Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer‐prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non‐transcribing sequences (‘genome‐overall’ repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP‐C defect in a cell‐free repair system containing UV‐damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N‐terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin‐like N‐terminus. The nature of the XP‐C defect implies that the complex exerts a unique function in the genome‐overall repair pathway which is important for prevention of skin cancer.

The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.

Background: Smad proteins are novel transcriptional regulators mediating the signalling of the transforming growth factor-␤ (TGF-␤) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, was shown to inhibit the signalling of TGF-␤. These findings raise the possibility that p300 may be involved in TGF-␤ signalling.

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Smad proteins are intracellular signalling mediators of transforming growth factor-β β β β (TGF-β β β β ) superfamily. In the nucleus, activated Smad complexes regulate transcriptional responses of the target genes in cooperation with transcriptional coactivators and corepressors. To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a coactivator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a coactivator for receptorregulated Smads (R-Smads) for TGF-β β β β signalling pathways. GCN5 functions like PCAF, in that it binds to TGF-β β β β -specific R-Smads, and enhances transcriptional activity induced by TGF-β β β β . In addition, GCN5, but not PCAF, interacts with R-Smads for bone morphogenetic protein (BMP) signalling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo . Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-β β β β . In conclusion we identified GCN5 as a Smad-binding transcriptional coactivator which positively regulates both TGF-β β β β and BMP signalling pathways.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as and at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express and, respectively. Fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in regulation is poorly understood. Here, we identified as an upstream repressor of was markedly upregulated in knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Furthermore, KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of KO blastocysts. These data demonstrate that suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HATcontaining coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.Transcription initiation of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, which comprises the TATA-binding protein (TBP) 1 and a series of TBP-associated factors (TAFs) (1-3). However, we have previously shown that initiation of polymerase II transcription can occur in a TFIID-independent manner in the presence of a novel human (h) multiprotein complex, termed TFTC for TBP-free TAF complex (4). TFTC is able to direct preinitiation complex assembly from different TATA box-containing and TATA-less promoters in vitro on naked DNA templates. TFTC contains no TBP but is composed of several TAFs and other proteins that have been shown to mediate transcriptional activation or are important in correct initiation site selection (4, 5). The three-dimensional structure of TFTC resembles a macromolecular clamp that contains five globular domains organized around a solvent-accessible groove of a size suitable to bind DNA (6). A large number of recent studies have provided a direct molecular link between histone acetylation and transcriptional activation (reviewed in Refs. 7 and 8). In these reports, it has been shown that several previously identified co-activators of transcription possess intrinsic HAT activity. Among these coactivators are yeast Gcn5 (9), human GCN5 (10), PCAF (11), TATA box-binding protein-associated factor 250 (TAF1; formerly TAF II 250) (12), p300/CBP (13), ACTR (14), and steroid receptor co-activator 1 (SRC-1) (15). Many of these chromatinmodifying activities have been found within large multiprotein complexes that also contain several components with homology or identity to known transc...