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New Problems in RNA Polymerase II Transcription Initiation: Matching the Diversity of Core Promoters with a Variety of Promoter Recognition Factors

2007; Elsevier BV; Volume: 282; Issue: 20 Linguagem: Inglês

10.1074/jbc.r700012200

1083-351X

Ferenc Muöller, Màté A. Demé ny, Làszlò Tora,

RNA and protein synthesis mechanisms

One of the key events in eukaryotic gene regulation is the assembly of general transcription factors (GTFs) 4The abbreviations used are: GTF, general transcription factor; Pol II, RNA polymerase II; PIC, pre-initiation complex; TBP, TATA-binding protein; TSS, transcription start site; TAF, TBP-associated factor; TRF, TBP-related factor; BP, broad peak TSS-containing promoter. and RNA polymerase II (Pol II) into a pre-initiation complex (PIC) at promoters. GTFs have been defined biochemically as a set of factors essential for accurate transcription initiation at a TATA box-containing viral promoter in vitro (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (856) Google Scholar, 2Roeder R.G. Nat. Med. 2003; 9: 1239-1244Crossref PubMed Scopus (56) Google Scholar). The textbook description states that the initiation of mRNA synthesis requires the recruitment and binding of the TATA-binding protein (TBP) as a component of the TFIID complex to a consensus sequence that is found in core promoters and is known as the TATA box (3Butler J.E. Kadonaga J.T. Genes Dev. 2002; 16: 2583-2592Crossref PubMed Scopus (458) Google Scholar). This view is now seriously challenged by a series of observations. (i) The TATA box is not a general component of all Pol II core promoters; (ii) not only TBP but different types of TBP-related factors can mediate Pol II transcription initiation; (iii) TBP- or TBP-related factor-independent Pol II transcription has been described; and (iv) TBP binding is not necessarily a prerequisite or even an indicator of promoter activation in vivo. The collapse of the old "general" dogma is a result of parallel developments in bioinformatic analysis of promoter sequences and the application of new technologies in studying the biology of transcription in metazoans. Here we review the emerging view suggesting that instead of TBP binding to a TATA box, in metazoans distinct sets of core promoter recognition factors recognize a huge diversity of core promoters (see Fig. 1). The core promoter was originally defined biochemically as the minimal DNA fragment that is sufficient to direct correct basal levels of transcription initiation by Pol II in vitro on naked DNA templates containing a single well defined transcription start site (TSS). According to this definition, the core promoter typically extends appreciatively 40–50 bp up- and downstream of the TSS and can contain several distinct core promoter sequence elements. An important problem in the understanding of core promoter function at the genomic level has been the difficulty of identifying core promoters in general. This is because they lack easily identifiable universal consensus motifs. Promoter prediction tools are numerous, but because of the lack of general identification features, high false positive and negative rates dampened their efficiency (4Bajic V.B. Tan S.L. Suzuki Y. Sugano S. Nat. Biotechnol. 2004; 22: 1467-1473Crossref PubMed Scopus (126) Google Scholar). In the past, only a few genes were analyzed sufficiently in depth to identify general core promoter features and TSSs. To get a better grip on the regulation of core promoters in general, a drastic improvement in identifying functional promoter sequences has become necessary. Promoter identification has improved drastically in the last several years because of the technological development in two areas of genomics: large scale sequencing of accurately determined 5′-ends of cDNAs (such as Cap analysis of gene expression, CAGE (5Shiraki T. Kondo S. Katayama S. Waki K. Kasukawa T. Kawaji H. Kodzius R. Watahiki A. Nakamura M. Arakawa T. Fukuda S. Sasaki D. Podhajska A. Harbers M. Kawai J. Carninci P. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15776-15781Crossref PubMed Scopus (576) Google Scholar)) and the availability of numerous genome sequences from yeast to human, which together allowed significant improvement in identifying and annotating TSSs on a large scale (6Cooper S.J. Trinklein N.D. Anton E.D. Nguyen L. Myers R.M. Genome Res. 2006; 16: 1-10Crossref PubMed Scopus (197) Google Scholar, 7Kimura K. Wakamatsu A. Suzuki Y. Ota T. Nishikawa T. Yamashita R. Yamamoto J. Sekine M. Tsuritani K. Wakaguri H. Ishii S. Sugiyama T. Saito K. Isono Y. Irie R. Kushida N. Yoneyama T. Otsuka R. Kanda K. Yokoi T. Kondo H. Wagatsuma M. Murakawa K. Ishida S. Ishibashi T. Takahashi-Fujii A. Tanase T. Nagai K. Kikuchi H. Nakai K. Isogai T. Sugano S. Genome Res. 2006; 16: 55-65Crossref PubMed Scopus (400) Google Scholar, 8Carninci P. Sandelin A. Lenhard B. Katayama S. Shimokawa K. Ponjavic J. Semple C.A. Taylor M.S. Engstrom P.G. Frith M.C. Forrest A.R. Alkema W.B. Tan S.L. Plessy C. Kodzius R. Ravasi T. Kasukawa T. Fukuda S. Kanamori-Katayama M. Kitazume Y. Kawaji H. Kai C. Nakamura M. Konno H. Nakano K. Mottagui-Tabar S. Arner P. Chesi A. Gustincich S. Persichetti F. Suzuki H. Grimmond S.M. Wells C.A. Orlando V. Wahlestedt C. Liu E.T. Harbers M. Kawai J. Bajic V.B. Hume D.A. Hayashizaki Y. Nat. Genet. 2006; 38: 626-635Crossref PubMed Scopus (1034) Google Scholar). The CAGE tag analysis of the mammalian promoterome provided the first truly global insight into the sequence structure of core promoters and the expression dynamics associated with core promoters. Strikingly, several classes of promoter types were identified on the basis of the differential usage of TSSs (8Carninci P. Sandelin A. Lenhard B. Katayama S. Shimokawa K. Ponjavic J. Semple C.A. Taylor M.S. Engstrom P.G. Frith M.C. Forrest A.R. Alkema W.B. Tan S.L. Plessy C. Kodzius R. Ravasi T. Kasukawa T. Fukuda S. Kanamori-Katayama M. Kitazume Y. Kawaji H. Kai C. Nakamura M. Konno H. Nakano K. Mottagui-Tabar S. Arner P. Chesi A. Gustincich S. Persichetti F. Suzuki H. Grimmond S.M. Wells C.A. Orlando V. Wahlestedt C. Liu E.T. Harbers M. Kawai J. Bajic V.B. Hume D.A. Hayashizaki Y. Nat. Genet. 2006; 38: 626-635Crossref PubMed Scopus (1034) Google Scholar). At the extreme ends of the spectrum, two significant classes of promoters can be defined: (a) single peak promoters, which have a relatively tightly defined TSS position within a few base pairs and (b) broad peak promoters, which show a random distribution of many TSSs in a 100-bp window. Strikingly, single peak promoters were found to be more likely associated with TATA boxes and to possess a higher frequency of transcription factor binding sites than broad peak promoters. The latter often carry CpG islands, are TATA-less, and are associated with less consensus binding sites. Moreover, it seems that TATA box-associated single peak promoters are involved in tight regulation of genes, which are often tissue-specific. In contrast, the broad peak promoters are often associated with ubiquitously expressed genes (8Carninci P. Sandelin A. Lenhard B. Katayama S. Shimokawa K. Ponjavic J. Semple C.A. Taylor M.S. Engstrom P.G. Frith M.C. Forrest A.R. Alkema W.B. Tan S.L. Plessy C. Kodzius R. Ravasi T. Kasukawa T. Fukuda S. Kanamori-Katayama M. Kitazume Y. Kawaji H. Kai C. Nakamura M. Konno H. Nakano K. Mottagui-Tabar S. Arner P. Chesi A. Gustincich S. Persichetti F. Suzuki H. Grimmond S.M. Wells C.A. Orlando V. Wahlestedt C. Liu E.T. Harbers M. Kawai J. Bajic V.B. Hume D.A. Hayashizaki Y. Nat. Genet. 2006; 38: 626-635Crossref PubMed Scopus (1034) Google Scholar, 9Schug J. Schuller W.P. Kappen C. Salbaum J.M. Bucan M. Stoeckert Jr., C.J. Genome Biol. 2005; 6: R33Crossref PubMed Google Scholar). In addition, from these studies it became clear that the TATA box, which was once believed to be a general feature of core promoters, is only present in less than 10% of all human Pol II promoters (10FitzGerald P.C. Shlyakhtenko A. Mir A.A. Vinson C. Genome Res. 2004; 14: 1562-1574Crossref PubMed Scopus (176) Google Scholar, 11Bajic V.B. Tan S.L. Christoffels A. Schonbach C. Lipovich L. Yang L. Hofmann O. Kruger A. Hide W. Kai C. Kawai J. Hume D.A. Carninci P. Hayashizaki Y. PLoS Genet. 2006; 2: e54Crossref PubMed Scopus (83) Google Scholar). The existence of TATA-less promoters regulating tissue-specific genes (12Latinkic B.V. Umbhauer M. Neal K.A. Lerchner W. Smith J.C. Cunliffe V. Genes Dev. 1997; 11: 3265-3276Crossref PubMed Scopus (167) Google Scholar) and TATA-containing promoters driving transcription of ubiquitously expressed genes (13Quitschke W.W. DePonti-Zilli L. Lin Z.Y. Paterson B.M. Mol. Cell. Biol. 1989; 9: 3218-3230Crossref PubMed Scopus (26) Google Scholar) demonstrates that the regulatory logic suggested by the above genome-wide tendencies cannot be universally applied to gene activation. Moreover, even when a TATA box is present in a core promoter, it may not be critical for defining promoter activity, and throughout the genome a consensus TATA element does not appear to be a major determinant of either TBP binding or gene expression in yeast (14Kim J. Iyer V.R. Mol. Cell. Biol. 2004; 24: 8104-8112Crossref PubMed Scopus (52) Google Scholar, 15Basehoar A.D. Zanton S.J. Pugh B.F. Cell. 2004; 116: 699-709Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Recent bioinformatic studies of vertebrates failed to identify a single core promoter element that would universally cluster close to the TSS for all, or at least the majority, of the analyzed core promoters. Nevertheless, a series of core promoter elements has been shown to exist in smaller subsets of core promoters with positional constraints in relation to the TSS and with specific transcription factors binding to them. These include the INR (initiator), BRE (TFIIB recognition element), DCE (downstream core element), DPE (downstream core promoter element), and MTE (mutation ten element) (Fig. 1) (see Refs. 3Butler J.E. Kadonaga J.T. Genes Dev. 2002; 16: 2583-2592Crossref PubMed Scopus (458) Google Scholar, 16Juven-Gershon T. Hsu J.Y. Kadonaga J.T. Biochem. Soc. Trans. 2006; 34: 1047-1050Crossref PubMed Scopus (65) Google Scholar, and 17Gross P. Oelgeschlager T. Biochem. Soc. Symp. 2006; 73: 225-236Crossref PubMed Scopus (42) Google Scholar for reviews). Interestingly, the INR with its degenerate sequence YYANWYY (18Smale S.T. Kadonaga J.T. Annu. Rev. Biochem. 2003; 72: 449-479Crossref PubMed Scopus (816) Google Scholar) does not seem to cluster at the TSSs (10FitzGerald P.C. Shlyakhtenko A. Mir A.A. Vinson C. Genome Res. 2004; 14: 1562-1574Crossref PubMed Scopus (176) Google Scholar). These elements can positively or negatively correlate with the presence of other consensus core promoter sequences; however the regulatory significance of these correlations is still unclear. For example the DPE-containing promoters are often, but not exclusively, TATA-less (3Butler J.E. Kadonaga J.T. Genes Dev. 2002; 16: 2583-2592Crossref PubMed Scopus (458) Google Scholar), and the newly described XCPE1 element (the X-gene core promoter element 1) has also been associated with TATA-less promoters (19Tokusumi Y. Ma Y. Song X. Jacobson R.H. Takada S. Mol. Cell. Biol. 2007; 27: 1844-1858Crossref PubMed Scopus (55) Google Scholar). The above described examples in which core promoters are considered as a combination of distinct motifs representing putative transcription factor binding sites may not be the only way to define the functionally relevant structure of core promoters. Chargaff's second parity rule states that the number of As equals the number of Ts and the number of Cs equals the number of Gs in a single strand over regions of sufficient size (20Chargaff E. Fed. Proc. 1951; 10: 654-659PubMed Google Scholar). This rule seems not to be valid in the immediate neighborhood of TSSs (11Bajic V.B. Tan S.L. Christoffels A. Schonbach C. Lipovich L. Yang L. Hofmann O. Kruger A. Hide W. Kai C. Kawai J. Hume D.A. Carninci P. Hayashizaki Y. PLoS Genet. 2006; 2: e54Crossref PubMed Scopus (83) Google Scholar, 21Aerts S. Thijs G. Dabrowski M. Moreau Y. De Moor B. BMC Genomics. 2004; 5: 34Crossref PubMed Scopus (60) Google Scholar). Furthermore, on the basis of mononucleotide distribution around TSSs four possible promoter classes can be defined in which the GC/AT distribution around the TSSs is significantly diverged (11Bajic V.B. Tan S.L. Christoffels A. Schonbach C. Lipovich L. Yang L. Hofmann O. Kruger A. Hide W. Kai C. Kawai J. Hume D.A. Carninci P. Hayashizaki Y. PLoS Genet. 2006; 2: e54Crossref PubMed Scopus (83) Google Scholar). However, at present it is not clear how these distinct classes of promoters with different GC/AT distribution can be recognized by transcription factors that direct transcription initiation site selection. Thus, the variations of sequence motif combinations and other yet unexplored sequence features in core promoters and the corresponding binding of diverse GTFs may function together in the determination of the transcriptional initiation site. The regulatory significance of the above described diversity of core promoters is not yet resolved. In the simplest scenario, distinct core promoter elements would create binding sites for different recognition factors (Fig. 1) that would allow the assembly of PICs with distinct factor composition. Thus, not all the GTFs will be present in every PIC, suggesting that several of the GTFs may not be appropriately called "general" (22Muller F. Tora L. EMBO J. 2004; 23: 2-8Crossref PubMed Scopus (79) Google Scholar). This suggestion is in good agreement with the observation that certain GTFs are not expressed in every cell type of a given organism (Refs. 22Muller F. Tora L. EMBO J. 2004; 23: 2-8Crossref PubMed Scopus (79) Google Scholar, 23Mohan I.W. Scheer E. Wendling O. Metzger D. Tora L. Mol. Cell. Biol. 2003; 23: 4307-4318Crossref PubMed Scopus (47) Google Scholar, 24Hiller M. Chen X. Pringle M.J. Suchorolski M. Sancak Y. Viswanathan S. Bolival B. Lin T.Y. Marino S. Fuller M.T. Development. 2004; 131: 5297-5308Crossref PubMed Scopus (161) Google Scholar, 25Xiao L. Kim M. DeJong J. Gene Expr. Patterns. 2006; 6: 409-419Crossref PubMed Scopus (40) Google Scholar and references therein). In the next paragraphs we will summarize findings about the known diversity of core promoter binding factors in support of the novel view that many structurally and functionally different PICs will assemble on a wide diversity of Pol II core promoters to direct the initiation of transcription. The TFIID complex, composed always of TBP and all the 13 or 14 TBP-associated factors (TAFs) (Ref. 26Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (197) Google Scholar and references therein), has long been viewed as the only core promoter recognition factor in Pol II transcription. However, TFIID complexes containing or lacking certain TAFs exist, generating a spectrum of variants of the prototype complex (reviewed in Refs. 22Muller F. Tora L. EMBO J. 2004; 23: 2-8Crossref PubMed Scopus (79) Google Scholar and 27Bell B. Tora L. Exp. Cell Res. 1999; 246: 11-19Crossref PubMed Scopus (109) Google Scholar). How do these different TFIID complexes form? Several experiments indicated that a subset of TAFs (TAF4, TAF5, TAF6, TAF8, TAF9, TAF10, and TAF12) forms a minimal TFIID core complex that lacks TBP and other TAFs (28Wright K.J. Marr 2nd, M.T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12347-12352Crossref PubMed Scopus (123) Google Scholar, 29Leurent C. Sanders S.L. Demeny M.A. Garbett K.A. Ruhlmann C. Weil P.A. Tora L. Schultz P. EMBO J. 2004; 23: 719-727Crossref PubMed Scopus (63) Google Scholar, 30Demé;ny M. Soutoglou E. Nagy Z. Scheer E. Janoshazi A. Richardot M. Argentini M. Kessler P. Tora L. PLoS ONE. 2007; 2: e316Crossref PubMed Scopus (44) Google Scholar). This stable TFIID core is suggested to associate with combinations of other TAFs and TBP (28Wright K.J. Marr 2nd, M.T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12347-12352Crossref PubMed Scopus (123) Google Scholar, 30Demé;ny M. Soutoglou E. Nagy Z. Scheer E. Janoshazi A. Richardot M. Argentini M. Kessler P. Tora L. PLoS ONE. 2007; 2: e316Crossref PubMed Scopus (44) Google Scholar) to form a large variety of different complexes. In agreement with a model according to which different submodules of TFIID complexes recognize distinct core promoter elements, several TAFs have been implicated in the recognition of the promoter DNA around the TSS (31Oelgeschlager T. Chiang C.M. Roeder R.G. Nature. 1996; 382: 735-738Crossref PubMed Scopus (149) Google Scholar). TAF1 and TAF2 can bind the INR (32Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (181) Google Scholar). TFIID complexes with or without TAF2 have been described (33Kaufmann J. Ahrens K. Koop R. Smale S.T. Muller R. Mol. Cell. Biol. 1998; 18: 233-239Crossref PubMed Scopus (54) Google Scholar, 34Martinez E. Ge H. Tao Y. Yuan C.X. Palhan V. Roeder R.G. Mol. Cell. Biol. 1998; 18: 6571-6583Crossref PubMed Scopus (62) Google Scholar), suggesting that only complexes that contain TAF1 and TAF2 will be recruited to the INR-containing class of core promoters. TAF1 was also shown to bind DCE-containing promoters (35Lee D.H. Gershenzon N. Gupta M. Ioshikhes I.P. Reinberg D. Lewis B.A. Mol. Cell. Biol. 2005; 25: 9674-9686Crossref PubMed Scopus (87) Google Scholar). Two domains of TAF1 are important for regulating largely non-overlapping sets of genes in vivo (36O'Brien T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2456-2461Crossref PubMed Scopus (54) Google Scholar), suggesting that even different domains of distinct TAFs may bind different core promoter regions. The TAF6/TAF9 and the TAF4b/TAF12 heterodimers have also been shown to bind DNA, and the TAF6/TAF9 pair was also found to contact the DPE-containing class of core promoters in vivo (3Butler J.E. Kadonaga J.T. Genes Dev. 2002; 16: 2583-2592Crossref PubMed Scopus (458) Google Scholar, 37Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (53) Google Scholar) (Fig. 1). The core TFIID model might imply that satellite TAFs are less broadly used than those that form the core. In agreement with the idea that TAF10 is part of the TFIID core, TAF10-lacking early mouse embryos seem to have an altered TFIID structure and have undetectable levels of Pol II transcription (23Mohan I.W. Scheer E. Wendling O. Metzger D. Tora L. Mol. Cell. Biol. 2003; 23: 4307-4318Crossref PubMed Scopus (47) Google Scholar). In contrast, later in development many genes are transcribed normally without TAF10, suggesting that TAF10-containing TFIID or other TFIID-like complexes are only required for the transcription of a subset of promoters at certain developmental stages or cell types (38Indra A.K. Mohan 2nd, W.S. Frontini M. Scheer E. Messaddeq N. Metzger D. Tora L. Dev. Biol. 2005; 285: 28-37Crossref PubMed Scopus (38) Google Scholar) (Fig. 2). To elucidate the contribution of TAFs to promoter selectivity in vivo a genome-wide promoter occupancy analysis has been performed (39Kim T.H. Barrera L.O. Zheng M. Qu C. Singer M.A. Richmond T.A. Wu Y. Green R.D. Ren B. Nature. 2005; 436: 876-880Crossref PubMed Scopus (762) Google Scholar). A tight correlation between promoter occupancy by TAF1, probably representing the binding of a TFIID subpopulation, and the activity of the corresponding gene was detected for only 75% of the genes tested. In this category of genes TFIID-containing PICs on promoters may lead to transcription as the classic model suggested. However, the remaining 25% of gene promoters either (i) did not bind TAF1 while being expressed or (ii) were bound by TAF1, but no transcription was detected (39Kim T.H. Barrera L.O. Zheng M. Qu C. Singer M.A. Richmond T.A. Wu Y. Green R.D. Ren B. Nature. 2005; 436: 876-880Crossref PubMed Scopus (762) Google Scholar). In the former case it is possible that TAF1 could bind alone without other components of the TFIID or that TAF1-containing TFIID only primed these genes for future activation of transcription (40Christova R. Oelgeschlager T. Nat. Cell Biol. 2002; 4: 79-82Crossref PubMed Scopus (131) Google Scholar, 41Marr 2nd, M.T. Isogai Y. Wright K.J. Tjian R. Genes Dev. 2006; 20: 1458-1469Crossref PubMed Scopus (76) Google Scholar). The genes that were transcribed without TAF1 binding may represent the activity of a variant TFIID core complex (28Wright K.J. Marr 2nd, M.T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12347-12352Crossref PubMed Scopus (123) Google Scholar) or that of a TBP-free transcription complex (see also below). In agreement with the idea that not every TAF is present at every active promoter, knockout or silencing of TAF genes in different metazoan cells influences the expression of only a small subset of genes (42Chen Z. Manley J.L. Mol. Cell. Biol. 2000; 20: 5064-5076Crossref PubMed Scopus (36) Google Scholar, 43Frontini M. Soutoglou E. Argentini M. Bole-Feysot C. Jost B. Scheer E. Tora L. Mol. Cell. Biol. 2005; 25: 4638-4649Crossref PubMed Scopus (52) Google Scholar), further suggesting that TFIID subunits are not generally involved in gene regulation at every promoter. Although there is a clear link between promoter occupancy by TBP and gene expression in yeast (44Kuras L. Struhl K. Nature. 1999; 399: 609-613Crossref PubMed Scopus (401) Google Scholar), this apparent correlation is not always true in metazoans. Overexpression of TBP increases transcription from TATA-containing promoters although it rather represses transcription from TATA-less promoters (45Colgan J. Manley J.L. Genes Dev. 1992; 6: 304-315Crossref PubMed Scopus (120) Google Scholar, 46Chong J.A. Moran M.M. Teichmann M. Kaczmarek J.S. Roeder R. Clapham D.E. Mol. Cell. Biol. 2005; 25: 2632-2643Crossref PubMed Scopus (39) Google Scholar), suggesting differential (even opposite) roles for TBP in Pol II transcription initiation at distinct promoters. Similarly, the TATA binding activity of TBP is not required for the functional recruitment of TFIID to certain natural promoters (47Martinez E. Chiang C.M. Ge H. Roeder R.G. EMBO J. 1994; 13: 3115-3126Crossref PubMed Scopus (161) Google Scholar). Also, no direct correlation between promoter occupancy by TBP and activation of a promoter by a strong viral enhancer in chicken DT40 cells was detected (48Chen Z. Manley J.L. Mol. Cell. Biol. 2003; 23: 7350-7362Crossref PubMed Scopus (26) Google Scholar). Thus, TBP may be present at the promoters but may not contribute to promoter recognition and/or transcription initiation events. TBP-free transcription initiation has also been suggested at zygotic developmental regulator genes, which were not affected by loss of TBP in vertebrate embryos (Ref. 22Muller F. Tora L. EMBO J. 2004; 23: 2-8Crossref PubMed Scopus (79) Google Scholar and references therein), and type I interferon responsive genes can be independently transcribed of TBP in human HeLa cells (49Paulson M. Press C. Smith E. Tanese N. Levy D.E. Nat. Cell Biol. 2002; 4: 140-147Crossref PubMed Scopus (94) Google Scholar). A recently identified set of TBP-related factors (TRFs) has been suggested to function similarly to TBP in Pol II promoter recognition and may indicate alternative mechanisms for TBP-free transcription initiation. Drosophila-specific TRF1 and vertebrate-specific TRF3/TBP2 were shown to bind the TATA box, interact with GTFs, and mediate Pol II transcription initiation from a limited set of promoters (50Holmes M.C. Tjian R. Science. 2000; 288: 867-870Crossref PubMed Scopus (92) Google Scholar, 51Bartfai R. Balduf C. Hilton T. Rathmann Y. Hadzhiev Y. Tora L. Orban L. Muller F. Curr. Biol. 2004; 14: 593-598Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 52Jallow Z. Jacobi U.G. Weeks D.L. Dawid I.B. Veenstra G.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13525-13530Crossref PubMed Scopus (83) Google Scholar, 53Persengiev S.P. Zhu X. Dixit B.L. Maston G.A. Kittler E.L. Green M.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14887-14891Crossref PubMed Scopus (62) Google Scholar). Thus, the specificity of TBP, TRF1, and TRF3/TBP2 to promoters may be determined by factors with which they form a complex and also by their differential expression patterns in the distinct cell types of a given organism. In contrast, the different DNA binding specificity of TRF2/TLF may explain its affinity to distinct set of promoters (Refs. 22Muller F. Tora L. EMBO J. 2004; 23: 2-8Crossref PubMed Scopus (79) Google Scholar, 46Chong J.A. Moran M.M. Teichmann M. Kaczmarek J.S. Roeder R. Clapham D.E. Mol. Cell. Biol. 2005; 25: 2632-2643Crossref PubMed Scopus (39) Google Scholar, and 54Dantonel J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar and references therein). Taken together, the recognition of promoters by multiple TBP-related factors or other TAF-containing complexes, lacking TBP, may represent an additional regulatory level acting on core promoters and may explain the diversity of core promoters observed in vertebrates. According to our present understanding the ultimate purpose of the binding of a PIC to the core promoter is to mark precisely the TSS for Pol II transcription initiation. However, a whole class of promoters, the broad peak TSS-containing promoters (BP) (8Carninci P. Sandelin A. Lenhard B. Katayama S. Shimokawa K. Ponjavic J. Semple C.A. Taylor M.S. Engstrom P.G. Frith M.C. Forrest A.R. Alkema W.B. Tan S.L. Plessy C. Kodzius R. Ravasi T. Kasukawa T. Fukuda S. Kanamori-Katayama M. Kitazume Y. Kawaji H. Kai C. Nakamura M. Konno H. Nakano K. Mottagui-Tabar S. Arner P. Chesi A. Gustincich S. Persichetti F. Suzuki H. Grimmond S.M. Wells C.A. Orlando V. Wahlestedt C. Liu E.T. Harbers M. Kawai J. Bajic V.B. Hume D.A. Hayashizaki Y. Nat. Genet. 2006; 38: 626-635Crossref PubMed Scopus (1034) Google Scholar), do not fit into this model, and their regulation may be fundamentally different. BPs are much more abundant than single peak promoters (55Carninci P. Trends Genet. 2006; 22: 501-510Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), yet the mechanisms underlying the initiation of transcription from them remain obscure. On these promoters multiple TSSs are distributed over a region of 100 bp. It is possible that on these usually TATA-less BPs (i) only one PIC assembles at a time and the same PIC "slides" to different sites in the same region for separate initiation events; (ii) distinct consecutively arriving PICs bind to slightly different sites; (iii) several PICs can be formed at the same time at different sites in these regions; or (iv) the distinct TSSs represent single or a low number of TSSs specific to each cell, but they appear as multiple TSSs in a cell population. This latter scenario has been suggested by a recent study, which indicated different TSS utilization at BPs in different cell types (56Kawaji H. Frith M.C. Katayama S. Sandelin A. Kai C. Kawai J. Carninci P. Hayashizaki Y. Genome Biol. 2006; 7: R118Crossref PubMed Scopus (69) Google Scholar) (Fig. 2). The fact that on these BPs many initiation sites have been detected suggests that the PICs binding to these regions do not have strong sequence specificity. Several studies suggested that active promoters are devoid of nucleosomes in a ∼150-bp to ∼200-bp region upstream of the TSSs (57Barrera L.O. Ren B. Curr. Opin. Cell Biol. 2006; 18: 291-298Crossref PubMed Scopus (95) Google Scholar). Thus, it is conceivable that PICs on the multiple TSS-containing promoters assemble at the nucleosome-free regions "non-specifically," because the BPs do not seem to contain well defined core promoter motifs. However, the fact that initiation from BPs seems to show a certain tissue specificity (56Kawaji H. Frith M.C. Katayama S. Sandelin A. Kai C. Kawai J. Carninci P. Hayashizaki Y. Genome Biol. 2006; 7: R118Crossref PubMed Scopus (69) Google Scholar) suggests that initiation from BPs may not be random involving some sequence motif-specific targeting mechanisms. In addition to multiple TSSs within a promoter region, an important development in the exploration of gene regulation at the core promoter level is the discovery of the widespread usage of alternative promoters of protein coding genes. Genome-wide promoter analyses in vertebrates suggest that 20–52% of the genes carry alternative promoters where each promoter is clearly separated by a genomic space of several hundred base pairs (7Kimura K. Wakamatsu A. Suzuki Y. Ota T. Nishikawa T. Yamashita R. Yamamoto J. Sekine M. Tsuritani K. Wakaguri H. Ishii S. Sugiyama T. Saito K. Isono Y. Irie R. Kushida N. Yoneyama T. Otsuka R. Kanda K. Yokoi T. Kondo H. Wagatsuma M. Murakawa K. Ishida S. Ishibashi T. Takahashi-Fujii A. Tanase T. Nagai K. Kikuchi H. Nakai K. Isogai T. Sugano S. Genome Res. 2006; 16: 55-65Crossref PubMed Scopus (400) Google Scholar, 39Kim T.H. Barrera L.O. Zheng M. Qu C. Singer M.A. Richmond T.A. Wu Y. Green R.D. Ren B. Nature. 2005; 436: 876-880Crossref PubMed Scopus (762) Google Scholar). The prevalence of alternative promoters is evident; however, their biological significance is largely unexplored with the exception of a small number of genes where alternative promoters were shown to receive differential signaling information to generate different protein isoforms (e.g. p53, p63, and p73 family (58Muller M. Schleithoff E.S. Stremmel W. Melino G. Krammer P.H. Schilling T. Drug Resist. Updat. 2006; 9: 288-306Crossref PubMed Scopus (122) Google Scholar)). Alternative promoter usage may provide a mechanism for tissue and developmental stage-specific activity of genes. For example, TATA box-containing promoters were shown to be utilized after embryonic development, and TATA-less promoters of the same genes were shown to be active during early embryo development (59Davis Jr., W. Schultz R.M. Dev. Biol. 2000; 218: 275-283Crossref PubMed Scopus (56) Google Scholar, 60Duan Z.J. Fang X. Rohde A. Han H. Stamatoyannopoulos G. Li Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5509-5514Crossref PubMed Scopus (32) Google Scholar, 61Majumder S. DePamphilis M.L. Mol. Cell. Biol. 1994; 14: 4258-4268Crossref PubMed Scopus (46) Google Scholar). A recent global analysis of mammalian promoters concluded that alternative promoters mostly play a role at highly regulated developmental genes and that single promoter genes are more likely involved in general cellular processes active in a broad range of tissues (62Baek D. Davis C. Ewing B. Gordon D. Green P. Genome Res. 2007; 17: 145-155Crossref PubMed Scopus (73) Google Scholar). Thus, PICs assembled at the alternative promoters may vary in a developmental stage or signaling pathway-specific manner. The above results also raise the issue of specificity of communication between core promoters and cis-regulatory elements through protein-protein interactions. The apparent specificity in promoter-enhancer interactions was demonstrated elegantly in Drosophila by the description of the differential ability of a DPE-containing promoter to interact with a set of enhancers (63Butler J.E. Kadonaga J.T. Genes Dev. 2001; 15: 2515-2519Crossref PubMed Scopus (205) Google Scholar) as compared with a TATA-containing promoter. The specificity of promoter-enhancer interactions enables promoters to distinguish between enhancers and the information displayed by them (64Kulkarni M.M. Arnosti D.N. Development. 2003; 130: 6569-6575Crossref PubMed Scopus (71) Google Scholar). This may be necessary when overlapping gene loci with multiple promoters are targeted by a single enhancer or when several enhancers may target a single promoter through long distance interactions. The molecular basis for the specificity of core promoter-enhancer interactions is unknown but likely involves different protein-protein interactions directed by variant complexes forming on different core promoters. The biological significance of the diversity of the core promoters and the binding of the corresponding complexes to them remains the ultimate question to be answered (Fig. 1). Analysis of molecular interactions between activator proteins and core promoter context-dependent PICs may argue for a model in which the core promoter is involved in processing information received on the cis-regulatory level of a gene. For example, different TATA box-containing promoters respond differentially to activators, suggesting that they do not bind the same TFIID and/or PIC complexes (65Simon M.C. Fisch T.M. Benecke B.J. Nevins J.R. Heintz N. Cell. 1988; 52: 723-729Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 66Wefald F.C. Devlin B.H. Williams R.S. Nature. 1990; 344: 260-262Crossref PubMed Scopus (149) Google Scholar). c-Fos and Elf-1 differentially activate transcription from TATA- and INR-containing core promoters (67Metz R. Kouzarides T. Bravo R. EMBO J. 1994; 13: 3832-3842Crossref PubMed Scopus (40) Google Scholar, 68Ernst P. Hahm K. Trinh L. Davis J.N. Roussel M.F. Turck C.W. Smale S.T. Mol. Cell. Biol. 1996; 16: 6121-6131Crossref PubMed Scopus (52) Google Scholar), whereas optimal induction by papilloma virus E2 and the artificial Gal-VP16 activator requires both TATA and INR elements (69Ham J. Steger G. Yaniv M. EMBO J. 1994; 13: 147-157Crossref PubMed Scopus (58) Google Scholar). As a parallel example, p53 specifically represses the TATA box but not TATA-less INR-containing promoters (70Mack D.H. Vartikar J. Pipas J.M. Laimins L.A. Nature. 1993; 363: 281-283Crossref PubMed Scopus (309) Google Scholar). On the other hand, DPE-containing IRF-1 and TAF7 promoters, but not the DCE-containing cyclin D promoter, require protein kinase CK2 and positive coactivator 4 (PC4) for transcription (71Lewis B.A. Sims 3rd, R.J. Lane W.S. Reinberg D. Mol. Cell. 2005; 18: 471-481Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Additional cellular signaling events that target TFIID components may provide the means for regulatory inputs to be transduced to the core promoter. An example of such a signaling mechanism is the observation that apoptotic stimuli can lead to alternative splicing of TAF6, resulting in a TFIID-type complex containing TAF6δ but lacking TAF9 (72Bell B. Scheer E. Tora L. Mol. Cell. 2001; 8: 591-600Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This alternative TFIID complex results in altered transcription programs. The fact that an inducible TAF isoform is capable of selectively altering TFIID composition and consequently gene expression programs underlines the biological significance of TFIID variants regulating gene activities at the core promoter level. In conclusion, a wealth of data gathered in recent years has established the diversity of core promoters and a variety of protein complexes that may bind to them. This diversity argues for a combined model for transcription regulation in multicellular organisms; differential input signals target both types of cis-regulatory elements such as enhancers as well as the core promoter. The resulting transcriptional output is dependent on the integration of both sets of signals. The input signals targeting either the enhancers or the core promoters may be qualitatively different and represent distinct aspects of cellular stimuli, thus necessitating the plasticity of core promoters and that of the complexes forming on them. Thus, the diversity of core promoters will facilitate additional plasticity in transcriptional outputs. Yet the regulatory logic that underlies the interactions between core promoters and the binding factor complexes remains mostly unexplored. It represents the next challenge in transcription biology, which will, without doubt, bring us many more surprises. This challenge is expected to be addressed by developing novel detection techniques to measure promoter occupancy by transcription factors in real time and detect core promoter activity and TSSs at a single cell level as well as to develop new bioinformatics tools to decipher the code embedded in core promoter sequences.