Single-cell transcriptomics identifies potential cells of origin of MYC rhabdoid tumors.

Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets.

Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells.

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.

κB-Ras and Ral GTPases regulate acinar to ductal metaplasia during pancreatic adenocarcinoma development and pancreatitis.

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 KRas-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival.

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation.

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.

Analyzing the Dynamics of Single TBP-DNA-NC2 Complexes Using Hidden Markov Models.

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.

The polynucleotide kinase 3'-phosphatase gene (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) previously related to MED25.

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).

Combined BRD4 and CDK9 inhibition as a new therapeutic approach in malignant rhabdoid tumors.

Rhabdoid tumors are caused by the deletion of , 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.

The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition.

CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes.

Cyclin-dependent kinase 7 controls mRNA synthesis by affecting stability of preinitiation complexes, leading to altered gene expression, cell cycle progression, and survival of tumor cells.

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.

Arsenic trioxide inhibits tumor cell growth in malignant rhabdoid tumors in vitro and in vivo by targeting overexpressed Gli1.

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.

Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor.

Background And Purpose: The cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9.

Experimental Approach: The selectivity of LDC000067 was established in functional kinase assays.

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells.

Background: Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches.

Methods: Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines.

Control of gene transcription by Mediator in chromatin.

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.

Structure and VP16 binding of the Mediator Med25 activator interaction domain.

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.

Basal core promoters control the equilibrium between negative cofactor 2 and preinitiation complexes in human cells.

Background: 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.

Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex.

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).

RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion.

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.

Identification of the variant Ala335Val of MED25 as responsible for CMT2B2: molecular data, functional studies of the SH3 recognition motif and correlation between wild-type MED25 and PMP22 RNA levels in CMT1A animal models.

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.

Regulation of nuclear import and export of negative cofactor 2.

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.

Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites.

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.

Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7.

RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. 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.

NC2 mobilizes TBP on core promoter TATA boxes.

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 Förster 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.

Global distribution of negative cofactor 2 subunit-alpha on human promoters.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters in vitro. Its association with TBP prevents the binding of TFIIB and leads to inhibition of preinitiation complex formation. Here, we investigate the association of NC2 subunit-alpha with human RNA polymerase II promoter regions by using gene-specific ChIP and genome-wide promoter ChIPchip analyses.

The VP16 activation domain establishes an active mediator lacking CDK8 in vivo.

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.