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How an organism uses its genome to construct a complex body network with many different cells, tissues and organs is a key area of research in the field of genetics. Certain elements are needed to complete the entire body assembly. These elements include regulatory proteins known as transcription factors (TFs) which are proteins that bind to specific regulatory DNA sequences on the same chromosome as the gene to be regulated (so called cis-elements, or cis-regulatory sequences) and promote binding of RNA polymerase to DNA for the initiation of transcription. TFs contain DNA binding domains (DBD) which bind to specific DNA sequence of the promoter regions of DNA (Stegmaier, Kel, & Wingender, 2004). A promoter determines the template strand (always read 3' to 5') and where transcription starts. Promoters are cis-elements, but because TFs can (but not necessarily) be encoded on a different chromosome, they are sometimes referred as trans-acting factors (Allison, 2007). The 10% of human genome approximately codes for TFs, so there are likely to be at least- 2000 TFs not including the variation created by alternative splicing (Brivanlou and Darnell, 2000). The possibility of different combinations of TFs being expressed at different times in different tissues interacting with each other and various co-regulators is the basis of the complexity of gene regulation and explains how a relatively small number of genes can give rise to such complexity as seen in higher organisms. Other eukaryotes like Arabidopsis have more than 5% of its genome that encodes for more than 1500 of TFs (Riechmann, Heard, Martin and Reuber, 2000). Methods like chromatin immunoprecipation and CpG island microarray analysis are used to identify TFs in the human genome (Weinmann, Yan, Oberley, Huang and Farnham, 2002).
The general transcription machinery consists of RNA pol II which contains general TFs like TFIIB which is a TF for RNA pol II, TFIID contains TBP (TATA binding protein) and TAF (TBP-associated factor), TFIIE which is a TF for RNA pol II E, TFIIF is a TF for RNA pol II F, TFIIH which is a TF for RNA pol II H (Allison, 2007). Other proteins that regulate transcription are given in Table 1. Some genes take more than one transcription factor to be switched on, for example, heme oxygenase-1 gene takes four TFs including NF-E2, HSF, AP-1 and NFkB to be activated (Alam, 2006). A mutation alters the availability of TFs as well as affects the gene expression if a cis-regulatory element where TFs bind is mutated. Transcription factors regulate the spatio-temporal expression of thousands of genes. Their action regulates the functions in an organism and its development. Alterations in TFs may lead to substantial changes in gene expression. A study on Drosophila fly suggested that bilateral animals have a core set of TFs that were inherited from a common ancestor from 500 million years ago and transcription factors that control the embryonic development are largely conserved (Hasia and Mcginis, 2003).
Table 1: Examples of proteins that regulate transcription (Retrieved from Allison, 2007).
ACTIVATORS AND REPRESSORS
CAAT binding factor
CAAT/enhancer binding protein
cAMP response element-binding protein
CCCTC binding factor
friend of GATA-1
nuclear factor erythoid -derived 2
nuclear factor of kappa light polypeptide enhancer in B cells
upstream stimulatory factor 1 and 2
Special AT-rich binding protein
SV40 early and late promoter-binding protein 1
COACTIVATORS AND COREPRESSORS
Chromatin modification complexes:-
CREB binding protein
Lysine specific demethylase 1
Chromatin remodelling complexes
Mating type switching defective /sucrose nonfermenter
Swi2/Snf2 related 1
Facilitates chromatin transcription
Transcription factor for RNA polymerase II S
Complexity of an organism is increased with complex transcription factors. A splicing process plays an important role in deriving complex transcription factors immediately after transcription. Introns in mRNA transcripts are removed by a process called splicing (Levine and Tjian, 2003). This process is controlled by an alternative splicing process that gives a variety of protein isoform products (Martine, Clark and Smith, 2005). When this splicing process takes place in genes that code for transcription factors, different isoforms of transcription factors are produced (Hawkins, 1995). Research studies have shown that splicing in a transcription factor gene coding-region is less compared to other genes in the genome. So alternative splicing regulates transcription factor function in a tissue-specific manner by producing tissue specific TF isoforms (Taneri, Snyder, Novoradovsky and Gaasterland, 2004). Alternative splicing is highly active during developmental stages that activate different transcription factors in cells. These cells possessing different TFs further develop and differentiate into other types of cells, tissues and organs. Cis- and trans-acting regulatory factors of splicing along with transcription factors control evolution changes in vertebrates (Kornblihtt, Mata and Fededa, 2004).
Some transcription factors are characterised by a helix-turn-helix DNA binding motif known as homeodomain. A number of inherited human disorders are caused by mutations in homeodomain proteins (Banerjee-Basu & Baxevanis, 2001). Some of the TF domains are classified below followed by TF mechanism.
Transcription Factor DNA Binding Domain Classification
TF DNA binding domains (DBD) are classified based on DNA binding domains into four categories: Basic domains, zinc-coordinating domains, helix-turn-helix domains and beta-scaffold domains with minor groove contacts (Stegmaier, 2004).
All human cells dissent from one single fertilised egg followed by subsequent events that direct some cells into various differentiated cells to be more specific and characterised by certain functions. Since all cells in the body share the same content of digital DNA information so what brings about the enormous differences among the human cells such as different tissues and organs? What is/are the mechanism/s that drive(s) the differentiation? The answer for these mysterious questions is that not all genes are expressed in all cells. In fact, there is a degree of variation in the number gene expression, for example, gene A is only expressed on nerve cells but not in muscle cells or other tissues. This is called tissue-specific gene and it forms the basic concept of differentiation. Beyond this simple answer, there is an unimaginable degree of complexity that reveals astonishing relationships between biochemical elements and gene regulatory circuits. Here comes a new branch of science called systems biology which is capable to explain some of this complexity between protein and DNA networks. Throughout its life, the cell receives a huge number of external signals, these signals are modified and transferred by signal transduction pathway to DNA resulting in initiation of a specific biological response. To be more specific, the key element in the entire process is the essence of interaction between transcription factors to its cis-elements DNA as well as the fluctuation concentration of certain transcription factors. As a consequence, the regulation level of the correspondent gene might be suppressed or activated. More interestingly, one single regulatory protein once activated it might generate a signal cascade that can influence the activity of other transcriptional factors either in negative or positive feedback (Hartwell et al., 2008).
A recent study on transcriptional factors of sea urchin that involved in endomesodermal development has revealed a complex network interaction of regulatory elements (proteins) and cis-elements (DNA). The interactions based on laboratory experiment results were modelled using a highly sophisticated computer software application. Result interpretations of gene regulatory interactions of gut development in sea urchin larva revealed that the network consists of over 50 genes containing at least 35 transcriptional factors (Figure 1). The cascade of reactions is initiated by maternal spatiality that activates the regulatory proteins in a fertilised egg. This inhered process considered as the first event in the chain reactions (Davidson et al., 2002).
Figure 1: Complex gene networks: A schematic diagram represents the cross-regulatory circuits involved in gut development of sea urchin (Retrieved from Davidson et al., 2002).
One example of tissue-specific transcriptional factors occurs in hepatocytes by which their genes are expressed during cell differentiation particularly all through the developmental stage. One study has identified a number of specific regulatory proteins. Homeodomain-containing proteins (HNF-1 alpha and beta) sharing some common molecular structures that bind precisely to DNA sequence target the vital function of liver transcriptional factor network and thus establish the physical characteristics of liver cells "phenotypic appearance" (Kyrmizi et al., 2006). Yeast, a eukaryotic cell, contains around 2500 proteins linked in 7000 complex interactions. A system approach study (gene knockout) of galactose metabolism outlined a remarkable dynamic behaviour of different protein-protein and protein-DNA interactions that occur in order to convert galactose into glucose. Some genes are specifically involved in galactose utilisation whereas others are not specific but may share some activity of various metabolic pathways (Chandra, 2001). Understanding the key events in regulatory networks theoretically makes it possible to reprogram the system by altering and modifying the stimuli as well as reengineering and targeting the transcriptional factor which may become a potential therapy for certain genetic diseases but how transcription factors recognise specific DNA sequence?
To regulate transcription, transcription factors bind to cis-regulatory part of DNA sequence known as response element. The mechanism of transcription factor binding to DNA is complex and there are many protein-DNA complexes available but there is no single code available between protein and DNA sequence specificity. To understand the mechanism by which transcription factors recognise DNA sequences, it has been divided into two categories, base and shape readouts. Base readout describes unique chemical signature of DNA bases recognised by proteins, whereas shape readout describes sequence dependent DNA shape distinguished by proteins. Based upon interactions between protein and DNA sequence, a complex is formed with different affinity and specificity. A particular protein binds specifically to a DNA sequence, for example, all homeodomains have asparagines at position 51 that specifically bind to AT rich region like TAAT. For example, homeodomain protein in TF containing (isoleucine) ILe 47 and (aspargine) Asp 51 that recognises ATTA site efficiently. More specifically, homeodomain containing engrailed transcription factor has (glutamine) Gln 50 that recognises CCATTA and CAATTA sequence. However if there is (serine) Ser 50 or (lysine) Lys 50 then transcription factor would not recognise CAATTA site. The Lys 50 binds to GGATTA with high affinity, while transcription factor with Gln 50 has low affinity for this particular site (White, 2001). However, lysine presence instead of glutamine causes TAATCC binding site to be recognised. Base readout studies are carried out in that a protein recognises chemical signature of base/base pair either in a major or minor groove of DNA. Such chemical signatures generally result in hydrogen bond or water-mediated hydrogen bond formation or hydrophobic interaction. As there is a unique pattern of hydrogen bond donor and acceptor, transcription factors confer more specificity in major groove than in minor groove of DNA (Figure 2) (Rohs et al., 2010).
Figure 2: Hydrogen bond donor and acceptor in major and minor grooves of DNA. Transcription factors have a sequence-specific ability to recognise hydrogen bond donor and acceptor in the major and minor grooves of DNA (Retrieved from Rohs et al., 2010).
Transcription factors bind to base sequence by HTH domain, zinc finger domain, immunoglobulin fold domain and N-terminal end of basic leucine zipper domain. Transcription factor binding specificity achieved through hydrogen bond formation depends on a number of contacts formed and hydrogen bonding geometry. Bidentate hydrogen bonds, that form two hydrogen bonds with different donor and acceptor, have the highest degree of specificity. Bifurcated hydrogen bonds, in that two hydrogen bonds share the same donor, have lower specificity than bidentate hydrogen bonds and higher specificity than single hydrogen bonds. Moreover, hydrophobic contacts also play a significant role in sequence-specific recognition and identification of different bases (Rohs et al., 2010). Transcription factors that bind in minor grooves by a hydrogen bond cannot distinguish AT and TA likewise GC and CG base sequence hence zinc finger protein with Cys2 Cys2 GATA like domain binds non specifically in both major and minor grooves. Thus the bases form unique hydrogen bond in major grooves but not specifically in minor grooves. Therefore, shape readout studies were carried out to illustrate DNA-transcription binding specificity in minor grooves (Rohs et al., 2009). In shape readout studies, there are deformities in the ideal structure of DNA. Shape readout is further divided into local shape readout mechanism in that DNA helix is deformed and global shape readout in that DNA-binding site is either deformed or deviated from ideal DNA conformation. Such deformities, including narrowing of minor grooves in DNA shape, are recognised by transcription factors for specificity (Rohs et al., 2010). These readout mechanisms show that narrow minor grooves (width <5 Ao) associated with AT-rich region enhance the negative electrostatic potential of DNA. A-track is associated with narrowing minor groove. TpA base sequence widens the minor groove as well as GC-rich region. Data showed that arginine is enriched in narrow minor groove. Arginine recognises the enhanced electrostatic potential and binds to the A-track specifically. For example, POU domain of OCT1-PORE transcription factor complex binds to A-track in such a way that it provides specific binding site for arginine. Thus A-track, TpA site, GC region, and other base pairs in the flanking region near the binding site can produce a complex minor landscape of the minor groove that provides numerous possibilities for transcription factors to bind specifically to DNA sequence (Rohs et al., 2009). In addition to this, if a change in 1 bp occurs, it will cause a change in the whole sequence, which in turn will disrupt the specificity for transcription factor binding and lead to wrong binding, ultimately resulting in mutation development.
Since transcription factors possess a regulatory role in development and function of normal cells, mutations in their genes have shown to contribute to disease formation (Latchman, 2008). The TF-related mutations are seen in the promoter regions within genes that cause wrong binding of transcription factors and affect rate of transcriptional level. In humans, acromegaly, which is characterised by excessive secretion of growth hormone by somatotrophic cells of the pituitary gland, has been linked to a mutation in Prophet of Pit-1 (PROP1) gene. Indeed, somatotroph growth and proliferation are largely regulated by this gene which controls Pit-1 transcription factor at the embryonic developmental stage. Pit-1 initiates growth hormone production upon binding to growth hormone promoter (Melmed, 2006). Once released, growth hormone binds to receptors, highly expressed in liver and cartilage, and thus promotes the intracellular signalling pathway such as activating Janus kinase 2 (JAK2) (Argetsinger et al., 1993). In addition, Pit-1 has a role in the development and proliferation of lactotrophoic, mammosomatotrophic and thyrotrophic cells. Along with Pit-1, oestrogen receptor (ER)ï€ ï¡ promotes prolactin secretion and thus mammosomatotroph growth and differentiation into lactotrophs. GATA-2, on the other hand, regulates thyrotroph growth and development (Asa and Ezzat, 2009). Moreover, inactivation of specific transcription factors may result in mutations leading to the formation of developmental abnormalities such as combined pituitary hormone deficiency (CPHD) which is caused by a mutation in the encoding Pit-1 transcription factor gene of POU family. As a consequence, production of growth hormone, prolactin and thyrotropin hormone is depleted leading to mental retardation and growth deficiency (Latchman, 1996). In addition, a mutation in PAX3 and PAX6 transcription factor genes of PAX family results in Waardenburg's syndrome and eye defects or aniridia, respectively. The transcription factor gene mutation may also affect non-coding DNA binding co-factors, for example, some of the receptors also act as transcription factors such as steroid-thyroid hormone receptors where a hormone binds to and activates these receptors followed by transcription event initiation. Receptor gene mutations may also affect transcription factor activity (Latchman, 2005). The structure of transcription factor is modular containing many different domains responsible for different functions. The activity of transcription factor can be enhanced by tissue-specific enhancers which trigger promoter activity within the gene in a particular tissue of its own or of another gene within the tissue. The fly Drosophila assessment study describes mutations in transcription factors which affect the body development and plan. The homeotic genes for Ftz (fush tarazu) transcription factor binds to a specific DNA sequence and thus helps in normal tissue growth of the fly. However, a mutation in 1 bp sequence affects Ftz binding and thus ultimately altering the production of the fly with half normal number of segments as shown in Figure 3 reference.
Binds to specific sequence TCAATTAAATGA
Ftz protein TCAATTAAATGA
b.) No binding of Ftz protein
Mutated sequence with mutated sequence.
Ftz protein TCAATTTAATGA
Figure 3: Developmental protein for the fly Drosophila. In panel (a) Ftz protein binds with specific sequence TCAATTAAATGA and results in normal transcription. In panel (b) if there is a 1 bp mutation in a particular sequence, for example, A is replaced by T, the sequence changes to TCAATTTAATGA (shown in red letters) and so no binding occurs as well as no normal transcription takes place.
The fly example shows how could a sequence effect have an influence on the gene expression and this has also been reported in POU domain which has Pit-Oct-Unc (POU) transcription factors. We discussed some points about Pit-1 above but a mutation in Unc-86 transcription factor gene which is normally involved in sensory neuron development fails to form specific sensory neurons. Another Drosophila study identifies certain other transcription factors like kruppel protein for abdominal and thoracic development which has four zinc finger motifs. A zinc finger motif contains two cysteine and two histidine amino acids and a zinc atom attached centrally. The overall structure of the motif has 30 amino acid repeat units. The structure is so specific that if a mutation in one residue, for example, cysteine replacement by serine, takes place, the zinc atom will not bind to the finger arrangement altering the normal function of the protein and thus resulting in a mutant fly (Figure 4). In contrast, mammals have Sp1 transcription factor that possesses three copies of zinc finger. Reference
Cys his Normal Transcription
Zn normal development
Serine his No Transcription
Zn Mutant fly
Serine replacing cysteine
Figure 4: Zinc finger assembly in normal development of Drosophila fly. (a) A zinc atom is bound to cysteine-histidine fingers whereas in one finger cysteine is replaced by serine so zinc atom is not properly bound to the arrangement (b) and so no transcription for normal development occurs resulting in mutant fly. Cys denotes cysteine, his histidine and Zn zinc atom.
Transcription factors also possess a key role in developing cancer in humans such as gene encoding transcription factor p53 gets mutated results in cancer. Oncogenes increase expression or activation of transcription factors. In fact, chromosomal translocation has been found to contribute to transcription factor activation such as the immunoglobulin gene relocation in B-cell leukaemia and T-cell-receptor gene relocation in T-cell leukaemia (Latchman, 1996). Three main transcription factor groups have shown linkage to caner pathology. Oestrogen and androgen, i.e. steroid receptors, are involved in breast and prostate cancers, respectively. Resident nuclear proteins such as MYC, c-FOS, c-JUN and ETS have shown contribution in cancer development. Phosphorylation of this transcription factor group occurs through activation of serine kinase cascades. Resident nuclear proteins persistently bind to DNA sequence and complex with other transcription factors leading to unregulated growth of cells. They may also interact with other gene-coactivating proteins (Darnell, 2002). MYC gene, located on chromosome 8, regulates normal cell proliferation. Its translocation on chromosome 14 causes unregulated cell proliferation due to the close proximity location to immunoglobulin heavy chain gene and thereby leading to leukaemia (Latchman, 1996). The third group involved in cancer development is latent cytoplasmic factors in which their activation requires receptor-ligand interactions. They may also get activated by other mechanisms such as proteolysis. Signal transducers and activators of transcription (STATs) as latent cytoplasmic factors remain inactive in the cytoplasm until stimulated by extracellular receptors. STATs increase transcription event via interactions with those receptors and/or other intracellular transcription factors such as the interaction of STAT5 with glucocorticoid receptor (GR) and STAT3 with GR and SP1 and PU.1 transcription factors and STAT3 with GR and c-JUN which results in activating interleukin (IL)-6 which in turn induces ï¡2-macroglobulin gene. Studies have shown that STAT3 is persistently active in head and neck cancers, leukaemia, lymphoma as well as multiple myelomas. Additionally, STAT5 is found to be persistently active in leukaemia and lymphoma. The persistently active STAT3 and STAT5 in cancer examples given above are perhaps due to signalling pathway dysregulation or mutations such as deletions and chromosomal translocations (Darnell, 2002).
Mutations in beta-cell transcription factor genes have been reported in diabetic individuals. The haploinsufficient defects are seen in transcription factor gene where one allele is inactivated and thus the level of encoding-protein is reduced by half. The heterogeneity effect is seen when different transcription factor genes are mutated such as mutation in transcription factors expressed in beta-cells results in maturity-onset diabetes of the youth (MODY). In MODY, mutation in transcription factor HNF-1Î± gene was found in 20-year old individuals, whereas HNF-4ï¡ gene mutation was identified in 26-year old individuals. Moreover, protein truncation was reported as a result of MODY-causing gene mutations (Frayling, 2001).
The mutation in genes encoding specific transcription factors results in a number of diseases identified at birth or shortly after birth. This includes MEF2A transcription factor gene mutation which may lead to coronary artery disease in middle aged patients. Various cardiac transcription factor gene mutations have been identified in congenital heart disease (CHD). FOG/ZFPM2 transcription factor modulates GATA4 activity. Two children out of 47 with CHD were diagnosed with heterozygous missense mutation within this gene. GATA4 transcription factor has a regulatory role in expressing other genes in the cardiac muscle. A mutation in its gene was found as an interstitial deletion in chromosomal region 8p23.1. Studies have suggested that GATA4 gene mutation is a loss-of-function mutation (Ramachandran and Benson, 2008). In addition, several other transcription factors like RunX and FOXO are also involved in several human diseases. The RunX1 transcription factor causes haematopoiesis and leukaemia whereas haploinsufficient RunX2 contributes to abnormal bone regulation. RunX3 is involved in human cancer as it hypermethylates RunX3 promoter region. Also it has been found that a deletion within RunX3 gene contributes to human cancer formation (Ken-ichi, Takashi, & Yoshiaki, 2008). Furthermore, FOXE3 and FOXP2 operates in eye development and for language acquisition, respectively. The FOXO protein also regulates cell cycle, DNA repair, apoptosis as well as oxidative stress resistance. It also upregulates cell cycle inhibitor p27kip1 for G1 arrest. FOXO protein acts upon response to insulin stimuli where it gets phosphorylated and inhibited by Akt pathway. So mutation in insulin receptor abolishes the phosphorylation of FOXO protein thus no inhibition by Akt and PI3 kinase extends the longevity of FOXO protein upto three folds (Carter & Brunet, 2007). In addition, a mutation in gene encoding glucocorticoid receptors contributes to steroid resistance where patient do not respond to glucocorticoid. The triplet repeats abnormalities are seen in huntington's and myotonic dystrophy in which abnormal RNA strand contains CUG triplet sequences that bind to transcription factors like Sp1, STAT1, STAT3 and retinoic acid receptors causing poor function of all factors. The mutation in TF genes causes disease and based on this TFs are considered as potential drug targets.
The possibility of developing rational strategy to design drugs targeting transcription factor have opened the door for TF based therapy, which is an ultimate goal of certain biopharmaceutical companies. Type II diabetes is a result of insulin resistance due to mutation in peroxisome proliferator activated receptor GAMA (PPAR). PPAR plays an important role in insulin response, so it is a good therapeutic target to enhance response in diabetes and obesity. The NFkB factor possesses a key role in immune response and blocking its activity would be beneficial in treating human diseases involving damaging inflammation. This includes increase inhibitory IkB protein by drugs that switch on patient's own IkB gene or by inhibiting NFkB synthesis itself. However, these approaches have certain limitations where delivery of the gene is not efficient and simple to human patients and are still under development. For this, it would be better to consider transcription factors themselves as potential drug targets. The possible targets for the drug are shown in figure 5.
Figure 5: Possible drug targets for gene specific transcription factors. The TF translocates to nucleus from cytoplasm (a), dimerise with the domain (b), binds with DNA(c), leads to transactivation (d), and also degradation of TF (e). The drug A (in star) can interfere at any level from a to d and prevent the function of TF. Drug A binds specifically to critical site within (a, b', c', d') to prevent the activity of TF (Retrieved from Papavassiliou, 1998).
Aspirin inhibits IkB phosphorylation which increases the association of NFkB and IkB to promote anti-inflammatory activity. The post-translational modification of transcription factors can also be a potential target as their activity is essential for activation. Also we can target protein-protein interactions that play a role in transcription factor activation such as small diffusible molecules which are easy to deliver and can disrupt specific protein-protein interaction, e.g. p53 is regulated by MDM2 molecule where part of p53 is inserted into the deep pocket of MDM2 molecule, to prevent this a chemical compound designed to fill this pocket thereby blocking p53-MDM2 binding and reactivating cell to induce apoptosis or to reduce tumor growth. Moreover, ligand binding for steroid-thyroid hormone family of transcription factors can also be a potential target for drug therapy such as tamoxifen and thiazolidinediones which are used for breast cancer treatment by competing with oestrogen to bind to receptor as well as for PPAR receptor activation, respectively. In addition, retinoic acid receptor (RAR)/PML fusion protein causes repression of transcription in leukaemia, so increase administration of retinoic acid could stimulate gene activation by RAR portion of the fusion protein. The repression can also be treated with Histone deacetylates (HDA) inhibitors to block gene repression caused by fusion protein as HDA is recruited for normal activity. The designer zinc fingers are also considered as therapeutic potential where they can bind to specific DNA sequence in target gene. Zinc fingers linkage to inhibitory domain switches off the target gene, whereas their linkage to activator domain would switch on the target gene for the expression. Expression of genes of human viral infections such as herpus simplex virus (HSV) and human immunodeficiency virus (HIV) can be inhibited by designer zinc finger which is linked with the inhibitory domain of transcription factor. Similarly, designer zinc finger can activate stimulation of vascular endothelial growth factor (VEGF) once it is bound to activator domain transcription factor for increase blood vessel growth. But designer zinc finger exhibits some limitations including inefficient and insecure delivery and to patients (Latchman, 2005). The novel approach which is considered to be the potential drug target is cis-element double stranded oligodeoxynucleotides (ODN) which act as decoy ODNs for anti-gene therapy. It is developed as an in-vivo therapy by cis-trans interaction that removes Trans factors from endogenous cis-element. The synthesis for sequence specific decoy is simple and targeted to specific tissue and for this there is no need to know the exact structure of transcription factor. The transcription factor decoy functions at reducing the promoter activity. The mechanism for decoy transcription factor is shown in Figure 6 below.
TF cis-element Normal transcription
Transcripiton factor-cis element binding
Decoy ODN cis-element No trancritipon.
Binding of decoy element with transcription factor in the nucleus before transcription factor binds to cis element in nucleus.
Figure 6: Mechanism of decoy oligodeoxynucleotides (ODNs). Normally transcription factor binds to cis element so that gene expression takes place (a), but decoy transcription factor which mimics the normal binding to cis element results in no transcription/expression (b). In panel (c) the binding of decoy ODNs to transcription factor occurs in nucleus before transcription factor binds to cis-element, so that no mRNA is formed in nucleus and the same is not entered in cytoplasm, so no expression occurs.
The inactivation of transcription factors is a result of inherited mutations that is incompatible with survival such as transactivation response gene (TAR) decoy which is short RNA oliginucleotide corresponds to HIV TAR sequence where it can inhibit the expression of HIV as well as blocks its replication by inhibiting the HIV recognition Tat protein binding with TAR region. The regulation of decoy expression is also a problem but it can be overcome by recruiting synthetic double stranded DNA as an in-vivo decoy cis-element where DNA exhibits high affinity for transcription factor, which upon binding with transcription factor causes cis-trans interaction and thereby blocks the activation of gene resulting in disease. Vascular smooth muscle cell (VSMC) decoy cis-element is developed to treat retinoblastoma (Rb). The decoy binds to E2F transcription factor which is persistently active in Rb causing cell cycle arrest, but there are chances of mismatch ODNs. Also, another decoy element for cardiovascular disease (CVDs) approved by Food and Drug Administration (FDA) was performed in vivo in rat with injured carotid arteries and the effect remained till 8 weeks for curing restenosis i.e. NFkB decoy cis element. The NFkB transcription factor activates cytokine as well as adhesion molecule gene as part of their normal function in inflammation and immune response, so the NFkB decoy cis element mimics the normal NFkB function and blocks the gene leading to reduction in damaged area. It is also useful in myocardial infarction and glomerulonephrities. In doing so NFkB decoy interferes with the normal NFkB function in inflammation and immune response, so these are the side effects taken into account for transcription factor-driven diseases (Morishita, Higaki, Tomita, & Ogihara, 1998). Small molecule inhibitors act by blocking activation site or by inhibiting protein DNA interaction. There are two approaches to develop small molecule agents. First is purification from natural source which gives many different anticancer drugs and few of them having reasonable activity toward TFs. The problem in using small molecule is they have low affinity and specificity to target and require high doses, which increase side effects. One approach carried out to inhibit the transcription factors binding, basically by modifying the DNA shape or sequence. If any change in minor groove or major groove has been done the cis-element cannot be recognised by TF. On the other hand Changing in the DNA sequence can be achieved by either alkylation or intercalation thereby further transcriptions could be inhibited. In vitro studies carried out for nitrogen mustard (HN2) and quinacrine mustard (QM). Result showed that nitrogen mustard inhibit NFxB transcription factor to interact with GC rich region while quinacrine mustard can inhibit NFxB transcription factor as well as OTF-1 transcription factor by modifying GC- rich as well as AT- rich region. Thus QM has potential risk of toxicity and cannot be used as a potential therapy. Moreover HN2 inhibit gene expression of CAT hence, there are also risk of cytotoxic effect by inhibiting DNA binding of some TFs that recognise the GC-rich sequance. So, further studies are required for selective inhibition of particular transcription factor (Fabbri, Prontera, Broggini, & D'Incalci, 1993). Oliginucleotide (ON) strategy shows convincing evidence in cell culture and animal model as well as in vivo stability is one of the challenges in using this method due to ON is degraded by endonuclease. To avoid degradation of phosphodiestrase ON, it is necessary to be converted into phosphorothionate backbone. Moreover, Phosphorothionate ON have lethal side effects such as coagulopathy. RNAi delivery in vivo is a great challenge that depends on choosing the delivery system. RNAi is instable and also highly susceptible for degradation by exonuclease. G-quartet method has certain disadvantage like delivery vehicle is required to get into cell (Redell & Tweardy, 2006). The small molecules are used to interfere with HIF-1 TF which will trigger neovascularisation of an ischemic tissue. For example hypoxic induceable factor-1 (HIF-1), responsible for initiating transcription of erythropoetine (EPO) gene, that regulates blood-O2 carrying capacity in response to hypoxic condition. It plays an essential role in disease pathology such as ischemic cardiovascular disease and cancer, so HIF-1 is considered as an interesting target for these diseases.. In addition to this it act as anticancer agent. But HIF-1 is involved with the regulation of oxygen homeostasis, it may not be a useful target as it may cause unintended side effects like aggravation in hypoxic condition, due to the inhibition of EPO gene that will cause dysregulation of oxygen homeostasis (Semenza, 2004). The signalling pathway such as Raf/Ras/ERK/Mek have a significant effect proliferative or anti-proliferative effect on downstream transcription factor including CREB, Ets-1, AP-1, c-Myc. All these TFs are activated by phosphorylation of ERKs cascade pathway. As this pathway transmits the signal from membrane receptors to TFs, it can be a therapeutic target for various diseases. The Raf inhibitors have been developed which are undergoing clinical trials such as chlobiocin, novobiocin,geldanamycin and Coumermycin A. So inhibition of certain Raf genes may be advantageous as well as inhibition of other Raf genes may be detrimental (,Chang et al., 2003). Specific artificial TF factors can be engineered to achieve target specific effect in human body. These artificial TF factors are composed of DNA binding domain that can recognise the specific DNA sequence and an effector domain that mediates transcriptional activation or repression. These synthetic DBDs have high level of affinity, permitting not only regulation of targeted promoter but it also show specific competition with endogenous TFs (Stanojevic & Youngs, 2002). The DNA effector domain have capacity to activate or repress the gene expression where as the specific zinc finger domain have capacity to modify the expression by the DNA modifying domain, which act by DNA methylation, DNA recombination and DNA cleavage.( Rebar & Pabo, 1994). Application of both TF and ZF we can achieve the highly specific gene regulation which can be used in functional genomics, molecular therapeutics, biotechnology and many other fields. This TF, ZF technology can be greatly useful in treatment of viral diseases such HIV and important in disorders such as cancer. However indigenous TF factor may block the binding sites. Some antisense agents have been shown to exert non-sequence-dependent effect through interaction with other micro molecules. The specific vitro technique has to develop to identify the action and specificity of TF on drug target.(Blancafort, Segal, & Barbas III, 2004). The conculsion remarks of report throughs focus on whether a particular drug action can be restricted within the tissue where TFs are targeted? Or the toxicity of the drug binding with other TFs then required be reduced? Whether the drugs therapeutic activity can be achieved in targeting TFs? All such questions which still needs answer, contributes to complexity of TFs as potential drug targets.