CSB Research

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Engineering Repressors with Coevolutionary Cues Facilitates Toggle Switches with a Master Reset
(Oxford University Press, 2019-04-24) Dimas, R. P.; Jiang, Xian-Li; Alberto de la Paz, Jose; Morcos, Faruck; Chan, C. T. Y.; Jiang, Xian-Li; Alberto de la Paz, Jose; Morcos, Faruck
Engineering allosteric transcriptional repressors containing an environmental sensing module (ESM) and a DNA recognition module (DRM) has the potential to unlock a combinatorial set of rationally designed biological responses. We demonstrated that constructing hybrid repressors by fusing distinct ESMs and DRMs provides a means to flexibly rewire genetic networks for complex signal processing. We have used coevolutionary traits among LacI homologs to develop a model for predicting compatibility between ESMs and DRMs. Our predictions accurately agree with the performance of 40 engineered repressors. We have harnessed this framework to develop a system of multiple toggle switches with a master OFF signal that produces a unique behavior: each engineered biological activity is switched to a stable ON state by different chemicals and returned to OFF in response to a common signal. One promising application of this design is to develop living diagnostics for monitoring multiple parameters in complex physiological environments and it represents one of many circuit topologies that can be explored with modular repressors designed with coevolutionary information. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
Inhibiting the Integrated Stress Response Pathway Prevents Aberrant Chondrocyte Differentiation Thereby Alleviating Chondrodysplasia
(eLife Sciences Publications Ltd) Wang, C.; Tan, Z.; Niu, B.; Tsang, K. Y.; Tai, A.; Chan, W. C. W.; Lo, R. L. K.; Leung, K. K. H.; Dung, N. W. F.; Itoh, N.; Zhang, Michael Q.; Chan, D.; Cheah, K. S. E.; 0000-0002-7408-1830 (Zhang, MQ); 161723084 (Zhang, MQ); Zhang, Michael Q.
The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders. © Wang et al.
Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis
(Cell Press, 2018-11-05) Shimada, Issei S.; Hwang, Sun-Hee; Somatilaka, Bandarigoda N.; Wang, Xin; Skowron, Patryk; Kim, Jiwoong; Kim, Min; Shelton, John M.; Rajaram, Veena; Xuan, Zhenyu; Taylor, Michael D.; Mukhopadhyay, Saikat; Xuan, Zhenyu
Sonic hedgehog (Shh) determines cerebellar granule cell (GC) progenitor proliferation and medulloblastoma pathogenesis. However, the pathways regulating GC progenitors during embryogenesis before Shh production by Purkinje neurons and their roles in tumorigenesis remain unclear. The cilium-localized G-protein-coupled receptor Gpr161 suppresses Shh-mediated signaling in the neural tube. Here, by deleting Gpr161 in mouse neural stem cells or GC progenitors, we establish Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. Irrespective of Shh production in the cerebellum, Gpr161 deletion increased downstream activity of the Shh pathway by restricting Gli3-mediated repression, causing more extensive generation and proliferation of GC progenitors. Moreover, earlier deletion of Gpr161 during embryogenesis increased tumor incidence and severity. GC progenitor overproduction during embryogenesis from Gpr161 deletion was cilium dependent, unlike normal development. Low GPR161 expression correlated with poor survival of SHH subtype medulloblastoma patients. Gpr161 restricts GC progenitor production by preventing premature and Shh-dependent pathway activity, highlighting the importance of basal pathway suppression in tumorigenesis.
Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella Enterica and Escherichia Coli
(2018-08-24) Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu; Reitzer, Lawrence J.; Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu; Reitzer, Lawrence J.
Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica Delta cyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by L-cysteine, D-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm formation. Cysteine inhibits several enzymes of amino acid synthesis; therefore, increasing cysteine concentrations could increase the levels of the inhibited enzymes. This inhibition implies that control of intracellular cysteine levels, which is the immediate product of sulfide assimilation, can affect several pathways and coordinate metabolism. For these and other reasons, cysteine and sulfide concentrations must be controlled, and this work shows that cysteine catabolism contributes to this control.
Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions
(2016-01-20) Li, Yi; Mendiratta, Saurabh; Ehrhardt, Kristina; Kashyap, Neha; White, Michael A.; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L)
CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wildtype alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.
Prediction of High-Risk Types of Human Papillomaviruses Using Statistical Model of Protein “Sequence Space”
(Hindawi Publishing Corporation, 2015-03-21) Wang, C.; Hai, Y.; Liu, X.; Liu, N.; Yao, Y.; He, P.; Dai, Qi
Discrimination of high-risk types of human papillomaviruses plays an important role in the diagnosis and remedy of cervical cancer. Recently, several computational methods have been proposed based on protein sequence-based and structure-based information, but the information of their related proteins has not been used until now. In this paper, we proposed using protein "sequence space" to explore this information and used it to predict high-risk types of HPVs. The proposed method was tested on 68 samples with known HPV types and 4 samples without HPV types and further compared with the available approaches. The results show that the proposed method achieved the best performance among all the evaluated methods with accuracy 95.59% and F1-score 90.91%, which indicates that protein "sequence space" could potentially be used to improve prediction of high-risk types of HPVs.
CRISPR-Based Self-Cleaving Mechanism for Controllable Gene Delivery in Human Cells
(Oxford University Press, 2014-12-18) Moore, Richard; Spinhirne, Alec; Lai, Michael J.; Preisser, Samantha; Li, Yi; Kang, Taek; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 2012076942‏ (Bleris, L)
Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery.;
Distinct and Predictive Histone Lysine Acetylation Patterns at Promoters, Enhancers, and Gene Bodies
(Genetics Society America, 2014-11-01) Rajagopal, Nisha; Ernst, Jason; Ray, Pradipta; Wu, Jie; Zhang, Michael Q.; Kellis, Manolis; Ren, Bing; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ (Zhang, MQ); Zhang, Michael Q.
In eukaryotic cells, histone lysines are frequently acetylated. However, unlike modifications such as methylations, histone acetylation modifications are often considered redundant. As such, the functional roles of distinct histone acetylations are largely unexplored. We previously developed an algorithm RFECS to discover the most informative modifications associated with the classification or prediction of mammalian enhancers. Here, we used this tool to identify the modifications most predictive of promoters, enhancers, and gene bodies. Unexpectedly, we found that histone acetylation alone performs well in distinguishing these unique genomic regions. Further, we found the association of characteristic acetylation patterns with genic regions and association of chromatin state with splicing. Taken together, our work underscores the diverse functional roles of histone acetylation in gene regulation and provides several testable hypotheses to dissect these roles.
Genome Wide Mapping of Foxo1 Binding-Sites in Murine T Lymphocytes
(Elsevier Inc, 2014-08-01) Liao, Will; Ouyang, Weiming; Zhang, Michael Q.; Li, Ming O.; Zhang, Michael Q.
The Forkhead box O (Foxo) family of transcription factors has a critical role in controlling the development, differentiation, and function of T cells. However, the direct target genes of Foxo transcription factors in T cells have not been well characterized. In this study, we focused on mapping the genome wide Foxo1-binding sites in naÃ¯ve CD4(+) T cells, CD8(+) T cells, and Foxp3(+) regulatory T (Treg) cells. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq), we identified Foxo1 binding sites that were shared among or specific to the three T cell populations. Here we describe the experiments, quality controls, as well as the deep sequencing data. Part of the data analysis has been published by Ouyang W et al. in Nature 20121] and Kim MV et al. in Immunity 20132], and the associated data set were uploaded to NCBI Gene Expression Omnibus.;
Nucleosome Eviction and Multiple Co-Factor Binding Predict Estrogen-Receptor-Alpha-Asociated Long-Range Interactions
(Oxford University Press, 2014-04-29) He, C.; Wang, X.; Zhang, Michael Q.; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ (Zhang, MQ); Zhang, Michael Q.
Many enhancers regulate their target genes via long-distance interactions. High-throughput experiments like ChIA-PET have been developed to map such largely cell-type-specific interactions between cis-regulatory elements genome-widely. In this study, we integrated multiple types of data in order to reveal the general hidden patterns embedded in the ChIA-PET data. We found characteristic distance features related to promoter-promoter, enhancer-enhancer and insulator-insulator interactions. Although a protein may have many binding sites along the genome, our hypothesis is that those sites that share certain open chromatin structure can accommodate relatively larger protein complex consisting of specific regulatory and 'bridging' factors, and may be more likely to form robust long-range deoxyribonucleic acid (DNA) loops. This hypothesis was validated in the estrogen receptor alpha (ERa) ChIA-PET data. An efficient classifier was built to predict ERa-associated long-range interactions solely from the related ChIP-seq data, hence linking distal ERa-dependent enhancers to their target genes. We further applied the classifier to generate additional novel interactions, which were undetected in the original ChIA-PET paper but were validated by other independent experiments. Our work provides a new insight into the long-range chromatin interactions through deeper and integrative ChIA-PET data analysis and demonstrates DNA looping predictability from ordinary ChIP-seq data.
Miror: A Method for Cell-Type Specific MicroRNA Occupancy Rate Prediction
(Royal Soc Chemistry, 2014-03-13) Xie, Peng; Liu, Yu; Li, Yanda; Zhang, Michael Q.; Wang, Xiaowo; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
MicroRNA (miRNA) regulation is highly cell-type specific. It is sensitive to both the miRNA-mRNA relative abundance and the competitive endogenous RNA (ceRNA) effect. However, almost all existing miRNA target prediction methods neglected the influence of the cellular environment when analyzing miRNA regulation effects. In this study, we proposed a method, MIROR (miRNA Occupancy Rate predictor), to predict miRNA regulation intensity in a given cell type. The major considerations were the miRNA-mRNA relative abundance and the endogenous competition between different mRNA species. The output of MIROR is the predicted miRNA occupancy rates of each target site. The predicted results significantly correlated with Ago HITS-CLIP experiment that indicated miRNA binding intensities. When applied to the analysis of the breast invasive carcinoma dataset, MIROR identified a number of differentially regulated miRNA-mRNA pairs with significant miRNA occupancy rate changes between tumor and normal tissues. Many of the predictions were supported by previous research studies, including the ones without a significant change in the mRNA expression level. These results indicate that MIROR provides a novel strategy to study the miRNA differential regulation in different cell types.
Assembly and Validation of Versatile Transcription Activator-Like Effector Libraries
(Nature Publishing Group, 2014-05-06) Li, Yi; Ehrhardt, Kristina; Zhang, Michael Q.; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 0000 0001 1707 1372 (Zhang, MQ); 2012076942‏ (Bleris, L); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
The ability to perturb individual genes in genome-wide experiments has been instrumental in unraveling cellular and disease properties. Here we introduce, describe the assembly, and demonstrate the use of comprehensive and versatile transcription activator-like effector (TALE) libraries. As a proof of principle, we built an 11-mer library that covers all possible combinations of the nucleotides that determine the TALE-DNA binding specificity. We demonstrate the versatility of the methodology by constructing a constraint library, customized to bind to a known p53 motif. To verify the functionality in assays, we applied the 11-mer library in yeast-one-hybrid screens to discover TALEs that activate human SCN9A and miR-34b respectively. Additionally, we performed a genome-wide screen using the complete 11-mer library to confirm known genes that confer cycloheximide resistance in yeast. Considering the highly modular nature of TALEs and the versatility and ease of constructing these libraries we envision broad implications for high-throughput genomic assays. ;
Complete Genome Analysis of Three Acinetobacter Baumannii Clinical Isolates in China for Insight into the Diversification of Drug Resistance Elements
(2013-06-24) Zhu, Lingxiang; Yan, Zhongqiang; Zhang, Zhaojun; Zhou, Qiming; Zhou, Jinchun; Wakeland, Edward K.; Fang, Xiangdong; Xuan, Zhenyu; Shen, Dingxia; Li, Quan-Zhen; Xuan, Zhenyu
Background: The emergence and rapid spreading of multidrug-resistant Acinetobacter baumannii strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant A. baumannii strains from Beijing using next-generation sequencing technology. Methodologies/Principal Findings: Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant blaOXA₂₃ and tetracycline (tet) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the ATPase gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying tra-locus and blaOXA₂₃ island, can be either inserted into one of the tniB gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both blaOXA-23 and blaOXA-58 genes on its genome. Besides, an Acinetobacter lwoffii adeABC efflux element was found inserted into the ATPase position in BJAB0715. Conclusions: Our comparative analysis on currently completed Acinetobacter baumannii genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.
OLego: Fast and Sensitive Mapping of Spliced mRNA-Seq Reads Using Small Seeds
(Oxford University Press, 2013-04) Wu, Jie; Anczuk©w, Olga; Krainer, Adrian R.; Zhang, Michael Q.; Zhang, Chaolin; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
A crucial step in analyzing mRNA-Seq data is to accurately and efficiently map hundreds of millions of reads to the reference genome and exon junctions. Here we present OLego, an algorithm specifically designed for de novo mapping of spliced mRNA-Seq reads. OLego adopts a multiple-seed-and-extend scheme, and does not rely on a separate external aligner. It achieves high sensitivity of junction detection by strategic searches with small seeds (âˆ¼14 nt for mammalian genomes). To improve accuracy and resolve ambiguous mapping at junctions, OLego uses a built-in statistical model to score exon junctions by splice-site strength and intron size. Burrows-Wheeler transform is used in multiple steps of the algorithm to efficiently map seeds, locate junctions and identify small exons. OLego is implemented in C++ with fully multithreaded execution, and allows fast processing of large-scale data. We systematically evaluated the performance of OLego in comparison with published tools using both simulated and real data. OLego demonstrated better sensitivity, higher or comparable accuracy and substantially improved speed. OLego also identified hundreds of novel micro-exons (<30 nt) in the mouse transcriptome, many of which are phylogenetically conserved and can be validated experimentally in vivo. OLego is freely available at http://zhanglab.c2b2.columbia.edu/index.php/OLego.;
FastDMA: An Infinium Humanmethylation450 Beadchip Analyzer
(2013-09-05) Wu, D.; Gu, J.; Zhang, Michael Q.; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
DNA methylation is vital for many essential biological processes and human diseases. Illumina Infinium HumanMethylation450 Beadchip is a recently developed platform studying genome-wide DNA methylation state on more than 480,000 CpG sites and a few CHG sites with high data quality. To analyze the data of this promising platform, we developed FastDMA which can be used to identify significantly differentially methylated probes. Besides single probe analysis, FastDMA can also do region-based analysis for identifying the differentially methylated region (DMRs). A uniformed statistical model, analysis of covariance (ANCOVA), is used to achieve all the analyses in FastDMA. We apply FastDMA on three large-scale DNA methylation datasets from The Cancer Genome Atlas (TCGA) and find many differentially methylated genomic sites in different types of cancer. On the testing datasets, FastDMA shows much higher computational efficiency than current tools. FastDMA can benefit the data analyses of large-scale DNA methylation studies with an integrative pipeline and a high computational efficiency. The software is freely available via http://bioinfo.au.tsinghua.edu.cn/software/fastdma/.
Synthetic Mammalian Transgene Negative Autoregulation
(2013-06-04) Shimoga, Vinay; White, Jacob T.; Li, Yi; Sontag, Eduardo; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L); 2012076942‏ (Bleris, L)
Biological networks contain overrepresented small-scale topologies, typically called motifs. A frequently appearing motif is the transcriptional negative-feedback loop, where a gene product represses its own transcription. Here, using synthetic circuits stably integrated in human kidney cells, we study the effect of negative-feedback regulation on cell-wide (extrinsic) and gene-specific (intrinsic) sources of uncertainty. We develop a theoretical approach to extract the two noise components from experiments and show that negative feedback results in significant total noise reduction by reducing extrinsic noise while marginally increasing intrinsic noise. We compare the results to simple negative regulation, where a constitutively transcribed transcription factor represses a reporter protein. We observe that the control architecture also reduces the extrinsic noise but results in substantially higher intrinsic fluctuations. We conclude that negative feedback is the most efficient way to mitigate the effects of extrinsic fluctuations by a sole regulatory wiring.;
ModuleRole: A Tool for Modulization, Role Determination and Visualization in Protein-Protein Interaction Networks
(Public Library of Science, 2014-05-01) Li, GuiPeng; Li, Ming; Zhang, YiWei; Wang, Dong; Li, Rong; Guimera, Roger; Gao, Juntao Tony; Zhang, Michael Q.; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
Rapidly increasing amounts of (physical and genetic) protein-protein interaction (PPI) data are produced by various high-throughput techniques, and interpretation of these data remains a major challenge. In order to gain insight into the organization and structure of the resultant large complex networks formed by interacting molecules, using simulated annealing, a method based on the node connectivity, we developed ModuleRole, a user-friendly web server tool which finds modules in PPI network and defines the roles for every node, and produces files for visualization in Cytoscape and Pajek. For given proteins, it analyzes the PPI network from BioGRID database, finds and visualizes the modules these proteins form, and then defines the role every node plays in this network, based on two topological parameters Participation Coefficient and Z-score. This is the first program which provides interactive and very friendly interface for biologists to find and visualize modules and roles of proteins in PPI network. It can be tested online at the website http://www.bioinfo.org/modulerole/index.php, which is free and open to all users and there is no login requirement, with demo data provided by "User Guide'' in the menu Help. Non-server application of this program is considered for high-throughput data with more than 200 nodes or user's own interaction datasets. Users are able to bookmark the web link to the result page and access at a later time. As an interactive and highly customizable application, ModuleRole requires no expert knowledge in graph theory on the user side and can be used in both Linux and Windows system, thus a very useful tool for biologist to analyze and visualize PPI networks from databases such as BioGRID. Availability: ModuleRole is implemented in Java and C, and is freely available at http://www.bioinfo.org/modulerole/index.php. Supplementary information (user guide, demo data) is also available at this website. API for ModuleRole used for this program can be obtained upon request.
Transcripts for Combined Synthetic MicroRNA and Gene Delivery
(2013-06-26) Kashyap, Neha; Pham, Bich; Xie, Zhen; Bleris, Leonidas; 0000 0001 2535 9739 (Bleris, L)
MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are produced from the same transcript by the microprocessor and the spliceosome protein complex respectively. The details and exact timing of the processing events have implications for downstream RNA interference (RNAi) efficiency and mRNA stability. Here we engineer and study in mammalian cells a range of synthetic intragenic miRNAs co-expressed with their host genes. Furthermore, we study transcripts which carry the target of the miRNA, thereby emulating a common regulation mechanism. We perform fluorescence microscopy and flow cytometry to characterize the engineered transcripts and investigate the properties of the underlying biological processes. Our results shed additional light on miRNA and pre-mRNA processing but importantly provide insight into engineering transcripts customized for combined delivery and use in synthetic gene circuits.;
Quantitative liver-specific protein fingerprint in blood: A signature for hepatotoxicity
(2014-01-14) Hu, Z.; Lausted, C.; Yoo, Hyuntae; Yan, X.; Brightman, A.; Chen, J.; Wang, W.; Bu, X.; Hood, L.
We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane- bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks.
Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells
(Oxford University Press, 2013-12-16) Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q.; 0000 0001 1707 1372 (Zhang, MQ); 99086074‏ ‎(Zhang, MQ); Zhang, Michael Q.
DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.;

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