Smads Transcriptional Activation

A: Interaction with p300/CBP, P/CAF and GCN5 Histon Acetyltransferases

Al: Overview

GAL4 fusion studies provided the first evidence that Smads are TGF-P family-regulated transcription factors (Liu et al., 1996). The C terminal domain together with a segment of the linker region from a receptor regulated Smad or Smad4 can activate transcription when fused to the GAL4 DNA binding domain (Liu et al., 1996). Full length Smads have very little activity in the GAL4 fusion assay, but their transcription activities are greatly increased by treatment with the corresponding agonists, BMP or TGF-P (Liu et al., 1996; Liu et al., 1997). Subsequent studies have shown that transcriptional activation by Smad3 and Smad2 occurs in part by their ability to recruit general transcriptional coactivator p300/CBP (Feng et al., 1998; Janknecht et al., 1998; Nishihara et al., 1998; Pouponnot et al., 1998; Shen et al., 1998; Topper et al., 1998). p300/CBP have intrinsic histone acetyltransferase activity (HAT) that facilitates transcription by altering nucleosome structure through histone acetylation and thereby remodeling the chromatin template (Roth et al., 2001). This interaction occurs through the MH2 and a segment of the linker region of Smad3 or Smad2 and the C-terminal domain of p300/CBP. In addition, P/CAF, another HAT-containing transcriptional co-activator, has been shown to associate with Smad3 and Smad2 upon TGF-P receptor activation and to enhance Smad3 and Smad2 transcriptional activity (Itoh et al., 2000). GCN5, another' co-activator containing the HAT, enhances transcriptional activation of a variety of genes. GCN5 is structurally related to P/CAF, and therefore is also designated P/CAF-B. Unlike P/CAF, which binds to only TGF-P activated Smad3 and Smad2, GCN5 binds to TGF-P activated Smad3 and Smad2 as well as BMP activated Smadl and Smad5, and enhancing both TGF-P- and BMP-mediated transcriptional responses (Kahata et al., 2004).

The transcriptional activity of receptor-regulated Smads is greatly increased in the presence of Smad4. This was shown by studies using Smad4 deficient colon cancer cells (Liu et al., 1997; Zhou et al., 1998a). In such cells, Smad2 and FAST-1 together have minimal ability to stimulate a typical activin/TGF-P reporter gene. In addition, GAL4-Smadl, GAL4-Smad2 have little transcriptional activities in the Smad4 deficient cells compared to the same cells with transfected Smad4 (Liu et al., 1997). Thus, Smad4 plays an important role in Smad-mediated transcriptional activation for many TGF-P-responsive genes. This is partly due to the presence of a unique Smad activation domain (SAD), a 48 amino acid proline-rich regulatory element in the linker region of Smad4 (de Caestecker et al., 2000). The crystal structure of a Smad4 fragment containing the SAD and the MH2 domain has been solved (Qin et al., 1999). The MH2 domain of Smad4 is highly homologous with that of Smad2 and Smad3 (50% identity), except that Smad4 has a unique insert of -35 amino acids which interacts with the C-terminal tail to form a TOWER-like structural extension from the core. The crystal structure suggests that SAD provides transcriptional capability by reinforcing the structural core and coordinating with the TOWER to present the proline rich surface and a glutamine-rich surface in the TOWER for interaction with transcription partners (Qin et al., 1999). It has been shown that the SAD domain physically interacts with the N-terminal domain of p300/CBP (de Caestecker et al., 2000).

The Smad3 linker region also contains an activation domain (Wang et al., 2005). When the linker region is fused to the GAL4 DNA binding domain, it has constitutive transcriptional activity, comparable to that of SAD in Smad4. In the context of full length Smad3, deletion of the linker region renders Smad3 unable to support TGF-P transcriptional activation, although it can still be phosphorylated by the TGF-P

receptor at the C-tail and has a markedly increased capacity to form a heteromeric complex with Smad4 (Wang et al., 2005). Further experiments using the GAL4 system indicate that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-p (Wang et al., 2005).

The transcriptional activity of the Smad3 linker region can be blocked by wild type El a, but not by an El a mutant that cannot bind to p300. Overexpression of p300 can partially but not completely rescue Ela-meidated repression. Immunoprecipitation analyses indicate that the linker region is capable of recruiting the p300 coactivator (Wang et al., 2005). Thus, these observations suggest that in addition to p300, other regulatory components may participate with the Smad3 linker region for transcriptional activation.

The Smad3 and Smad2 linker region contains demonstrated as well as suspected phosphorylation sites for multiple kinases, such as the cyclin-dependent kinase (CDK), ERK mitogen activated protein (MAP) kinase, c-Jun N-terminal kinase, p38 MAP kinase, and Ca2+-calmodulin-dependent kinase II (CamKII) (Fig. 11.3) (Kretzschmar et al. 1999; Wicks et al. 2000; Derynck and Zhang 2003; Matsuura et al. 2004; Mori et al. 2004). For example, it has been shown that CDK phosphoryaltion of Smad3 inhibits its transcriptional activity and antiproliferative function (Matsuura et al., 2004). It will be very interesting to elucidate the exact mechanism by which CDK phosphorylation of Smad3 inhibits its transcriptional activity. Phosphorylation of the linker region by the various kinases may differentially influence Smad3 transcriptional activity in a context-dependent manner. The C-terminal domain of Smad3 is regulated by TGF-P receptor phosphorylation. The C-terminal domain of Smad3 is also a protein-protein interaction domain, responsible for homo-trimerization, hetero-trimerization with Smad4, and also interaction with a number of DNA binding proteins (Fig. 11.3). Under different conditions, the linker region and the C-terminal domain may functionally interact differentially and therefore display varying transcriptional activities, leading to distinct biological responses in a cell-context dependent manner.

A2: Recruitment of p300 by Smad4 Coactivator MSG1

MSG1 is a potent transcriptional activator (Shioda et al., 1997). It was originally identified as a candidate pigmentation-related gene in melanocytes (Shioda et al., 1996). Its possible involvement in differentiation and development was suggested based on its restricted and developmentally regulated expression (Shioda et al., 1996). MSG1 lacks an intrinsic DNA binding activity

(Shioda et al., 1997). In a yeast two-hybrid screen using MSG1 as bait, it was found that MSG1 interacted with Smad4. Subsequent studies indicate that MSG1 associates with p300, recruits p300 to Smad4, and enhance Smad-mediated transcriptional activiation in the presence of TGF-p. Thus, MSG1 functions as a co-activator of Smad4 (Shioda et al., 1998; Yahata et al., 2000).

A3: Requirement of p300/CBP for the Effect of Smad4 Coactivator SMIF

SMIF is another coactivor of Smad4. SMIF is a ubiquitously expressed protein that contains an EVH1/ WH1 (enabled VASP homology 1 /WASP homology 1) domain (Bai et al., 2002). The EVH1/WH1 domain is a protein interaction module for binding to proline-rich regions (Callebaut, 2002). SMIF interacts with Smad4 but not with other Smads. The interaction occurs through the EVH1/WH1 domain of SMIF and the SAD domain of Smad4. TGF-P induces the formation of a SMIF-Smad4 complex, which translocates to the nucleus. Whereas SMIF does not directly bind to p300/CBP, SMIF possesses strong TGF-P-inducible Smad4 and p300-dependent transcriptional activity (Bai et al., 2002).

A4: Recruitment of p300 by PIAS3

The family of protein inhibitor of activated STAT (PIAS) represents a group of proteins that play an important role in regulating a variety of signaling pathways and transcriptional responses (Schmidt and Muller, 2003). The acronym PIAS stems from the initial observation that members of the PIAS family inhibit the DNA binding activity of activated STATs (see Chapter 10). Subsequent studies indicate that PIAS proteins interact with a variety of transcription factors and regulate diverse cellular processes. The mammalian PIAS family includes five members: PIAS1, PIAS3, PIASxa, PIASxP, and PIASy. The PIAS proteins contain a SAP domain in the N-terminal domain. The term SAP refers to three of the founding members of SAP-containing proteins: scaffold attachment factor (SAF), acinus, and PIAS. A common feature of SAP-containing proteins is their ability to bind to chromatin. PIAS proteins contain a RING domain. All PIAS proteins possess SUMO E3 ligase activity, and the RING domain is essential for this activity (Schmidt and Muller, 2003).

PIAS3 can activate transcription of a Smad-dependent reporter gene, and TGF-P treatment markedly increases the transcriptional response (Long et al., 2004b). PIAS3 interacts with Smad proteins. The strongest interaction is with Smad3 and it occurred through the C-terminal domain of Smad3. The interaction can be detected at endogenous protein levels. PIAS3 can also interact with p300/CBP. Interestingly, the RING domain of PIAS3, which is essential for the SUMO E3 ligase activity of PIAS3, is also necessary for interaction with p300/CBP and for activation of Smad-dependent transcription. Importantly, PIAS3, Smad3 and p300 can form a ternary complex. This complex formation is significantly increased in the presence of TGF-P, which help explain that PIAS3 activation of Smad-dependent transcription is significantly increased in the presence of TGF-P (Long et al., 2004b), p300 does not interact with all PIAS proteins. For example, PIASy, which inhibits Smad3 transcriptional activity and other transcriptional responses, cannot bind to p300/CBP (Long et al., 2004b). Since PIAS3 has SUMO E3 ligase activity, whether sumoylation of certain target is necessary for the stimulatory effect of PIAS3 remains to be elucidated. It is possible that PIAS3 regulates transcription through both sumoylation-dependent and sumoylation-independent mechanisms.

B: Interaction with ARC105

Activator-recruited co-factor (ARC) and the related or identical metazoan Mediator play an important role in transcriptional regulation (Taatjes et al., 2004). These complexes contain multiple polypeptides, and can associate with many transcription factors and activate transcription in vitro. ARC 105, a component of the ARC/mediator complex, is essential for signaling by TGF-P, activin and nodal, a member of the TGF-P superfamily (Kato et al., 2002). ARC 105 interacts with the C-terminal domains of Smad2, Smad3, and Smad4, and the interaction is induced or increased in the presence of ligand. Moreover, ARC 105 is recruited to the responsive promoters as shown by chromatin immunoprecipitation assay. Overexpression of ARC 105 enhances TGF-p/activin/nodal-inducible transcription. Conversely, knockdown ARC 105 by siRNA inhibited the transcription. Thus, ARC 105 links the ARC/Mediator complex with Smad-mediated transcriptional control (Kato et al., 2002).

Smads Transcriptional Repression

A: Multiple Repression Mechanisms

Smads can inhibit transcription through recruitment of corepressors, which is described in detail below. In addition, Smads, in particular Smad3, can repress transcription through other modes. For instances, TGF-P inhibits osteoblast, skeletal muscle, and adipocyte differentiation. Smad3 mediates the TGF-P inhibitory effects. Smad3 inhibits osteoblast differentiation by physically interacts with CBFA1, which is a key transcription factor for osteoblast differentiation, and prevents CBFA1 from activating osteocalcin and the CBFA1 promoter itself (Alliston et al, 2001). This inhibition is both cell type- and promoter-dependent. It occurs in mesenchymal cells but not in epithelial cells (Alliston et al., 2001). For myogenesis, Smad3 inhibits myogenic differentiation through interaction with MyoD and MEF2 (Liu et al., 2001a; Liu et al., 2004). Smad3 physically interacts with the HLH domain of MyoD, thus inhibiting MyoD heterodimerization with an E-box binding protein (such as E12 and E47) and subsequent binding of the heterodimer to the E-box (Liu et al., 2001a). Smad3 interacts with MEF2 and inhibits its transcriptional activity (Liu et al., 2004). Similarly, Smad3 inhibits adipocyte differentiation through interaction with C/EBP and inhibits its transactivation function (Choy and Derynck, 2003). Thus, Smad3 is a key regulator for TGF-P-mediated differentiation control.

Smad3 also plays an important role to inhibit the expression of genes that are necessary for the TGF-P cytostatic effects. c-Myc downregulation is essential for the TGF-P-mediated growth inhibitory responses (Pietenpol et al., 1990; Chen et al., 2001; Shi and Massague, 2003). TGF-P treatment induces a preassembled complex containing Smad3, E2F4/5 and DPI, and the Rb-related factor pi07 to move into the nucleus, associate with Smad4, bind to a compository Smad-E2F site for repression (Chen et al., 2002a; Yagi et al., 2002; Frederick et al., 2004). Inhibition of Idl expression is also a general feature of the TGF-P-induced growth inhibition. TGF-P-activated Smad3 directly induces the expression of ATF3, a transriptional repressor. ATF3, Smad3 and Smad4 then form a complex that directly mediates Idl repression (Kang et al., 2003).

Smad3 can also compete for DNA binding. Smad3 can inhibit the expression of the goosecoid gene (Labbe et al., 1998). This is thought to occur through Smad3 competing with Smad4 binding to a GC-rich sequence. While binding of Smad4 in complex with Smad2 and FAST-2 to this GC-rich sequence activates transcription, binding of Smad3 to this sequence may alter the conformation of the DNA binding complex, thus leading to inhibition of transcription (Labbe et al., 1998).

Smad3 has been reported to interact with HDAC through its MH1 domain (Liberati et al, 2001). Although it is not clear whether the interaction is direct, one possibility is that direct interaction of Smad3 with HDAC may contribute to some of the repression examples described above. Future studies are necessary to explore this interesting possibility.

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