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Gene-selective developmental roles of general transcription factors
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Gene-selective developmental roles of general transcription factors Gert Jan C. Veenstra and Alan P. Wolffe Innumerable transcription factors integrate cellular and intercellular signals to generate a profile of expressed genes that is characteristic of the biochemical and cellular properties of the cell. This profile of expressed genes changes dynamically along with the developmental stage and differentiation state of the cell. The biochemical machinery upon which transcription factors integrate their signals is referred to as the general transcription machinery. However, this machinery is not of universal composition, and variants of the general transcription factors play specific roles in embryonic development, reflecting the constraints and requirements of developmental gene regulation.
24 30 80 250
40 150
110
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Gert Jan C. Veenstra Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. e-mail: VeenstrG@ exchange.nih.gov Alan P. Wolffe served as Senior Vice President of Sangamo Biosciences, Richmond, CA, USA, from April 2000 until May 2001.
Transcription in eukaryotic cells is mediated by three RNA polymerases: RNA polymerase I (Pol I) transcribes rRNA genes, RNA polymerase II (Pol II) transcribes protein-encoding genes and some small nuclear RNA (snRNA) genes, and RNA polymerase III (Pol III) is involved in the transcription of small RNA genes such as tRNA and snRNA. Core promoters are defined as the minimal sequences required to recruit the appropriate RNA polymerase and initiate transcription [1]. Core promoter elements of Pol II-dependent genes can include a TATA box (at –20 to –30 relative to the transcription start site), an initiator element (at the transcription start site) and a downstream promoter element (DPE, at +30 relative to the transcription start site). TFIID, a high molecular weight complex comprising TATA-binding protein (TBP) and more than ten TBP-associated factors (TAFIIs), plays an important role in recruiting the transcription machinery to several wellcharacterized core promoters (Fig. 1). The TATA box can be bound by TBP, the initiator element is contacted by TFIID subunits TAFII150 and TAFII250, and Drosophila TAFII40 and TAFII60 have been shown to interact with a DPE [2]. Although TBP binds the TATA box of TATA-box-containing genes (Fig. 2a), this factor is often recruited to TATA-less core promoters through protein–protein interactions (Fig. 2b). This is also true for genes transcribed by RNA polymerases I and III, as TBP has been implicated in transcription mediated by all three polymerases [3]. Other general transcription factors (GTFs), such as TFIIA, TFIIB, TFIIE and TFIIF, are recruited to Pol II promoters, either along with TBP, or subsequent to TBP binding. An important question, however, concerns the extent to which the general transcription machinery is universally used. With this in mind, this review focuses on the complexity and versatility of the core promoter transcription machinery, and on its dependence on cellular and/or developmental context. http://tibs.trends.com
TBP
TATA
Inr
DPE Ti BS
Fig. 1. Core promoter elements of RNA polymerase II (Pol II)-dependent promoters recognized by TFIID subunits. Core promoter elements can include a TATA box (recognized by TATA binding protein, TBP), an initiator (Inr) element (recognized by TBP-associated factors TAFII250 and TAFII150) and a downstream promoter element (DPE, recognized by the Drosophila TBP-associated factors dTAFII60 and dTAFII40). Core promoter elements can differ in sequence, are not present in all promoters and can be present in various combinations. Numbers refer to TAFII names of Drosophila TFIID (Table 1), represented schematically in this figure [4]. The core promoter elements, and the proteins binding these elements are color-coded: blue, TATA box and TBP; green, Inr element, TAFII250 and TAFII150; orange, DPE, dTAFII60 and dTAFII40; yellow, other TAFIIs.
Detailed coverage of other aspects of the general transcription machinery can be found in several excellent reviews [1,3–6]. Gene selectivity by multiple transcription complexes and variant transcription factors
The variety of combinations of core promoter elements found in natural genes, as well as the sequence variations of these elements, suggests that the architecture and composition of core promoter nucleoprotein complexes is not universal but instead involves a variey of protein–DNA and protein–protein interaction interfaces. Indeed, several GTF complexes of varying composition have been identified. For example, TBP is found not only in TFIID, but also in several other complexes, for example Mot1 and NC2. The NC2 complex represses TATA-box-containing promoters, but activates promoters containing a
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(a) TATA box: TBP
(b) Inr and/or DPE: TAFIIs
TAFII250 IIA
TAFII150
TBP
(c) TLF core promoter: TLF, no TBP
dTAFII 60,40
IIB TATA
IIA
TLF IIB
Inr
DPE Ti BS
Fig. 2. Gene-selective function of general transcription factors (GTFs) is mediated in part by core promoter elements. Factors binding to core promoter elements are shown [blue, TBP; purple, TBP-like factor (TLF); green, TBP-associated factors TAFII250 and TAFII150; orange, dTAFII60 and dTAFII40]. TFIIA and TFIIB, which increase the affinity of TBP for the TATA box are also shown (gray). (a) Core promoters featuring a TATA box require TBP for core promoter recognition and generally do not require TAFIIs for basal transcription in vitro. TATA-box-containing promoters can recruit TFIID (TBP plus TAFIIs) in vivo, but do not necessarily recruit TAFIIs – a subset of promoters has been shown to recruit TBP but no TAFIIs [13,14]. The in vivo requirements for TAFIIs most likely depend on both core promoter elements and interactions with transcriptional activators [37]. (b) Core promoters featuring an initiator (Inr) or downstream promoter element (DPE), or both, in many cases recruit and require TBP along with the TAFIIs that bind to these core promoter elements. Of the TAFIIs, only TAFII250, TAFII150, dTAFII60 and dTAFII40 are shown. (c) TLF-dependent core promoters do not require TBP in vivo. It is not known which core promoter elements are required for selective TLF recruitment and whether GTFs other than TFIIA and TFIIB are involved in TLF-dependent transcription and/or promoter recognition. It should be noted that core promoter elements can exist in different combinations and show sequence variation, and that additional core promoter elements could possibly exist, further diversifying core promoter architecture beyond the examples shown in this figure.
DPE [7]. TBP is not the only factor found in multiple complexes; some of the TAFIIs of TFIID are also found in other biochemical entities, for example in the TBP-free TAFII-containing complex TFTC, which is similar to the yeast SAGA and human PCAF complexes (reviewed in Ref. [5]). These complexes contain a histone acetyl transferase (HAT) activity, which is implicated in transcriptional activation. TFTC is also capable of directing transcription from a core promoter in vitro, apparently in the absence of TBP [8]. However, TFTC might not be as broadly involved in transcription initiation as is TFIID; indeed, TFTC is preferentially recruited to UV-damaged DNA and might play a role in DNA repair [9]. In yeast, individual TAFII subunits have selective effects on gene transcription [10–14]. Different complexes can also compete for, or coexist at, some promoters. The TATA-box-containing promoters that are repressed by NC2 are presumably transcribed in a TFIID-dependent fashion. Furthermore, NC2 versus TFIID recruitment, and TFIID- versus SAGA-dependence, are not mutually exclusive in yeast [10,12]. The use of different transcription complexes could also play metazoan-specific roles. TAC is a mammalian TAFII-free complex that includes, among other factors, TBP and unprocessed TFIIA. TFIIA normally consists of three subunits: α, β and γ. The α and β subunits are produced from a http://tibs.trends.com
single gene by cleavage of the αβ precursor but, in TAC, the αβ precursor is not cleaved. TAC is capable of mediating trans-activation, and is present in embryonic stem cells but not in several differentiated cell lines [15]. This suggests that the use of different complexes in metazoans might contribute to cell-typespecificity in gene regulation. A second source of diversity in the transcription machinery is provided by GTF variants, often encoded by distinct genes (paralogs), which show a high degree of homology to the originally identified transcription factor. For example, the human genome contains single-copy genes for Pol II, TFIIB, TFIIE, TFIIF and TFIIH, generally without evidence of related genes [16,17]. By contrast, multiple sequences related to TFIIA and TFIID subunits are found within the human genome, potentially providing a variety of GTFs not found in yeast. Although the presence of sequence homology in the genome does not necessarily imply the existence of a functional gene, some of the paralogs have been characterized and shown to play gene-selective developmental roles. There are several ways in which variant GTFs can act in a gene-selective manner. First, variant transcription factors such as TBP-like factors or variant TAFIIs could potentially recognize alternative core promoter elements, diversifying the core promoter architecture (Fig. 2; Table 1). Second, variant GTFs can diversify the transcription machinery by competing with related factors for participation in some of the known transcription complexes, thereby substituting for one or more subunits of these complexes (Fig. 3). Alternatively, these factors could form novel complexes. These novel and variant complexes could potentially mediate selective interactions with transcription activators binding to distal promoter or enhancer sequences. Third, some GTFs are expressed in specific cell types or at specific developmental stages, diversifying the regulatory properties of the general transcription machinery in space and time within the metazoan organism. The literature reviewed here confirms and highlights the complex roles of alternative transcription factors in the regulation of the metazoan genome. The TBP family
The TBP subunit of TFIID was once thought to be essential for all transcription in the eukaryotic cell.
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Table 1. TBP-associated factors (TAFIIs)a TAFIIb
TAFIIs found in TFIID and HAT complexesd
Related TAFIIs (species: number – name)c
TFIID
hTAFII250(CCG1) dTAFII250(230)
h: 1 d: 0
hTAFII250 dTAFII250
hTAFII150 dTAFII150
h: 0 d: 0
hTAFII150 dTAFII150
hTAFII150
hTAFII140 dBIP2(dTAFII155)
ND
hTAFII140 dBIP2
hTAFII140
hTAFII130(135) dTAFII110
h: 1 – hTAFII105 d: 1 – nht
hTAFII130, hTAFII105 dTAFII110
hTAFII130
hTAFII100 dTAFII80
h: 1 – hPAF65β d: 1 – can
hTAFII100 dTAFII80
hTAFII100, hPAF65β dTAFII80
hTAFII80(70) dTAFII60(62)
h: 1 – hPAF65α d: 1 – dTAFII60-2 (mia)
hTAFII80 dTAFII60
hTAFII80, hPAF65α dTAFII60
hTAFII68 d-cabeza
h: 0 d: 0
hTAFII68
hTAFII55 dTAFII55
h: 1 d: 0
hTAFII55
hTAFII55
hTAFII32 dTAFII40(42)
h: 6 d: 0
hTAFII32 dTAFII40
hTAFII32 dTAFII40
hTAFII30 dTAFII16
h: 0 d: 1 – dTAFII24
hTAFII30 dTAFII16, dTAFII24
hTAFII30 dTAFII24
hTAFII28 dTAFII30β
h: 0 d: 0
hTAFII28 dTAFII30β
hTAFII20(15) dTAFII30α(28)
h: 0 d: 1 – dTAFII30α-2 (rye)
hTAFII20 dTAFII30α
hTAFII20 dTAFII30α
hTAFII18 dTAFII18
h: 1 – hSPT3 d: 0
hTAFII18
hSPT3
(h)ENL (d)ENL/AF-9
h: 1 – AF-9 d: 0
ENL/AF-9
HAT
Core promoter interactione Inr DPE hTAFII250 dTAFII250 hTAFII150 dTAFII150
dTAFII60
dTAFII40
aAbbreviations:
d, Drosophila melanogaster; DPE, downstream promoter element; h, Homo sapiens; HAT, histone acetyl transferase; Inr, initiator element; ND, not determined; TBP, TATA-binding protein. bPutative or confirmed orthologs in different species. Names of orthologs vary across species as many TAF s derive their name from their II biochemical association with TBP and their molecular weight. For more information on TAFIIs, see Refs [6,17,49] and the online supplement of Ref. [16]. cThe number of related TAF s, encoded by paralogs, is different in different species and varies for each TAF . II II dTAF s are found with TBP in the TFIID transcription factor, but also in complexes lacking TBP, such as the HATs TFTC, SAGA and PCAF. II eSeveral TAF s bind core promoter elements such as the Inr or DPE (Fig. 1). II
Indeed, TBP is essential for transcription in yeast; however, metazoans have at least one TBP-related protein. The Caenorhabditis elegans genome encodes only two members of the TBP family: TBP and TBP-like factor (TLF, also referred to as TRF2 in some species). The Drosophila genome encodes three members of the TBP family: TBP and the TBP-related factors TRF1 and TRF2. The human genome possibly also encodes a third TBP family member, in addition to TBP and TLF/TRF2 [16]. It will be important to determine whether this putative novel member is a functional gene and, if so, to characterize the functions of this gene. TBP itself is developmentally regulated, at least in the mouse and the amphibian Xenopus laevis. In mice, the nuclear TBP concentration declines during oocyte growth, is low in the fertilized egg and increases at the two-cell stage [18]. In Xenopus, TBP protein is virtually undetectable by western blotting in the full-grown oocyte, but TBP levels increase http://tibs.trends.com
dramatically during blastula stages as maternally stored TBP mRNA gets translated [19]. In both mice and frogs, the increase of TBP protein correlates with the onset of embryonic transcription. Early embryonic development of many species is characterized by a period during which the embryonic genome is transcriptionally silent. The stage at which embryonic transcription starts varies between species. In the mouse, this is the two-cell stage (20 h post fertilization), whereas in Xenopus it is the midblastula stage (4000 cells, 5 h of development at 24°C). Transcriptional repression before the onset of embryonic gene expression involves multiple layers of regulation, including those operating at the levels of chromatin and transcription factor availability. Chromatin remodeling by the nucleosomal ATPase ISWI might play a role by removing TBP from chromatin in Xenopus eggs [20], thereby preventing recruitment of the transcription machinery by the small amounts of TBP that are present in eggs and
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TFTC
TAFIIs
TFIID Mot1
Variant TAFIIs TBP
TBP
TBP
Novel complexes?
TLF
TBP NC2
α/β TAC TBP
α γ
TFIIA
β γ
α
β
ALF Ti BS
Fig. 3. Combinatorial diversity generated by protein–protein interactions of general transcription factors (GTFs). Some related GTFs share interaction interfaces, competing for protein–protein interactions, but could potentially also form novel transcription complexes. Transcription complexes of variable composition might compete for the same target genes (merely modulating the activity of the involved promoters) or regulate distinct sets of genes. TBP (blue) interacts with TAFIIs (yellow) to form TFIID. Some of the TAFIIs in TFIID can be replaced (symbolized by ↔) by variant TAFIIs (green), for example hTAFII105 can substitute for hTAFII130. Conversely, both TAFIIs and related variant TAFIIs can participate in histone acetyl transferase (HAT) complexes such as TFTC, for example hTAFII100 and hPAF65β, and hTAFII80 and hPAF65α (Table 1). Both TFIIA and the unprocessed form of TFIIA in the TAC complex are capable of binding TBP. Similarly, TFIIA-like factor (ALF, red) can substitute for TFIIAα/β (orange) in binding to TBP. TBP is not only found in TFIID and TAC, but also in the Mot1 and NC2 complexes. So far TBP-like factor (TLF, magenta) has not been found in TFIID, TAC, Mot1 or NC2. However, TLF can compete with TBP for TFIIA binding (symbolized by ↔). Therefore, substitutions are selective, and not all theoretically possible substitution reactions happen in vivo because of divergence of sequence and function of related GTFs. In addition, inclusion of related factors into high molecular weight complexes is not necessarily mutually exclusive.
early embryos. Experimental manipulation of TBP levels in the early embryo indicated that TBP levels are rate-limiting for transcription before the midblastula transition (MBT) [19,21], and that developmentally regulated translation of maternal TBP mRNA is required for the onset of transcription of a subset of genes [19,22]. TBP is not universally required for transcription in the early vertebrate embryo: in both Xenopus and zebrafish, transcription of a subset of early embryonic genes is independent of TBP [22,23]. Frog embryos lacking detectable levels of TBP are capable of initiating gastrulation (for which transcription is required) but arrest before completing this process. Normal levels of TBP are also required for normal cell division in cultured chicken cells [24]. In these cells, even a moderate decrease of TBP levels by disruption of one copy of the TBP gene caused a delay in mitosis. Although TBP is not required for transcription of all metazoan genes, this http://tibs.trends.com
factor is clearly an essential protein in yeast and metazoans alike. Drosophila TRF1 is initially expressed throughout the embryo, but gradually becomes restricted to the central nervous system and the gonads [25]. Chromosomal localization studies showed that TRF1 binds a limited number of genes in fly salivary gland cells. TRF1 binds relatively well to the TATA-box and exhibits GTF requirements similar to TBP in an in vitro transcription assay [25]. However, transcription of the in vivo target gene tudor was dependent on an 11-bp TC box rather than a TATA box [26]. In vivo, the majority of TRF1 is associated with BRF, a core component of the Pol III transcription factor TFIIIB. In Drosophila, TRF1 rather than TBP is required for Pol III-dependent transcription of tRNA, 5S rRNA and U6 snRNA [27]. So far, TRF1 has only been identified in flies. TLF/TRF2 is found in many metazoans and is more distantly related to TBP than is TRF1 [28]. Although the protein interacts with TFIIA and TFIIB similar to TBP (Fig. 3), unlike TBP it does not bind well to canonical TATA boxes [29–31]. TLF reduces the activity of several TBP-dependent promoters in vitro and in transfection assays, probably by sequestration of TFIIA [29–31]. However, transcription from the SV40 promoter is moderately stimulated by TLF overexpression. This promoter features a variant TATA box (TATTTAT) for which TLF exhibits modest affinity [31]. Interestingly, a similar TATT (Tat-tee) box is present in the promoters of the brachyury and GS17 genes, which are dependent on TLF for their expression in Xenopus laevis embryos. These promoters might be regulated by TLF directly because they exhibit TLF-dependence early after the onset of transcription [22]. However, in vivo TLF-binding sites in these promoters are not known, and it will be important to analyze the mechanisms by which TLF functions and to determine the promoter elements that mediate TLF-dependent transcriptional regulation. Negative regulation of TBP-dependent promoters by TLF-mediated sequestration of TFIIA is potentially relevant in vivo, as it has been observed that transcription of some genes is derepressed in tlf-1 [RNAi] embryos of C. elegans, suggesting a negative role for TLF in regulation of a subset of genes [32]. However, a positive role of TLF in the in vivo transcription regulation of a subset of genes has also been observed [22,23,32–35] (Fig. 2c). TLF-dependent genes have been shown to play an important role in early embryogenesis of C. elegans, Xenopus and zebrafish [22,23,32,33] whereas, in the mouse, TLF-dependent genes play a role in spermatogenesis but not early embryogenesis [34,35]. In the frog and fish, TLF and TBP appear to play complementary roles in that some genes are TLF-dependent, some genes are TBP-dependent, and another group of genes appears to require both factors for normal expression [22,23]. Dependence on both TBP and TLF
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is most likely caused by indirect effects rather than a mechanistic requirement for both factors. The apparent requirement of TLF at different stages of the life cycle in different species (mouse versus worm, fish and frog) is remarkable, and raises the question of whether there is a unifying mechanistic or developmental principle underlying these differences. It will be important to identify specific pathways of TBP- and TLF-dependent gene regulation in different species to understand the different biological roles in the context of common mechanisms of gene regulation. TBP-associated factors
The TBP-associated factors implicated in Pol II transcription (TAFIIs) play a role in transcriptional activation, core promoter interactions and enzymatic protein modification [4]. Although initially identified as subunits of TFIID, some TAFIIs are also part of HAT complexes such as PCAF and TFTC (Table 1). TAFIIs can function in a gene-selective manner in yeast [10–14]; for example, several TAFIIs bind as subunits of TFIID to core promoter elements present in some, but not all, promoters (Figs 1,2; Table 1). Variant TAFIIs encoded by metazoan paralogs of these yeast proteins could act to further diversify core promoter recognition and increase gene selectivity. Furthermore, TAFIIs activate specific genes by mediating protein–protein interactions with specific transcriptional activators binding to distal promoter elements of these genes [4,36]. Further diversifying the general transcription machinery, and increasing the potential for geneselective effects, are selective substitution reactions, in which a subunit of a transcription complex is replaced by a related factor (Fig. 3). The gene-selective action of both the unique TAFIIs and the TAFIIs for which related factors have been identified (Table 1), is reflected by the variety of biological functions of TAFIIs in the cell, and in metazoan development. Mammalian TAFII250 and its yeast homolog yTAFII145 are important for progression through the G1/S transition of the cell cycle. In hamster cells, TAFII250 is selectively important for transcription from the cyclin A and D1 promoters. Two mechanisms have been implicated in gene-selective activation of cyclin A, as both activator-mediated regulation of cyclin A expression and cyclin A core promoter function are dependent on TAFII250 [37]. In Drosophila, TAFII250 is essential for viability. Weak loss-of-function alleles cause pleiotropic defects in a manner consistent with roles for TAFII250 in regulation of the cell cycle, cell differentiation, proliferation and survival [38]. Human TAFII150 is also important for the cell cycle, being essential for progression through the G2/M transition in cultured cells [39]. The human genome contains more than 12 potential alternative genes for the various TAFIIs; flies and yeast have five and three alternative TAFII http://tibs.trends.com
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genes, respectively [16,17] (Table 1). For example, the human (h) TBP-associated factor hTAFII32 has six putative paralogs in the genome, whereas Drosophila (d) has no genes related to dTAFII40/42, the homolog of hTAFII32. As dTAFII40 has been implicated in core promoter recognition, it will be important to determine whether or not the human paralogs have a similar function. The gene encoding dTAFII40/42 is essential for viability, although flies carrying insertions in dTAFII40/42 that do not fully abolish dTAFII40/42 function are viable and female sterile [40]. In other cases, for example that of hTAFII30, the human genome does not contain related genes, whereas the Drosophila genome encodes two related TAFIIs (dTAFII24 and dTAFII16) [16]. Both dTAFII24 and dTAFII16 interact with TBP in a Drosophila TFIID complex, suggesting that these proteins can substitute for one another in TFIID (Table 1, Fig. 3). However, this might not be the case for their function in HAT complexes as dTAFII24, but not dTAFII16, was found to be part of a TBP-free TAF–HAT complex [41]. Immunostaining of salivary gland polytene chromosomes revealed that dTAFII24 and dTAFII16 localize to distinct, yet overlapping sets of genes. Combined with the distinct spatio-temporal expression patterns of these TAFIIs during embryogenesis, it is possible that two related genes are required to meet the demands for differential regulation of distinct sets of target genes during fly embryogenesis. The single homolog identified so far in the mouse, mTAFII30, is required for cell-cycle progression and viability in embryonal carcinoma cells [42]. Similar to wild-type embryonal carcinoma cells, TAFII30-null cells were shown to be capable of retinoic acid-induced differentiation into primitive endoderm, which also rescued viability. By contrast, differentiation into parietal endoderm required TAFII30. These results suggest that mTAFII30 is required for the proper transcription of a subset of genes essential for specific cell types and differentiation pathways. Both hTAFII130 and its Drosophila homolog dTAFII110 have a single relative: hTAFII105 and nht, respectively. The mouse homolog of hTAFII105 is an alternative TFIID subunit (Fig. 3), which is relatively abundant in testis, ovary and B cells, and is required for ovarian development and expression of a subset of genes [43]. By contrast, Nht expression is restricted to the testis and plays a role in Drosophila spermatogenesis (M. Hiller et al., pers. commun.). dTAFII110 is more broadly expressed than its relative nht, and mutation of dTAFII110 revealed that it is involved in activation by the embryonic transcription factors Dorsal and Twist [44,45]. A similar situation has been found for dTAFII60 and mia, the Drosophila homologs of hTAFII80 and hPAF65α (PCAF-associated factor 65 α). Similar to nht, mia is required for spermatogenesis (M. Hiller et al., pers. commun.), whereas mutation of dTAFII60
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Table 2. The metazoan minimal transcription machinerya,b General components
Variable components
RNA polymerase IIc
TBP, TLF, TAFIIs, variant TAFIIs
TFIIB
TFIIA, ALF
TFIIE
TFIIHd
TFIIF aAbbreviations: ALF, TFIIAαβ-like factor; TAF, TBP-associated factor; TBP, TATA-binding protein; TF, transcription factor; TLF, TBP-like factor. bThe metazoan minimal transcription machinery is the combination of general and variable factors necessary and sufficient for transcription from a given core promoter. This combination of factors consists of general, universally required components, and variable components that might function in gene-selective ways. cSome subunits have paralogs in the human genome [16] and might not be universally required. dThe kinase activity of TFIIH is not universally required in yeast [50]. In addition, several subunits have potential paralogs in metazoan genomes [16].
revealed that this TAF, similar to dTAFII110, plays a role in Dorsal-mediated activation [44,45]. A single relative exists for the hTAFII100 and dTAFII80 genes, called PAF65β and can in human and fly, respectively. Whereas dTAFII80 is widely expressed, can is selectively expressed in primary spermatocytes, and is required for testis-specific gene transcription and male fertility [46]. It will be important to determine whether can exerts its tissuespecific effects as part of a TFIID complex or as part of a TBP-free HAT complex, which would help elucidate the mechanisms underlying the gene-selective role of this variant cell-type-specific TAF. TFIIA and ALF
Acknowledgements This review is dedicated to the memory of Alan P. Wolffe, who died in an accident on 26 May, 2001. I thank I.B. Dawid, D.L. Weeks and D.A. Wassarman for critical reading of the manuscript, and M. Hiller, J. Pringle and M. Fuller for communicating data before publication.
TFIIA consists of three subunits (α, β and γ), which are encoded by two genes: those encoding TFIIAαβ and TFIIAγ. The human genome contains a gene related to TFIIAαβ, referred to as TFIIAαβ-like factor (ALF) or TFIIAτ [47,48]. Similar to TFIIAαβ, ALF plus TFIIAγ promotes binding of TBP to DNA, and ALF can also substitute for TFIIA-α/β in an in vitro transcription system [47,48] (Fig. 3). ALF is expressed in a variety of tissues but is most abundant in the testis and, therefore, a role in spermatogenesis has been suggested. Although this is an interesting possibility, no experimental data have, as yet, been reported to indicate whether ALF is redundant with TFIIAαβ or whether it plays a unique role in the transcription of a distinct set of genes. Conclusions and prospects
The eukaryotic transcription machinery is a biochemical entity of variable composition and function in both yeast and metazoans. However, metazoans exhibit a greater variability and gene selectivity of GTFs, reflecting a wide variety of
References 1 Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 2 Burke, T.W. et al. (1997) The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila. Genes Dev. 11, 3020–3031 http://tibs.trends.com
regulatory constraints and transcriptional requirements during development and differentiation. It should be noted that the general transcription machinery is far less diverse than the thousands of transcription factors that activate or repress transcription from proximal or distal cis-acting elements. Nevertheless, rather than viewing the general transcription machinery as the common biochemical entity upon which myriad sequence-specific trans-acting transcription factors integrate their regulatory signals, it would be fair to say that this multitude of transcriptional regulators integrate their signals on a common RNA polymerase through a non-universal, regulated and partly variable ‘minimal transcription machinery’ (Table 2). This minimal transcription machinery consists of general components (Pol II, TFIIB, TFIIE, and TFIIF) and variable components (TBP, TLF, TAFIIs, TFIIA and ALF), and is defined as the combination of general and variable factors that is necessary and sufficient for correct initiation of transcription from a given core promoter. It is useful to distinguish these transcription factors from cofactors and sequence-specific trans-acting factors that only modulate the relative efficiency of initiation by activation or repression. It is interesting to note that many GTFs are enriched in testis (e.g. TBP, TLF, Drosophila TAFIIs, TFIIA, ALF, TFIIE, the large subunit of Pol II), and several of these (murine TLF and Drosophila TAFIIs) have been shown to play a role in spermatogenesis. Apparently, the basal transcription requirements of the testis are distinct from those of many other organs. TBP and some of the TAFIIs for which no paralogs exist in the human genome, are important for cell-cycle progression. This does not necessarily mean that these factors are not important for other processes, as it is possible that the cell cycle is very sensitive to alterations in levels of GTFs. For a clearer understanding of gene regulation, it will be important to gain comprehensive information on GTF requirements for the genes in the metazoan genome in general, and for developmental pathways in particular. Biochemical analysis is needed to reveal the nature and identity of the core promoter elements that determine gene-selective recruitment of variant transcription factors, and the various nucleoprotein complexes that comprise these factors. Finally, it will be important to determine the implications of a variable minimal transcription machinery for promoter-selective interactions between this machinery and activators and repressors that modulate transcriptional activity.
3 Hernandez, N. (1993) TBP, a universal eukaryotic transcription factor? Genes Dev. 7, 1291–1308 4 Albright, S.R. et al. (2000) TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1–13 5 Pugh, B.F. (2000) Control of gene expression through regulation of the TATA-binding protein. Gene 255, 1–14
6 Gangloff, Y. et al. (2001) The histone fold is a key structural motif of transcription factor TFIID. Trends Biochem. Sci. 26, 250–257 7 Willy, P.J. et al. (2000) A basal transcription factor that activates or represses transcription. Science 290, 982–984 8 Wieczorek, E. et al. (1998) Function of TAF(II)-containing complex without TBP in
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transcription by RNA polymerase II. Nature 393, 187–191 Brand, M. et al. (2001) UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation. EMBO J. 20, 3187–3196 Holstege, F.C. et al. (1998) Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 Apone, L.M. et al. (1998) Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA. Mol. Cell 2, 653–661 Geisberg, J.V. et al. (2001) Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo. Mol. Cell. Biol. 21, 2736–2742 Li, X.Y. et al. (2000) Distinct classes of yeast promoters revealed by differential TAF recruitment. Science 288, 1242–1244 Kuras, L. et al. (2000) TAF-containing and TAFindependent forms of transcriptionally active TBP in vivo. Science 288, 1244–1248 Mitsiou, D.J. et al. (2000) TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAαβ precursor and the TFIIAγ subunit. Mol. Cell 6, 527–537 Tupler, R. et al. (2001) Expressing the human genome. Nature 409, 832–833 (Supplementary information: http://www.nature.com/nature/ journal/v409/n6822/suppinfo/409832a0.html) Aoyagi, N. et al. (2000) Genes encoding Drosophila melanogaster RNA polymerase II general transcription factors: diversity in TFIIA and TFIID components contributes to gene-specific transcriptional regulation. J. Cell Biol. 150, F45–F50 Worrad, D.M. et al. (1994) Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP. Development 120, 2347–2357 Veenstra, G.J.C. et al. (1999) Translation of maternal TBP mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition. Mol. Cell. Biol. 19, 7972–7982 Kikyo, N. et al. (2000) Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI. Science 289, 2360–2362 Prioleau, M-N. et al. (1994) Competition between chromatin and transcription complex assembly
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regulates gene expression during early development. Cell 77, 439–449 Veenstra, G.J.C. et al. (2000) Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus. Science 290, 2312–2315 Muller, F. et al. (2001) TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Curr. Biol. 11, 282–287 Um, M. et al. (2001) Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis. Mol. Cell. Biol. 21, 2435–2448 Hansen, S.K. et al. (1997) Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 91, 71–83 Holmes, M.C. et al. (2000) Promoter-selective properties of the TBP-related factor TRF1. Science 288, 867–870 Takada, S. et al. (2000) A TRF1:BRF complex directs Drosophila RNA polymerase III transcription. Cell 101, 459–469 Dantonel, J.C. et al. (1999) The TBP-like factor: an alternative transcription factor in metazoa? Trends Biochem. Sci. 24, 335–339 Rabenstein, M.D. et al. (1999) TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family. Proc. Natl. Acad. Sci. U. S. A. 96, 4791–4796 Teichmann, M. et al. (1999) Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells. Proc. Natl. Acad. Sci. U. S. A. 96, 13720–13725 Moore, P.A. et al. (1999) A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators. Mol. Cell. Biol. 19, 7610–7620 Dantonel, J.C. et al. (2000) TBP-like factor is required for embryonic RNA polymerase II transcription in C. elegans. Mol. Cell 6, 715–722 Kaltenbach, L. et al. (2000) The TBP-like factor CeTLF is required to activate RNA polymerase II transcription during C. elegans embryogenesis. Mol. Cell 6, 705–713 Martianov, I. et al. (2001) Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene. Mol. Cell 7, 509–515 Zhang, D. et al. (2001) Spermiogenesis deficiency in mice lacking the Trf2 gene. Science 292, 1153–1155 Verrijzer, C.P. et al. (1996) TAFs mediate transcriptional activation and promoter
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selectivity. Trends Biochem. Sci. 21, 338–342 Wang, E.H. et al. (1997) TAFII250-dependent transcription of cyclin A is directed by ATF activator proteins. Genes Dev. 11, 2658–2669 Wassarman, D.A. et al. (2000) TAF250 is required for multiple developmental events in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 97, 1154–1159 Martin, J. et al. (1999) Human transcription factor hTAF(II)150 (CIF150) is involved in transcriptional regulation of cell cycle progression. Mol. Cell. Biol. 19, 5548–5556 Soldatov, A. et al. (1999) TAFII40 protein is encoded by the e(y)1 gene: biological consequences of mutations. Mol. Cell. Biol. 19, 3769–3778 Georgieva, S. et al. (2000) Two novel Drosophila TAFIIs have homology with human TAFII30 and are differentially regulated during development. Mol. Cell. Biol. 20, 1639–1648 Metzger, D. et al. (1999) Mammalian TAF(II)30 is required for cell cycle progression and specific cellular differentiation programmes. EMBO J. 18, 4823–4834 Freiman, R.N. et al. (2001) Requirement of tissue-selective TBP-associated factor TAF(II)105 in ovarian development. Science 293, 2084–2087 Zhou, J. et al. (1998) TAFII mutations disrupt Dorsal activation in the Drosophila embryo. Proc. Natl. Acad. Sci. U. S. A. 95, 13483–13488 Pham, A-D. et al. (1999) Mesoderm-determining transcription in Drosophila is alleviated by mutations in TAFII60 and TAFII110. Mech. Dev. 84, 3–16 Hiller, M.A. et al. (2001) Developmental regulation of transcription by a tissue-specific TAF homolog. Genes Dev. 15, 1021–1030 Ozer, J. et al. (2000) A testis-specific transcription factor IIA (TFIIAtau) stimulates TATA-binding protein-DNA binding and transcription activation. J. Biol. Chem. 275, 122–128 Upadhyaya, A.B. et al. (1999) Identification of a general transcription factor TFIIAαβ homolog selectively expressed in testis. J. Biol. Chem. 274, 18040–18048 Gangloff, Y.G. et al. (2001) The TFIID components human TAF(II)140 and Drosophila BIP2 (TAF(II)155) are novel metazoan homologues of yeast TAF(II)47 containing a histone fold and a PHD finger. Mol. Cell. Biol. 21, 5109–5121 Lee, D. et al. (1998) Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo. Nature 393, 389–392
Nobel Prize winners This years Nobel Prize for Physiology or Medicine has been awarded to Tim Hunt, Paul Nurse and Leland Hartwell for their pioneering work on aspects of the cell cycle. We would like to extend our warmest congratulations to all three scientists. A special mention goes to Tim Hunt, who served on the TiBS editorial board for over 20 years and was Editor-in-Chief on the journal from 1992–2000. Congratulations Tim! Axel Innis is a PhD student at the Dept of Biochemistry, University of Cambridge, UK.
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Gene-selective developmental roles of general transcription factors Gert Jan C. Veenstra and Alan P. Wolffe Innumerable transcription factors integrate cellular and intercellular signals to generate a profile of expressed genes that is characteristic of the biochemical and cellular properties of the cell. This profile of expressed genes changes dynamically along with the developmental stage and differentiation state of the cell. The biochemical machinery upon which transcription factors integrate their signals is referred to as the general transcription machinery. However, this machinery is not of universal composition, and variants of the general transcription factors play specific roles in embryonic development, reflecting the constraints and requirements of developmental gene regulation.
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Gert Jan C. Veenstra Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. e-mail: VeenstrG@ exchange.nih.gov Alan P. Wolffe served as Senior Vice President of Sangamo Biosciences, Richmond, CA, USA, from April 2000 until May 2001.
Transcription in eukaryotic cells is mediated by three RNA polymerases: RNA polymerase I (Pol I) transcribes rRNA genes, RNA polymerase II (Pol II) transcribes protein-encoding genes and some small nuclear RNA (snRNA) genes, and RNA polymerase III (Pol III) is involved in the transcription of small RNA genes such as tRNA and snRNA. Core promoters are defined as the minimal sequences required to recruit the appropriate RNA polymerase and initiate transcription [1]. Core promoter elements of Pol II-dependent genes can include a TATA box (at –20 to –30 relative to the transcription start site), an initiator element (at the transcription start site) and a downstream promoter element (DPE, at +30 relative to the transcription start site). TFIID, a high molecular weight complex comprising TATA-binding protein (TBP) and more than ten TBP-associated factors (TAFIIs), plays an important role in recruiting the transcription machinery to several wellcharacterized core promoters (Fig. 1). The TATA box can be bound by TBP, the initiator element is contacted by TFIID subunits TAFII150 and TAFII250, and Drosophila TAFII40 and TAFII60 have been shown to interact with a DPE [2]. Although TBP binds the TATA box of TATA-box-containing genes (Fig. 2a), this factor is often recruited to TATA-less core promoters through protein–protein interactions (Fig. 2b). This is also true for genes transcribed by RNA polymerases I and III, as TBP has been implicated in transcription mediated by all three polymerases [3]. Other general transcription factors (GTFs), such as TFIIA, TFIIB, TFIIE and TFIIF, are recruited to Pol II promoters, either along with TBP, or subsequent to TBP binding. An important question, however, concerns the extent to which the general transcription machinery is universally used. With this in mind, this review focuses on the complexity and versatility of the core promoter transcription machinery, and on its dependence on cellular and/or developmental context. http://tibs.trends.com
TBP
TATA
Inr
DPE Ti BS
Fig. 1. Core promoter elements of RNA polymerase II (Pol II)-dependent promoters recognized by TFIID subunits. Core promoter elements can include a TATA box (recognized by TATA binding protein, TBP), an initiator (Inr) element (recognized by TBP-associated factors TAFII250 and TAFII150) and a downstream promoter element (DPE, recognized by the Drosophila TBP-associated factors dTAFII60 and dTAFII40). Core promoter elements can differ in sequence, are not present in all promoters and can be present in various combinations. Numbers refer to TAFII names of Drosophila TFIID (Table 1), represented schematically in this figure [4]. The core promoter elements, and the proteins binding these elements are color-coded: blue, TATA box and TBP; green, Inr element, TAFII250 and TAFII150; orange, DPE, dTAFII60 and dTAFII40; yellow, other TAFIIs.
Detailed coverage of other aspects of the general transcription machinery can be found in several excellent reviews [1,3–6]. Gene selectivity by multiple transcription complexes and variant transcription factors
The variety of combinations of core promoter elements found in natural genes, as well as the sequence variations of these elements, suggests that the architecture and composition of core promoter nucleoprotein complexes is not universal but instead involves a variey of protein–DNA and protein–protein interaction interfaces. Indeed, several GTF complexes of varying composition have been identified. For example, TBP is found not only in TFIID, but also in several other complexes, for example Mot1 and NC2. The NC2 complex represses TATA-box-containing promoters, but activates promoters containing a
0968-0004/01/$ – see front matter. Published by Elsevier Science Ltd. PII: S0968-0004(01)01970-3
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(a) TATA box: TBP
(b) Inr and/or DPE: TAFIIs
TAFII250 IIA
TAFII150
TBP
(c) TLF core promoter: TLF, no TBP
dTAFII 60,40
IIB TATA
IIA
TLF IIB
Inr
DPE Ti BS
Fig. 2. Gene-selective function of general transcription factors (GTFs) is mediated in part by core promoter elements. Factors binding to core promoter elements are shown [blue, TBP; purple, TBP-like factor (TLF); green, TBP-associated factors TAFII250 and TAFII150; orange, dTAFII60 and dTAFII40]. TFIIA and TFIIB, which increase the affinity of TBP for the TATA box are also shown (gray). (a) Core promoters featuring a TATA box require TBP for core promoter recognition and generally do not require TAFIIs for basal transcription in vitro. TATA-box-containing promoters can recruit TFIID (TBP plus TAFIIs) in vivo, but do not necessarily recruit TAFIIs – a subset of promoters has been shown to recruit TBP but no TAFIIs [13,14]. The in vivo requirements for TAFIIs most likely depend on both core promoter elements and interactions with transcriptional activators [37]. (b) Core promoters featuring an initiator (Inr) or downstream promoter element (DPE), or both, in many cases recruit and require TBP along with the TAFIIs that bind to these core promoter elements. Of the TAFIIs, only TAFII250, TAFII150, dTAFII60 and dTAFII40 are shown. (c) TLF-dependent core promoters do not require TBP in vivo. It is not known which core promoter elements are required for selective TLF recruitment and whether GTFs other than TFIIA and TFIIB are involved in TLF-dependent transcription and/or promoter recognition. It should be noted that core promoter elements can exist in different combinations and show sequence variation, and that additional core promoter elements could possibly exist, further diversifying core promoter architecture beyond the examples shown in this figure.
DPE [7]. TBP is not the only factor found in multiple complexes; some of the TAFIIs of TFIID are also found in other biochemical entities, for example in the TBP-free TAFII-containing complex TFTC, which is similar to the yeast SAGA and human PCAF complexes (reviewed in Ref. [5]). These complexes contain a histone acetyl transferase (HAT) activity, which is implicated in transcriptional activation. TFTC is also capable of directing transcription from a core promoter in vitro, apparently in the absence of TBP [8]. However, TFTC might not be as broadly involved in transcription initiation as is TFIID; indeed, TFTC is preferentially recruited to UV-damaged DNA and might play a role in DNA repair [9]. In yeast, individual TAFII subunits have selective effects on gene transcription [10–14]. Different complexes can also compete for, or coexist at, some promoters. The TATA-box-containing promoters that are repressed by NC2 are presumably transcribed in a TFIID-dependent fashion. Furthermore, NC2 versus TFIID recruitment, and TFIID- versus SAGA-dependence, are not mutually exclusive in yeast [10,12]. The use of different transcription complexes could also play metazoan-specific roles. TAC is a mammalian TAFII-free complex that includes, among other factors, TBP and unprocessed TFIIA. TFIIA normally consists of three subunits: α, β and γ. The α and β subunits are produced from a http://tibs.trends.com
single gene by cleavage of the αβ precursor but, in TAC, the αβ precursor is not cleaved. TAC is capable of mediating trans-activation, and is present in embryonic stem cells but not in several differentiated cell lines [15]. This suggests that the use of different complexes in metazoans might contribute to cell-typespecificity in gene regulation. A second source of diversity in the transcription machinery is provided by GTF variants, often encoded by distinct genes (paralogs), which show a high degree of homology to the originally identified transcription factor. For example, the human genome contains single-copy genes for Pol II, TFIIB, TFIIE, TFIIF and TFIIH, generally without evidence of related genes [16,17]. By contrast, multiple sequences related to TFIIA and TFIID subunits are found within the human genome, potentially providing a variety of GTFs not found in yeast. Although the presence of sequence homology in the genome does not necessarily imply the existence of a functional gene, some of the paralogs have been characterized and shown to play gene-selective developmental roles. There are several ways in which variant GTFs can act in a gene-selective manner. First, variant transcription factors such as TBP-like factors or variant TAFIIs could potentially recognize alternative core promoter elements, diversifying the core promoter architecture (Fig. 2; Table 1). Second, variant GTFs can diversify the transcription machinery by competing with related factors for participation in some of the known transcription complexes, thereby substituting for one or more subunits of these complexes (Fig. 3). Alternatively, these factors could form novel complexes. These novel and variant complexes could potentially mediate selective interactions with transcription activators binding to distal promoter or enhancer sequences. Third, some GTFs are expressed in specific cell types or at specific developmental stages, diversifying the regulatory properties of the general transcription machinery in space and time within the metazoan organism. The literature reviewed here confirms and highlights the complex roles of alternative transcription factors in the regulation of the metazoan genome. The TBP family
The TBP subunit of TFIID was once thought to be essential for all transcription in the eukaryotic cell.
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Table 1. TBP-associated factors (TAFIIs)a TAFIIb
TAFIIs found in TFIID and HAT complexesd
Related TAFIIs (species: number – name)c
TFIID
hTAFII250(CCG1) dTAFII250(230)
h: 1 d: 0
hTAFII250 dTAFII250
hTAFII150 dTAFII150
h: 0 d: 0
hTAFII150 dTAFII150
hTAFII150
hTAFII140 dBIP2(dTAFII155)
ND
hTAFII140 dBIP2
hTAFII140
hTAFII130(135) dTAFII110
h: 1 – hTAFII105 d: 1 – nht
hTAFII130, hTAFII105 dTAFII110
hTAFII130
hTAFII100 dTAFII80
h: 1 – hPAF65β d: 1 – can
hTAFII100 dTAFII80
hTAFII100, hPAF65β dTAFII80
hTAFII80(70) dTAFII60(62)
h: 1 – hPAF65α d: 1 – dTAFII60-2 (mia)
hTAFII80 dTAFII60
hTAFII80, hPAF65α dTAFII60
hTAFII68 d-cabeza
h: 0 d: 0
hTAFII68
hTAFII55 dTAFII55
h: 1 d: 0
hTAFII55
hTAFII55
hTAFII32 dTAFII40(42)
h: 6 d: 0
hTAFII32 dTAFII40
hTAFII32 dTAFII40
hTAFII30 dTAFII16
h: 0 d: 1 – dTAFII24
hTAFII30 dTAFII16, dTAFII24
hTAFII30 dTAFII24
hTAFII28 dTAFII30β
h: 0 d: 0
hTAFII28 dTAFII30β
hTAFII20(15) dTAFII30α(28)
h: 0 d: 1 – dTAFII30α-2 (rye)
hTAFII20 dTAFII30α
hTAFII20 dTAFII30α
hTAFII18 dTAFII18
h: 1 – hSPT3 d: 0
hTAFII18
hSPT3
(h)ENL (d)ENL/AF-9
h: 1 – AF-9 d: 0
ENL/AF-9
HAT
Core promoter interactione Inr DPE hTAFII250 dTAFII250 hTAFII150 dTAFII150
dTAFII60
dTAFII40
aAbbreviations:
d, Drosophila melanogaster; DPE, downstream promoter element; h, Homo sapiens; HAT, histone acetyl transferase; Inr, initiator element; ND, not determined; TBP, TATA-binding protein. bPutative or confirmed orthologs in different species. Names of orthologs vary across species as many TAF s derive their name from their II biochemical association with TBP and their molecular weight. For more information on TAFIIs, see Refs [6,17,49] and the online supplement of Ref. [16]. cThe number of related TAF s, encoded by paralogs, is different in different species and varies for each TAF . II II dTAF s are found with TBP in the TFIID transcription factor, but also in complexes lacking TBP, such as the HATs TFTC, SAGA and PCAF. II eSeveral TAF s bind core promoter elements such as the Inr or DPE (Fig. 1). II
Indeed, TBP is essential for transcription in yeast; however, metazoans have at least one TBP-related protein. The Caenorhabditis elegans genome encodes only two members of the TBP family: TBP and TBP-like factor (TLF, also referred to as TRF2 in some species). The Drosophila genome encodes three members of the TBP family: TBP and the TBP-related factors TRF1 and TRF2. The human genome possibly also encodes a third TBP family member, in addition to TBP and TLF/TRF2 [16]. It will be important to determine whether this putative novel member is a functional gene and, if so, to characterize the functions of this gene. TBP itself is developmentally regulated, at least in the mouse and the amphibian Xenopus laevis. In mice, the nuclear TBP concentration declines during oocyte growth, is low in the fertilized egg and increases at the two-cell stage [18]. In Xenopus, TBP protein is virtually undetectable by western blotting in the full-grown oocyte, but TBP levels increase http://tibs.trends.com
dramatically during blastula stages as maternally stored TBP mRNA gets translated [19]. In both mice and frogs, the increase of TBP protein correlates with the onset of embryonic transcription. Early embryonic development of many species is characterized by a period during which the embryonic genome is transcriptionally silent. The stage at which embryonic transcription starts varies between species. In the mouse, this is the two-cell stage (20 h post fertilization), whereas in Xenopus it is the midblastula stage (4000 cells, 5 h of development at 24°C). Transcriptional repression before the onset of embryonic gene expression involves multiple layers of regulation, including those operating at the levels of chromatin and transcription factor availability. Chromatin remodeling by the nucleosomal ATPase ISWI might play a role by removing TBP from chromatin in Xenopus eggs [20], thereby preventing recruitment of the transcription machinery by the small amounts of TBP that are present in eggs and
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TFTC
TAFIIs
TFIID Mot1
Variant TAFIIs TBP
TBP
TBP
Novel complexes?
TLF
TBP NC2
α/β TAC TBP
α γ
TFIIA
β γ
α
β
ALF Ti BS
Fig. 3. Combinatorial diversity generated by protein–protein interactions of general transcription factors (GTFs). Some related GTFs share interaction interfaces, competing for protein–protein interactions, but could potentially also form novel transcription complexes. Transcription complexes of variable composition might compete for the same target genes (merely modulating the activity of the involved promoters) or regulate distinct sets of genes. TBP (blue) interacts with TAFIIs (yellow) to form TFIID. Some of the TAFIIs in TFIID can be replaced (symbolized by ↔) by variant TAFIIs (green), for example hTAFII105 can substitute for hTAFII130. Conversely, both TAFIIs and related variant TAFIIs can participate in histone acetyl transferase (HAT) complexes such as TFTC, for example hTAFII100 and hPAF65β, and hTAFII80 and hPAF65α (Table 1). Both TFIIA and the unprocessed form of TFIIA in the TAC complex are capable of binding TBP. Similarly, TFIIA-like factor (ALF, red) can substitute for TFIIAα/β (orange) in binding to TBP. TBP is not only found in TFIID and TAC, but also in the Mot1 and NC2 complexes. So far TBP-like factor (TLF, magenta) has not been found in TFIID, TAC, Mot1 or NC2. However, TLF can compete with TBP for TFIIA binding (symbolized by ↔). Therefore, substitutions are selective, and not all theoretically possible substitution reactions happen in vivo because of divergence of sequence and function of related GTFs. In addition, inclusion of related factors into high molecular weight complexes is not necessarily mutually exclusive.
early embryos. Experimental manipulation of TBP levels in the early embryo indicated that TBP levels are rate-limiting for transcription before the midblastula transition (MBT) [19,21], and that developmentally regulated translation of maternal TBP mRNA is required for the onset of transcription of a subset of genes [19,22]. TBP is not universally required for transcription in the early vertebrate embryo: in both Xenopus and zebrafish, transcription of a subset of early embryonic genes is independent of TBP [22,23]. Frog embryos lacking detectable levels of TBP are capable of initiating gastrulation (for which transcription is required) but arrest before completing this process. Normal levels of TBP are also required for normal cell division in cultured chicken cells [24]. In these cells, even a moderate decrease of TBP levels by disruption of one copy of the TBP gene caused a delay in mitosis. Although TBP is not required for transcription of all metazoan genes, this http://tibs.trends.com
factor is clearly an essential protein in yeast and metazoans alike. Drosophila TRF1 is initially expressed throughout the embryo, but gradually becomes restricted to the central nervous system and the gonads [25]. Chromosomal localization studies showed that TRF1 binds a limited number of genes in fly salivary gland cells. TRF1 binds relatively well to the TATA-box and exhibits GTF requirements similar to TBP in an in vitro transcription assay [25]. However, transcription of the in vivo target gene tudor was dependent on an 11-bp TC box rather than a TATA box [26]. In vivo, the majority of TRF1 is associated with BRF, a core component of the Pol III transcription factor TFIIIB. In Drosophila, TRF1 rather than TBP is required for Pol III-dependent transcription of tRNA, 5S rRNA and U6 snRNA [27]. So far, TRF1 has only been identified in flies. TLF/TRF2 is found in many metazoans and is more distantly related to TBP than is TRF1 [28]. Although the protein interacts with TFIIA and TFIIB similar to TBP (Fig. 3), unlike TBP it does not bind well to canonical TATA boxes [29–31]. TLF reduces the activity of several TBP-dependent promoters in vitro and in transfection assays, probably by sequestration of TFIIA [29–31]. However, transcription from the SV40 promoter is moderately stimulated by TLF overexpression. This promoter features a variant TATA box (TATTTAT) for which TLF exhibits modest affinity [31]. Interestingly, a similar TATT (Tat-tee) box is present in the promoters of the brachyury and GS17 genes, which are dependent on TLF for their expression in Xenopus laevis embryos. These promoters might be regulated by TLF directly because they exhibit TLF-dependence early after the onset of transcription [22]. However, in vivo TLF-binding sites in these promoters are not known, and it will be important to analyze the mechanisms by which TLF functions and to determine the promoter elements that mediate TLF-dependent transcriptional regulation. Negative regulation of TBP-dependent promoters by TLF-mediated sequestration of TFIIA is potentially relevant in vivo, as it has been observed that transcription of some genes is derepressed in tlf-1 [RNAi] embryos of C. elegans, suggesting a negative role for TLF in regulation of a subset of genes [32]. However, a positive role of TLF in the in vivo transcription regulation of a subset of genes has also been observed [22,23,32–35] (Fig. 2c). TLF-dependent genes have been shown to play an important role in early embryogenesis of C. elegans, Xenopus and zebrafish [22,23,32,33] whereas, in the mouse, TLF-dependent genes play a role in spermatogenesis but not early embryogenesis [34,35]. In the frog and fish, TLF and TBP appear to play complementary roles in that some genes are TLF-dependent, some genes are TBP-dependent, and another group of genes appears to require both factors for normal expression [22,23]. Dependence on both TBP and TLF
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is most likely caused by indirect effects rather than a mechanistic requirement for both factors. The apparent requirement of TLF at different stages of the life cycle in different species (mouse versus worm, fish and frog) is remarkable, and raises the question of whether there is a unifying mechanistic or developmental principle underlying these differences. It will be important to identify specific pathways of TBP- and TLF-dependent gene regulation in different species to understand the different biological roles in the context of common mechanisms of gene regulation. TBP-associated factors
The TBP-associated factors implicated in Pol II transcription (TAFIIs) play a role in transcriptional activation, core promoter interactions and enzymatic protein modification [4]. Although initially identified as subunits of TFIID, some TAFIIs are also part of HAT complexes such as PCAF and TFTC (Table 1). TAFIIs can function in a gene-selective manner in yeast [10–14]; for example, several TAFIIs bind as subunits of TFIID to core promoter elements present in some, but not all, promoters (Figs 1,2; Table 1). Variant TAFIIs encoded by metazoan paralogs of these yeast proteins could act to further diversify core promoter recognition and increase gene selectivity. Furthermore, TAFIIs activate specific genes by mediating protein–protein interactions with specific transcriptional activators binding to distal promoter elements of these genes [4,36]. Further diversifying the general transcription machinery, and increasing the potential for geneselective effects, are selective substitution reactions, in which a subunit of a transcription complex is replaced by a related factor (Fig. 3). The gene-selective action of both the unique TAFIIs and the TAFIIs for which related factors have been identified (Table 1), is reflected by the variety of biological functions of TAFIIs in the cell, and in metazoan development. Mammalian TAFII250 and its yeast homolog yTAFII145 are important for progression through the G1/S transition of the cell cycle. In hamster cells, TAFII250 is selectively important for transcription from the cyclin A and D1 promoters. Two mechanisms have been implicated in gene-selective activation of cyclin A, as both activator-mediated regulation of cyclin A expression and cyclin A core promoter function are dependent on TAFII250 [37]. In Drosophila, TAFII250 is essential for viability. Weak loss-of-function alleles cause pleiotropic defects in a manner consistent with roles for TAFII250 in regulation of the cell cycle, cell differentiation, proliferation and survival [38]. Human TAFII150 is also important for the cell cycle, being essential for progression through the G2/M transition in cultured cells [39]. The human genome contains more than 12 potential alternative genes for the various TAFIIs; flies and yeast have five and three alternative TAFII http://tibs.trends.com
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genes, respectively [16,17] (Table 1). For example, the human (h) TBP-associated factor hTAFII32 has six putative paralogs in the genome, whereas Drosophila (d) has no genes related to dTAFII40/42, the homolog of hTAFII32. As dTAFII40 has been implicated in core promoter recognition, it will be important to determine whether or not the human paralogs have a similar function. The gene encoding dTAFII40/42 is essential for viability, although flies carrying insertions in dTAFII40/42 that do not fully abolish dTAFII40/42 function are viable and female sterile [40]. In other cases, for example that of hTAFII30, the human genome does not contain related genes, whereas the Drosophila genome encodes two related TAFIIs (dTAFII24 and dTAFII16) [16]. Both dTAFII24 and dTAFII16 interact with TBP in a Drosophila TFIID complex, suggesting that these proteins can substitute for one another in TFIID (Table 1, Fig. 3). However, this might not be the case for their function in HAT complexes as dTAFII24, but not dTAFII16, was found to be part of a TBP-free TAF–HAT complex [41]. Immunostaining of salivary gland polytene chromosomes revealed that dTAFII24 and dTAFII16 localize to distinct, yet overlapping sets of genes. Combined with the distinct spatio-temporal expression patterns of these TAFIIs during embryogenesis, it is possible that two related genes are required to meet the demands for differential regulation of distinct sets of target genes during fly embryogenesis. The single homolog identified so far in the mouse, mTAFII30, is required for cell-cycle progression and viability in embryonal carcinoma cells [42]. Similar to wild-type embryonal carcinoma cells, TAFII30-null cells were shown to be capable of retinoic acid-induced differentiation into primitive endoderm, which also rescued viability. By contrast, differentiation into parietal endoderm required TAFII30. These results suggest that mTAFII30 is required for the proper transcription of a subset of genes essential for specific cell types and differentiation pathways. Both hTAFII130 and its Drosophila homolog dTAFII110 have a single relative: hTAFII105 and nht, respectively. The mouse homolog of hTAFII105 is an alternative TFIID subunit (Fig. 3), which is relatively abundant in testis, ovary and B cells, and is required for ovarian development and expression of a subset of genes [43]. By contrast, Nht expression is restricted to the testis and plays a role in Drosophila spermatogenesis (M. Hiller et al., pers. commun.). dTAFII110 is more broadly expressed than its relative nht, and mutation of dTAFII110 revealed that it is involved in activation by the embryonic transcription factors Dorsal and Twist [44,45]. A similar situation has been found for dTAFII60 and mia, the Drosophila homologs of hTAFII80 and hPAF65α (PCAF-associated factor 65 α). Similar to nht, mia is required for spermatogenesis (M. Hiller et al., pers. commun.), whereas mutation of dTAFII60
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Table 2. The metazoan minimal transcription machinerya,b General components
Variable components
RNA polymerase IIc
TBP, TLF, TAFIIs, variant TAFIIs
TFIIB
TFIIA, ALF
TFIIE
TFIIHd
TFIIF aAbbreviations: ALF, TFIIAαβ-like factor; TAF, TBP-associated factor; TBP, TATA-binding protein; TF, transcription factor; TLF, TBP-like factor. bThe metazoan minimal transcription machinery is the combination of general and variable factors necessary and sufficient for transcription from a given core promoter. This combination of factors consists of general, universally required components, and variable components that might function in gene-selective ways. cSome subunits have paralogs in the human genome [16] and might not be universally required. dThe kinase activity of TFIIH is not universally required in yeast [50]. In addition, several subunits have potential paralogs in metazoan genomes [16].
revealed that this TAF, similar to dTAFII110, plays a role in Dorsal-mediated activation [44,45]. A single relative exists for the hTAFII100 and dTAFII80 genes, called PAF65β and can in human and fly, respectively. Whereas dTAFII80 is widely expressed, can is selectively expressed in primary spermatocytes, and is required for testis-specific gene transcription and male fertility [46]. It will be important to determine whether can exerts its tissuespecific effects as part of a TFIID complex or as part of a TBP-free HAT complex, which would help elucidate the mechanisms underlying the gene-selective role of this variant cell-type-specific TAF. TFIIA and ALF
Acknowledgements This review is dedicated to the memory of Alan P. Wolffe, who died in an accident on 26 May, 2001. I thank I.B. Dawid, D.L. Weeks and D.A. Wassarman for critical reading of the manuscript, and M. Hiller, J. Pringle and M. Fuller for communicating data before publication.
TFIIA consists of three subunits (α, β and γ), which are encoded by two genes: those encoding TFIIAαβ and TFIIAγ. The human genome contains a gene related to TFIIAαβ, referred to as TFIIAαβ-like factor (ALF) or TFIIAτ [47,48]. Similar to TFIIAαβ, ALF plus TFIIAγ promotes binding of TBP to DNA, and ALF can also substitute for TFIIA-α/β in an in vitro transcription system [47,48] (Fig. 3). ALF is expressed in a variety of tissues but is most abundant in the testis and, therefore, a role in spermatogenesis has been suggested. Although this is an interesting possibility, no experimental data have, as yet, been reported to indicate whether ALF is redundant with TFIIAαβ or whether it plays a unique role in the transcription of a distinct set of genes. Conclusions and prospects
The eukaryotic transcription machinery is a biochemical entity of variable composition and function in both yeast and metazoans. However, metazoans exhibit a greater variability and gene selectivity of GTFs, reflecting a wide variety of
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regulatory constraints and transcriptional requirements during development and differentiation. It should be noted that the general transcription machinery is far less diverse than the thousands of transcription factors that activate or repress transcription from proximal or distal cis-acting elements. Nevertheless, rather than viewing the general transcription machinery as the common biochemical entity upon which myriad sequence-specific trans-acting transcription factors integrate their regulatory signals, it would be fair to say that this multitude of transcriptional regulators integrate their signals on a common RNA polymerase through a non-universal, regulated and partly variable ‘minimal transcription machinery’ (Table 2). This minimal transcription machinery consists of general components (Pol II, TFIIB, TFIIE, and TFIIF) and variable components (TBP, TLF, TAFIIs, TFIIA and ALF), and is defined as the combination of general and variable factors that is necessary and sufficient for correct initiation of transcription from a given core promoter. It is useful to distinguish these transcription factors from cofactors and sequence-specific trans-acting factors that only modulate the relative efficiency of initiation by activation or repression. It is interesting to note that many GTFs are enriched in testis (e.g. TBP, TLF, Drosophila TAFIIs, TFIIA, ALF, TFIIE, the large subunit of Pol II), and several of these (murine TLF and Drosophila TAFIIs) have been shown to play a role in spermatogenesis. Apparently, the basal transcription requirements of the testis are distinct from those of many other organs. TBP and some of the TAFIIs for which no paralogs exist in the human genome, are important for cell-cycle progression. This does not necessarily mean that these factors are not important for other processes, as it is possible that the cell cycle is very sensitive to alterations in levels of GTFs. For a clearer understanding of gene regulation, it will be important to gain comprehensive information on GTF requirements for the genes in the metazoan genome in general, and for developmental pathways in particular. Biochemical analysis is needed to reveal the nature and identity of the core promoter elements that determine gene-selective recruitment of variant transcription factors, and the various nucleoprotein complexes that comprise these factors. Finally, it will be important to determine the implications of a variable minimal transcription machinery for promoter-selective interactions between this machinery and activators and repressors that modulate transcriptional activity.
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Nobel Prize winners This years Nobel Prize for Physiology or Medicine has been awarded to Tim Hunt, Paul Nurse and Leland Hartwell for their pioneering work on aspects of the cell cycle. We would like to extend our warmest congratulations to all three scientists. A special mention goes to Tim Hunt, who served on the TiBS editorial board for over 20 years and was Editor-in-Chief on the journal from 1992–2000. Congratulations Tim! Axel Innis is a PhD student at the Dept of Biochemistry, University of Cambridge, UK.
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