An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II.

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Regulation of the RNA polymerase II pre-initiation complex by its associated coactivators.

The RNA polymerase II (Pol II) pre-initiation complex (PIC) is a critical node in eukaryotic transcription regulation, and its formation is the major rate-limiting step in transcriptional activation. Diverse cellular signals borne by transcriptional activators converge on this large, multiprotein assembly and are transduced via intermediary factors termed coactivators. Cryogenic electron microscopy, multi-omics and single-molecule approaches have recently offered unprecedented insights into both the structure and cellular functions of the PIC and two key PIC-associated coactivators, Mediator and TFIID. Here, we review advances in our understanding of how Mediator and TFIID interact with activators and affect PIC formation and function. We also discuss how their functions are influenced by their chromatin environment and selected cofactors. We consider how, through its multifarious interactions and functionalities, a Mediator-containing and TFIID-containing PIC can yield an integrated signal processing system with the flexibility to determine the unique temporal and spatial expression pattern of a given gene.

Mediator: A Drawbridge across the Enhancer-Promoter Divide.

Two papers in this issue of Molecular Cell provide insights into how the multisubunit Mediator coactivator complex dynamically links enhancer-bound activators to the RNA polymerase II machinery at the core promoter.

Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth.

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

AID-RNA polymerase II transcription-dependent deamination of IgV DNA.

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.

Mediator-regulated transcription through the +1 nucleosome.

Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing.

Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II.

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

Core promoter-selective function of HMGA1 and Mediator in Initiator-dependent transcription.

The factors and mechanisms underlying the differential activity and regulation of eukaryotic RNA polymerase II on different types of core promoters have remained elusive. Here we show that the architectural factor HMGA1 and the Mediator coregulator complex cooperate to enhance basal transcription from core promoters containing both a TATA box and an Initiator (INR) element but not from "TATA-only" core promoters. INR-dependent activation by HMGA1 and Mediator requires the TATA-binding protein (TBP)-associated factors (TAFs) within the TFIID complex and counteracts negative regulators of TBP/TATA-dependent transcription such as NC2 and Topoisomerase I. HMGA1 interacts with TFIID and Mediator and is required for the synergy of TATA and INR elements in mammalian cells. Accordingly, natural HMGA1-activated genes in embryonic stem cells tend to have both TATA and INR elements in a synergistic configuration. Our results suggest a core promoter-specific regulation of Mediator and the basal transcription machinery by HMGA1.

Impact of Toupet Versus Nissen Fundoplication on Dysphagia in Patients With Gastroesophageal Reflux Disease and Associated Preoperative Esophageal Dysmotility: A Systematic Review and Meta-Analysis.

BACKGROUND: Controversy exists regarding the best surgical approach for the management of gastroesophageal reflux disease (GORD) and associated preoperative esophageal dysmotility. Our aim was to conduct a systematic review and meta-analysis to compare the outcomes of Toupet fundoplication (TF) and Nissen fundoplication (NF) in patients with GORD and coexistent preoperative esophageal dysmotility. METHODS: We conducted a systematic search of electronic information sources, including MEDLINE, EMBASE, CINAHL, CENTRAL, ClinicalTrials.gov , and bibliographic reference lists. We applied a combination of free text search and controlled vocabulary search adapted to thesaurus headings, search operators, and limits in each of the above-mentioned databases. Postoperative dysphagia and improvement in dysphagia were primary outcome parameters. RESULTS: We identified 3 randomized controlled trials and 1 observational study reporting a total of 220 patients, of whom 126 underwent TF and the remaining 94 patients had NF. Despite the existence of significantly higher preoperative dysphagia in the TF group (29.3% vs 4.2%, P = .05), TF was associated with significantly lower postoperative dysphagia (odds ratio [OR] = 0.31, P = .002) with low between-study heterogeneity ( I2 = 11%, P = .34), and significantly higher improved dysphagia (OR = 10.32, P < .0001) with moderate between-study heterogeneity ( I2 = 31%, P = .23) compared with NF. CONCLUSION: TF may be associated with significantly lower postoperative dysphagia than NF in patients with GORD and associated preoperative esophageal dysmotility. However, no definite conclusions can be drawn as the best available evidence comes mainly from a limited number of heterogeneous randomized controlled trials. Future studies are encouraged to include patients with similar preoperative dysphagia status and report the outcomes with respect to recurrence of acid reflux symptoms.

Irrigation Versus Suction Alone in Laparoscopic Appendectomy: Is Dilution the Solution to Pollution? A Systematic Review and Meta-Analysis.

OBJECTIVES: To investigate outcomes of peritoneal irrigation versus suction without irrigation in patients undergoing emergency laparoscopic appendectomy. METHODS: We performed a systematic review and conducted a search of electronic information sources to identify all randomized controlled trials (RCTs) and observational studies investigating outcomes of irrigation versus suction alone in patients undergoing emergency laparoscopic appendectomy. We used the Cochrane risk of bias tool and the Newcastle-Ottawa scale to assess the risk of bias of RCTs and observational studies, respectively. Random-effects models were applied to calculate pooled outcome data. RESULTS: We identified 3 RCTs and 2 retrospective observational studies, enrolling 2511 patients. Our results suggested that there was no difference between peritoneal irrigation and suction alone in terms of intraabdominal abscess rate (odds ratio = 2.39, 95% confidence interval [CI] = 0.49-11.74, P = .28), wound infection (risk difference = 0.00, 95% CI = -0.04 to 0.05, P = .85), and length of stay (mean difference = -1.02, 95% CI = -3.10 to 1.07, P = .34); however, peritoneal irrigation was associated with longer operative time (mean difference = 7.12, 95% CI = 4.33 to 9.92, P < .00001). Our results remained consistent when randomized trials, adult patients, and pediatric patients were analyzed separately. CONCLUSIONS: The best available evidence suggests that the peritoneal irrigation with normal saline during laparoscopic appendectomy does not provide additional benefits compared with suction alone in terms of intraabdominal abscess, wound infection, and length of stay but it may prolong the operative time. The quality of the best available evidence is moderate; therefore, high-quality RCTs, which are adequately powered, are required to provide more robust basis for definite conclusions.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Epigenetics? Mediator does that too!

Adding to the concept that Mediator acts as an "integrative hub,"Ding et al. (2008) report in this issue of Molecular Cell that, by facilitating the recruitment of the G9a methyl transferase, Mediator can also promote epigenetic silencing of selected genes.

The RNA polymerase II transcriptional machinery and its epigenetic context.

RNA polymerase II (Pol II) is the main engine that drives transcription of protein-encoding genes in eukaryotes. Despite its intrinsic subunit complexity, Pol II is subject to a host of factors that regulate the multistep transcription process. Indeed, the hallmark of the transcription cycle is the dynamic association of Pol II with initiation, elongation and other factors. In addition, Pol II transcription is regulated by a series of cofactors (coactivators and corepressors). Among these, the Mediator has emerged as one of the key regulatory factors for Pol II. Transcription by Pol II takes place in the context of chromatin, which is subject to numerous epigenetic modifications. This chapter mainly summarizes the various biochemical mechanisms that determine formation and function of a Pol II preinitiation complex (PIC) and those that affect its progress along the gene body (elongation). It further examines the various epigenetic modifications that the Pol II machinery encounters, especially in certain developmental contexts, and highlights newer evidence pointing to a likely close interplay between this machinery and factors responsible for the chromatin modifications.

Effectiveness of conservative treatment in the management of secretory otitis media.

BACKGROUND: Secretory otitis media is a very important and common ENT disease, especially in the children. The aetiology of Secretory otitis media is multifocal, and the treatment is initially medical or conservative and if it fails then surgery is indicated. The objective of the study was to determine the efficacy of medical treatment in the management of Secretory otitis media. METHODS: This cross sectional descriptive study was conducted at the outpatient department of ENT, Ayub Medical Institute (AMI) Abbottabad, from Mar to Sep 2013. A total of 40 patients were included in this study and standard medical treatment of secretory otitis media was given. The patients were followed up at 2nd and then 4th week and results were analysed. RESULTS: Both clinical and audiological diagnosis of secretory otitis media was made and patients were followed up at 2nd and 4th week to see the effect of treatment. Among 40 patients, 26 (65%) completely recovered from the disease while 14 patients (35%) did not improve. CONCLUSION: Conservative treatment is effective in the management of Secretory otitis media.

Laser and incisional myringotomy in otitis media with effusion-a comparative study.

BACKGROUND: Otitis media with effusion (OME) or middle ear effusion (MEE) is a common cause of hearing difficulty in children. MEE must be detected early and managed properly to prevent conductive hearing loss in children. It was aimed to compare results of laser myringotomy and conventional myringotomy in terms of hearing improvement, recurrence of MEE and time to put ventilation tube. METHODS: This randomized control trial was conducted from February 2012 to April 2014. Children of 4 years age or older with MEE were included in the study. These children were investigated with Pure tone audiometry (PTA) and tympanometry to confirm conductive hearing loss. X-Ray nasopharynx lateral view was performed for adenoids. Sixty-six patients were randomly assigned in to 2 groups, (1) treated with laser myringotomy and (2) treated with classical myringotomy. The ears were evaluated for MEE, for presence of perforation and level of hearing. RESULTS: A total of 98 ears in 66 patients underwent intervention. Middle ear effusion cleared in 44 out of 48 ears with laser myringotomy (LM) as compared to34 out of 50 ears with incisional myringotomy. The perforation was still patent in 36 ears treated with LM while it was found closed in all 50 ears with conventional myringotomy after 2 weeks. The hearing level improved with LM by 10-15 dB after first 3 months. CONCLUSION: The aim of management in otitis media with effusion is ventilation of tympanic cavity. Laser myringotomy is a best alternative to conventional one. It also has comparable results with ventilation tubes (VT). The ears with refractory or recurrent MEE should have VT insertion.

Anatomical relationship between recurrent laryngeal nerve and inferior thyroid artery in thyroidectomy patients.

BACKGROUND: Thyroid surgery is performed very frequently now a day. Previously it was used to be associated with high morbidity especially hoarseness. This complication is now almost negligible as most of the surgeons are well acquainted with the anatomical knowledge of the nerves in relation to the gland. The objective of this study was to find out variable anatomical relationships between Recurrent Laryngeal Nerve and Inferior Thyroid Artery in patients undergoing thyroid surgery. METHODS: This cross-sectional retrospective study was conducted in Government Lady Reading Hospital Peshawar and Abasyn Hospital (Private) Peshawar from May 2010 to June 2014. Patients undergoing surgery for benign goiters, T1, T2 well differentiated thyroid cancers without lymph node involvement was included. Data on various types of relationships between RLN and ITA were recorded. RESULTS: In total 271 patients operated and included in the study, 117 were male and 154 were female. Total of 398 RLNs were identified in 416 sides operated. In 55.27% cases the nerve was found to be anterior to inferior thyroid artery while it was posterior to the artery in 34.67% cases. In the remaining 10.05% cases the nerve was observed passing within the branches of inferior thyroid artery. CONCLUSIONS: The anatomical relationship between Recurrent Laryngeal Nerve RLN and Inferior Thyroid Artery ITA is highly variable. For all head and neck surgeons to perform safe surgery on thyroid, it is necessary to have sound anatomical knowledge of these variable relationships between recurrent laryngeal nerve and inferior thyroid artery.

Kinetic and adsorption isotherm studies of Malachite Green dye onto surfactant-tailored alginate hydrogel beads: An influence of surfactant hydrophobicity.

This study details the synthesis and characterization of surfactant-modified sodium alginate hydrogel beads crosslinked with Ba2+ ions through ionotropic gelation. Cationic surfactants such as, dodecyltrimethylammonium bromide (DTAB), didodecyldimethylammonium bromide (DDAB), and butanediyl-α,ω-bis-(dimethyldodecylammonium bromide) (GEM), were employed in the modification process. The surfactant-modified ALG-DTAB, ALG-DDAB, and ALG-GEM beads were investigated for the removal of cationic dye Malachite Green (MG) to elucidate the impact of hydrophobicity of amphiphiles on the adsorption process. The characterizations were carried out using Rheometry, Field Emission Scanning Electron Microscopy (FESEM), Infrared Spectroscopy (IR), and Energy Dispersive X-ray Spectroscopy (EDX). Under optimized conditions, ALG-GEM and ALG-DDAB demonstrated highest maximum adsorption capacity (Qmax > 700 mgg-1). The adsorption data fitted well to pseudo-second order kinetic and Langmuir adsorption models, suggesting the involvement of chemisorption phenomena with notable contributions from pore diffusion. The effects of pH, initial dye concentration, adsorbent dose, temperature, and competing ions on the removal of MG were investigated. Interestingly, ALG-GEM beads exhibited an increase in adsorption capacity with rising pH and a subsequent decrease with increasing temperature, showcasing optimal adsorption at pH 7.0 and 25 °C. The study proposes that ALG beads modified with cationic surfactants with higher hydrophobicity could offer a promising avenue in wastewater treatment processes.

Two target gene activation pathways for orphan ERR nuclear receptors.

Estrogen-related receptors (ERRα/ß/γ) are orphan nuclear receptors that function in energy-demanding physiological processes, as well as in development and stem cell maintenance, but mechanisms underlying target gene activation by ERRs are largely unknown. Here, reconstituted biochemical assays that manifest ERR-dependent transcription have revealed two complementary mechanisms. On DNA templates, ERRs activate transcription with just the normal complement of general initiation factors through an interaction of the ERR DNA-binding domain with the p52 subunit of initiation factor TFIIH. On chromatin templates, activation by ERRs is dependent on AF2 domain interactions with the cell-specific coactivator PGC-1α, which in turn recruits the ubiquitous p300 and MED1/Mediator coactivators. This role of PGC-1α may also be fulfilled by other AF2-interacting coactivators like NCOA3, which is shown to recruit Mediator selectively to ERRß and ERRγ. Importantly, combined genetic and RNA-seq analyses establish that both the TFIIH and the AF2 interaction-dependent pathways are essential for ERRß/γ-selective gene expression and pluripotency maintenance in embryonic stem cells in which NCOA3 is a critical coactivator.

Complex regulation of the transactivation function of hypoxia-inducible factor-1 alpha by direct interaction with two distinct domains of the CREB-binding protein/p300.

Activation of transcription in response to low oxygen tension is mediated by the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of two proteins: aryl hydrocarbon receptor nuclear translocator and the oxygen-regulated HIF-1 alpha. The C-terminal activation domain of HIF-1 alpha has been shown to interact with cysteine/histidine-rich region 1 (CH1) of the coactivator CBP/p300 in a hypoxia-dependent manner. However, HIF forms lacking C-terminal activation domain (naturally occurring or genetically engineered) are still able to activate transcription of target genes in hypoxia. Here, we demonstrate that the N-terminal activation domain (N-TAD) of HIF-1 alpha interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1 alpha N-TAD with CBP/p300 is mediated by the CH3 region of CBP known to interact with, among other factors, p53. Using fluorescence resonance energy transfer experiments, we demonstrate that N-TAD interacts with CH3 in vivo. Coimmunoprecipitation assays using endogenous proteins showed that immunoprecipitation of CBP in hypoxia results in the recovery of a larger fraction of HIF-1 alpha than of p53. Chromatin immunoprecipitation demonstrated that at 1% O(2) CBP is recruited to a HIF-1 alpha but not to a p53 target gene. Upon activation of both pathways, lower levels of chromatin-associated CBP were detected at either target gene promoter. These results identify CBP as the coactivator directly interacting with HIF-1 alpha N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1 alpha is a major CBP-interacting transcription factor that may compete with other CBP-dependent factors, including p53, for limiting amounts of this coactivator, underscoring the complexity in the regulation of gene expression by HIF-1 alpha.

Correlation of operative findings with pre-operative clinical signs and analysis of x-rays in patients with airway foreign bodies.

BACKGROUND: Foreign body aspiration is one of the commonly encountered emergencies in ENT and still it remains a significant cause of mortality and morbidity. However with the development of modern endoscopic techniques and controlled anaesthesia, most foreign bodies can be removed safely with a bronchoscope. METHODS: This study was carried out at Department of ENT, Head and Neck Surgery, Ayub Medical College, Abbottabad from 1st Jan 2003 to 30th June 2005. Total 81 patients were registered in the study. Two patients, in whom the foreign body could not be removed, were excluded from the study. RESULTS: The most consistent finding was decreased air entry on chest auscultation on the side of impacted foreign body which was present in 72 (91.1%) patients. The second most consistent finding was audible wheeze on the side of impacted foreign body found in 42 (53.2%) patients. The most common finding on Chest X-ray was emphysema found in 19 (61.3%) patient, followed by atelactasis in 9 (28%) patients while 3 (9.7%) patients had normal Chest x-ray. CONCLUSION: The pre-operative clinical signs in patient with aspirated foreign body give an idea about the site of foreign body in an airway. Although chest x-ray gives an idea about the pathological changes in respiratory tract it has little impact in the management of a patient with aspirated foreign body.