PC3, functionally equivalent to DNA topoisomerase I (Topo I; Kretzschmar et al., 1993; Merino et al, 1993), consists of 765 amino acids with a molecular size around 91 kDa (Champoux, 2001). This protein is well known for its role in relaxing DNA by transiently introducing nicks, allowing strand passage and religation (Wang, 1996). Topo I has an unstructured N-terminal region, which contains nuclear targeting signals and available surfaces for interaction with various transcription factors, such as p53 (Gobert et al., 1999), that is dispensable for relaxing DNA in vitro (Stewart et al., 1996). The enzymatic activity of Topo I is regulated posttranslationally by casein kinase II- and protein kinase C-mediated phosphorylation, leading to increased relaxation activity and in responding to mitogenic stimuli (Wang, 1996). When Topo I encounters damaged DNA, it is stalled and must be removed from the lesions in order to prevent formation of irreversible single or double strand breaks that impair genomic integrity. In this regard, PARP-1 can target Topo I for poly(ADP-ribosyl)ation and facilitate Topo I to remove itself from cleaved DNA and close the resulting gap (Malanga and Althaus, 2004).

Aside from its role in modulating DNA topology,

Topo I can function as a transcriptional coactivator by contacting directly with activators, such as Gal4-AH (Kretzschmar et al, 1993) and p53 (Gobert et al., 1999), and with components of the general transcription machinery, such as TBP (Merino et al., 1993) in order to stimulate transcription. The coactivator function of Topo I is in part due to its ability to enhance TFIID-TFIIA-promoter complex formation (Shykind et al., 1997). In the absence of activator, Topo I functions as a repressor in inhibiting basal transcription. Although the transcription activity of Topo I is separated from its DNA relaxation activity (Merino et al., 1993; Kretzschmar et al., 1993), the precise mechanism by which Topo I regulates transcription remains to be elucidated. This is an area currently underexplored.

PC4 is a protein consisting of 127 amino acids with an N-terminal regulatory domain spanning amino acids 1 to 62 and a C-terminal single-stranded DNA-binding and dimerization domain located between amino acids 63 and 127 (Ge and Roeder, 1994a; Kretzschmar et al., 1994; Brandsen et al., 1997). The N-terminal region, important for PC4 interaction with distinct activation domains (Ge and Roeder, 1994a) and binding to double-stranded DNA in a non-sequence-specific manner (Kaiser et al., 1995), contains two serine-enriched acidic (SEAC) domains located respectively at amino acids 9 to 22 and 50 to 61, separated by a lysine-rich region lying between amino acids 23 to 41 (Kaiser et al., 1995). Both SEAC domains are susceptible to phosphorylation by several protein kinases, in particular casein kinase II (CKII), leading to inactivation of the coactivator function of PC4 likely by preventing PC4 interaction with TBP-bound TFIIA on the promoter region (Ge et al, 1994; Kretzschmar et al, 1994) and with transcriptional activators, such as the HIV Tat protein (Holloway et al, 2000). The lysine-rich region, proposed to bind nonspecific double-stranded DNA (Kaiser et al, 1995), may serve as acetylation sites for p300-enhanced PC4 binding to double-stranded DNA, consistent with the observation that CKlI-mediated phosphorylation on the SEAC domains inhibits p300-dependent acetylation on PC4 (Kumar et al, 2001). The C-terminal region, in which the structure has been resolved by X-ray crystallography at 1.74 A resolution (Brandsen et al, 1997), forming a dimer with each monomer composed of four antiparallel (3-strands followed by a kinked a-helix, is able to bind with high affinity (Kd ~ 0.07 nM) two single strands of DNA running in opposite directions, as found in internally melted DNA duplexes. In the full-length protein, this single-stranded DNA-binding region is normally masked by intramolecular interaction with the N-terminal region and only becomes exposed after conformational changes induced, for instance, by CKII-mediated phosphorylation of the SEAC domains (Kaiser et al, 1995), thereby leading to inactivation of the coactivator function of PC4 and further inhibition of transcription mediated by the single-stranded DNA-binding activity of PC4 (Werten et al., 1998).

The inhibitory activity of PC4 allows it to function as a repressor in suppressing basal transcription when activators are absent (Malik et al, 1998; Werten et al, 1998; Wu and Chiang, 1998). This inhibition usually occurs prior to PIC assembly in the absence of TAFs (Wu and Chiang, 1998) and can be alleviated by adding increasing amounts of TFIID, TFIIH and pol II holoenzyme in the transcription reaction, correlating with the ability of these multiprotein complexes to phosphorylate PC4 (Kershnar et al., 1998; Malik et al., 1998). However, since inactivation of the ATP-binding site of ERCC3 helicase, but not ERCC2 helicase or CDK7 kinase, impairs the ability of recombinant TFIIH to overcome PC4-mediated repression (Fukuda et al., 2003), it remains unclear the precise mechanism used by components of the general transcription machinery to antagonize PC4 repressing activity.

The coactivator function of PC4 was evidenced by its ability to substitute for a crude USA fraction in mediating activator-dependent transcription in a cellfree transcription system reconstituted with recombinant general transcription factors (TFIIB, TBP, TFIIE, and TFIIF) and epitope-tagged multiprotein complexes (TFIID, TFIIH, and pol II; Wu et al, 1998). In this system, where TAFs and Mediator are not essential for activator-dependent transcription, PC4 is the only general cofactor indispensable for transcriptional activation mediated by Gal4-VP16 (Wu et al, 1998), and human papillomavirus E2 (Wu and Chiang, 2001; Hou et al., 2002; Wu et al., 2003). Not surprisingly, PC4 can interact with both transcriptional activators, such as Gal4-VP16 (Ge and Roeder, 1994a), BRCA1 (Haile and Parvin, 1999), AP2 (Kannan and Tainsky, 1999), HIV Tat (Holloway et al, 2000), human papillomavirus E2 (Wu and Chiang, 2001), and p53 (Banerjee et al, 2004), and components of the general transcription machinery, such as TFIIA (Ge and Roeder, 1994a), TFIIH (Fukuda et al., 2004), and pol II (Malik et al, 1998), thereby serving as a bridging molecule to facilitate activator-dependent transcription likely through enhancement of PIC assembly on the promoter region. In addition, PC4 may promote sequence-specific DNA-binding activity of some activators, such as p53 (Banjeree et al, 2004), stimulate promoter escape in a TFIIA- and TAF-dependent manner (Fukuda et al., 2004), or enhance pol II elongation by modulating TFIIH kinase and Fcpl phosphatase activity on CTD phosphorylation (Calvo and Manley, 2005). Clearly, PC4 can work in conjunction with other general cofactors, such as TAFs (Wu and Chiang, 1998; Wu et al., 1998) and Mediator (Fondell et al., 1999; Malik et al., 2000; Wu et al., 2003), to synergistically mediate activator-dependent transcription. Whether phosphorylation of PC4, which accounts for 95% of total PC4 in the cell (Ge et al., 1994), by TFIID, TFIIH, pol II holoenzyme (Kershnar et al., 1998) and Mediator (Gu et al., 1999), plays a role in different steps of the transcriptional process remains to be further defined.

Besides being a transcriptional coactivator, PC4 has also been implicated in other cellular processes, such as DNA repair and DNA replication. In the aspect of DNA repair, PC4 can prevent mutagenesis arising from oxidative DNA damage caused by the interaction of reactive oxygen species (ROS) with DNA, depending upon its single-stranded DNA-binding activity (Wang et al., 2004). The involvement of PC4 in DNA replication appears to be more complicated, as PC4 can interact with replication protein A (RPA) on single-stranded DNA and facilitate T-antigen-mediated unwinding of DNA containing SV40 origin of replication, while it also inhibits RNA primer synthesis and DNA polymerase 8-catalyzed DNA chain elongation (Pan et al., 1996). The biological significance of these in vitro reactions performed in the presence of PC4 warrants further investigations.

NCI, also known as HMG1 or HMGB1 with a molecular size around 25 kDa, is a member of the highly conserved chromatin-associated proteins that bend DNA and bind preferentially to distorted DNA structures (Bustin, 2001; Thomas and Travers, 2001). HMGB1 is structurally divided into three domains: two homologous DNA-binding HMG-box domains A and B each containing approximately 80 amino acids, and a C-terminal tail containing a stretch of 30 acidic residues. Boxes A and B each forms an "L-shaped" structure with three a-helices constituting a minor-groove DNA-binding domain that preferentially binds distorted DNA, such as four-way junctions, cisplatin-modified DNA and bulged DNA, and induces DNA bending without sequence specificity (Thomas, 2001; Thomas and Travers, 2001). Both domains A and B contain an additional basic extension of amino acid residues that enhance the DNA-binding affinity of the HMG box.

The C-terminal acidic tail modulates the DNA-binding activity of HMGB1 and seems to be inhibitory toward HMG box binding to DNA.

The transcriptional role of HMGB1 is similar to other USA-derived cofactors in that it normally functions as a repressor in the absence of an activator, but acts as a coactivator in activator-dependent transcription. The repressing activity of HMGB1 appears to work by promoting the formation of a stable HMGB1-TBP-promoter complex that prevents TFIIB entry (Ge and Roeder, 1994b), as the presence of HMGB1 increases the affinity of TBP for the TATA box by 20-fold (Das and Scovell, 2001). The interaction domains were mapped to the HMG box A of HMGB1 (Sutrias-Grau et al., 1999) and the glutamine-rich region of TBP (Das and Scovell, 2001). TFIIA, as an antirepressor (see Chapter by Hou and Chiang), can displace HMGB1 from the ternary complex and overcome HMGB1-mediated inhibition of PIC formation, thereby restoring transcription activity (Ge and Roeder, 1994b). The coactivator function of HMGB1 is attributed to its direct interaction with transcriptional activators, such as p53, steroid hormone receptors, Oct-1, HOX and Rel proteins (Thomas and Travers, 2001; Agresti and Bianchi, 2003), and with components of the general transcription machinery, including TBP (Ge and Roeder, 1994b) and TAF10 (Verrier et al, 1997). Undoubtedly, the architectural role of HMGB1 in bending DNA will further contribute to its coactivator function typically by enhancing sequence-specific recognition by these DNA-binding proteins.


Transcription in higher eukaryotes is a complex process involving a diverse set of protein factors acting through specific sequence elements surrounding the promoter region. The fact that the promoter itself can be recognized by TBP, TRFs, TFIID, and other TAF-containing complexes already lends flexibility for interaction with distinct transcriptional regulators as well as general cofactors which typically possess dual activities in repressing basal transcription and enhancing activator-dependent transcription in response to environmental changes. While tissue-specific TAFs and TRFs play an important role in regulating transcription during development, it still remains a mystery what roles positive cofactors and Mediator play in embryonic development. Without doubt, the presence of TAF variants and multiple pathways for regulating PIC assembly provide an additional way to fine-tune the transcription events occurring on individual genes. For

TAF-independent pathways, the alternative usage of other general cofactors, such as Mediator and USA-derived cofactors, help transduce regulatory signals between activators and the general transcription machinery. Considering that many of the general cofactors discussed in this chapter also exhibit multiple enzymatic activities and can function synergistically to activate transcription, it would be exciting to explore how these general cofactors communicate with one another in order to suppress their inhibitory activity during the activation process. The issue of functional redundancy and transcriptional synergy among these general cofactors warrant intensive studies for the years to come.


We are grateful for Drs. Parminder Kaur, Samuel Y. Hou and Shwu-Yuan Wu for their comments on this chapter. The research conducted in Dr. Chiang's laboratory is currently sponsored by grants CA103867 and GM59643 from the National Institutes of Health in the United States.

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