type of PTM can initiate another type of PTM, as can be seen in how ubiquitination tags proteins for degradation through proteolysis.
Proteolysis, other than being involved in breaking down proteins, is
also important in activating and deactivating them, and in regulating
biological processes such as DNA transcription and cell death.
Measurement
Measuring gene expression is an important part of many life sciences,
as the ability to quantify the level at which a particular gene is
expressed within a cell, tissue or organism can provide a lot of
valuable information. For example, measuring gene expression can:
Identify viral infection of a cell (viral protein expression).
Determine an individual's susceptibility to cancer (oncogene expression).
Similarly, the analysis of the location of protein expression is a
powerful tool, and this can be done on an organismal or cellular scale.
Investigation of localization is particularly important for the study of
development
in multicellular organisms and as an indicator of protein function in
single cells. Ideally, measurement of expression is done by detecting
the final gene product (for many genes, this is the protein); however,
it is often easier to detect one of the precursors, typically mRNA and to infer gene-expression levels from these measurements.
mRNA quantification
Levels of mRNA can be quantitatively measured by northern blotting, which provides size and sequence information about the mRNA molecules. A sample of RNA is separated on an agarose gel
and hybridized to a radioactively labeled RNA probe that is
complementary to the target sequence. The radiolabeled RNA is then
detected by an autoradiograph.
Because the use of radioactive reagents makes the procedure time
consuming and potentially dangerous, alternative labeling and detection
methods, such as digoxigenin and biotin chemistries, have been
developed. Perceived disadvantages of Northern blotting are that large
quantities of RNA are required and that quantification may not be
completely accurate, as it involves measuring band strength in an image
of a gel. On the other hand, the additional mRNA size information from
the Northern blot allows the discrimination of alternately spliced
transcripts.
Another approach for measuring mRNA abundance is RT-qPCR. In this technique, reverse transcription is followed by quantitative PCR. Reverse transcription first generates a DNA template from the mRNA; this single-stranded template is called cDNA. The cDNA template is then amplified in the quantitative step, during which the fluorescence emitted by labeled hybridization probes or intercalating dyes changes as the DNA amplification
process progresses. With a carefully constructed standard curve, qPCR
can produce an absolute measurement of the number of copies of original
mRNA, typically in units of copies per nanolitre of homogenized tissue
or copies per cell. qPCR is very sensitive (detection of a single mRNA
molecule is theoretically possible), but can be expensive depending on
the type of reporter used; fluorescently labeled oligonucleotide probes
are more expensive than non-specific intercalating fluorescent dyes.
For expression profiling, or high-throughput analysis of many genes within a sample, quantitative PCR may be performed for hundreds of genes simultaneously in the case of low-density arrays. A second approach is the hybridization microarray.
A single array or "chip" may contain probes to determine transcript
levels for every known gene in the genome of one or more organisms.
Alternatively, "tag based" technologies like Serial analysis of gene expression (SAGE) and RNA-Seq, which can provide a relative measure of the cellular concentration
of different mRNAs, can be used. An advantage of tag-based methods is
the "open architecture", allowing for the exact measurement of any
transcript, with a known or unknown sequence. Next-generation sequencing
(NGS) such as RNA-Seq
is another approach, producing vast quantities of sequence data that
can be matched to a reference genome. Although NGS is comparatively
time-consuming, expensive, and resource-intensive, it can identify single-nucleotide polymorphisms,
splice-variants, and novel genes, and can also be used to profile
expression in organisms for which little or no sequence information is
available.
RNA profiles in Wikipedia
The RNA expression profile of the GLUT4 Transporter (one of the main glucose transporters found in the human body)
Profiles like these are found for almost all proteins listed in Wikipedia. They are generated by organizations such as the Genomics Institute of the Novartis Research Foundation and the European Bioinformatics Institute.
Additional information can be found by searching their databases (for
an example of the GLUT4 transporter pictured here, see citation).
These profiles indicate the level of DNA expression (and hence RNA
produced) of a certain protein in a certain tissue, and are color-coded
accordingly in the images located in the Protein Box on the right side
of each Wikipedia page.
Protein quantification
For
genes encoding proteins, the expression level can be directly assessed
by a number of methods with some clear analogies to the techniques for
mRNA quantification.
One of the most commonly used methods is to perform a Western blot against the protein of interest. This gives information on the size of the protein in addition to its identity. A sample (often cellular lysate) is separated on a polyacrylamide gel, transferred to a membrane and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase
for imaging and/or quantification. The gel-based nature of this assay
makes quantification less accurate, but it has the advantage of being
able to identify later modifications to the protein, for example
proteolysis or ubiquitination, from changes in size.
mRNA-protein correlation
Quantification
of protein and mRNA permits a correlation of the two levels. The
question of how well protein levels correlate with their corresponding
transcript levels is highly debated and depends on multiple factors.
Regulation on each step of gene expression can impact the correlation,
as shown for regulation of translation or protein stability. Post-translational factors, such as protein transport in highly polar cells, can influence the measured mRNA-protein correlation as well.
Localisation
In situ-hybridization of Drosophilaembryos at different developmental stages for the mRNA responsible for the expression of hunchback. High intensity of blue color marks places with high hunchback mRNA quantity.
Analysis of expression is not limited to quantification; localisation
can also be determined. mRNA can be detected with a suitably labelled
complementary mRNA strand and protein can be detected via labelled
antibodies. The probed sample is then observed by microscopy to identify
where the mRNA or protein is.
The three-dimensional structure of green fluorescent protein.
The residues in the centre of the "barrel" are responsible for
production of green light after exposing to higher energetic blue light.
From PDB: 1EMA.
By replacing the gene with a new version fused to a green fluorescent protein (or similar) marker, expression may be directly quantified in live cells. This is done by imaging using a fluorescence microscope.
It is very difficult to clone a GFP-fused protein into its native
location in the genome without affecting expression levels so this
method often cannot be used to measure endogenous gene expression. It
is, however, widely used to measure the expression of a gene
artificially introduced into the cell, for example via an expression vector.
It is important to note that by fusing a target protein to a
fluorescent reporter the protein's behavior, including its cellular
localization and expression level, can be significantly changed.
The enzyme-linked immunosorbent assay works by using antibodies immobilised on a microtiter plate
to capture proteins of interest from samples added to the well. Using a
detection antibody conjugated to an enzyme or fluorophore the quantity
of bound protein can be accurately measured by fluorometric or colourimetric
detection. The detection process is very similar to that of a Western
blot, but by avoiding the gel steps more accurate quantification can be
achieved.
Expression system
Tet-ON inducible shRNA system
An expression system is a system specifically designed for the
production of a gene product of choice. This is normally a protein
although may also be RNA, such as tRNA or a ribozyme. An expression system consists of a gene, normally encoded by DNA, and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein
using the reagents provided. In the broadest sense this includes every
living cell but the term is more normally used to refer to expression as
a laboratory tool. An expression system is therefore often artificial
in some manner. Expression systems are, however, a fundamentally natural
process. Viruses are an excellent example where they replicate by using
the host cell as an expression system for the viral proteins and
genome.
In
addition to these biological tools, certain naturally observed
configurations of DNA (genes, promoters, enhancers, repressors) and the
associated machinery itself are referred to as an expression system.
This term is normally used in the case where a gene or set of genes is
switched on under well defined conditions, for example, the simple
repressor switch expression system in Lambda phage and the lac operator
system in bacteria. Several natural expression systems are directly
used or modified and used for artificial expression systems such as the Tet-on and Tet-off expression system.
Gene networks
Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors,
and outputs being the level of gene expression. The node itself
performs a function, and the operation of these functions have been
interpreted as performing a kind of information processing within cells and determines cellular behavior.
Gene networks can also be constructed without formulating an
explicit causal model. This is often the case when assembling networks
from large expression data sets. Covariation and correlation of expression is computed across a large sample of cases and measurements (often transcriptome or proteome
data). The source of variation can be either experimental or natural
(observational). There are several ways to construct gene expression
networks, but one common approach is to compute a matrix of all
pair-wise correlations of expression across conditions, time points, or
individuals and convert the matrix (after thresholding at some cut-off
value) into a graphical representation in which nodes represent genes,
transcripts, or proteins and edges connecting these nodes represent the
strength of association.
Commonly used protein production systems include those derived from bacteria, yeast,baculovirus/insect, mammalian cells, and more recently filamentous fungi such as Myceliophthora thermophila. When biopharmaceuticals are produced with one of these systems, process-related impurities termed host cell proteins also arrive in the final product in trace amounts.
Cell-based systems
The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vector,
its cloned DNA, and the host for the vector that provide a context to
allow foreign gene function in a host cell, that is, produce proteins at
a high level".Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype.
There are many ways to introduce foreign DNA
to a cell for expression, and many different host cells may be used for
expression — each expression system has distinct advantages and
liabilities. Expression systems are normally referred to by the host and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria (such as E.coli, B. subtilis), yeast (such as S.cerevisiae) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses (such as baculovirus, retrovirus, adenovirus), plasmids, artificial chromosomes and bacteriophage (such as lambda). The best expression system depends on the gene involved, for example the Saccharomyces cerevisiae is often preferred for proteins that require significant posttranslational modification. Insect or mammal
cell lines are used when human-like splicing of mRNA is required.
Nonetheless, bacterial expression has the advantage of easily producing
large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.
Because bacteria are prokaryotes,
they are not equipped with the full enzymatic machinery to accomplish
the required post-translational modifications or molecular folding.
Hence, multi-domain eukaryotic proteins expressed in bacteria often are
non-functional. Also, many proteins become insoluble as inclusion bodies
that are difficult to recover without harsh denaturants and subsequent
cumbersome protein-refolding.
To address these concerns, expressions systems using multiple
eukaryotic cells were developed for applications requiring the proteins
be conformed as in, or closer to eukaryotic organisms: cells of plants
(i.e. tobacco), of insects or mammalians (i.e. bovines) are transfected
with genes and cultured in suspension and even as tissues or whole
organisms, to produce fully folded proteins. Mammalian in vivo
expression systems have however low yield and other limitations
(time-consuming, toxicity to host cells,..). To combine the high
yield/productivity and scalable protein features of bacteria and yeast,
and advanced epigenetic features of plants, insects and mammalians
systems, other protein production systems are developed using
unicellular eukaryotes (i.e. non-pathogenic 'Leishmania' cells).
Bacterial systems
Escherichia coli
E. coli, one of the most popular hosts for artificial gene expression.
E. coli is one of the most widely used expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli
are well developed and work by increasing the number of copies of the
gene or increasing the binding strength of the promoter region so
assisting transcription.
For example, a DNA sequence for a protein of interest could be cloned or subcloned into a high copy-number plasmid containing the lac (often LacUV5) promoter, which is then transformed into the bacterium E. coli. Addition of IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest.
E. coli strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack lon and OmpT proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides T7 RNA polymerase (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead.
Corynebacterium
Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine, components of human food, animal feed and pharmaceutical products.
Expression of functionally active human epidermal growth factor has been done in C. glutamicum,
thus demonstrating a potential for industrial-scale production of human
proteins. Expressed proteins can be targeted for secretion through
either the general, secretory pathway (Sec) or the twin-arginine translocation pathway (Tat).
The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein.
Eukaryotic systems
Yeasts
Expression systems using either S. cerevisiae or Pichia pastoris
allow stable and lasting production of proteins that are processed
similarly to mammalian cells, at high yield, in chemically defined media
of proteins.
Filamentous fungi
Filamentous fungi, especially Aspergillus and Trichoderma, but also more recently Myceliophthora thermophila C1 have been developed into expression platforms for screening and production of diverse industrial enzymes.
The expression system C1 shows a low viscosity morphology in submerged
culture, enabling the use of complex growth and production media.
Baculovirus-infected cells
Baculovirus-infected insect cells (Sf9, Sf21, High Five strains) or mammalian cells (HeLa, HEK 293) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.
It is useful for production of proteins in high quantity. Genes are not
expressed continuously because infected host cells eventually lyse and
die during each infection cycle.
Non-lytic insect cell expression
Non-lytic
insect cell expression is an alternative to the lytic baculovirus
expression system. In non-lytic expression, vectors are transiently or
stably transfected into the chromosomal DNA of insect cells for subsequent gene expression. This is followed by selection and screening of recombinant clones.
The non-lytic system has been used to give higher protein yield and
quicker expression of recombinant genes compared to baculovirus-infected
cell expression. Cell lines used for this system include: Sf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells. With this system, cells do not lyse and several cultivation modes can be used. Additionally, protein production runs are reproducible. This system gives a homogeneous product. A drawback of this system is the requirement of an additional screening step for selecting viable clones.
Leishmania tarentolae
(cannot infect mammals) expression systems allow stable and lasting
production of proteins at high yield, in chemically defined media.
Produced proteins exhibit fully eukaryotic post-translational
modifications, including glycosylation and disulfide bond formation.
Mammalian systems
The most common mammalian expression systems are Chinese Hamsterovary (CHO) and Human embryonic kidney (HEK) cells.
Cell-free production of proteins is performed in vitro
using purified RNA polymerase, ribosomes, tRNA and ribonucleotides.
These reagents may be produced by extraction from cells or from a
cell-based expression system. Due to the low expression levels and high
cost of cell-free systems, cell-based systems are more widely used.
