The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase

A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis.

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression(1). AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation(2). AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis(3-6), making it important to identify co-regulatory factors that 'read' the NHR2 oligomerization and contribute to leukaemogenesis(4). Here we show that, in human leukaemic cells, AML1-ETO resides in and functions through a stable AML1-ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia

TATA-Binding Protein-Like Protein (TLP/TRF2/TLF) Negatively Regulates Cell Cycle Progression and Is Required for the Stress-Mediated G(2) Checkpoint

The TATA-binding protein (TBP) is a universal transcription factor required for all of the eukaryotic RNA polymerases. In addition to TBP, metazoans commonly express a distantly TBP-related protein referred to as TBP-like protein (TLP/TRF2/TLF). Although the function of TLP in transcriptional regulation is not clear, it is known that TLP is required for embryogenesis and spermiogenesis. In the present study, we investigated the cellular functions of TLP by using TLP knockout chicken DT40 cells. TLP was found to be dispensable for cell growth. Unexpectedly, TLP-null cells exhibited a 20% elevated cell cycle progression rate that was attributed to shortening of the G(2) phase. This indicates that TLP functions as a negative regulator of cell growth. Moreover, we found that TLP mainly existed in the cytoplasm and was translocated to the nucleus restrictedly at the G(2) phase. Ectopic expression of nuclear localization signal-carrying TLP resulted in an increase (1.5-fold) in the proportion of cells remaining in the G(2)/M phase and apoptotic state. Notably, TLP-null cells showed an insufficient G(2) checkpoint when the cells were exposed to stresses such as UV light and methyl methanesulfonate, and the population of apoptotic cells after stresses decreased to 40%. These phenomena in G(2) checkpoint regulation are suggested to be p53 independent because p53 does not function in DT40 cells. Moreover, TLP was transiently translocated to the nucleus shortly (15 min) after stress treatment. The expression of several stress response and cell cycle regulatory genes drifted in a both TLP- and stress-dependent manner. Nucleus-translocating TLP is therefore thought to work by checking cell integrity through its transcription regulatory ability. TLP is considered to be a signal-transducing transcription factor in cell cycle regulation and stress response

Application of Bayesian Probability Network to Music Scene Analysis

We propose a process model for hierarchical perceptual sound organization, which recognizes perceptual sounds included in incoming sound signals. We consider perceptual sound organization as a scene analysis problem in the auditory domain. Our current application is a music scene analysis system, which recognizes rhythm, chords, and source-separated musical notes included in incoming music signals. Our process model consists of multiple processing modules and a probability network for information integration. The structure of our model is conceptually based on the blackboard architecture. However, employment of a Bayesian probability network has facilitated integration of multiple sources of information provided by autonomous modules without global control knowledge. 1 Introduction We humans recognize or understand existence, localization and movements of external entities through five senses. We call this function "scene analysis". Scene analysis is viewed here as an information pr..

Organization of Hierarchical Perceptual Sounds: Music Scene Analysis with Autonomous Processing Modules and a Quantitative Information Integration Mechanism

We propose a process model for hierarchical perceptual sound organization, which recognizes perceptual sounds included in incoming sound signals. We consider perceptual sound organization as a scene analysis problem in the auditory domain. Our model consists of multiple processing modules and a hypothesis network for quantitative integration of multiple sources of information. When input information for each processing module is available, the module rises to process it and asynchronously writes output information to the hypothesis network. On the hypothesis network, individual information is integrated and an optimal internal model of perceptual sounds is automatically constructed. Based on the model, a music scene analysis system has been developed for acoustic signals of ensemble music, which recognizes rhythm, chords, and source-separated musical notes. Experimental results show that our method has permitted autonomous, stable and effective information integration to construct the..

Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

<div><p>The c-<i>fos</i> gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R) facilitates transcription reinitiation of the c-<i>fos</i> promoter <i>in vitro</i> in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH) as well as TFIIB, which recruits RNA polymerase II (Pol II) and TFIIF to Scaffold. The cooperative action of hnRNP R and Mediator is diminished by the cyclin-dependent kinase 8 (CDK8) module, which is comprised of CDK8, Cyclin C, MED12 and MED13 of the Mediator subunits. Interestingly, we find that the length of the G-free cassettes, and thereby their transcripts, influences the hnRNP R-mediated facilitation of reinitiation. Indeed, indicative of a possible role of the transcript in facilitating transcription reinitiation, the RNA transcript produced from the G-free cassette interacts with hnRNP R through its RNA recognition motifs (RRMs) and arginine-glycine-glycine (RGG) domain. Mutational analyses of hnRNP R indicate that facilitation of initiation and reinitiation requires distinct domains of hnRNP R. Knockdown of hnRNP R in mouse cells compromised rapid induction of the <i>c-fos</i> gene but did not affect transcription of constitutive genes. Together, these results suggest an important role for hnRNP R in regulating robust response of the <i>c-fos</i> gene.</p></div

Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

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

The Mediator containing the CDK8 module does not facilitate transcription reinitiation.

<p>(A) Med(0.5) and Med(0.85) were purified from HeLa nuclear extract expressing 3xFLAG-tagged MED10 by using phosphocellulose (P11) and anti-FLAG® M2 affinity gel. The purified Med(0.5) and Med(0.85) were separated by SDS-PAGE and silver-stained. The positions of the molecular mass marker are indicated on the left. Mediator was detected via 3xFLAG-tagged MED10 using an anti-FLAG antibody. (B) Western blot analyses were performed using anti-CDK8, anti-Cyclin C, anti-MED12, anti-MED13 or anti-MED17 antibody. (C) <i>In vitro</i> transcription was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072496#pone-0072496-g001" target="_blank">Figure 1A</a>. hnRNP R, Med(0.5) and Med(0.85) were added as shown in the figure. The arrows indicate the first-round (1st) and second-round (2nd) transcripts, and the values (means ± SE) of the quantified data from four independent experiments are shown in the top (1st round) and middle (2nd round) panels on the right. The bottom panel on the right shows the ratios of the second-round to first-round transcripts.</p

Functional domains of hnRNP R required for facilitating initiation and reinitiation.

<p>(A) Schematic diagram of the domain structure of hnRNP R and its deletion mutants are shown. hnRNP R is comprised of the acidic domain (acidic), three RNA recognition motifs (RRMs), a nuclear localization signal (NLS), arginine-glycine-glycine-rich domain (RGG) and the glutamine-asparagine-rich domain (QN). The results of RNA binding assays (shown in B) and <i>in vitro</i> transcription (shown in C) for the mutants are summarized on the right. An <i>in vitro</i> transcription assay for Δ1-446 was not done (nd) because the mutant was insoluble unless expressed as a GST-fusion protein. The domains required for initiation (1st round) and reinitiation (multiple round) are indicated above the diagram. (B) Binding of the hnRNP R to the RNA derived from the 390-nt G-free cassette was assayed using immobilized GST (lane 3), GST-hnRNP R (lane 4), or GST-hnRNP R mutants (lanes 5–10). The bound RNA was analyzed by electrophoresis and autoradiography, and lanes 1 and 2 contained 10% and 5% of the input RNA. (C) <i>In vitro</i> transcription was preformed using the purified FLAG-tagged hnRNP R mutants. pfMC2AT harboring the 390-nt G-free cassette was used as a template. The positions of the first-round and second-round transcripts are indicated by arrows on the right, and the relative levels of the first-round transcript are indicated below each lane.</p