Search

Rhabdoid tumors (RT) are embryonal neoplasms occurring most frequently in the central nervous system where they are termed atypical teratoid rhabdoid tumor (ATRT). A common hallmark of RT is homozygous loss of the BAF complex subunit SMARCB1. RT patients have a poor prognosis with an overall survival time of 17 months and >60% of patients suffer from relapses. The lack of an optimal treatment strategy could be attributed to the heterogeneity within and between different subgroups of ATRT. Despite the recent advancements in characterizing RT at a molecular level, the cellular origin of RT remains elusive. Thus, this study focused on the identification of the cellular origin of MYC-RT and underlying epigenetic deregulations which account for the cellular heterogeneity in these tumors. We showed that Smarcb1 abrogation in Sox2-positive progenitor cells at E6.5 give rise to RT of the MYC and SHH subgroup in genetically engineered mouse models (GEMM). To uncover distinct cells of origin (COO) for the SHH and MYC subgroups, unbiased computational approaches were used to compare single-cell transcriptomes of GEMMs with single-cell reference maps of murine early embryogenesis. While SHH tumors arise from mid/hindbrain progenitor cells, primordial germ cells (PGCs) emerge as COO of both intracranial and extracranial MYC tumors. PGCs as COO of MYC-RT were validated in vivo by using PGC-specific Smarcb1 knockout mouse model. We further characterized a deregulated transcriptome in MYC-RT compared to PGCs, which is sustained by a subset of epigenetically driven tumor cells. Deregulated expression of genes driving methylation/demethylation processes in MYC tumors and regression of these tumors upon treatment with decitabine in vitro and in vivo, indicates that DNA methylation plays a key role in cellular transformation and development of MYC-RT.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1’s ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1’s catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

Charcot-Marie-Tooth disease (CMT) represents a heterogeneous group of hereditary peripheral neuropathies. We previously reported a CMT locus on chromosome 19q13.3 segregating with the disease in a large Costa Rican family with axonal neuropathy and autosomal recessive pattern of inheritance (CMT2B2). We proposed a homozygous missense variant in the Mediator complex 25 (MED25) gene as causative of the disease. Nevertheless, the fact that no other CMT individuals with MED25 variants were reported to date led us to reevaluate the original family. Using exome sequencing, we now identified a homozygous nonsense variant (p.Gln517ter) in the last exon of an adjacent gene, the polynucleotide kinase 3'-phosphatase (PNKP) gene. It encodes a DNA repair protein recently associated with recessive ataxia with oculomotor apraxia type 4 (AOA4) and microcephaly, seizures, and developmental delay (MCSZ). Subsequently, five unrelated Costa Rican CMT2 subjects initially identified as being heterozygous for the same MED25 variant were found to be also compound heterozygote for PNKP. All were heterozygous for the same variant found homozygous in the large family and a second one previously associated with ataxia (p.Thr408del). Detailed clinical reassessment of the initial family and the new individuals revealed in all an adult-onset slowly progressive CMT2 associated with signs of cerebellar dysfunction such as slurred speech and oculomotor involvement, but neither microcephaly, seizures, nor developmental delay. We propose that PKNP variants are the major causative variant for the CMT2 phenotype in these individuals and that the milder clinical manifestation is due to an allelic effect.

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.

Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling., The molecular mechanisms of acinar-to-ductal metaplasia (ADM) in the course of pancreatitis and cancer development are unclear. Here, the authors show that loss of κB-Ras and consequent Ral activation promotes tumour initiation and progression through persistent ADM and enhanced cell proliferation

Single-pair Forster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Forster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.

PURPOSE: Atypical teratoid rhabdoid tumors (ATRT), characterized by SMARCB1 loss, have been classified by DNA methylation and gene expression profiling into three distinct molecular subgroups (ATRT-SHH, -MYC, -TYR). We hypothesize that ATRT from distinct subgroups have a different cell of origin. METHODS: Multiple precursor cell specific, constitutive and inducible Smarcb1 knockout mouse strains were established by using a Cre-loxP system. Murine tumors were analyzed by histology and gene expression profiling. Unsupervised hierarchical clustering and differential expression analyses were performed. RESULTS: Rhabdoid tumor (RT) development was detected only when Smarcb1 abrogation occurred in a very restricted time frame during embryonic development and only under the control of an ubiquitous (Rosa26) or Sox2 promoter. Unsupervised hierarchical clustering of Affymetrix gene expression profiles of these murine and of published human ATRT classified tumors of the Rosa26cre(ERT2)::Smarcb1(Fl/Fl) model either as ATRT-MYC or as ATRT-SHH. In contrast, ATRT of Sox2cre(ERT2)::Smarcb1(Fl/Fl) mice were assigned to the ATRT-MYC subgroup only. Mouse strains in which the Smarcb1 knockout was driven by Nestin-, hGFAP-, Math1-, Olig1- Cre recombinase presented other phenotypes but not RT. CONCLUSION: Subgroup-specific RT genesis depends on the cell of origin. Here we identify early Sox2-positive progenitors as precursor cells of ATRT-MYC subgroup. We further demonstrate that Smarcb1 abrogation during a specific, short time period and in a specific targeted cell population is crucial for induction of ATRT-MYC.

Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6-Rb-, the WNT- and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients.

Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator. Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding.

In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.

The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and γH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair.

Mediator (TRAP/ARC/PC2) is a large (22-28 subunit) protein complex that binds RNA polymerase II and controls transcription from class II genes. The evolutionarily conserved core of Mediator is found in all eukaryotes. It binds RNA polymerase II and is probably critical for basal transcription but it also mediates activation and repression of transcription. During evolution the complex has acquired additional species-specific subunits. These serve as an interface for regulatory factors and support specific signalling pathways. Recent mechanistic studies are consistent with the hypothesis that Mediator marks genes for binding by RNA polymerase II whereupon it subsequently activates the preinitiation complex. It is further likely that Mediator coordinates the recruitment of chromatin-modifying cofactor activities.

Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF(II)170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2alpha (DRAP1) and NC2beta (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2alpha subunit interacts with BTAF1. In contrast, the NC2beta subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2alpha or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2alpha. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2alpha in TBP regulation and provide a framework to understand transcription functions of NC2alpha and NC2beta in vivo.

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 HAT-containing 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.

Background: The Herpes Simplex Virion Protein 16 (VP16) contains a strong activation domain which can be subdivided into two regions, H1 and H2, both of which independently activate transcription in vivo. Several components of the basal transcription machinery have been shown to interact with the activation domain of VP16, mostly through the H1 region. Results: We show that the H2 region binds directly to histone acetyltransferase, CBP (CREB (cAMP Responsive Element Binding Protein) Binding Protein) both in vivo and in vitro. The sites of interaction with the H2 region were mapped to both the amino- and carboxy-terminal segments of CBP. A mutation in the H2 region disrupts the interaction with CBP and abolishes the ability of VP16 to mediate in vitro transactivation from chromatin templates in an acetyl-CoA dependent manner. In contrast, human Mediator, another co-activator complex, binds specifically to both the H1 and H2 regions. Conclusion: The H1 and H2 regions of the VP16 activation domain activate transcription via distinct pathways. The H2 requires CBP for activation, whereas the H1 may function through Mediator and general transcription factors.

Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.

Human Mediator complexes have been described as important bridging factors that enhance the effect of activators in purified systems and in chromatin. Here we report a novel basal function of a human Mediator complex. A monoclonal antibody was generated that depleted the majority of Mediator components from crude cell extracts. The removal of human Mediator abolished transcription by RNA polymerase II. This was observed on all genes tested, on TATA-containing and TATA-less promoters, both in the presence and absence of activators. To identify the relevant complex a combined biochemical and immunopurification protocol was applied. Two variants termed Mediator and basal Mediator were functionally and structurally distinguished. Basal Mediator function relies on additional constraints, which is reflected in the observation that it is essential in crude but not in purified systems. We conclude that basal Mediator is a novel general transcription factor of RNA polymerase II.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

The Vβ 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vβ promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer–promoter interplay is mediated through the conserved regions of the Vβ promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter–enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.

RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 . Differential phosphorylation of serine-2 and serine-5 at the 5′ and 3′ regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.

PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF. In addition, CBP associates with two other TSs, P/CAF and SRC1. We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation. We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. We also find that the HAT domains of CBP and P/CAF share sequence similarity. Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.

Activation of the immediate-early gene c-fos through MAP kinases is a hallmark of growth factor signaling. In this issue of Molecular Cell, Roy and colleagues (Hakre et al., 2006) show that TFII-I isoforms play differential roles in this process.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro . The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD + , probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.

Using crude in vitro systems, we have previously shown that RXR/RAR heterodimers are able to activate transcription from the RARβ2 promoter in a retinoid-dependent manner. Here we demonstrate that cofactors distinct from general transcription factors or receptors are required to mediate retinoic acid-dependent transcription in vitro.

Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6-Rb-, the WNT- and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients.

The human general co-factors werediscovered during biochemical fractionation of mammalian nuclear extracts in functional in vitro assays. They appear to act in concert with other coactivators that bind tightly to the TATA-binding protein and RNA polymerase II. Several co-factors have been shown to interact with general transcription factors, leading either to activation or repression of transcription. At least one subgroup of co-factors that enhance the effects of activators on transcription are DNA-binding proteins located in the chromatin. In fact, one co-factor, the repressor NC2, is structurally related to histones. The understanding of the molecular interplay of such components of the initiation complex in the chromatin—including general co-factors, other co-factors, general factors and activators—will be a major challenge in the future.

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.

The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes. Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system. Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor. A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors. The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments. PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation. However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID. Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

The Mediator complex serves as an adaptor for regulatory factors, recruits and controls RNA polymerase II promotes preinitiation complex formation and functions post initiation. There is increasing evidence for further coordinating roles of the Mediator complex in chromatin. Here we summarize interactions with regulatory, general and accessory factors that function in transcription and chromatin.

The transcriptional activation of eukaryotic class II genes by sequence-specific regulatory proteins requires cofactors in addition to the general transcription factors. One cofactor (termed PC3) was purified from HeLa cells and identified by sequence analysis and functional assays as human DNA topoisomerase I (EC5.99.1.2). Under identical conditions PC3 mediates both a net activation of transcription by the acidic activator GAL4-AH and repression of basal transcription, thereby leading to a large induction of transcription by the activator. PC3-mediated activation of transcription is dependent on the presence of both the GAL4-AH activation domain and the TATA-binding protein (TBP)-associated-factors (TAFs) in natural transcription factor TFIID, while repression of basal transcription is observed with either TFIID or the derived TBP alone. These results suggest novel functions, apparently through distinct mechanisms, for human DNA topoisomerase I in the regulation of transcription initiation by RNA polymerase II.

NF-kappa B, purified from HeLa cell cytosol, and a recombinant p50 subunit of NF-kappa B alone (expressed in and purified from bacteria) both stimulated transcription from the HIV-1 promoter in vitro (at least up to 15-fold). A deletion analysis of the p50 subunit revealed that transcriptional activation was mediated by the conserved c-rel-related domain. I kappa B-beta (or a related protein), which binds to the p65 but not the p50 subunit of NF-kappa B, inhibited stimulation by natural NF-kappa B but not by recombinant p50. Experiments employing a purified transcription system revealed that efficient induction of transcription by both natural NF-kappa B or recombinant p50 required a cofactor fraction in addition to the general initiation factors. Combined with DNA-binding experiments, these studies suggest a role of p50 homodimers in transcriptional activation of certain promoters, with a possible preference for those carrying symmetric NF-kappa B recognition sites, and a potential role of I kappa B-beta in direct transcriptional regulation within the nucleus.

The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. All mendelian patterns of inheritance have been described. We identified a homozygous p.A335V mutation in the MED25 gene in an extended Costa Rican family with autosomal recessively inherited Charcot-Marie-Tooth neuropathy linked to the CMT2B2 locus in chromosome 19q13.3. MED25, also known as ARC92 and ACID1, is a subunit of the human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator. MED25 was identified by virtue of functional association with the activator domains of multiple cellular and viral transcriptional activators. Its exact physiological function in transcriptional regulation remains obscure. The CMT2B2-associated missense amino acid substitution p. A335V is located in a proline-rich region with high affinity for SH3 domains of the Abelson type. The mutation causes a decrease in binding specificity leading to the recognition of a broader range of SH3 domain proteins. Furthermore, Med25 is coordinately expressed with Pmp22 gene dosage and expression in transgenic mice and rats. These results suggest a potential role of this protein in the molecular etiology of CMT2B2 and suggest a potential, more general role of MED25 in gene dosage sensitive peripheral neuropathy pathogenesis. © Springer-Verlag 20 La enfermedad de Charcot-Marie-Tooth (CMT) es un trastorno clínica y genéticamente heterogéneo. Se han descrito todos los patrones mendelianos de herencia. Identificamos una mutación homocigota p.A335V en el gen MED25 en una familia extensa costarricense con neuropatía de Charcot-Marie-Tooth de herencia autosómica recesiva ligada al locus CMT2B2 en el cromosoma 19q13.3. MED25, también conocido como ARC92 y ACID1, es una subunidad del cofactor humano reclutado por activador (ARC), una familia de grandes complejos coactivadores transcripcionales relacionados con el mediador de levadura. MED25 se identificó en virtud de la asociación funcional con los dominios activadores de múltiples activadores transcripcionales celulares y virales. Su función fisiológica exacta en la regulación transcripcional sigue siendo oscura. La sustitución de aminoácidos sin sentido asociada a CMT2B2 p. A335V se encuentra en una región rica en prolina con alta afinidad por los dominios SH3 de tipo Abelson. La mutación provoca una disminución en la especificidad de unión que conduce al reconocimiento de una gama más amplia de proteínas del dominio SH3. Además, Med25 se expresa coordinadamente con la dosificación y expresión del gen Pmp22 en ratones y ratas transgénicas. Estos resultados sugieren un papel potencial de esta proteína en la etiología molecular de CMT2B2 y sugieren un papel potencial más general de MED25 en la patogénesis de la neuropatía periférica sensible a la dosis génica. © Springer-Verlag 20 Universidad Nacional, Costa Rica. Escuela de Ciencias Biológicas

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2alpha and NC2beta, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2alpha and NC2beta depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin alpha/beta heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2beta. In contrast, NC2alpha does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2alpha masks the NES in NC2beta, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin alpha/beta-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits.

A novel activity (USA) stimulated activator-dependent transcription in a reconstituted system in conjunction with natural TFIID, resulting in 10- to 50-fold levels of induction by regulatory factors. USA mediated a modest induction by USF in conjunction with either recombinant human TFIID, intact yeast TFIID, or the evolutionarily conserved C-terminal portion of yeast TFIID. Upon further purification, USA was resolved into two components that had opposite effects on core promoter activity and that in combination potentiated activator function. Gel mobility shift experiments indicated physical interactions between the inhibitory activity and TFIID, suggesting that the additional components (cofactors) associate with the preinitiation complex both to reduce promoter activity in the absence and to increase promoter activity in the presence of transcriptional activators.

Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Forster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box–binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Forster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP–DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

VP16 has been widely used to unravel the mechanisms underlying gene transcription. Much of the previous work has been conducted in reconstituted in vitro systems. Here we study the formation of transcription complexes at stable reporters under the control of an inducible Tet-VP16 activator in living cells. In this simplified model for gene activation VP16 recruits the general factors and the cofactors Mediator, GCN5, CBP, and PC4, within minutes to the promoter region. Activation is accompanied by only minor changes in histone acetylation and H3K4 methylation but induces a marked promoter-specific increase in H3K79 methylation. Mediated through contacts with VP16 several subunits of the cleavage and polyadenylation factor (CPSF/CstF) are concentrated at the promoter region. We provide in vitro and in vivo evidence that VP16 activates transcription through a specific MED25-associated Mediator, which is deficient in CDK8.

The identification of functional proteomes is a major challenge in proteomic research. Here we describe a method for the detection and isolation of nuclear (localization sequence containing) proteins using a derepressed import receptor (DIRE) as a synthetic antibody. We demonstrate that the DIRE method specifically detects nuclear localization sequence containing proteins. Application to activation of primary T-lymphocytes exemplifies the potential use of DIRE for comparative proteomics and for diagnostics.

Positive cofactor 4 (PC4) is a coactivator that strongly augments transcription by various activators, presumably by facilitating the assembly of the preinitiation complex (PIC). However, our previous observation of stimulation of promoter escape in GAL4-VP16-dependent transcription in the presence of PC4 suggested a possible role for PC4 in this step. Here, we performed quantitative analyses of the stimulatory effects of PC4 on initiation, promoter escape, and elongation in GAL4-VP16-dependent transcription and found that PC4 possesses the ability to stimulate promoter escape in response to GAL4-VP16 in addition to its previously demonstrated effect on PIC assembly. This stimulatory effect of PC4 on promoter escape required TFIIA and the TATA box binding protein-associated factor subunits of TFIID. Furthermore, PC4 displayed physical interactions with both TFIIH and GAL4-VP16 through its coactivator domain, and these interactions were regulated distinctly by PC4 phosphorylation. Finally, GAL4-VP16 and PC4 stimulated both initiation and promoter escape to similar extents on the promoters with three and five GAL4 sites; however, they stimulated promoter escape preferentially on the promoter with a single GAL4 site. These results provide insight into the mechanism by which PC4 permits multiply bound GAL4-VP16 to attain synergy to achieve robust transcriptional activation.