A nivel local y nacional

Gene expression is the process by the one dimensional genetic information in the genome is transcribed and translated to synthesis the three dimensional functional protein. In eukaiyotic cells this process involves transcription o f the gene to messenger RNA (mRNA) in the nucleus, transportation of mRNA to the cytoplasm and translation of the message to the primary amino acid sequence of the protein at the endoplasmic reticulum. Although post-translational processing does contribute to the production o f the functional active protein (Clapham, 1995), transcription o f the gene to its mRNA by the enzyme RNA polymerase is the primary rate-limiting step in this complex multi-step process.

Transcription is an active process occurring on an elaborate multi-protein temaiy complex comprising of DNA protein and newly synthesised RNA molecule. This large multi-protein complex consists of numerous DNA-binding transcription factors, stabilising accessory protein factors and the enzyme RNA Polymerase. In addition to the catalytic activity of RNA polymerase in the complex, various accessory proteins o f the complex also have enzymatic activity such as helicase activity for "melting" the DNA duplex and ATP hydrolysis in ATP/ADP exchange reactions which energise the process (reviewed by Conaway and Conaway, 1993).

Three different types of RNA polymerases, each responsible for transcribing a different class of genes, are found in the eukaiyotic cells (La Thangue and Rigby, 1988). The class H genes which encode for all cellular proteins and snRNA (except the U6 snRNA) are transcribed by RNA polymerase II (Pol U) (Sawadogo and Sentenac, 1990). Class I genes are primarily transcribed by RNA polymerase I and encode for the ribosomal RNA genes. RNA polymerase III transcribe class III genes which comprise o f tRNA 5S ribosomal RNA and U6 snRNA genes. Transcription of cellular genes by RNA Pol II is the most intensely studied class of genes.

1.5 (i) Eukaiyotic lYanscnptional Apparatus

At the level o f the transcriptional unit (i.e. the gene along linear DNA) there are similarities between the structural organisation of the eukaryotic promoter and the prokaryotic operon first discovered by Jacob and Monod (1961) (reviewed by Reznikoff et al., 1985). As in the prokaryote promoter, the transcription pre-initiation complex, comprising of RNA polymerase and accessory proteins, form within a segment o f DNA o f approximately 100 bps present upstream o f the gene (von Hippel et al., 1984). As in prokaiyotics, eukaryotic transcription involves the formation o f the initiation complex at the promoter of the gene, elongation of the synthesised RNA as the complex precesses 5' to 3' along the primary sequences o f the gene and termination o f transcription at the 3' end of the transcriptional unit.

However, unlike prokaryotic transcription where purified RNA polymerase can in the presence o f ribonucleotides and promoter containing DNA sequences catalyze the synthesis o f RNA in vitro, eukaryotic transcription requires additional protein factors present in nuclear extracts to stabilise the formation of the initiation complex (Conaway and Conaway, 1993). In addition to this difference, regulation of eukaryotic transcription is not as simple as the negative regulation observed in the lac operon where transcription is simply blocked by the binding of repressor proteins to operator sequences present adjacent to the promoter (Reznikoff et al., 1985). Regulation o f eukaryotic transcription involves binding of trans-acting factors to multiple cis- regulatory elements which operate fi*om various distances fi*om the promoter of

transcribed genes (La Thangue and Rigby, 1988).

The assembly o f the transcription initiation complex at the promoter is dependent on the DNA sequence around the initiation site. DNA sequence comparison of promoters o f many cellular genes have shown that with exception of the conservation o f 4-6 nucleotide sequences at approximal -70 and -30 bps from the transcriptional initiation site, there is a general lack of conservation of sequence between promoters (Corden et al., 1980; McKnight and Kingsbury, 1982). The conservation o f an AT-rich region at approximately -30 bps of the promoter, known as the TATA box, which is also conserved in the prokaryotic transcriptional unit (known as the Pribnow box at -10 bps) is essential for correct initiation in many genes which posses this motif (Maniatis et al., 1987). However, not all eukaiyotic cellular genes have this conserved TATA motif. For example, certain genes usually house-keeping genes lack the TATA m otif but posses GC-rich motifs. In eukaryotic promoters there are additional conserved oligo-nucleotide sequences (6-12 bps) upstream of the TATA box at approximal -70 bps known as upstream promoter elements (Maniatis et al., 1987).

The functional importance of these conserved nucleotide sequences for the basal activity o f the transcriptional unit have been assessed using mutagenesis experiments (Corden et al., 1980; McKnight and Kingsbury, 1982). For example saturation mutagenesis of the (Lglobin minimal promoter, in which every nucleotide o f the promoter is mutated and transcriptional activity of the mutated promoter is compared to the basal activity of the wild type promoter in HeLa cell transfection oq^eriments revealed the biological importance of these conserved oligo-nucleotide DNA elements (Maniatis et al., 1987). In general mutation of theses sequences resulted in the reduction of the basal activity o f the promoter.

DNA footprinting experiments of prokaryotic promoters using purified E. coli. RNA polymerase have shown that the enzyme directly interacts with promoter DNA (Siebenlist et al., 1980). These experiments have shown that the enzyme binds the DNA asymmetrical by making extensive contact with the non-template strand over the length o f the promoter spanning 50 bps of DNA. The enzyme make extensive contact

over the Pribnow box at -10 bps and an upstream conserved hexa-nucleotide sequence at -35 bps. However, in eukaiyotic transcription RNA Pol II is not en ab le of specifically binding the promoter and initiating transcription autonomously (Sawadogo and Sentenac, 1990). Eukaryotic cells have evolved a number o f protein factors which complex with the cis-elements of the promoter and recruit and stabilise the enzyme into the complex to initiate transcription (Conaway and Conaway, 1993). These additional factors called basal transcription factors have been identified, purified and cloned by the fi:uctionation of nuclear extracts in in vitro transcription assays using minimal promoters (Conaway and Conaway, 1993). These experiments generally utilised TATA-containing minimal promoter such as the (Tglobin minimal promoter; consequently molecular organisation of the initiation complex of TATA-containing promoters are better understood than TATA-less promoters. However, recently a ITbp m otif sufficient for accurate basal transcription, known as the initiator, have been identified in a TATA-less promoter (Smale and Baltimore, 1989).

DNA footprinting experiments to study DNA-Protein interactions, kinetic experiments to determine binding affinities, gel retardation electrophoresis to study binding specificity and nuclear extract reconstitution e?q)eriments to study function, have been deployed to analyze the dynamics of the assembly and the function of the basal transcription factors in the formation o f the eukaryotic transcription initiation complex. These studies have shown that the TATA box is important for the formation of the initiation complex (Buratowski, 1994). This involves the initial binding o f the transcription factor TFIID to the TATA box (Fig. 1.6) (Sharp, 1992). TFIID is itself a multi-component factor comprising of the Tata Binding Protein (TBP) and a number of associated polypeptides known as TAFs (TBP Associated Factors) which co-purify as a single complex (Rigby, 1993). TBP can bind the TATA box and initiate fromation of the preinitiation complex by itself However the TAFs are thought to be necessary for mediating the modulating activity of upstream activating factors (Hernandez, 1993). The carboxyl-terminus DNA binding domain o f TBP is highly conserved in many organisms (Nikolov et al., 1992).

Fîg 1.7 The formation o f the transcription initiation site at the TATA box o f an eukaryotic gene promoter.

Reproduced from Gilbert (1991).

m R N A initiation site +30 +10 +50 TFIID m R N A initiation site TFIID TFIIA m R N A initiation site TFIID TFIIA TFIIB m R N A initiation site R N A p o l y m e r a s e II TFIID TFIIA TFIIB m R N A initiation site R N A p o l y m e r a s e II TFIIF TFIID TFIIE TFII

The binding o f TFIID to the TATA box has a short half-life and it requires the binding o f TFIIA to form the stable complex (Fig. 1.3). The recruitment of RNA pol n to form the committed initiation complex requires the binding o f TFIIB to form the DAB ternary complex (Fig. 1.3). DNase protection and footprinting analysis have shown that TFIIB binds asymmetrical to one strand of the promoter DNA and extend past the transcription initiation site. This is similar to the DNase protection pattern observed for RNA polymerase in the prokaryotic transcriptional initiation complex (Siebenlist et al., 1980). Since RNA Pol II will not bind to the promoter in the absence o f the DA binary complex, TFIIB have been suggested to function as a bridging factor between TFIID and RNA Pol II (Buratowski et al., 1989). The entry o f PTF, TFIIF, TFIIH and TFIIJ into the complex initiates the synthesis and elongation of the RNA (Zawel and Reinberg, 1992). The DNA dependent ATPase activity o f PTF is th o u ^ t to supply the energy requirements of this process and the helicase activity associated with THIIF is thought to be necessary for unwinding the DNA duplex (Zawel and Reinberg, 1992).

However, this classically accepted model, derived from in vitro biochemical data, have been challenged recently by genetic and biochemical evidence from suppressor o f yeast RNA polymerase (Koleske and Young, 1994; Carey, 1994). The c-terminal domain (CTD) of the largest subunit of the mammalian RNA Pol II is essential for its function. The CTD consists of multiple repeats (52 in mammalian RNA Pol II) of a consensus heptapeptide m otif which is subject to phosphorylation. Pol II mutants that delete portions o f the CTD are lethal in yeast. Suppressor of the mutation which rescue the phenotype have been isolated and shown to co-purify with Pol II and basal transcription factors (TFIIH, TFIIF & TFIIB) at expected stoichiometric ratios. The complex was also shown to support transcription in vitro when supplemented with TBP and TFIIB. Based on this data Koleske and Young (1994) suggested the possible presence of a pre-assembled initiation complex termed the holoenzyme which can interact with transacting factors and recruit the promoter of the gene (Koleske and Young, 1995).

Once the initiation complex has assembled the process of transcription begins. The

molecular details o f the precession of RNA PolII and associated factors along the gene (or visa versa) is largely unknown. The transition from the initiation to elongation is an active ATP requiring process called promoter clearance. In the model suggested by Goodrich and Tjian the kinase and helicase activities associated with TFIIH o f the initiation complex is necessaiy for promoter clearance (Goodrich and Tjian, 1994). It has been suggested that the helicase activity melt the duplex DNA in the direction of transcription and the kinase activity associated with TFIIH phosphorylate the CTD of Pol II empowering it to process along the gene (Corden, 1993).

After promoter clearance, TFIIF is thought to be the only basal factor which remain associated with the polymerase during elongation. After the disassociation o f the initiation complex during elongation some components o f the complex remain associated with the promoter (Corden, 1993). This suggests that the promoter remains primed for further multiple re-initiation from a single committed complex.

1.5 (ii) Modular Nature of Eukaiyotic Qs-Regulation

The regulative capacity of a gene is dependent on the number and the type o f cis- regulatoiy elements present within the gene promoter and flanking sequences of the locus. The influence of a cis-element on the regulation o f its cognate gene is dependent on the availability of active transcription factors within a given cell. Therefore tissue and developmental stage specific expression of a gene is dependent on the induction o f specific transcription factors and their functional activation by post translational processing (e.g. phosphorylation, glycosylation etc.) (Clapham, 1995).

The cis-regulatory elements, i.e. the oligo-nucleotide sequences present distal to the promoter, function via the binding of trans-acting factors to modulate the activity of the basal transcription rate. This modulation is thought to be mediated through the interaction of TBP and the associated TAFs which are the corhponents o f TFIID (Hernandez, 1993). The cis-elements can act as both positive and negative regulators of transcription and can effect their influence from a range of distances and orientations from the promoter (Maniatis et al., 1987). Structural analysis o f these

motifs have shown that they share common consensus sequences. Therefore they are said have a modular structure comprising o f multiple and sometimes overlapping consensus motifs which bind specific transcription factors (Dynan, 1989).

1.5 (ii) (a) Short Range Q s Elements: Upstream Promoter Elements (UPEs)

Whilst the TATA box and related elements function in the formation o f the transcription initiation complex and regulate the position o f transcription initiation, elements upstream o f the promoter (UPE) fimction in regulating the rate of transcription (M cK ni^t and Kingsbury, 1982; Maniatis et al., 1987). The UPEs are conserved cis-regulatory elements found upstream of the TATA box usually at around -70 bps from the transcription initiation site. Many different UPEs have been identified and characterised. These include the commonly occurring CCAAT box and SPl binding GC boxes which are found in many eukaryotic genes and within viral promoters (McKnight and Tjian, 1986).

The UPEs can function in an orientation independent manner in relation to the TATA box. However, variation o f the distance between the TATA box and the UPE results in significant reduction in the transcription rate (Takahashi et al., 1986). It was found that insertions constituting half a helical turn of DNA resulted in a greater loss of transcription activation than insertions which constituted full helical turns o f DNA. This observation suggests that the interaction between the trans-acting factors bound to the UPEs and the initiation complex is dependent on the stereospecific alignment of the proteins on the DNA helix (Takahashi et al., 1986).

1.5 (ii) (b) Long Range G s Elements: Enhances & Silencers

Enhancer and silencer motifs, as their names imply, are positive and negative regulators o f the rate o f transcription from cis-linked promoters. Enhancers were originally isolated from viral genomes as DNA sequences which were capable of enhancing transcription from cis-linked promoters in cell transfection experiments (Baneiji et al., 1981; McKnight and Tjian, 1986). Similar cell transfection assay have

been utilised to isolate and characterise enhancer and silencer motife from cellular genes (McKnight and Tjian, 1986). These elements are frmctionally distinct from the UPEs in that they are able to modulate the transcriptional activity o f cis-linked genes in an orientation independent manner from greater distances from the linked promoter. However, at the molecular level the composition of UPEs and enhancers and silencers are very similar in that they possesses transcription factor binding consensus sequences (Dynan, 1989; Baneiji et al., 1981). Furthermore the multimerisation of individual transcription factor binding consensus sequences have been demonstrated to produce enhancer activity in transient transfection assays (McKnight and Tjian, 1986; Ondek et al., 1988). Therefore, the operational difference between enhancer and UPE elements may be the number and combination of different transcription factor binding sites, rather than a fimdamental difference within these elements (Fromental et al.,

198&X

It was not until the advent of transgenic mice technology (Jaenisch, 1988) that it was possible to frilly analyze the functional significance of proximal regulatory elements o f a gene in vivo (Dillon and Grosveld, 1993). This analysis showed that specific cellular enhancer elements function in regulating the tissue and temporal specific induction of cellular genes. These inducible enhancer motifs are not always necessarily localised within the 5' flanking sequences of the locus but can also be present in intronic and 3' flanking sequences of genes. For example independent regulatory elements present within the first and second introns o f the nestin gene have been shown to regulate expression in muscle precursors cells and in the CNS (Zimmerman et al., 1994). Other examples of genes which contain intronic enhancers include the murine Thy-1 (Spanopoulou et al., 1991), IL-4 genes (Henkel et al., 1992), the human T cell receptor 8 gene (Redondo et al., 1990). Example of genes possessing 3' enhancers include the human p-globin gene (Antoniou et al., 1988), the TCR genes (Winoto and Baltimore, 1989), the CD34 gene (Bum et al., 1992).

1.5 (ii) (c) Dominant Elements: LCRs and Insulators

mice (Magram et a l, 1985; Chada et al., 1985; Townes et al., 1985). Initial analysis showed the expression o f cis-linked genes in transgenic mice was h i ^ y depend on the position of the integration of the transgene in the genome (Chada et al., 1985; Townes et al., 1985). The proportion of the transgenes which expressed the gene and the level of expression was low and varied between different transgenic lines. This revealed that a large proportion of the genome was in a transcriptional repressed state. However, the integration o f the transgene to a transcriptional permissive site enable the expression o f the transgene. This ejq)ression was in general regulated by the proximal elements present within the transgene. However, the expression o f the transgene did not show stoichiometric relationship with the copy number o f the integrated transgene (Kollias et al., 1986). In addition to transcriptional repression of the integrated transgenes i.e. a negative position effects, transgene expression was also shown to be influenced by positive position effects occasionally. In this situation the activity o f the transgene was influenced by the transcriptional activity of the site of integration (Chada et al., 1985).

These observations implied that in addition to the short range UPEs o f the promoter and long range enhancer elements of the gene, additional proximal elements in the vicinity o f the locus contributed significantly to the transcriptional regulation of the gene (Grosveld et al., 1987). However, presumptive evidence for the possible existence of such global regulatory mechanisms have been observed three decades prior to the advent transgenic mice technology. These were the identification o f the giant polytene chromosomal puffs in the cells of the salivary gland of dipteran flies and the pictures of lambrush chromosomes in amphibian oocytes.

The notion that the genome was physically altered during gene induction lead to the development o f the DNAase I hpersensitivity assay for localising the putative regulatory regions o f cellular genes (Gross and Garrard, 1988). This assay measures the susceptibility of the flanking DNA sequences of a gene to endonuclease digestion by pancreatic or Staphylococcal deoxyribonuclease I. Purified nuclear extracts were incubated with the enzyme for varying length of time before isolating the genomic DNA and assessing the DNase sensitivity of the locus of interest by southem blot

analysis. The correlation between gene induction and the DNAasel sensitivity was illustrated first for the chicken globin (Weintraub and Groudine, 1976) and ovalbumin genes (Garel and Axel, 1976). The globin genetic locus was shown to be preferentially sensitive towards DNAasel digestion in erythrocytes in which the gene is actively transcribed compared to fibroblast nuclei in the gene is inactive. Similarly, DNAasel sensitivity in the ovalbumin locus was shown to be present in nuclei isolated from chicken oviducts, but not in nuclei isolated from chicken liver or erythrocytes in which the gene is never expressed.

The site o f the DNAasel sensitivity of a genetic locus correlates with the site o f the