BHasIt Muhcal Btdltun (1991) Vol. 47, No. 1, pp. 115-135 © The Brituh Council 1991
G Evan Imperial Cancer Research Laboratories, St. Bartholomew's Hospital, London, UK
A fundamental tenet of biology is that the phenotype of an organism is ultimately determined by its complement of genes. In multicellular organisms, it is the regulated pattern of expression of genes which determines the proliferation and differentiation of individual cell lineages and hence establishes the adult phenotype. It is therefore no surprise that both neoplasia and many developmental pathologies involve lesions in the regulation of specific genes. For this reason, an understanding of how genes are regulated has become an area of intense interest in both medicine and biology.
Much of our knowledge of the basic molecular principles of the control of gene expression originated with the historic work of Jacob and Monod in the early 1960s1 who studied the regulation of the genes involved in lactose metabolism in Escherichia coli. By a series of elegant genetic experiments they identified many of the fundamental components responsible for gene regulation, and their work has proved a basis for our current understanding of the complexities of gene regulation in eukaryotes. CONTROL OF EUKARYOTIC GENE EXPRESSION Eukaryotic genes share a basic common structure. All contain a number of regulatory elements that lie upstream of the coding region. This coding region is usually fragmented into exons interspersed with non-coding introns. Genes encoding proteins are transcribed by the enzyme RNA polymerase II (polll) whereas ribosomal and other RNA genes are transcribed by RNA polymerases I and III. It appears that the major point of control of eukaryotic gene expression is at the level of initiation of transcrip-
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
Regulation of gene expression
REGULATION OF GENE EXPRESSION
117
REGULATORY ELEMENTS OF EUKARYOTIC GENES Two major DNA elements are involved in the control of eukaryotic gene expression, promoters and enhancers. Promoters contain sites at which RNA polymerase binds to DNA and hence fix the start of transcription. Thus, they are always found in specific positions and orientations with respect to the gene they govern. Genes may have multiple promoters each of which can function with differing efficiency and be responsive to different forms of regulation. Frequently, promoters are characterised by the presence of certain DNA sequence motifs which are recognized by polypeptide co-factors necessary for RNA polll to function. One of the commonest is the TATA box which is the binding site for the transcription factor TFIID. Upstream of the TATA box are frequently found other motifs such as the CCAAT and GC boxes which also each bind specific regulatory co-factors. Enhancers are also DNA sequences which regulate transcription of genes, although always in a promoter-dependent manner.5 Unlike promoters, enhancers are largely independent of orientation or position with respect to the gene they control and can exert their effects over long distances (thousands of bases). There is, however, a degree of functional overlap between enhancers and promoters. Many sequence motifs are common to both, suggesting that similar regulatory factors can interact with either element. Moreover, multimerisation of some promoter sequences can give rise to elements with positionindependent enhancer activity. Ultimately, the regulation of a specific gene is likely to depend upon its own particular array of
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
tion, and this will therefore be the major subject of this review. Nonetheless, other regulatory mechanisms exist. Attenuation, the process whereby the transcription machinery prematurely terminates and generates incomplete transcripts, is an important regulatory system in many genes although its molecular basis is obscure.2 In addition, the fragmentation of many eukaryotic genes necessitates that the initial RNA transcript must be processed prior to translation, in order to splice together exon sequences. This processing uses a complex machinery whose regulation is imperfectly understood,3 but which introduces yet another possible tier of gene regulation. Rates of processing of particular transcripts can radically alter the degree of expression of particular genes, whilst differential splicing of various exons within one gene can generate related, but functionally discrete, polypeptides.4
118
THE CANCER CELL
Transcription factors Over the last few years, a large number of sequence-specific polypeptide factors have been identified that are implicated in control of transcription of various genes. These are called transcription factors.6 Comparison of the sequences and predicted structures of known transcription factors, both within and across species, has shown that many are highly related and can be grouped into families.7'8 Mutagenesis studies have identified regions of transcription factors responsible both for sequence-specific DNA-binding and also for transcriptional modulation, although little is presently known about the precise molecular interactions by which such modulation is mediated. Some transcription factors are expressed ubiquitously, but many are specific to certain cell types and are presumably responsible for governing the differentiated phenotype of various cell lineages. Still others are expressed widely in cells, but only exert their effect in response to specific signals. Although substantial overlap exists between them, these three categories nonetheless provide a useful framework for discussing transcription factor function. Common and ubiquitous transcription factors A number of upstream regulatory DNA sequences are common to many genes and the factors binding them are very widespread in many cell types. Accordingly, many of these factors comprise part of a more general transcription regulatory machinery. At least five accessory factors are known to be required for initiation of transcription by RNA polymerase, although their precise molecular functions are presently unknown. The accessory factor TFIID specifically binds to the TATA sequence present in the majority of promoters. This interaction is expedited by the factor TFIIA. The complex then binds the factors TFIIB, followed by TFIIE which is probably a DNA-dependent ATPase. Both TFIIB and TFIIE have been shown to interact directly with polll. The resultant TFII-DNA complex can promote initiation of transcription in vitro in the presence of ATP and ribonucleotides. It is presumed that the TFII complex provides a linkage
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
promoter and enhancer elements which dictate the repertoire of available positive and negative regulatory factors which can activate or repress transcription.
REGULATION OF GENE EXPRESSION
119
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
between upstream regulatory factors and RNA polymerase in vivo.9 In genes lacking a TATA sequence it is unclear what fulfils the function of TFIID. The transcription factor SP1 activates transcription by binding to a GC rich element commonly found upstream of the TATA box.10 The DNA-binding region of SP1 lies at its C terminus and contains three zinc fingers, structures first identified in the 5S RNA gene transcription factor TFIIIA. The SP1 zinc ringer motif comprises a sequence of some 30 amino acids that contains two cysteine and two histidine residues, appropriately spaced, which tetrahedrally coordinate a Zn 2 + ion.11 This 'finger' is necessary and sufficient for DNA binding. The zinc ringer motif, in various forms, is found in a number of other transcription factors including the steroid hormone receptor family (see Table 1). Zinc fingers have also been found in some proteins not known to bind DNA where their function may be to interact with other proteins. Mutagenesis studies have been used to identify four domains of SP1 responsible for transcription activation. The two most active are glutamine-rich (25% Gin) regions containing very few charged residues. The third is a basic domain near the zinc fingers, whilst the fourth is a region at the extreme C terminus with no obvious specific features. SP1 is extensively O-glycosylated, containing some 10 sugar linkages per molecule,12 but the functional significance of this modification is unknown. The CTF/NF-I factor binds the GCCAAT box present in many genes.13 The DNA-binding region lies towards the N-terminus of this
G Evan Imperial Cancer Research Laboratories, St. Bartholomew's Hospital, London, UK
A fundamental tenet of biology is that the phenotype of an organism is ultimately determined by its complement of genes. In multicellular organisms, it is the regulated pattern of expression of genes which determines the proliferation and differentiation of individual cell lineages and hence establishes the adult phenotype. It is therefore no surprise that both neoplasia and many developmental pathologies involve lesions in the regulation of specific genes. For this reason, an understanding of how genes are regulated has become an area of intense interest in both medicine and biology.
Much of our knowledge of the basic molecular principles of the control of gene expression originated with the historic work of Jacob and Monod in the early 1960s1 who studied the regulation of the genes involved in lactose metabolism in Escherichia coli. By a series of elegant genetic experiments they identified many of the fundamental components responsible for gene regulation, and their work has proved a basis for our current understanding of the complexities of gene regulation in eukaryotes. CONTROL OF EUKARYOTIC GENE EXPRESSION Eukaryotic genes share a basic common structure. All contain a number of regulatory elements that lie upstream of the coding region. This coding region is usually fragmented into exons interspersed with non-coding introns. Genes encoding proteins are transcribed by the enzyme RNA polymerase II (polll) whereas ribosomal and other RNA genes are transcribed by RNA polymerases I and III. It appears that the major point of control of eukaryotic gene expression is at the level of initiation of transcrip-
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
Regulation of gene expression
REGULATION OF GENE EXPRESSION
117
REGULATORY ELEMENTS OF EUKARYOTIC GENES Two major DNA elements are involved in the control of eukaryotic gene expression, promoters and enhancers. Promoters contain sites at which RNA polymerase binds to DNA and hence fix the start of transcription. Thus, they are always found in specific positions and orientations with respect to the gene they govern. Genes may have multiple promoters each of which can function with differing efficiency and be responsive to different forms of regulation. Frequently, promoters are characterised by the presence of certain DNA sequence motifs which are recognized by polypeptide co-factors necessary for RNA polll to function. One of the commonest is the TATA box which is the binding site for the transcription factor TFIID. Upstream of the TATA box are frequently found other motifs such as the CCAAT and GC boxes which also each bind specific regulatory co-factors. Enhancers are also DNA sequences which regulate transcription of genes, although always in a promoter-dependent manner.5 Unlike promoters, enhancers are largely independent of orientation or position with respect to the gene they control and can exert their effects over long distances (thousands of bases). There is, however, a degree of functional overlap between enhancers and promoters. Many sequence motifs are common to both, suggesting that similar regulatory factors can interact with either element. Moreover, multimerisation of some promoter sequences can give rise to elements with positionindependent enhancer activity. Ultimately, the regulation of a specific gene is likely to depend upon its own particular array of
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
tion, and this will therefore be the major subject of this review. Nonetheless, other regulatory mechanisms exist. Attenuation, the process whereby the transcription machinery prematurely terminates and generates incomplete transcripts, is an important regulatory system in many genes although its molecular basis is obscure.2 In addition, the fragmentation of many eukaryotic genes necessitates that the initial RNA transcript must be processed prior to translation, in order to splice together exon sequences. This processing uses a complex machinery whose regulation is imperfectly understood,3 but which introduces yet another possible tier of gene regulation. Rates of processing of particular transcripts can radically alter the degree of expression of particular genes, whilst differential splicing of various exons within one gene can generate related, but functionally discrete, polypeptides.4
118
THE CANCER CELL
Transcription factors Over the last few years, a large number of sequence-specific polypeptide factors have been identified that are implicated in control of transcription of various genes. These are called transcription factors.6 Comparison of the sequences and predicted structures of known transcription factors, both within and across species, has shown that many are highly related and can be grouped into families.7'8 Mutagenesis studies have identified regions of transcription factors responsible both for sequence-specific DNA-binding and also for transcriptional modulation, although little is presently known about the precise molecular interactions by which such modulation is mediated. Some transcription factors are expressed ubiquitously, but many are specific to certain cell types and are presumably responsible for governing the differentiated phenotype of various cell lineages. Still others are expressed widely in cells, but only exert their effect in response to specific signals. Although substantial overlap exists between them, these three categories nonetheless provide a useful framework for discussing transcription factor function. Common and ubiquitous transcription factors A number of upstream regulatory DNA sequences are common to many genes and the factors binding them are very widespread in many cell types. Accordingly, many of these factors comprise part of a more general transcription regulatory machinery. At least five accessory factors are known to be required for initiation of transcription by RNA polymerase, although their precise molecular functions are presently unknown. The accessory factor TFIID specifically binds to the TATA sequence present in the majority of promoters. This interaction is expedited by the factor TFIIA. The complex then binds the factors TFIIB, followed by TFIIE which is probably a DNA-dependent ATPase. Both TFIIB and TFIIE have been shown to interact directly with polll. The resultant TFII-DNA complex can promote initiation of transcription in vitro in the presence of ATP and ribonucleotides. It is presumed that the TFII complex provides a linkage
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
promoter and enhancer elements which dictate the repertoire of available positive and negative regulatory factors which can activate or repress transcription.
REGULATION OF GENE EXPRESSION
119
Downloaded from https://academic.oup.com/bmb/article-abstract/47/1/116/392175 by University of Winnipeg user on 24 January 2019
between upstream regulatory factors and RNA polymerase in vivo.9 In genes lacking a TATA sequence it is unclear what fulfils the function of TFIID. The transcription factor SP1 activates transcription by binding to a GC rich element commonly found upstream of the TATA box.10 The DNA-binding region of SP1 lies at its C terminus and contains three zinc fingers, structures first identified in the 5S RNA gene transcription factor TFIIIA. The SP1 zinc ringer motif comprises a sequence of some 30 amino acids that contains two cysteine and two histidine residues, appropriately spaced, which tetrahedrally coordinate a Zn 2 + ion.11 This 'finger' is necessary and sufficient for DNA binding. The zinc ringer motif, in various forms, is found in a number of other transcription factors including the steroid hormone receptor family (see Table 1). Zinc fingers have also been found in some proteins not known to bind DNA where their function may be to interact with other proteins. Mutagenesis studies have been used to identify four domains of SP1 responsible for transcription activation. The two most active are glutamine-rich (25% Gin) regions containing very few charged residues. The third is a basic domain near the zinc fingers, whilst the fourth is a region at the extreme C terminus with no obvious specific features. SP1 is extensively O-glycosylated, containing some 10 sugar linkages per molecule,12 but the functional significance of this modification is unknown. The CTF/NF-I factor binds the GCCAAT box present in many genes.13 The DNA-binding region lies towards the N-terminus of this
