Name: high-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4c-seq)
Uploaded: 2018-10-05T13:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq at JoVE.com

This work was supported by NIAMS (R01AR065523).

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The authors declare that they have no competing financial interests.

4C results have the potential to reveal chromatin interactions that can identify previously unknown regulatory elements and/or target genes that are important in a specific biological context24,25,26. However, technical hurdles may limit the data obtained from these experiments. PCR bias stemming from over-amplification of template in 4C protocols is likely. This protocol addresses this issue by utilizing qPCR to determine the o...

The identification of regulatory elements for gene expression has been facilitated by the Encyclopedia of DNA Elements (ENCODE) Project that comprehensively annotated functional activity for 80% of the human genome1,2. The identification of the sites for in vivo transcription factor binding, DNaseI hypersensitivity, and epigenetic histone and DNA methylation modifications in individual cell types paved the way for the functional analyses of candidate regulatory elements for target gene expression. Armed with these findings, we are faced with the challenge of determining the functional interconnectivity between regulatory elements and genes. Specifically, what is the relationship between a given target gene and its enhancer(s)? The chromatin conformation capture (3C) method directly addresses this question by identifying physical, and likely functional, interactions between a region of interest and candidate interacting sequences through captured events in fixed chromatin3. As our understanding of chromatin interactions has increased, however, it is clear that the investigation of preselected candidate loci is insufficient to provide a complete understanding of gene-enhancer interactions. For example, ENCODE used the high-throughput chromosomal conformation capture carbon copy (5C) method to examine a small portion of the human genome (1%, pilot set of 44 loci) and reported complex interconnectivity of the loci. Genes and enhancers with identified interactions averaged 2&#8211;4 different interacting partners, many of which were hundreds of kilobases away in linear space4. Further, Li et al. used Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) to analyze whole-genome promoter interactions and found that 65% of RNA polymerase II binding sites were involved in chromatin interactions. Some of these interactions resulted in large, multi-gene complexes spanning hundreds of kilobases of genomic distance and containing, on average, 8-9 genes each5. Together, these findings highlight the need for unbiased whole-genome methods for interrogating chromatin interactions. Some of these methods are reviewed in Schmitt et al.6.
More recent methods for chromatin conformation capture studies coupled with next-generation sequencing (Hi-C and 4C-seq) enable the discovery of unknown sequences interacting with a region of interest6. Specifically, circular chromosome conformation capture with next-generation sequencing (4C-seq) was developed to identify loci interacting with a sequence of interest in an unbiased manner7 by sequencing DNA from captured chromatin proximal to the region of interest in 3D space. Briefly, chromatin is fixed to preserve its native protein-DNA interactions, cleaved with a restriction enzyme, and subsequently ligated under dilute conditions to capture biologically relevant &#34;tangles&#34; of interacting loci (Figure 1). The cross links are reversed to remove the protein, thus leaving the DNA available for additional cleavage with a second restriction enzyme. A final ligation generates smaller circles of interacting loci. Primers to the sequence of interest are then used to generate an amplified library of unknown sequences from the circularized fragments, followed by downstream next generation sequencing.
The protocol presented here, which focuses on sample preparation, makes two major alterations to existing 4C-seq methods8,9,10,11,12. First, it uses a qPCR-based method to empirically determine the optimal number of amplification cycles for 4C-seq library preparation steps and thus mitigates the potential for PCR bias stemming from over-amplification of libraries. Second, it uses an additional restriction digest step in an effort to reduce the uniformity of known &#34;bait&#34; sequences that hinders accurate base-calling by the sequencing instrument and, hence, maximizes the unique, informative sequence in each read. Other protocols circumvent this issue by pooling many (12-15)8 4C-seq libraries with different bait sequences and/or restriction sites, a volume of experiments which may not be achievable by other laboratories. The modifications presented here allow a small number of experiments, samples, and/or replicates to be indexed and pooled into a single lane.

<table><tbody><tr><td>HindIII</td><td>NEB</td><td>R0104S</td><td></td></tr><tr><td>CviQI</td><td>NEB</td><td>R0639S</td><td></td></tr><tr><td>DNA oligonucleotide primers</td><td>IDT</td><td></td><td>To be designed by the reader</td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Fisher Scientific</td><td>06-443-19</td><td></td></tr><tr><td>1.7 mL microcentrifuge tubes</td><td>MidSci</td><td>AVSS1700</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher</td><td>14190-136</td><td></td></tr><tr><td>Formaldehyde, methanol free</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Nutator</td><td>VWR</td><td>15172-203</td><td></td></tr><tr><td>Glycine</td><td>JT Baker</td><td>4059-00</td><td></td></tr><tr><td>Benchtop centrifuge</td><td></td><td></td><td></td></tr><tr><td>Refrigerated microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>ED2SS</td><td></td></tr><tr><td>20% SDS solution</td><td>Sigma-Aldrich</td><td>05030</td><td></td></tr><tr><td>Trypan Blue</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Glass slides</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Cover slips</td><td>VWR</td><td>16004-094</td><td></td></tr><tr><td>Light microscope</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Alfa Aesar</td><td>A16046</td><td></td></tr><tr><td>Shaking heat block</td><td></td><td></td><td></td></tr><tr><td>2 M Tris-HCl</td><td>Quality Biological</td><td>351-048-101</td><td></td></tr><tr><td>Proteinase K</td><td>NEB</td><td>P8107S</td><td></td></tr><tr><td>Phenol:chloroform:isoamyl alcohol (25:24:1)</td><td>Sigma-Aldrich</td><td>P2069</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>M5661</td><td></td></tr><tr><td>20 mg/mL glycogen</td><td>Thermo Fisher</td><td>R0561</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-223</td><td></td></tr><tr><td>Nuclease-free water</td><td>Fisher Scientific</td><td>MT-46-000-CM</td><td></td></tr><tr><td>qPCR cycler</td><td>Thermo Fisher</td><td>4453536</td><td></td></tr><tr><td>qPCR plates</td><td>Thermo Fisher</td><td>4309849</td><td></td></tr><tr><td>Thermocycler</td><td>Thermo Fisher</td><td>4375786</td><td></td></tr><tr><td>PCR strip tubes</td><td>MidSci</td><td>AVSST-FL</td><td></td></tr><tr><td>1 M Magnesium chloride</td><td>Quality Biological</td><td>351-033-721</td><td></td></tr><tr><td>Dithiothreotol</td><td>Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>Adenosine triphosphate</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A6013</td><td></td></tr><tr><td>RNase A</td><td>Thermo Fisher</td><td>EN0531</td><td></td></tr><tr><td>Qiaquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>MinElute PCR Purification kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Expand Long Template PCR System</td><td>Sigma-Aldrich</td><td>11681834001</td><td></td></tr><tr><td>dNTP mix</td><td>Thermo Fisher</td><td>R0191</td><td></td></tr><tr><td>SYBR Green I</td><td>Sigma-Aldrich</td><td>S9430</td><td></td></tr><tr><td>ROX</td><td>BioRad</td><td>172-5858</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>End-It DNA End-Repair Kit</td><td>Lucigen</td><td>ER0720</td><td></td></tr><tr><td>LigaFast Rapid DNA Ligation System</td><td>Promega</td><td>M8221</td><td></td></tr><tr><td>SYBR Safe</td><td>Thermo Fisher</td><td>S33102</td><td></td></tr><tr><td>Taq Polymerase</td><td>NEB</td><td>M0267S</td><td></td></tr><tr><td>UltraSieve Agarose</td><td>IBI Scientific</td><td>IB70054</td><td></td></tr><tr><td>Qiaquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr></tbody></table>

high-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4c-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

Primary human keratinocytes were isolated from 2&#8211;3 discarded neonatal foreskins, pooled, and cultured in KSFM supplemented with 30 &#181;g/mL bovine pituitary extract, 0.26 ng/mL recombinant human epidermal growth factor, and 0.09 mM calcium chloride (CaCl2) at 37 &#176;C, 5% carbon dioxide. The cells were split into two flasks, and one flask was differentiated by the addition of CaCl2 to a final concentration of 1.2 mM for 72 h. 107 cells each from ...

Watch this Scientific Journal Video about High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq at JoVE.com

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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10.2K Views.  Washington University School of Medicine. The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Video: High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial patterns of gene expression. Embryoid bodies (EBs) derived from pluripotent stem cells have been a popular model to study the differentiation of the main three germ layers and to define regulatory circuits during cell fate specification. Although it is well-known that tissue-specific enhancers play an important role in these networks by interacting with promoters, assigning them to their relevant target genes still remains challenging. To make this possible, quantitative approaches are needed to study enhancer-promoter contacts and their dynamics during development. Here, we adapted a 4C method to define enhancers and their contacts with cognate promoters in the EB differentiation model. The method uses frequently cutting restriction enzymes, sonication, and a nested-ligation-mediated PCR protocol compatible with commercial DNA library preparation kits. Subsequently, the 4C libraries are subjected to high-throughput sequencing and analyzed bioinformatically, allowing detection and quantification of all sequences that have contacts with a chosen promoter. The resulting sequencing data can also be used to gain information about the dynamics of enhancer-promoter contacts during differentiation. The technique described for the EB differentiation model is easy to implement.

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

We report the application of quantitative chromosome conformation capture followed by high-throughput sequencing in embryoid bodies generated from embryonic stem cells. This technique allows to identify and quantitate the contacts between putative enhancers and promoter regions of a given gene during embryonic stem cell differentiation.

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

A Comprehensive Approach to Studying the Promoter Interactome in Scarce Cells

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Author Spotlight: An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have revolutionized the study of gene regulation. A lack of standardization in the analysis of the highly dimensional datasets generated by these techniques has made reproducibility difficult to achieve, leading to discrepancies in the published, processed data. Part of this problem is due to the diverse range of bioinformatic tools available for the analysis of these types of data. Secondly, a number of different bioinformatic tools are required sequentially to convert raw data into a fully processed and interpretable output, and these tools require varying levels of computational skills. Furthermore, there are many options for quality control that are not uniformly employed during data processing. We address these issues with a complete assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) upstream pipeline (CATCH-UP), an easy-to-use, Python-based pipeline for the analysis of bulk ChIP-seq and ATAC-seq datasets from raw fastq files to visualizable bigwig tracks and peaks calls. This pipeline is simple to install and run, requiring minimal computational knowledge. The pipeline is modular, scalable, and parallelizable on various computing infrastructures, allowing for easy reporting of methodology to enable reproducible analysis of novel or published datasets.

ATAC-seq and ChIP-seq allow detailed investigation of gene regulation; however, processing these data types is challenging and often inconsistent between research groups. We present CATCH-UP: an easy-to-use computational pipeline that allows standardized and reproducible data processing and analysis of new and published ATAC/ChIP-seq datasets.

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Biology

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

Summary

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Chapters in this video

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

Capturing Chromosome Conformation Across Length Scales

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq