Dihydrofolate Reductase (DHFR) enzyme is responsible for the reaction in which dihydrofolate is reduced to tetrahydrofolate. Tetrahydrofolate is vital for synthesis of Purines and Thymidylate. For this reason the regulation of DHFR expression in the cell is imperative in order for cells to grow and proliferate.. DHFR is an extensively studied as a model gene due to some of its distinctive properties. These include lack of a TATA promoter,interactions with transcription factors at different stages of the cell cycle, autoregulation of translation. This review summarizes the current understanding of the regulation of human dihydrofolate reductase expression in the cell. Initially the structure and function of the enzyme are discussed in order to establish an understanding as to its importance in the cell. Next the review looks at regulation of the gene from many levels; Transcription, Post- Transcriptional modifications and Translation.
Dihydrofolate reductase is one of the key enzymes in the folate metabolism pathway.
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Figure 1 represents a simplified version of the folate metabolism pathway. Genes and proteins can be seen in blue while the substrates are the green rounded rectangles. The area of interest to this review is denoted by an arrow. (Leeder and Mitchel, 2007).
Dihydrofolate reductase (DHFR) enzyme catalyses the reduction of 7,8-dihydrofolate (H2F) 5,6,7,8-tetrafolate (H4F) utilizing NADPH as a cofactor (Abali et al, 2008). This reaction is depicted in figure 2 below.
Figure 2:adapted from http://research.chem.psu.edu/sjbgroup/projects/dihydro.htm
Â DHFR is the only enzyme in the pathway capable of maintaining the pool of tetrahydrofolate (H4F). Tetrahydrofolate is essential in the synthesis of thymidylate, purines and some amino acids, including methionine, glycine and serine.( Abali et al, 2008). Thymidylate and purines are the structural components of the DNA molecule.
The inhibition of DHFR results in DNA synthesis cessation and cell death. The enzyme has been exploited as a target in a number of diseases such as cancer and arthritis. Extensive research has been conducted since the 50s on the structure and function of DHFR. The drug Methotrexate is a structural analogue which inhibits DHFR by competitively binding to the active site. It is used as an anticancer drug as well as an antinflammatory agent in the treatment of arthritis. (B.Schweitzer et al, 1990).
The focus of this review is the regulation of human DHFR at the transcriptional and translational level. Currently a complete understanding of the regulatory control of the DHFR gene has not been established however it is an area of increased research activity in recent years.
Structure and Mechanism of action of the enzyme
Figure 3: The 3D structure of the dihydrofolate reductase enzyme. Its substrate (DHF) and cofactor (NADPH) can be seen in red.(Wong et al, 2005).
The DHFR enzyme has been extensively studied, initially in E.coli and more recently in mammals in order to determine its structure and understand how it functions. The amino acids involved in catalysis and the secondary structure of the molecule have been conserved throughout evolution in bacteria and mammalian species. The main difference between Bacterial and human DHFR is the flexibility of the molecules. Human DHFR is a more rigid molecule, and this stems from the DHFR lacking subdomain rotation structures. (Abali et al, 2008 and Sawaya and Kraut, 1997).
The DHFR gene is approximately 30 kb long and is located in chromosome region q11.2-q13.3 of chromosome 5. After transcription the most abundant transcript is 3800 nucleotides in length and the other 2 transcripts are 800 and 1,000 nucleotides long. The reason for the different lengths of the transcripts lies on the fact that three different polyadenylation signals are found on exon 6. A polyadenylation signal is the sequence on the mRNA at which the post-transcriptional modification process polyadenylation occurs; this is the addition of a polyA tail to the transcript. (Abali et al, 2008, Chen et al, 1984, Maurer et al, 1984).
Figure 4 depicts the location of human DHFR on chromosome 5. The area surrounded by the blue frame indicates the location of the DHFR gene at position q11.2-q13.3. The red line on the chromosome indicates the position of one of the DHFR pseudogenes. (Adapted from image at: http://www.genecards.org/cgi-bin/carddisp.pl?gene=DHFR)
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DHFR is a housekeeping gene, a gene that is constantly expressed because it codes for a protein that is always required by the cell. As with many housekeeping genes DHFR lacks a TATA box promoter, instead it possesses multiple GC boxes (Abali et al, 2008).TATA and GC boxes are types of promoters, a group of short sequence elements located upstream of the coding sequence of the gene. Promoters act as recognition sites for transcription factors. The TATA box is simply a sequence of approximately 20 base pairs of T and A bases with a core sequence of: 5'-TATAAA-3'. Human genes commonly have GC boxes as their core promoters and the sequence is rich in guanine and cytosine residues.
Pseudogenes can be defined as non-functional, intronless versions of a functional gene. Anagnou and his team discovered that there were at least 4 pseudogene versions of the DHFR gene, mostly located on different chromosomes. These were identified as pseudogenes as they are composed of sequences homologous to the functional DHFR exons but lacking introns. (Anagnou N.P. et al, 1984)
Regulation of Transcription and Translation
The central dogma of molecular biology is the transcription of DNA to mRNA and finally its translation to protein. This dogma underlies the expression of every gene in the genome and is characterised in 4 major steps; transcription, RNA processing, translation and post translational modification.
Figure 5: shows how eukaryotic DNA is transcribed to RNA and then translated to a protein. (Available at: http://www.accessexcellence.org/RC/VL/GG/central.php)
Only a small portion of the entire DNA contained in cells is ever transcribed.
The synthesis of RNA is accomplished by an RNA polymerase, with DNA as a template strand and ATP, CTP, GTP and UTP as RNA precursors. There are three eukaryotic RNA polymerases each transcribe a specific class of RNAs. RNA polymerase II is the polymerase that functions to transcribe all protein coding genes.
RNA polymerases cannot initiate transcription independently, they require transcription factors; proteins that bind to specific DNA sequences in order to guide the polymerases.
Short sequence elements located in the immediate vicinity of a gene, often upstream of the coding sequence, collectively constitute a promoter. Promoters act as recognition sites for transcription factors.
Transcription is regulated by repressor proteins which repress transcription by blocking the attachment of RNA polymerases to the promoter.
The mRNA transcript undergoes a number of modifications before it is translated to a protein. These modifications include splicing; the removal of exons and joining of the introns together, Capping; addition of 7-methyguanosine triphosphate to the 5' end of the mRNA and polyadenylation; addition of a poly A tail to the 3'end.
Once the mRNA transcript has been created and modified it moves out to the cytoplasm where it interacts with ribosomes to produce a chain of polypeptides. The correct folding of the protein into its 3D structure is directly related to the overall function of the molecule.
The proteins are finally modified by the addition of carbohydrate groups to the side chains of certain amino acids in a process termed glycosylation. Glycosylation helps the protein to fold correctly and confers stability to the molecule. (Strachan and Read 2004).
The expression of a gene is regulated at many levels. The conformation and structure of the DNA can affect the accessibility of RNA polymerase, the rate at which transcription occurs and the stability of the RNA relate to the levels of RNA available for translation and the modifications the resultant protein undergoes. However, the principal means by which DHFR levels are regulated in the cell is attributable to transcriptional control. (Slansky and Farnham, 1996).
Regulation of Transcription
As mentioned previously DHFR lacks a TATA box promoter, instead it possesses 2 bidirectional promoters which consists of two initiator elements and multiple GC boxes. (Fujii et al, 1992).The major transcripts produced by the downstream promoter are responsible for the transcription of 99% of the mRNA. (Masters and Attardi, 1985)The upstream, minor transcript initiates 400 nucleotides upstream from the major transcription site. (Blume et al. 2003)
Regulation at chromosomal level
As mentioned previously transcription is regulated by transcription factors but at another level it is also regulated by complexes that re-model chromatin structure in order to regulate the expression of individual genes. The chromatin -remodelling complex is defined as "A polypeptide complex that can compact or relax the secondary and tertiary structure of chromatin". This re-modeling of chromatin is based on the acetylation, methylation and phosphorylation of histones, the structural components of chromatin. Once the transcription factors have bound to the DNA histone acetyl transferases(HAT) are attracted to the site enhancing relaxation of the chromatin thereby increasing the level of transcription. Repression of transcription can also occur through interaction of the transcription factors with histone deacetylases (HDAC) that remove acetyl groups and hence stabilize the nucleosomal structure. (Roberts and Orkin, 2004). Hence transcriptional activity is modulated by the balance between histone acetylation and deacetylation. (Park et al, 2003).
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Regulation during cell cycle by Sp1 and E2F
As discussed earlier Transcription factors are essential for the regulation of transcription at the cellular level. There are two key transcription factor families that play a major role in the modulation of DHFR levels; Sp and E2F. The most important members of these families; Sp1 and E2F1 are responsible for the regulation of the DHFR promoter. Sp1 and E2F are located in the vicinity of the DHFR transcription initiation region. (Jensen et al, 1997) Sp1 recognizes the consensus GC box sequence; 5'GGGGCGGGGC 3' in the DHFR promoter. (Abali et al, 2008). The determination of the importance of SP1 to transcription was discovered from many in vitro and in vivo studies. The DHFR promoter uniquely contains four binding sites for Sp1.( Park et al,2003).
E2F1 is mediated by the sequence 5' TTTCGCGCCAAA 3' on DHFR, which is close to a transcription start site.(Abali et al, 2008)
Figure 6: Schematic representation of the DHFR major promoter. The consensus binding sites for Sp1 and E2F can be seen. The arrow indicates the direction and location of the major transcription start site. ( Wells et al, 1997).
DHFR is continually transcribed at low levels throughout the cell cycle but the rate of transcription increase dramatically during G1/S phase. (Slansky and Farnham,1996).
Figure 7: The above diagram represents the 4 phases of the cell cycle. (Sadava et al, 2008).
Studies in 1996 suggested 3 key components involved in the regulation of transcription of DHFR; CTD, SP1 and E2F. Slansky suggested a model for transcription of DHFR during the cell cycle. This model proposes that binding of multiple transcription factors to the promoter is necessary for increased transcription levels at the G1/S phase of the cycle.(Slansky and Farnham, 1996).
It is apparent that Sp1 forms a basal complex and continuous low levels of DHFR are transcribed through all stages of the cell cycles.(Slansky and Farnham, 1996).
The E2F family of transcription factors however was discovered to have more complex interactions with other proteins that can either repress or increase the level of transcription at different phases in the cycle.It has been determined that there are seven members of the E2F family; 5 different E2Fs and 2 different DPs. In order for efficient binding to occur heterodimers must form between a member of the E2Fsub-group and the DP sub-group. (Bandara et al, 1993, Helin et al, 1993 and Krek et al, 1993). Following on from this discovery it was ascertained that levels of certain E2F family members increase at the G1/S boundary, implying that changes in E2F binding to the DHFR promoter could be responsible for regulation of transcription. (Slansky and Farnham, 1996).
At the G0/G1 boundary E2F interacts with negative regulators of cell growth; retinoblastoma (Rb), p107 and p130 causing the formation of a repressor complex. This complex then binds to the promoter ensuring a very low level of DHFR transcription. (Cobrinik, 1995). At the G1/S phase the negative regulators become phosphorylated and are released from the E2F complex, allowing E2F to interact with other proteins as well as the basal transcription complex. (Slansky and Farnham, 1996). After G1/S phase transcription from the DHFR promoter is only activated by the SP1 sites. E2F is bound to an inhibitory protein and the level of transcription of DHFR returns to the same level as during early G0/G1. (Slansky and Farnham, 1996 and Cobrinik, 1995).
RNA polymerase II contains a C-terminal domain (CTD) which has been shown to be essential for in vitro transcription of DHFR in mice; Slansky suggests that this is also the case for human DHFR. A number of observations indicate that the phosphorylation state of the CTD determines its structure and these observations were confirmed by in vitro and in vivo cross linking experiments. A hypothesis emerged that the CTD must be in an unphosphorylated state in order to form a stable transcription complex and then must become phosphoylated to allow elongation of transcription. (Dahmus, 1994). During the G0/G1 phase the phosphorylation of CTD by TFIIH (present in all transcription complexes) allows transcription to occur at low levels. At the G1/S boundary a sudden increase of transcription of DHFR is observed, this may be explained by the interaction of E2F with a cyclin that caused an increase in the phosphorylation of CTD. (Slansky and Farnham, 1996).
Although Slansky's study was well researched and employed numerous experimental methods to prove its validity the model proposed remains a hypothesis.
The exact roles of the transcription factors SP1 and E2F in the regulation of transcription are not yet clear and have sparked considerable debate.
Following on from Slansky's work, recent studies by Sahin and Sladek have proposed a dual role for E2F-1 depending on the cell cycle. They suggest that in growing cells E2F-1 binding to retinoblastoma protein (pRb) causes repression of DHFR expression while in cells re-entering the cell cycle E2F-1 acts as an activator of expression of DHFR.( Sahin and Sladek, 2010).
Figure 8: models for the roles of E2F and pRb in gene expression of a number of genes including our gene of interest human DHFR. (Sahin and Sladek, 2010).
In vitro studies have demonstrated that mutations within the E2F-binding sites cause little effect to the levels of transcription from the DHFR promoter. However, point mutations in the E2F sites result in loss of the transcriptional increase at the G1/S boundary. Through experiments involving serum stimulation it was determined that it is unlikely that E2F could account for the increase in DHFR activity at the G1/S phase.(Means et al, 1991)
It has also been showed that in the absence of functional Sp1 proteins or Sp1-binding sites in the promoter, DHFR transcription is almost completely prevented. (ref modulation ofâ€¦) As well as further highlighting the importance of Sp1 in the transcription of DHFR this also indicates that E2F is a very weak activator of transcription. (ref modulation ofâ€¦ park) V. Noe, C. Chen, C. Alemany, M. Nicolas, I. Caragol, L.A. Chasin and C.J. Ciudad, Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1. Eur. J. Biochem. 249 (1997), pp. 13-20
Furthers studies have shown it is necessary for a number of elements to interact with each other in order to regulate transcription. Sp1 and E2F interact with each other and with the master regulator, tumour suppressor proteins, Rb and two other Rb homologues, p107 and p130 in order to regulate the expression of the DHFR. (Nevins 2001).
New Layer of Regulations-transcriptional repression
Martianov discovered a phenomenon by which the regulatory transcript produced from the DHFR minor promoter plays a critical role in the modification of gene expression through repression of transcription. As discussed previously the downstream major promoter is responsible for transcription of 99% of DHFR RNA. By testing the effect of the non-coding transcript in trans, a repressive effect in the major promoter was evident, indicating the sequence of the major promoter is the essential part of the regulatory RNA.The analysis of Martianov and his team revealed an RNA-dependent mechanism of transcriptional repression reliant on the production of the regulatory transcript from the minor promoter.( Martianov et al, 2007)
Enhancement of Transcription
An inverted repeat sequence has been shown to enhance transcription of the DHFR gene by adding stability to E2F binding. This overlapping inverted repeat sequence 5'-TTTCGCGCCAAA-3' is highly conserved near the transcription start site in the promoters of 3 mammalian genes that encode DHFR. The sequence is composed of 2 overlapping, oppositely orientated sites that match the E2F consensus sequence. It was demonstrated that mutations in the motif that eliminate the nature of the inverted repeat significantly decrease the binding stability of E2F. (Wade et al, 1995).
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Post transcriptional modifications are an integral part of the regulation of eukaryotic genes. There are numerous mechanisms that operate post transcriptionally to regulate the expression of any gene. The process of post transcriptional modification mainly relies on proteins interacting with the mRNA transcript produced at the transcription stage.(Tai et al, 2008). These RNA-protein interactions result in either targeted degradation of the mRNA or prevent the ribosome from accessing the translation start codon. Proper processing of the mRNA involves splicing, 5'- and 3' end modifications and export as well as their half life and translation rate. .(Tai et al, 2008) The majority of the sequences responsible for post-transcriptional regulation are localized in the untranslated regions (UTRs).
MicroRNAs (miRNAs) are a relatively new discovery in molecular biology and are considered to play a vital role in gene regulation by a post-transcriptional mechanism. It is predicted that 30% of all human genes are regulated by MicroRNAs by targeting sequences in their 3'UTR. (Mishra et al, 2007) miRNAs have been shown to regulate gene expression through translational repression, mRNA cleavage, mRNA deadenylation, or transcriptional silencing. (Abali et al,2008). Mishra have experimentally determined that a specific microRNA, miR-24 is partly responsible for the regulation of DHFR mRNA and protein levels. miR-24 is located adjacent to a single nucleotide polymorphism(i.e a change in base pair at one specific position on the genetic sequence which may or may not lead to an effect on cell function). The SNP is a change from cytosine to thymine at position 829 on the sequence (829Câ†’T). In individuals who have the normal allele 829C miR-24 downregulates DHFR protein, while people who carry the SNP 829T, over expression of DHFR is caused by interfering with miR-24 function. People carry SNP 829Câ†’T tend to have increased resistance to methotrexate when treated with the drug than those who have the wild type DHFR (i.e the typical form).This evidence could explain the reason why certain patients do not respond to treatment with MTX. Hence the SNP 829Câ†’T acts as a loss-of -function mutation. (Mishra et al, 2007)
Regulation of gene expression in eukaryotes is controlled at many levels. During the last decade the importance of translational regulation in gene expression has become increasingly clear. Translational control is an energy efficient process that functions to regulation the expression of a given protein and it helps maintain the ordered nature of the cell cycle. (liu et al, 2002).
It was Bertino who first identified an increased level of DHFR in patients being treatment with antifolates. He observed a six fold increase that he tested using a partially purified enzyme from patients resistant to the drug and comparing it to cells from the same patient before treatment, confiming his findings that the levels of DHFR increase upon treatment with Methotrexate. (Bertino et al, 1962). Following on from this it was determined that a translational regulation process underlies this increase in DHFR levels. Hillcoat predicted this stating that an increase in DHFR activity during log phase of cell growth to be due to a stable species of mRNA. (Hillcoat et al, 1967).
DHFR regulates its own translation by binding to its own cognate mRNA. This phenomenon of autoregulation of translation is a well-established mechanism in prokaryotes but had not been observed in eukaryotes. DHFR is one of the first eukaryotic genes whose expression has been shown to adopt translational autoregulation. (Tai et al, 2004). Translational autoregulation poses major advantages to the cell as it gives them a swift and proficient method of altering gene expression in response to external stimuli and cytotoxic stress. (Tai et al, 2004).
Through methods such as in vitro RNA-binding and filter-binding assays and in vivo experiments such as transfection, a cis-acting element in the protein coding region that interacts with high affinity to DHFR protein has been identified. This element has been found to be localized to a 164 nucleotide RNA sequence corresponding to nucleotides 401-564. This sequence binds to DHFR protein with a similar affinity to that of full-length DHFR mRNA. The 164-nt sequence was cloned with the reporter plasmid luciferase and was transfected into human cells to determine its biological activity. The findings from this experiment illustrated that the 401-564 sequence was affected by alterations in DHFR protein expression and was therefore a true DHFR-response element. (Tai et al, 2004). The element was further localized to an 82 nt sequence related to nucleotides 401-482. Further in vitro studies identified 2 other sequences contained within the coding region that DHFR might also interact with. These sequences correspond to DHFR:1-200 and DHFR:220-370. New studies have even further localized this cis element to a 27nt sequence which binds with high affinity to human DHFR and forms a ribonucleoprotein complex. They confirmed that DHFR27 RNA sequence is a true DHFR response element and that an intact DHFR protein is necessary for its translation regulatory effects. (Tai et al, 2008).
Figure 9: Translational autoregulation (Tai et al, 2004).
The above model represents the autoregulation of translation of DHFR. When cells are in the quiescent phase i.e. the G0 phase of the cell cycle, DHFR binds to its cognate mRNA and hence represses translation. However when growth requirements or substrate levels increase or when cells are exposed to a cytotoxic stress as in the case of treatment with methotrexate the DHFR protein is no longer able to bind to its target DHFR mRNA ,thereby relieving translational autoregulation and allowing translation and synthesis of additional DHFR protein to occur. (Tai et al, 2004 and Emine et al, 1997). This process has huge biological significance given that it is an efficient mechanism by which DHFR levels in a cell can be accurately controlled. As well as having biological importance the process of translational autoregulation also explains why some patients being treated with an inhibitory compound of DHFR, such as Methotrexate, develop resistance to the drug.(Tai et al, 2004).
The enzyme DHFR is highly regulated at many levels. One of the most important of these is transcription. There is still a huge amount of research to be carried out-exact roles of Sp1 and E2F
DHFR has a number of fascinating and unique properties- this is the reason why it is studied as a model.
First target for cancer therapeutics.
Still provides a rich area for research.
Understanding the regulation of transcription and translation of the dihydrofolate gene may present novel therapeutic targets for human diseases. (and improve upon previously established ones).