Molecular biology: How proteins find their DNA target
Nature 507, 7492 (2014). doi:10.1038/507277a
Researchers have captured images of individual proteins searching for their DNA-binding sites, and have quantified parts of this process.Zhe Liu and his team at the Howard Hughes Medical Institute in Ashburn, Virginia, looked at transcription factors — proteins that bind to specific genes to
Nature Protocols 9, 871 (2014). doi:10.1038/nprot.2014.056
Authors: Sung Min Kim, Hannah Flaßkamp, Andreas Hermann, Marcos Jesús Araúzo-Bravo, Seung Chan Lee, Sung Ho Lee, Eun Hye Seo, Seung Hyun Lee, Alexander Storch, Hoon Taek Lee, Hans R Schöler, Natalia Tapia & Dong Wook Han
Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of
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Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs.
Genome Biol. 2014 Mar 14;15(3):R51
Authors: Martin HC, Wani S, Steptoe AL, Krishnan K, Nones K, Nourbakhsh E, Vlassov A, Grimmond SM, Cloonan N
Abstract
BACKGROUND: MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5[prime] end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs.
RESULTS: We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artefact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions.
CONCLUSIONS: Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.
PMID: 24629056 [PubMed - as supplied by publisher]
MeCP2 Caught Moonlighting as a Suppressor of MicroRNA Processing.
Dev Cell. 2014 Mar 10;28(5):477-8
Authors: Woo JS, Kim VN
Abstract
MeCP2 is a transcriptional regulator important for neurodevelopment and is involved in Rett syndrome and autism. In this issue of Developmental Cell, Cheng and colleagues (2014) report that MeCP2 also regulates microRNA biogenesis. MeCP2 phosphorylation induces a direct interaction with DGCR8, leading to reduced microRNA processing and retardation of dendritic growth.
PMID: 24636253 [PubMed - in process]
Navigating and Mining modENCODE Data.
Methods. 2014 Mar 14;
Authors: Boley N, Wan KH, Bickel PJ, Celniker SE
Abstract
modENCODE was a 5 yr NHGRI funded project (2007- 2012) to map the function of every base in the genomes of worms and flies characterizing positions of modified histones and other chromatin marks, origins of DNA replication, RNA transcripts and the transcription factor binding sites that control gene expression. Here we describe the Drosophila modENCODE datasets and how best to access and use them for genome wide and individual gene studies.
PMID: 24636835 [PubMed - as supplied by publisher]
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Convergent transcription induces dynamic DNA methylation at disiRNA loci.
PLoS Genet. 2013;9(9):e1003761
Authors: Dang Y, Li L, Guo W, Xue Z, Liu Y
Abstract
Cytosine methylation of DNA is an important epigenetic gene silencing mechanism in plants, fungi, and animals. In the filamentous fungus Neurospora crassa, nearly all known DNA methylations occur in transposon relics and repetitive sequences, and DNA methylation does not depend on the canonical RNAi pathway. disiRNAs are Dicer-independent small non-coding RNAs that arise from gene-rich part of the Neurospora genome. Here we describe a new type of DNA methylation that is associated with the disiRNA loci. Unlike the known DNA methylation in Neurospora, disiRNA loci DNA methylation (DLDM) is highly dynamic and is regulated by an on/off mechanism. Some disiRNA production appears to rely on pol II directed transcription. Importantly, DLDM is triggered by convergent transcription and enriched in promoter regions. Together, our results establish a new mechanism that triggers DNA methylation.
PMID: 24039604 [PubMed - indexed for MEDLINE]
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Unlocking the vault: next-generation museum population genomics.
Mol Ecol. 2013 Dec;22(24):6018-32
Authors: Bi K, Linderoth T, Vanderpool D, Good JM, Nielsen R, Moritz C
Abstract
Natural history museum collections provide unique resources for understanding how species respond to environmental change, including the abrupt, anthropogenic climate change of the past century. Ideally, researchers would conduct genome-scale screening of museum specimens to explore the evolutionary consequences of environmental changes, but to date such analyses have been severely limited by the numerous challenges of working with the highly degraded DNA typical of historic samples. Here, we circumvent these challenges by using custom, multiplexed, exon capture to enrich and sequence ~11,000 exons (~4 Mb) from early 20th-century museum skins. We used this approach to test for changes in genomic diversity accompanying a climate-related range retraction in the alpine chipmunks (Tamias alpinus) in the high Sierra Nevada area of California, USA. We developed robust bioinformatic pipelines that rigorously detect and filter out base misincorporations in DNA derived from skins, most of which likely resulted from postmortem damage. Furthermore, to accommodate genotyping uncertainties associated with low-medium coverage data, we applied a recently developed probabilistic method to call single-nucleotide polymorphisms and estimate allele frequencies and the joint site frequency spectrum. Our results show increased genetic subdivision following range retraction, but no change in overall genetic diversity at either nonsynonymous or synonymous sites. This case study showcases the advantages of integrating emerging genomic and statistical tools in museum collection-based population genomic applications. Such technical advances greatly enhance the value of museum collections, even where a pre-existing reference is lacking and points to a broad range of potential applications in evolutionary and conservation biology.
PMID: 24118668 [PubMed - indexed for MEDLINE]
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Single-cell analysis of the transcriptome and its application in the characterization of stem cells and early embryos.
Cell Mol Life Sci. 2014 Mar 21;
Authors: Liu N, Liu L, Pan X
Abstract
Cellular heterogeneity within a cell population is a common phenomenon in multicellular organisms, tissues, cultured cells, and even FACS-sorted subpopulations. Important information may be masked if the cells are studied as a mass. Transcriptome profiling is a parameter that has been intensively studied, and relatively easier to address than protein composition. To understand the basis and importance of heterogeneity and stochastic aspects of the cell function and its mechanisms, it is essential to examine transcriptomes of a panel of single cells. High-throughput technologies, starting from microarrays and now RNA-seq, provide a full view of the expression of transcriptomes but are limited by the amount of RNA for analysis. Recently, several new approaches for amplification and sequencing the transcriptome of single cells or a limited low number of cells have been developed and applied. In this review, we summarize these major strategies, such as PCR-based methods, IVT-based methods, phi29-DNA polymerase-based methods, and several other methods, including their principles, characteristics, advantages, and limitations, with representative applications in cancer stem cells, early development, and embryonic stem cells. The prospects for development of future technology and application of transcriptome analysis in a single cell are also discussed.
PMID: 24652479 [PubMed - as supplied by publisher]
Nature advance online publication 23 March 2014. doi:10.1038/nature13119
Authors: Xi Chen, Dimitrios Iliopoulos, Qing Zhang, Qianzi Tang, Matthew B. Greenblatt, Maria Hatziapostolou, Elgene Lim, Wai Leong Tam, Min Ni, Yiwen Chen, Junhua Mai, Haifa Shen, Dorothy Z. Hu, Stanley Adoro, Bella Hu, Minkyung Song, Chen Tan, Melissa D. Landis, Mauro Ferrari, Sandra J. Shin, Myles Brown, Jenny C. Chang, X. Shirley Liu & Laurie H. Glimcher
Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)—a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)—is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.
Summary: With the advances of RNA sequencing technologies, scientists need new tools to analyze transcriptome data. We introduce RNAseqViewer, a new visualization tool dedicated to RNA-Seq data. The program offers innovative ways to represent transcriptome data for single or multiple samples. It is a handy tool for scientists who use RNA-Seq data to compare multiple transcriptomes, for example, to compare gene expression and alternative splicing of cancer samples or of different development stages.
Availability and implementation: RNAseqViewer is freely available for academic use at http://bioinfo.au.tsinghua.edu.cn/software/RNAseqViewer/
Contact: zhangxg@tsinghua.edu.cn
Supplementary information: Supplementary data are available at Bioinformatics online.
Motivation: High-throughput ChIP-seq studies typically identify thousands of peaks for a single transcription factor (TF). It is common for traditional motif discovery tools to predict motifs that are statistically significant against a naïve background distribution but are of questionable biological relevance.
Results: We describe a simple yet effective algorithm for discovering differential motifs between two sequence datasets that is effective in eliminating systematic biases and scalable to large datasets. Tested on 207 ENCODE ChIP-seq datasets, our method identifies correct motifs in 78% of the datasets with known motifs, demonstrating improvement in both accuracy and efficiency compared with DREME, another state-of-art discriminative motif discovery tool. More interestingly, on the remaining more challenging datasets, we identify common technical or biological factors that compromise the motif search results and use advanced features of our tool to control for these factors. We also present case studies demonstrating the ability of our method to detect single base pair differences in DNA specificity of two similar TFs. Lastly, we demonstrate discovery of key TF motifs involved in tissue specification by examination of high-throughput DNase accessibility data.
Availability: The motifRG package is publically available via the bioconductor repository.
Contact: yzizhen@fhcrc.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Nature Chemical Biology 10, 291 (2014). doi:10.1038/nchembio.1452
Authors: Sai Pradeep Velagapudi, Steven M Gallo & Matthew D Disney
The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.
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Biobanking practice: RNA storage at low concentration affects integrity.
Biopreserv Biobank. 2014 Feb;12(1):46-52
Authors: Olivieri EH, de Andrade Franco L, Pereira RG, Carvalho Mota LD, Campos AH, Carraro DM
Abstract
A critical issue in defining protocols for biobanking practices is the preservation of total RNA for assessing the whole transcriptome and ensuring that it can be utilized in clinically oriented studies. Storage conditions, such as temperature and the length of time that tissues and purified RNA stay frozen, may directly impact RNA preservation. In this study, we evaluated a) the quality of RNA (as measured by RNA Integrity Number) purified from head and neck tumor tissues stored at -140°C for distinct time intervals of up to 7 years, and b) the quality of their respective RNAs stored for 4 years at -80°C when diluted at either 250 ng/μL or 25 ng/μL, with repeated freezing and thawing. Additionally, we generated a profile of the RNA collection of human tumors from different body sites stored at the AC Camargo Biobank. Our results showed no significant change in RIN values according to length of storage at -140°C. With respect to RNA aliquots stored at -80°C, RNA integrity at 250 ng/μL was preserved, while statistically significant degradation was observed at 25 ng/μL after only 8 months of storage. The RNA collection from most of the human tumors stored at the AC Camargo Biobank exhibited high quality, with average RIN around seven. However, ovary and stomach samples had the greatest RNA degradation. Taken together, the results show that both the temperature of preservation and the concentration of RNA should be strictly controlled by the biobank staff involved in macromolecule purification. Moreover, the RNAs from our biobank can be useful for the most demanding methods of gene expression analysis by virtue of adherence to optimal standard operating procedures for both tissue and macromolecule laboratories.
PMID: 24620769 [PubMed - in process]
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Nonsense-mediated mRNA decay immunity can help identify human polycistronic transcripts.
PLoS One. 2014;9(3):e91535
Authors: Shahaf G, Shweiki D
Abstract
Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript.
PMID: 24621851 [PubMed - in process]
Nature advance online publication 16 March 2014. doi:10.1038/nature12962
Authors: James B. Brown, Nathan Boley, Robert Eisman, Gemma E. May, Marcus H. Stoiber, Michael O. Duff, Ben W. Booth, Jiayu Wen, Soo Park, Ana Maria Suzuki, Kenneth H. Wan, Charles Yu, Dayu Zhang, Joseph W. Carlson, Lucy Cherbas, Brian D. Eads, David Miller, Keithanne Mockaitis, Johnny Roberts, Carrie A. Davis, Erwin Frise, Ann S. Hammonds, Sara Olson, Sol Shenker, David Sturgill, Anastasia A. Samsonova, Richard Weiszmann, Garret Robinson, Juan Hernandez, Justen Andrews, Peter J. Bickel, Piero Carninci, Peter Cherbas, Thomas R. Gingeras, Roger A. Hoskins, Thomas C. Kaufman, Eric C. Lai, Brian Oliver, Norbert Perrimon, Brenton R. Graveley & Susan E. Celniker
Nature advance online publication 16 March 2014. doi:10.1038/nature13069
Authors: Kate M. Creasey, Jixian Zhai, Filipe Borges, Frederic Van Ex, Michael Regulski, Blake C. Meyers & Robert A. Martienssen
In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide ‘epigenetically activated’ small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence.
Nature Protocols 9, 828 (2014). doi:10.1038/nprot.2014.047
Authors: Dan Su, Clement T Y Chan, Chen Gu, Kok Seong Lim, Yok Hian Chionh, Megan E McBee, Brandon S Russell, I Ramesh Babu, Thomas J Begley & Peter C Dedon
Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for