COMMENTARY 2895

TAFII250: a toolbox

David A. Wassarman1 and Frank Sauer2,* 1National Institutes of Health, National Institute of Child Health and Human Development, Cell Biology and Metabolism Branch, Building 18T, Room 101, Bethesda, MD 20892, USA 2Zentrum fur Molekulare Biologie der Universtät Heidelberg (ZMBH), Im Neunheimer Feld 282, Heidelberg 69120, Germany *Author for correspondence (e-mail: [email protected])

Journal of Cell Science 114, 2895-2902 (2001) © The Company of Biologists Ltd

Summary Activation of RNA-polymerase-II-dependent transcription particular promoters, TAFII250 regulates binding of TBP involves conversion of signals provided by gene-specific to DNA, TAFII250 binds core initiator elements, proteins into the synthesis of messenger RNA. TAFII250 binds acetylated lysine residues in core , This conversion requires dynamic structural changes in and TAFII250 possesses protein kinase, - and assembly of general transcription factors activating/conjugating and acetylase activities that modify (GTFs) and RNA polymerase II at core promoter sequence histones and GTFs. We speculate that these activities elements surrounding the transcription start site of achieve two goals - (1) they aid in positioning and genes. One hallmark of transcriptional activation is the stabilizing TFIID at particular promoters, and (2) they interaction of DNA-bound activators with coactivators alter chromatin structure at the promoter to allow such as the TATA-box binding protein (TBP)-associated assembly of GTFs - and we propose a model for how factors (TAFIIs) within the GTF TFIID. TAFII250 possesses TAFII250 converts activation signals into active a variety of activities that are likely to contribute to the transcription. initial steps of RNA polymerase II transcription. TAFII250 is a scaffold for assembly of other TAFIIs and TBP into Key words: TAFII250, TFIID, acetyltransferase, Kinase, TFIID, TAFII250 binds activators to recruit TFIID to Ubiquitination, Chromatin, Transcription

Introduction TFIIA, TFIIB, TFIIE, TFIIF and TFIIH (Orphanides et al., The expression of thousands of genes in eukaryotic cells is 1996; Hampsey, 1998). The nucleating function of TFIID is regulated at the level of transcription. This regulation involves thought to comprise several distinct activities: (1) activator- the orchestrated interplay of chromatin-packaged genes with dependent recognition of core promoter DNA sequence multiprotein complexes that control chromatin dynamics, elements; (2) the generation of a chromatin environment that transcription initiation and transcription elongation. The is favorable to PIC assembly and transcription initiation; and inability of general transcription factors (GTFs) to access (3) structural modification of GTFs to facilitate PIC assembly chromatin, DNA wrapped around core histones H2A, H2B, H3 and transcription initiation. TAFII250 contributes to each of and H4 in repeating subunits called , implies that these TFIID activities. Recent excitement about TAFII250 a requisite step in transcription initiation is the alteration of stems from its involvement in regulating the association of chromatin structure (Wolffe, 1998). Two general classes of TFIID with the core promoter and its intrinsic complex/enzyme have been implicated in chromatin-altering enzymatic activities that post-translationally modify GTFs and events: ATP-dependent -remodeling complexes histones. Here, we review recent studies of TAFII250 and (e.g. SWI/SNF, RCS, ACS, CHRAC, NURF, and Mi-2/ suggest a model for how TAFII250 activities contribute to gene- NURD) and histone-modifying enzymes (e.g. histone specific transcriptional activation in the context of chromatin. acetyltransferases (HATs) and histone deacetylases (HDACs); Strahl and Allis, 2000; Vignali et al., 2000). Although the order of action of chromatin-remodeling/modifying complexes TAFII250 is a broadly acting regulator of during transcriptional activation may be gene specific, their transcription interdependent activities appear to precede and facilitate TAFII250 is an essential protein in yeast, fruit flies, hamster binding of TFIID, which, along with TFIIB, are the only cell lines and probably all eukaryotic organisms (Talavera components of the preinitiation complex (PIC) that can bind and Basilico, 1977; Nishimoto et al., 1982; Poon et al., 1995; specifically to core promoter DNA (Lagrange et al., 1998; Wassarman et al., 2000). Homologs are designated TAFII130 Cosma et al., 1999; Krebs et al., 1999; Agalioti et al., 2000). and TAFII145 in yeast, TAFII230 and TAFII250 in Drosophila, TAFII250 is one of 10-12 TATA-binding protein (TBP)- and TAFII250 and gene 1 (CCG1) in mammals associated factors (TAFIIs) that are complexed with TBP in (Aoyagi and Wassarman, 2000). Here, we use TAFII145 and TFIID (Albright and Tjian, 2000; Aoyagi and Wassarman, TAFII250 to refer to the yeast and metazoan proteins, 2000). Binding of TFIID to a core promoter surrounding the respectively. Inactivation of TAFII145 in yeast or TAFII250 in transcription start site of a gene nucleates assembly of the PIC, hamster cell lines results in arrest in G1 phase of the cell cycle, which contains RNA polymerase II (RNA pol II) and GTFs and null mutations in Drosophila TAFII250 result in lethality 2896 JOURNAL OF CELL SCIENCE 114 (16) late in embryogenesis or early in larval development (Talavera TAFIIs are components of other transcriptional regulatory and Basilico, 1977; Nishimoto et al., 1982; Walker et al., 1996; complexes, which makes it difficult to resolve the contribution Wassarman et al., 2000). Furthermore, homozygous mutant of a TAFII to TFIID activity (Bell and Tora, 1999). However, clones of TAFII250 cannot be generated in Drosophila, which even in the case of TAFII250, which appears to be unique to suggests that it has a role in or cell survival TFIID, this is a difficult question to address, since there are (Wassarman et al., 2000). factors that can bypass a requirement for some TAFII250 The essential nature of TAFII250 can be attributed to its functions. broad requirement during RNA-pol-II-dependent transcription. A growing list of factors can rescue cell cycle arrest of, or In Drosophila, TAFII250 is expressed in all nuclei and binds to block apoptosis of, ts13 or tsBN462 hamster cells, which a large number of euchromatic sites on polytene contain an identical glycine-to-aspartate missense mutation in (Rabenstein et al., 1999; Wassarman et al., 2000). In yeast, TAFII250 (Hayashida et al., 1994). This list includes viral genome-wide microarray analysis indicates that expression of proteins (simian virus 40 (SV40) large T antigen (Damania and 14-27% of RNA-pol-II-transcibed genes is downregulated Alwine, 1996), hepatitis B virus pX (Haviv et al., 1998), human >twofold upon TAFII145 inactivation (Lee et al., 2000). cytomegalovirus (HCMV) immediate-early (IE) proteins Finally, in ts13 hamster cells that harbor a temperature- IEP86 and IEP72 (Lukak and Alwine, 1999), and human sensitive allele of TAFII250, similar analysis indicates that the papilloma virus (HPV) 16 E7 (Sekiguchi et al., 1999)) and transcription of 18% of genes transcribed by RNA pol II is cellular proteins (E2F-1 (Sekiguchi et al., 1999), CIITA (Raval affected >twofold at the non-permissive temperature (O’Brien et al., 2001) and D-type cyclins (Sekiguchi et al., 1999)). Two and Tjian, 2000). As anticipated, given the phenotype of distinct mechanisms are involved: overexpression of viral TAFII250 mutants, many genes showing TAFII250-dependent proteins or CTIIA rescues cells by activating TAFII250- expression encode proteins that are involved in cell cycle dependent promoters in the absence of functional TAFII250, regulation and growth control (Wang and Tjian, 1994; Walker whereas overexpression of E2F-1 and D-type cyclins rescues et al., 1997; Lee et al., 2000; O’Brien and Tjian, 2000). cells by providing the downstream targets of TAFII250 activity. TAFII250 could play a more extensive role in transcription SV40 large T antigen, HCMV IE and CIITA proteins bind to than these studies predict, since temperature-sensitive TAFIIs in vitro and in vivo, and mutations that affect TAFII mutations might not inactivate all TAFII250 functions (Dikstein interactions are unable to rescue the ts13 or tsBN462 et al., 1996; Dunphy et al., 2000; Lee et al., 2000; Tsukihashi transcriptional defects (Damania and Alwine, 1996; Mahanta et al., 2000). There is considerable disagreement among et al., 1997; Lukak and Alwine, 1999). Furthermore, rescue by researchers about the extent to which TAFIIs in general are CTIIA depends on its acetyltransferase activity (Raval et al., required for RNA pol II transcription (Moqtaderi et al., 1996; 2001). Since TAFII250 acetyltransferase activity is impaired in Walker et al., 1996; Komarnitsky et al., 1999; Lee et al., 2000). tsBN462 cells (discussed below), this suggests that CIITA Part of this controversy results from the fact that a subset of substitutes for TAFII250 by performing two of its functions:

Substrates E F A H4 F H3 H2B H1 F

NTK HAT E1/E2 Bromo Bromo CTK Interaction partners

Acetyl TBP TAT RAP74 H4 Acetyl RB RB Interacting region known Interacting region unknown

30α 150 60 110 80 30β

TAFIIs

Fig. 1. A schematic diagram of metazoan TAFII250. Locations of enzymatic domains (N-terminal kinase domain (NTK), C-terminal kinase domain (CTK), histone acetyltransferase domain (HAT), and ubiquitin-activating/conjugating domain (E1/E2)) and (Bromo) are indicated within the linear TAFII250 protein. Substrates for the enzymatic activities are indicated above the TAFII250 protein, and interacting proteins are indicated below the protein. Substrates and interacting partners are abbreviated as follows: TFIIA (A), TFIIE (E), TFIIF (F), TATA- binding protein (TBP), (RB), HIV Tat (TAT) and TFIIFα (RAP74). TAF II250: a transcription toolbox 2897 association with TFIID and of proteins. Thus, other TAFII80, TAFII110 and TAFII150 (Chen et al., 1994; Fig. 1). viral and cellular proteins that rescue ts13 or tsBN462 Immobilized TAFII250 can serve as a scaffold for assembly of cell defects and associate with TFIID might also be TFIID subcomplexes and holo-TFIID from recombinant acetyltransferases or affiliate with an acetyltransferase. subunits, which suggests that assembly and integrity of TFIID are dependent on TAFII250. Accordingly, inactivation of TAFII250 in yeast leads to degradation of other TFIID subunits TAFII250 is not the only scaffold for TFIID in vivo (Walker et al., 1996). However, inactivation of all TAFIIs tested The TFIID complex is held together by TAFII-TAFII and TAFII- to date results in degradation of other TAFIIs, which suggests TBP interactions. In particular, Drosophila TAFII250 engages that every TAFII is necessary for the integrity of TFIID in vivo in strong interactions with TBP, TAFII30α, TAFII30β, TAFII60, (Walker et al., 1996; Michel et al., 1998; Moqtaderi et al., 1998).

1) Recruitment

TAFII250-activator interaction TFIID250 150 110 A TBP A Core promoter 250 Gene x Nucleosome{ 2) Nucleosome and core promoter recognition and binding Binding to nucleosome and core promoter

Acetyl 250 250 Acetyl A Double 150 TBP Bromo H4 Core promoter Gene x Fig. 2. A four-step model for how TAFII250 3) Chromatin dynamics contributes to transcriptional activation. Histone modification In each step, the panel on the left depicts the general event and the panel on the H2B H3 right depicts the specific A TFIID250 250 Acetylation contribution of TAFII250 to HAT H4 Acetyl the event. Activators are Acetyl H4 H3 denoted by a red circle E1/E2 labeled A; TFIIA is H2A H2B Gene x Ubiquitination H1 denoted by a green oval H1 labeled A; TFIIE is denoted by a green circle labeled E; ubiquitin TFIIF is denoted by a blue 4) Initiation and elongation of transcription circle labeled F; histones are labeled H1, H2A, H2B, Modification of GTFs H3 or H4; and TFIID is denoted by a collection of objects labeled 250, 150 250 E F Acetylation and TBP. The balance of HAT the general transcription A F TFIID250 machinery, which is not E NTK GTM specifically identified, is A A F indicated by a collection of objects labeled GTM. Core promoter Gene x 2898 JOURNAL OF CELL SCIENCE 114 (16)

TAFII250 function is regulated by activators TAFII250 binds to core promoter initiator sequences How do TAFIIs increase the rate of transcription initiation? The DNA-crosslinking experiments have revealed that TAFII250 initial finding that TFIID, but not TBP, can mediate activator- intimately contacts the core promoter initiator element both in directed transcription in a reconstituted RNA pol II system TATA-box-containing and TATA-less core promoters (Purnell indicated that one function of TAFIIs is to respond to - et al., 1994; Sypes and Gilmour, 1994; Verrijzer et al., 1995; bound activators (Pugh and Tjian, 1990; Dynlacht et al., 1991). Wu et al., 2001; Fig. 2). The initiator (Inr) is a conserved In the case of TAFII250, activator interactions increase sequence element that encompasses the start site of promoter occupancy of TFIID (i.e. recruitment) and modulate transcription and can direct accurate transcription initiation in TAFII250 regulatory and enzymatic activities (i.e. regulation). the absence of a TATA box (Smale, 1997). A role for TAFIIs Physical interactions between TAFII250 and activators are a in recognizing the Inr was suggested by the finding that TFIID critical component of these mechanisms. TAFII250 binds containing a TBP subunit that cannot bind to DNA cannot activators (e.g. HIV Tat (Weissman et al., 1998), adenovirus function on TATA-only promoters but can support transcription E1A (Geisberg et al., 1995), Herpes simplex virus type 1 ICP4 from TATA-less, Inr-containing promoters (Martinez et al., (Carozza and DeLuca, 1996) and JUN (Lively et al., 2001)), 1994). Mutational analysis suggests that a region C-terminal to and other transcriptional regulators (retinoblastoma tumor the HAT domain of yeast TAFII145 is required for promoter suppressor protein RB (Seigert and Robbins, 1999), the binding in vivo (Mencia and Struhl, 2001). In vitro, MDM2 proto-oncogene (Leveillard and Wasylyk, 1997) and recombinant TAFII250-TAFII150, TBP-TAFII250 and TBP- cyclin D (Seigert et al., 2000)) (Fig. 1). TAFII250-TAFII150 complexes efficiently bind Inr-containing Distinct lines of evidence point to a mechanism by which promoters (Chen et al., 1994; Verrijzer et al., 1995). Moreover, activators bind to TAFII250 to tether TFIID to particular a TAFII250-TAFII150 complex can support Inr-mediated promoters (Fig. 2). (1) The region of ICP4 required for transcription and specifically binds sequences that match the transcriptional activation is also required for TAFII250 Inr consensus sequence from a pool of random sequence association (Carrozza and DeLuca, 1996). (2) Gene-specific oligonucleotides (Verrijzer et al., 1995; Chalkley and Verrijzer, promoter occupancy of TAFII145 in yeast is elevated in 1999). These results imply that TAFII250, together with response to an activation signal and is reduced after removal TAFII150, mediates binding of TFIID to the Inr and that TBP of enhancer sites (Kuras et al., 2000; Li et al., 2000). (3) is dispensable for this activity. This hypothesis is further Enhancers can confer TAFII250 dependence on promoters that supported by the identification of TBP-free TFIID complexes are normally TAFII250 independent (Wang et al., 1997). Once from mammalian cells that support transcription from TATA- recruited to a promoter, TAFII250 could participate in PIC less and TATA-containing promoters that contain Inr elements assembly by binding to the RAP74 subunit (also designated (Wieczorek et al., 1998). TFIIFα) of TFIIF, as suggested by the finding that TAFII250 Analysis of core promoters of genes whose expression is mutants that fail to interact with RAP74 are unable to rescue affected by TAFII250 mutations does not clarify the role of the the ts13 cell cycle defect (Ruppert and Tjian, 1995). Tethering TATA element in specifying TAFII250 dependence. Some of TFIID to particular genes through activator-TAFII250 TAFII250-dependent promoters contain a nonconsensus TATA binding is advantageous because TFIID is limiting in cells element, whereas others contain a consensus TATA element (Walker et al., 1996). The importance of TAFII250 interactions (Moqtaderi et al., 1996; Shen and Green, 1997). Insertion of a with both activators and GTFs argues that TAFII250 functions canonical TATA element into a TATA-less promoter can bypass as a classically defined that bridges activators to a dependence on TAFII145, but mutation of a weak TATA PIC assembly. element to the consensus sequence does not change the There is also ample support for a mechanism in which requirement for TAFII145 (Shen and Green, 1997; Tsukihashi activators modify TAFII250 activities to enhance formation or et al., 2000). Thus, there appears to be a sophisticated code stability of the PIC on promoter DNA (Fig. 2). Competitive composed of contributions from TATA and Inr core promoter interplay between activators and TAFII250 could control elements that specifies TAFII250 dependence. binding of TFIID to core promoters. The N-terminus of TAFII250 binds to the DNA-binding surface of TBP and inhibits TBP-DNA interactions (Kotani et al., 1998). However, TAFII250 post-translationally modifies histones and acidic activators, JUN and TFIIA compete with TAFII250 for GTFs binding to TBP, thus altering the DNA-binding properties of Functional characterization of TFIID and searches for enzymes TFIID (Kokubo et al., 1998; Ozer et al., 1998; Kotani et al., that post-translationally modify histones have demonstrated 2000; Lively et al., 2001). Moreover, some inhibit that TAFII250 possesses protein kinase, HAT, and ubiquitin- TAFII250 enzymatic activities (discussed below) that may activating and -conjugating enzymatic activities (Figs 1, 2). promote PIC assembly by modifying the structure of GTFs and Interestingly, TAFII250 does not have significant sequence chromatin. Binding of HIV Tat, an activator and of similarity to other members of these enzyme families, which viral gene transcription, to TAFII250 inhibits TAFII250 HAT raises the possibility that other TAFIIs, and other proteins in activity (Weissman et al., 1998). Similarly, binding of RB to general, possess enzymatic domains that remain unidentified TAFII250 inhibits TAFII250 N-terminal kinase activity (Siegert because they do not conform to defined primary sequence and Robbins, 1999; Solow et al., 2001). In contrast, E1A and motifs. cyclin D1 suppress the TAFII250-kinase-inhibitory effect of RB (Siegert et al., 2000). Thus, regulation of TAFII250 TAF II250 is a bipartite protein kinase activities by interactions with activators and GTFs is integral TAFII250 contains two independent serine/threonine protein to the process of transcriptional activation. kinase domains: one at the N-terminus (NTK) and the other at TAF II250: a transcription toolbox 2899 the C-terminus (CTK; Dikstein et al., 1996; O’Brien and Tjian, (1) transcriptional activation is temperature sensitive from non- 1998; Fig. 1). Kinase activity has been demonstrated in vitro chromatin templates in ts13 cells extracts; and (2) inhibition of for the Drosophila, human and yeast proteins. Yeast is unusual TAFII250 acetyltransferase activity by HIV Tat represses in that the kinase domains reside in two separate proteins. transcription from non-chromatin templates in HeLa cell TAFII145 and factor 1 (Bdf1) contain the NTK extracts (Wang and Tjian, 1994; Weissman et al., 1998). Thus, and CTK, respectively (Mantangkasonbut et al., 2000). the TAFII250 acetyltransferase activity is necessary in vitro The TAFII250 NTK and CTK domains autophosphorylate, even in the absence of histones. Furthermore, TFIIEβ and and the NTK domain transphosphorylates RAP74 and the large TFIIF might not be relevant substrates in vitro, because no subunit of TFIIA (TFIIA-L) in vitro (Dikstein et al., 1996; proteins are acetylated in reconstituted transcription reactions Solow et al., 2001). RAP74 and TFIIA are reasonable containing purified GTFs (including TFIID, TFIIEβ and candidates as substrates for TAFII250 kinase activity TFIIF), RNA pol II and an activator (Galasinski et al., 2000). in vivo: endogenous RAP74 is hyperphosphorylated; However, it remains an open question whether GTFs and dephosphorylation of RAP74 reduces its ability to support histones are acetylated by TAFII250 in vivo. Acetylation of transcription elongation in vitro; and phosphorylation of TFIIA histones by TAFII250 in vivo might produce a localized change stimulates TFIIA-TBP-TATA-element complex formation in in chromatin structure to enhance accessibility and binding of vitro (Kitajima et al., 1994; Solow et al., 2001; Fig. 2). TFIID and other PIC components (Fig. 2). Autophosphorylation by TAFII250 might also play a regulatory In addition to acetylating proteins, TAFII250 binds role in transcription, as has been shown for TFIIF (Rossignol to multiply acetylated histones through two tandem et al., 1999). Despite the lack of definitive substrates, it is clear bromodomains located in the C-terminal region of the protein that kinase activity is required in vivo, because a recombinant (Jacobson et al., 2000; Fig. 1). The bromodomain is an ~120- TAFII250 protein that lacks the NTK domain cannot rescue residue motif present in a variety of proteins that associate with ts13 phenotypes (O’Brien and Tjian, 1998; O’Brien and Tjian, chromatin (Jeanmougin et al., 1997). The TAFII250 double 2000). Moreover, deletion of the CTK in Drosophila results bromodomain (DBD) binds most tightly to in lethality (V. Jo, J. G. Shanklin, E. M. Schlag and D.A.W., acetylated at lys5 and lys12 (Jacobson et al., 2000). This is unpublished). consistent with the crystal structure of the DBD, which shows that the binding pockets for acetyllysine span a distance TAF II250 is a histone acetyltransferase and binds to equivalent to seven residues. Therefore, the TAFII250 acetylated histones bromodomains may target TFIID to chromatin-packaged Mizzen et al. using an in-gel activity assay, showed that promoters (Fig. 2). TAFII250 has HAT activity (Mizzen et al., 1996). The HAT domain maps to the central, most conserved portion of TAF II250 is a ubiquitin-activating/conjugating enzyme metazoan TAFII250 and the C-terminal portion of yeast Most recently, TAFII250 has been demonstrated to mediate TAFII145 (Fig. 1). Many studies have established a correlation monoubiquitination of , a linker histone that binds between acetylation of highly conserved lysine residues in the to DNA between adjacent nucleosomes (Crane-Robinson, N-terminal tails of histones and transcriptional activation 1999; Pham and Sauer, 2000). In vitro, monoubiquitination (Strahl and Allis, 2000). One model to explain how histone requires the sequential activity of ubiquitin-activating (E1) and acetylation affects gene expression proposes that the extent of ubiquitin-conjugating (E2) enzymes (Ciechanover et al., 2000). chromatin condensation is directly related to the level of TAFII250 contains both of these activities: it becomes histone acetylation. Accordingly, hyperacetylation reduces the covalently linked, via a thioester bond, to ubiquitin in an ATP- affinity of histone tails for DNA, resulting in less compact dependent manner (E1 activity), and it transfers activated chromatin and increased accessibility of transcription factors ubiquitin to histone H1 via an isopeptide bond (E2 activity; to DNA. Pham and Sauer, 2000). The E1 and E2 activities of Drosophila In vitro, Drosophila TAFII250 acetylates free histones and and human TAFII250 reside in the central region of the protein nucleosomal histones weakly, relative to yeast HAT1 and (Fig. 1). Most intriguingly, this region is absent from yeast human P/CAF (Mizzen et al., 1996; Wassarman et al., 2000). TAFII145 and Bdf1, and, whereas a histone-H1-like protein Lys14 of is the preferred site of acetylation, (HHO1) has been reported in yeast, it is not known whether it although other lysine residues in H3 and H4 are good is functionally homologous to metazoan H1 proteins (Ushinsky substrates for TAFII250 (Mizzen et al., 1996). TAFII250 also et al., 1997). acetylates TFIIEβ and TFIIF in vitro (Imhof et al., 1997) Mutations in Drosophila TAFII250 that impair histone H1 Evidence for the in vivo importance of TAFII250 HAT activity ubiquitination activity in vitro and in vivo were isolated in a comes from ts13 and tsBN462 cells. The ability of mutant genetic screen for genes that modulate the ability of the Ras TAFII250 to acetylate histones in vitro is temperature sensitive, GTPase to specify cell fates (Karim et al., 1996; Wassarman et suggesting that the enzymatic activity is compromised in ts13 al., 2000). Gene targets for TAFII250 in Ras signaling pathways cells at the nonpermissive temperature and that cell cycle and have not been identified, but TAFII250 mutations do affect transcriptional defects observed at the nonpermissive transcription in the Drosophila embryo, reducing the temperature result from reduced HAT activity (Dunphy et al., expression of Dorsal and Caudal target genes (Pham and Sauer, 2000). 2000; A.-D. Pham and F.S., unpublished). It is unclear how Although histones and GTFs are substrates for TAFII250 monoubiquitination of histone H1 by TAFII250 mediates acetyltransferase activity in vitro, it is unclear whether they are transcription. One attractive model, based on the ability of H1 transcriptionally relevant substrates. Two lines of evidence to facilitate the folding of nucleosomal arrays into higher-order indicate that histones might not be relevant substrates in vitro: structures, is that ubiquitination changes the chromatin-binding 2900 JOURNAL OF CELL SCIENCE 114 (16) properties of H1 and thereby destabilizes both local and higher- In summary, this simplistic model describes how individual order chromatin structures and alters core histone-DNA tools contained within a TAFII250 ‘toolbox’ contribute to the interactions (Crane-Robinson, 1999; Mizzen and Allis, 2000; conversion of activation signals received by TFIID into the Fig. 2). Indeed, binding of histone H1 to chromatin is highly enzymatic synthesis of messenger RNA. We anticipate that dynamic in living cells, and modulation of H1 binding activity the model will undergo numerous refinements as in vivo is thought to be an important step in regulating access of the substrates for TAFII250 activities are defined, the molecular transcriptional machinery to DNA (Lever et al., 2000; Misteli consequences of these modifications are determined, and high- et al., 2000). resolution structures of TAFII250, TFIID and the PIC are While TAFII250 does not require an E3 ubiquitin-ligase to solved (Andel et al., 1999). monoubiquitinate histone H1 in vitro, it is possible that it utilizes one to ubiquitinate other proteins in vivo. An excellent This work was supported by the Intramural Program in the National candidate for this enzyme is the MDM2 ubiquitin ligase, whose Institute of Child Health and Human Development (D.A.W.) and the DFG (F.S.). We thank R. Kamakaka, L. 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