The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying ...enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255-1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.
Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor ...alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.
Histone deacetylases (HDACs) catalyze the hydrolysis of Ɛ-acetyl-lysine residues of histones. Removal of acetyl groups results in condensation of chromatin structure and repression of gene ...expression. Human class I, II, and IV HDACs are said to be zinc-dependent in that they require divalent zinc ions to catalyze the deacetylase reaction. HDACs are considered potential targets for the treatment of cancer due to their role in regulating transcription. They are also thought to play important roles in the development of organisms such as honey bees. The fatty acid, 10-hydroxy-2E-decenoic acid (10-HDA), which can account for up to 5% of royal jelly composition has been reported as an HDAC inhibitor. The crystal structure of the HDAC3:SMRT complex possesses two monovalent cations (MVCs) labeled as potassium with one MVC binding site near the active site Zn(II) and the second MVC binding site ≥20 Å from the active site Zn(II). We report here the inhibitory effects of excess Zn(II) on the catalytic activity of histone deacetylase 3 (HDAC3) bound to the deacetylase activating domain of nuclear receptor corepressor 2 (NCOR2). We also report the effects of varying concentrations of potassium ions where K+ up to 10 mM increase HDAC3 activity with a maximum kcat/KM of approximately 80,000 M-1s-1 while K+ above 10 mM inhibit HDAC3 activity. The inhibition constant (Ki) of 10-HDA was determined to be 5.32 mM. The regulatory effects of zinc, potassium, and 10-HDA concentration on HDAC3 activity suggest a strong correlation between these chemical species and epigenetic control over Apis mellifera caste differentiation among other control mechanisms.
The white-backed planthopper Sogatella furcifera is an economically important rice pest distributed throughout Asia. It damages rice crops by sucking phloem sap, resulting in stunted growth and plant ...virus transmission. We aimed to obtain the full-length transcriptome data of S. furcifera using PacBio single-molecule real-time (SMRT) sequencing. Total RNA extracted from S. furcifera at various developmental stages (egg, larval, and adult stages) was mixed and used to generate a full-length transcriptome for SMRT sequencing. Long non-coding RNA (lncRNA) identification, full-length coding sequence prediction, full-length non-chimeric (FLNC) read detection, simple sequence repeat (SSR) analysis, transcription factor detection, and transcript functional annotation were performed. A total of 12,514,449 subreads (15.64 Gbp, clean reads) were generated, including 630,447 circular consensus sequences and 388,348 FLNC reads. Transcript cluster analysis of the FLNC reads revealed 251,109 consensus reads including 29,700 high-quality reads. Additionally, 100,360 SSRs and 121,395 coding sequences were identified using SSR analysis and ANGEL software, respectively. Furthermore, 44,324 lncRNAs were annotated using four tools and 1,288 transcription factors were identified. In total, 95,495 transcripts were functionally annotated based on searches of seven different databases. To the best of our knowledge, this is the first study of the full-length transcriptome of the white-backed planthopper obtained using SMRT sequencing. The acquired transcriptome data can facilitate further studies on the ecological and viral-host interactions of this agricultural pest.
DNA‐Forensik Kremer, Kerstin; Fritzsch, Sabrina; Stahl, Frank
Chemie in unserer Zeit,
December 2017, Letnik:
51, Številka:
6
Journal Article
Recenzirano
Zusammenfassung
Der Begriff Forensik fasst die wissenschaftlichen Arbeitsmethoden zusammen, mit denen Straftaten untersucht, analysiert und aufgeklärt werden können. Dienen der Aufklärung DNA‐Spuren, ...die am Tatort vorgefunden wurden, spricht man von DNA‐Forensik oder forensischer Genetik. Häufig liegt am Tatort nicht nur die DNA‐Spur des Täters vor, sondern es sind auch die DNA von Opfer, Zeugen und berechtigten Personen (Ersthelfer, Polizeikräfte, etc.) usw. aufzufinden. Dann wird die Analyse komplex und das Verhältnis der DNA‐Spuren zueinander bedeutsam. Seit einigen Jahren steht die DNA‐Forensik vor völlig neuen Möglichkeiten, die neu entwickelte, moderne Sequenzierungstechniken, die zusammengefasst als Next Generation Sequencing (NGS) oder inzwischen sogar als Third Generation Sequencing (SMRT) bezeichnet werden, bieten. Diese Techniken erlauben eine schnelle Sequenzierung des gesamten Genoms und damit Merkmale wie Augenfarbe und Haarfarbe einer Person zu bestimmen. So könnte aus dem abstrakten DNA‐Profil in den nächsten Jahren ein plastisches DNA‐Phantombild werden. Die Entwicklungen erfordern eine Anpassung der gesetzlichen Regelungen und öffentliche Akzeptanz. Im vorliegenden Artikel werden die aktuellen Praktiken der DNA‐Forensik am Tatort und in den Laboratorien beschrieben und ein Ausblick auf zukünftige Möglichkeiten der forensischen DNA‐Analyse gegeben werden.
Summary
Forensics deals with the scientific methods to gather information at a crime scene for solving criminal actions. DNA forensics uses genetic material for these purposes. DNA fingerprinting is established as an important method for police detective work since the end of the 1980s. Currently, DNA forensics faces completely new possibilities through the application of more efficient high‐throughput sequencing methods, summarized as Next Generation Sequencing (NGS). Using NGS it could be possible to predict numerous externally visible characteristics including the complete facial shape of an unknown perpetrator. This article aims at presenting practices of forensic DNA analyses used to date and extending the picture for future possibilities and challenges.
Die Ausstrahlung von Fernsehserien wie CSI: Miami, oder Navy CIS fördert ein öffentliches Interesse an den forensischen Wissenschaften und schafft Alltagsvorstellungen vom forensischen Arbeiten. Häufig wirken die in den Serien verwendeten analytischen Methoden jedoch im Vergleich zu den wirklich im Labor angewendeten Techniken wie Science Fiction. So führt in den Fernsehserien eine in wenigen Minuten durchgeführte DNA‐Analyse zur direkten Identifizierung des Täters mit komplettem Strafregister und aktueller Adresse. Diese Unterhaltungsserien bilden die Komplexität im kriminaltechnischen Labor und bei der Ermittlung jedoch nicht real ab, wodurch dem Zuschauer ein falscher Eindruck vermittelt wird. Hieraus kann sich eine Einstellung gegenüber der Forensik in der echten Kriminalistik ergeben, die nicht auf den tatsächlichen wissenschaftlichen Möglichkeiten beruht. Ziel dieses Beitrages ist es, aus wissenschaftlicher aber auch aus rechtlicher und bewertender Perspektive die aktuell realen Möglichkeiten und Grenzen der DNA‐Forensik aufzuzeigen.
Objective:
The aim of this project was to implement long-read sequencing for BCR-ABL1 TKI resistance mutation screening in a clinical setting for patients undergoing treatment for chronic myeloid ...leukemia.
Materials and Methods:
Processes were established for registering and transferring samples from the clinic to an academic sequencing facility for long-read sequencing. An automated analysis pipeline for detecting mutations was established, and an information system was implemented comprising features for data management, analysis and visualization. Clinical validation was performed by identifying BCR-ABL1 TKI resistance mutations by Sanger and long-read sequencing in parallel. The developed software is available as open source via GitHub at https://github.com/pharmbio/clamp
Results:
The information system enabled traceable transfer of samples from the clinic to the sequencing facility, robust and automated analysis of the long-read sequence data, and communication of results from sequence analysis in a reporting format that could be easily interpreted and acted upon by clinical experts. In a validation study, all 17 resistance mutations found by Sanger sequencing were also detected by long-read sequencing. An additional 16 mutations were found only by long-read sequencing, all of them with frequencies below the limit of detection for Sanger sequencing. The clonal distributions of co-existing mutations were automatically resolved through the long-read data analysis. After the implementation and validation, the clinical laboratory switched their routine protocol from using Sanger to long-read sequencing for this application.
Conclusions:
Long-read sequencing delivers results with higher sensitivity compared to Sanger sequencing and enables earlier detection of emerging TKI resistance mutations. The developed processes, analysis workflow, and software components lower barriers for adoption and could be extended to other applications.
Nuclear functions for IκB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors ...concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKα associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKα restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.
The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, ...NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.
Fish mortality caused by
is a major economic problem in aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by
through handling of contaminated fish. In this ...study, we present the complete genome sequence of
strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of
QMA0248 carries a 32 kb prophage, a 12 kb genomic island and 92 discrete insertion sequence (IS) elements. These include nine novel IS types that belong mostly to the IS
family. Comparative and phylogenetic analysis between
QMA0248 and publicly available complete
genomes revealed discrepancies that are probably due to misassembly in the genomes of isolates ISET0901 and ISNO. Long-range PCR confirmed five rRNA loci in the PacBio assembly of QMA0248, and, unlike
89353, no tandemly repeated rRNA loci in the consensus genome. However, we found sequence read evidence that the tandem rRNA repeat existed within a subpopulation of the original QMA0248 culture. Subsequent nanopore sequencing revealed that the tandem rRNA repeat was the most prevalent genotype, suggesting that there is selective pressure to maintain fewer rRNA copies under uncertain laboratory conditions. Our study not only highlights assembly problems in existing genomes, but provides a high-quality reference genome for
QMA0248, including manually curated mobile genetic elements, that will assist future
comparative genomic and evolutionary studies.
Non-planar di-
-substituted PCB 153 (2,2’,4,4’,5,5’-hexachlorobiphenyl), one of the most abundant PCB congeners in the environment and in biological and human tissues, has been identified as ...potential endocrine disruptor affecting the reproductive and endocrine systems in rodents, wildlife, and humans. The aim of this study was to gain a deeper insight into its mode/mechanism of action in Chinese hamster ovary K1 cells (CHO-K1). PCB 153 (10–100 μmol/L) inhibited CHO-K1 cell proliferation, which was confirmed with four bioassays (Trypan Blue, Neutral Red, Kenacid Blue, and MTT), of which the MTT assay proved the most sensitive. PCB 153 also induced ROS formation in a dose-dependent manner. Apoptosis was seen after 6 h of exposure to PCB 153 doses ≥50 μmol/L, while prolonged exposure resulted in the activation of the necrotic pathway. PCB 153-induced disturbances in normal cell cycle progression were time-dependent, with the most significant effects occurring after 72 h.
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