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Single-pair Förster 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 Förster 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.

Schizophrenia or schizoaffective disorders are quite common features in patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS) as a result of hemizygosity of chromosome 22q11.2. We evaluated the PCQAP gene, which maps within the DGS/VCFS interval, as a potential candidate for schizophrenia susceptibility. PCQAP encodes for a subunit of the large multiprotein complex PC2, which exhibits a coactivator function in RNA polymerase II mediated transcription. Using a case–control study, we searched association between schizophrenia and the intragenic coding trinucleotide polymorphism. The distribution of the CAG repeat alleles was significantly different between patients and controls with the Mann-Whitney test (z = −2.5694, P = 0.0051; schizophrenics: n = 378, W = 161,002.5, Mean rank = 425.9325; controls: n = 444, W = 177,250.5, Mean rank = 399.2128). This result may indicate a possible involvement of the multiprotein complex PC2 in schizophrenia susceptibility. © 2003 Wiley-Liss, Inc.

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 vitroon 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.

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 PcqapcDNA. 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 PCQAPin DGS/VCFS, its developmental expression pattern was analyzed. In situhybridization of mouse embryos at different developmental stages revealed that Pcqapis 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 PCQAPgene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAPin 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-mycis 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-mycchromatin 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-mycactivity, 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-mycpromoters. 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-mycpromoter in vivoif the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-mycpromoter 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 CREB-CREM transcription factors are the main gene regulatory effectors of the cAMP signaling pathway. The investigations of this family of transcription factors had a profound impact on the understanding of signaling-induced gene transcription. Here we discuss some key aspects of the underlying biology, review transcriptional activation by CREB proteins through transcription cofactors and present novel insights into the context- and position-specific function of CREB on complex genes.

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 vitroand in vivoby 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 vitrobinding 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 vivoand in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

The general transcriptional cofactor PC4 enhances transcription from various promoters and functions with a wide range of transcriptional activators. Earlier studies have suggested that this enhancement originates mostly from stabilization of the TATA-box/TFIID/TFIIA complex by simultaneous interaction of PC4 with transactivation domains of upstream-binding factors and the basal factor TFIIA. However, the C-terminal half of the protein also has been shown to exhibit substantial ssDNA binding properties, to which as yet no clear function has been assigned. We have investigated the interaction of this domain with various DNA structures and report that high-affinity binding, characterized by an equilibrium dissociation constant in the nanomolar range, requires either a heteroduplex containing a minimum of about eight mismatches, or alternatively a single-stranded DNA molecule consisting of 16 to 20 nucleotides. Furthermore, both juxtaposed single strands of a heteroduplex are protected by the C-terminal domain of PC4 in DNase I footprinting experiments, whereas the double-stranded regions do not appear to be contacted. We conclude from these observations that the role of PC4 ssDNA binding is likely to involve simultaneous interaction with opposing strands in internally melted duplexes, or the induction of a pronounced distortion in the local structure of ssDNA that results in a similar juxtaposed arrangement of single strands. In addition, we have observed that both the PC4 C-terminal domain and the intact PC4 destabilize dsDNA and we discuss the possible involvement of PC4 in promoter opening and other strand displacement events.

The crystal structure of human replication and transcription cofactor PC4CTDreveals a dimer with two single-stranded (ss)DNA binding channels running in opposite directions to each other. This arrangement suggests a role in establishment or maintenance of melted DMA at promoters or origins of replication.

The T-cell receptor (TCR) β-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 β gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vβ 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.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5′‐flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen‐bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5′-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

ABSTRACTThe transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2α/DRAP1 and NC2β/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2α in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

ABSTRACTThe transcription factor AREB6 contains a homeodomain flanked by two clusters of Kru¨ppel type C2H2zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2a/DRAP1 and NC2ß/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2a in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-κB directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB. A functional interaction of TFIIB with p65 was evident from assays in yeast cells. Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene. The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65. A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-κB by at least one cofactor, PC1, isolated from HeLa cells. These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.

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.

Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB).L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses.We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest.In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins.No relevant conflicts of interest to declare.

Abstract 5110

The general transcription factor TFIIB and its antagonist negative cofactor 2 (NC2) are hallmarks of RNA polymerase II (RNAPII) transcription. Both factors bind TATA box-binding protein (TBP) at promoters in a mutually exclusive manner. Dissociation of NC2 is thought to be followed by TFIIB association and subsequent preinitiation complex formation. TFIIB dissociates upon RNAPII promoter clearance, thereby providing a specific measure for steady-state preinitiation complex levels. As yet, genome-scale promoter mapping of human TFIIB has not been reported. It thus remains elusive how human core promoters contribute to preinitiation complex formation in vivo.

Nuclear factor I (NFI) was purified to homogeneity from porcine liver by DNA-affinity chromatography and displays a single band with a molecular weight of 36 kDa in SDS-polyacrylamide gels. The purified protein was used to determine absolute equilibrium binding constants by gel retardation techniques for a variety of DNA fragments with genuine or mutated NFI binding sites and a number of DNA fragments derived from various eukaryotic promoters carrying the CCAAT-box as a half-site for NFI binding. We present a model which allows prediction of the functional significance of mutated NFI binding-sites from sequence data. The data suggest that the single molecular species of NFI from porcine liver may not be able to recognize and activate the -CCAAT- promoter element in vivo without additional interactions, e.g. with other proteins.

We have identified a number of as yet unknown structural abnormalities of the NF I-DNA binding site within the inverted terminal repetition of adenovirus DNA by probing it with a hydroxyl radical footprinting technique. NF I binding alters the accessibility of the deoxyribose moieties to hydroxyl radicals both at the 3′ and at the 5′ side of the recognition sequence 5′-TGG(N)6GCCAA-3′. A smooth bend at the 5′ side of the binding sequence is already present in naked linear DNA and it is further enhanced by protein binding. This could be demonstrated not only by hydroxyl radical footprinting but also by studying the temperature dependent mobility during gel electrophoresis of DNA fragments carrying the NF I binding site at circularly permutated positions. We propose that the bent conformation at this site is responsible for facilitating protein/DNA interactions.

Human NC2 utilizes a unique mechanism of repression of transcription by associating with TBP and inhibition of preinitiation complex formation. Here we have cloned two genes from Saccharomyces cerevisiae and functionally characterized them as yeast NC2. We show that yeast NC2 binds to TBP as a heterodimer and represses RNA polymerase II transcription during assembly of the preinitiation complex. Yeast NC2 is highly homologous to its human counterpart within histone fold domains. C-Terminal regions previously discussed to be important for repression in man are in part not conserved. The human α but not the β subunit efficiently heterodimerizes and represses transcription in combination with the corresponding yeast subunit. Yeast and human NC2 inhibit transcription in the presence of yeast and human TBP. However, repression is optimal within one species. The N-terminus of human TBP supports repression of transcription by human but not by yeast NC2.

The long control region (LCR) of human papillomaviruses (HPV) encompasses 5–12% of the viral genome and contains an intricate network of cis responsive elements. In the LCR of seven unrelated HPV-types, namely HPV-1, 6, 8, 11, 16, 18 and 33, we have identified clusters of 4 to 75-TTGGC-3 motifs suggesting nuclear factor I (NFI) binding sites. We randomly selected 20 (out of a total of 38) of these motifs and showed that pure NFI from porcine liver protects virtually the same nucleotides as a factor present in crude HeLa nuclear extracts. The footprints obtained with HeLa extracts in the LCR of HPV-16 are eliminated in competition experiments by an oligonucleotide representing the palindromic adenovirus NFI binding site. Restriction fragments from the genome of HPV-11, 16 and 18, which contain this cluster of NFI binding sites associated with binding sites of unrelated transcription factors, function as transcriptional enhancers. In contrast, a fragment from HPV-8 exhibiting exclusively NFI binding sites, or polymerized NFI sites from HPV-16, are functionally inactive. NFI seems to be necessary but not sufficient for HPV enhancer activation.