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Author: Maxwell Robert Mumbach
Publisher:
ISBN:
Category :
Languages : en
Pages :
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If you unpack all of the DNA in a single cell and lay it out flat, it's about six feet long -- and yet it can all fit into a micron-scale nucleus. Not only does it fit, but it's folded within the nucleus precisely to allow for the same DNA sequence across cell types to be specifically accessed for distinct functions. My thesis sought to investigate how DNA is folded within a cell, and how this folding impacts gene regulation and human disease. There are currently two main approaches to measure DNA folding: imaging and chromosome conformation capture (3C) methods. Although imaging is incredibly powerful, it is limited in throughput and resolution. 3C relies on a proximity ligation -- generating a hybrid "contact" fragment of two pieces of DNA that are near each other in 3D space. While there are many 3C-based methods, arguably the most transformative is Hi-C, which applies next-generation sequencing to a 3C library and has identified multiple DNA organizational features within a cell. However, Hi-C is inherently restricted due to sampling space: all possible 3D interactions are being measured. Because of this, billions of sequencing reads are needed to start observing confident DNA interactions over background. To combat this sampling space issue, we developed a method called HiChIP, which combines chromatin immunoprecipitation (ChIP) with Hi-C, allowing for a directed view of long-range contacts associated with a protein factor of interest. HiChIP improves the yield of contact reads by over 10-fold and lowers the input requirement over 100-fold relative to that of previous methods. As a proof of principle, we performed HiChIP on cohesin, a protein complex that has been reported to stabilize chromatin loops. Cohesin HiChIP identified a similar set of loops as Hi-C maps with increased signal-to-background and ten-fold less sequencing. The HiChIP technology we developed allowed, for the first time, DNA folding to be measured in disease-relevant patient samples. Precisely measuring how the immune system operates is central to our understanding of autoimmune disease and cancer. However, due to technological limitations the principles that govern regulatory 3D interactions in disease-relevant patient samples have been incompletely understood. This gap in understanding is particularly problematic for interpreting the molecular functions of inherited risk factors for common human diseases, which reside in intergenic enhancers or other non-coding DNA features in up to 90% of cases. We therefore performed HiChIP on the enhancer and promoter-associated histone mark H3K27ac in sorted T cell subsets and examined DNA interactions to map autoimmune disease variants present in non-coding regions and identify which genes they contact with. We found that the majority of disease-associated enhancers interact with genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for these autoimmune disease SNPs. While protein-factors are well-known to regulate the 3D structure of the genome, less is known regarding RNA. However, specific RNAs, such as XIST and HOTTIP, have been reported to utilize the topology of the genome in order to carry out their functions. We therefore sought to modify the HiChIP method to enrich for DNA interactions focused around an RNA of interest (HiChIRP). Interestingly, we identified many RNAs that engage in different types of 3D interactions. For example, we found that the RNA lncRNA-EPS binds to the boundaries of topological domains, a layer of DNA organization, to then engage in 3D interactions and regulate target genes contained within the domain. In summary, my thesis provided an unprecedented view of the 3D genome through the development of novel genomic tools for the research community. Importantly, these tools enable 3D measurements: (1) efficiently with less sequencing needed, (2) in disease-relevant systems that could not be assayed previously, and (3) focused on regulatory RNAs, whose roles in DNA structure are incompletely understood. I have then applied these tools across many collaborations to better understand the roles of DNA folding in gene regulation and disease.
Author: Maxwell Robert Mumbach
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
If you unpack all of the DNA in a single cell and lay it out flat, it's about six feet long -- and yet it can all fit into a micron-scale nucleus. Not only does it fit, but it's folded within the nucleus precisely to allow for the same DNA sequence across cell types to be specifically accessed for distinct functions. My thesis sought to investigate how DNA is folded within a cell, and how this folding impacts gene regulation and human disease. There are currently two main approaches to measure DNA folding: imaging and chromosome conformation capture (3C) methods. Although imaging is incredibly powerful, it is limited in throughput and resolution. 3C relies on a proximity ligation -- generating a hybrid "contact" fragment of two pieces of DNA that are near each other in 3D space. While there are many 3C-based methods, arguably the most transformative is Hi-C, which applies next-generation sequencing to a 3C library and has identified multiple DNA organizational features within a cell. However, Hi-C is inherently restricted due to sampling space: all possible 3D interactions are being measured. Because of this, billions of sequencing reads are needed to start observing confident DNA interactions over background. To combat this sampling space issue, we developed a method called HiChIP, which combines chromatin immunoprecipitation (ChIP) with Hi-C, allowing for a directed view of long-range contacts associated with a protein factor of interest. HiChIP improves the yield of contact reads by over 10-fold and lowers the input requirement over 100-fold relative to that of previous methods. As a proof of principle, we performed HiChIP on cohesin, a protein complex that has been reported to stabilize chromatin loops. Cohesin HiChIP identified a similar set of loops as Hi-C maps with increased signal-to-background and ten-fold less sequencing. The HiChIP technology we developed allowed, for the first time, DNA folding to be measured in disease-relevant patient samples. Precisely measuring how the immune system operates is central to our understanding of autoimmune disease and cancer. However, due to technological limitations the principles that govern regulatory 3D interactions in disease-relevant patient samples have been incompletely understood. This gap in understanding is particularly problematic for interpreting the molecular functions of inherited risk factors for common human diseases, which reside in intergenic enhancers or other non-coding DNA features in up to 90% of cases. We therefore performed HiChIP on the enhancer and promoter-associated histone mark H3K27ac in sorted T cell subsets and examined DNA interactions to map autoimmune disease variants present in non-coding regions and identify which genes they contact with. We found that the majority of disease-associated enhancers interact with genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for these autoimmune disease SNPs. While protein-factors are well-known to regulate the 3D structure of the genome, less is known regarding RNA. However, specific RNAs, such as XIST and HOTTIP, have been reported to utilize the topology of the genome in order to carry out their functions. We therefore sought to modify the HiChIP method to enrich for DNA interactions focused around an RNA of interest (HiChIRP). Interestingly, we identified many RNAs that engage in different types of 3D interactions. For example, we found that the RNA lncRNA-EPS binds to the boundaries of topological domains, a layer of DNA organization, to then engage in 3D interactions and regulate target genes contained within the domain. In summary, my thesis provided an unprecedented view of the 3D genome through the development of novel genomic tools for the research community. Importantly, these tools enable 3D measurements: (1) efficiently with less sequencing needed, (2) in disease-relevant systems that could not be assayed previously, and (3) focused on regulatory RNAs, whose roles in DNA structure are incompletely understood. I have then applied these tools across many collaborations to better understand the roles of DNA folding in gene regulation and disease.
Author: Institute of Medicine
Publisher: National Academies Press
ISBN: 0309224187
Category : Science
Languages : en
Pages : 354
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Book Description
Technologies collectively called omics enable simultaneous measurement of an enormous number of biomolecules; for example, genomics investigates thousands of DNA sequences, and proteomics examines large numbers of proteins. Scientists are using these technologies to develop innovative tests to detect disease and to predict a patient's likelihood of responding to specific drugs. Following a recent case involving premature use of omics-based tests in cancer clinical trials at Duke University, the NCI requested that the IOM establish a committee to recommend ways to strengthen omics-based test development and evaluation. This report identifies best practices to enhance development, evaluation, and translation of omics-based tests while simultaneously reinforcing steps to ensure that these tests are appropriately assessed for scientific validity before they are used to guide patient treatment in clinical trials.
Author: Bruce Alberts
Publisher:
ISBN: 9780815332183
Category : Cytology
Languages : en
Pages : 0
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Author: Nils Stein
Publisher: Springer
ISBN: 3319925288
Category : Science
Languages : en
Pages : 394
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Book Description
This book presents an overview of the state-of-the-art in barley genome analysis, covering all aspects of sequencing the genome and translating this important information into new knowledge in basic and applied crop plant biology and new tools for research and crop improvement. Unlimited access to a high-quality reference sequence is removing one of the major constraints in basic and applied research. This book summarizes the advanced knowledge of the composition of the barley genome, its genes and the much larger non-coding part of the genome, and how this information facilitates studying the specific characteristics of barley. One of the oldest domesticated crops, barley is the small grain cereal species that is best adapted to the highest altitudes and latitudes, and it exhibits the greatest tolerance to most abiotic stresses. With comprehensive access to the genome sequence, barley’s importance as a genetic model in comparative studies on crop species like wheat, rye, oats and even rice is likely to increase.
Author: Manfred Heinlein
Publisher: Humana
ISBN: 9781071607145
Category : Science
Languages : en
Pages : 490
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Book Description
This book provides a compendium of state-of-the-art methods for the labeling, detection, and purification of RNA and RNA-protein complexes and thereby constitutes an important toolbox for researchers interested in understanding the complex roles of RNA molecules in development, signaling, and disease. Beginning with a section on in situ detection of RNA molecules using FISH techniques, the volume continues with parts exploring in vivo imaging of RNA transport and localization, imaging and analysis of RNA uptake and transport between cells, identification and analysis of RNA-binding proteins, guide RNAs in genome editing, as well as other specific analytical techniques. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, RNA Tagging: Methods and Protocols serves as a vital reference for researchers looking to further the increasingly important research in RNA biology.
Author: Mahesh Uttamchandani
Publisher: Methods in Molecular Biology
ISBN: 9781493982424
Category : Science
Languages : en
Pages : 274
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Book Description
Author: National Research Council
Publisher: National Academies Press
ISBN: 0309168503
Category : Science
Languages : en
Pages : 240
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Book Description
Biologists communicate to the research community and document their scientific accomplishments by publishing in scholarly journals. This report explores the responsibilities of authors to share data, software, and materials related to their publications. In addition to describing the principles that support community standards for sharing different kinds of data and materials, the report makes recommendations for ways to facilitate sharing in the future.
Author: Marian Bemer
Publisher: Humana
ISBN: 9781493984510
Category : Science
Languages : en
Pages : 0
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Book Description
This volume provides a comprehensive collection of protocols that can be used to study plant chromatin structure and composition. Chapters divided into three sections detail the profiling of chromatin features in relation to epigenetic regulation, investigate the interaction between chromatin modifications and gene regulation, and explore the 3D spatial organization of the chromatin inside the nucleus. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Plant Chromatin Dynamics: Methods and Protocols aims to ensure successful results in the further study of this vital field.
Author: Mousumi Debnath
Publisher: Springer Science & Business Media
ISBN: 9048132614
Category : Medical
Languages : en
Pages : 527
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Book Description
A rapid development in diverse areas of molecular biology and genetic engineering resulted in emergence of variety of tools. These tools are not only applicable to basic researches being carried out world over, but also exploited for precise detection of abnormal conditions in plants, animals and human body. Although a basic researcher is well versed with few techniques used by him/her in the laboratory, they may not be well acquainted with methodologies, which can be used to work out some of their own research problems. The picture is more blurred when the molecular diagnostic tools are to be used by physicians, scientists and technicians working in diagnostic laboratories in hospitals, industry and academic institutions. Since many of them are not trained in basics of these methods, they come across several gray areas in understanding of these tools. The accurate application of molecular diagnostic tools demands in depth understanding of the methodology for precise detection of the abnormal condition of living body. To meet the requirements of a good book on molecular diagnostics of students, physicians, scientists working in agricultural, veterinary, medical and pharmaceutical sciences, it needs to expose the reader lucidly to: Give basic science behind commonly used tools in diagnostics Expose the readers to detailed applications of these tools and Make them aware the availability of such diagnostic tools The book will attract additional audience of pathologists, medical microbiologists, pharmaceutical sciences, agricultural scientists and veterinary doctors if the following topics are incorporated at appropriate places in Unit II or separately as a part of Unit-III in the book. Molecular diagnosis of diseases in agricultural crops Molecular diagnosis of veterinary diseases. Molecular epidemiology, which helps to differentiate various epidemic strains and sources of disease outbreaks. Even in different units of the same hospital, the infections could be by different strains of the same species and the information becomes valuable for infection control strategies. Drug resistance is a growing problem for bacterial, fungal and parasitic microbes and the molecular biology tools can help to detect the drug resistance genes without the cultivation and in vitro sensitivity testing. Molecular diagnostics offers faster help in the selection of the proper antibiotic for the treatment of tuberculosis, which is a major problem of the in the developing world. The conventional culture and drug sensitivity testing of tuberculosis bacilli is laborious and time consuming, whereas molecular diagnosis offers rapid drug resistant gene detection even from direct clinical samples. The same approach for HIV, malaria and many more diseases needs to be considered. Molecular diagnostics in the detection of diseases during foetal life is an upcoming area in the foetal medicine in case of genetic abnormalities and infectious like TORCH complex etc. The book will be equally useful to students, scientists and professionals working in the field of molecular diagnostics.
Author: M.R.S. Rao
Publisher: Springer
ISBN: 9811052034
Category : Medical
Languages : en
Pages : 336
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Book Description
This contributed volume offers a comprehensive and detailed overview of the various aspects of long non-coding RNAs and discusses their emerging significance. Written by leading experts in the field, it motivates young researchers around the globe, and offers graduate and postgraduate students fascinating insights into genes and their regulation in eukaryotes and higher organisms.