Name: 3d multicolor dna fish tool to study nuclear architecture in human primary cells
Uploaded: 2020-01-25T13:00:00
Description: Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

The authors acknowledge the technical assistance of the INGM Imaging Facility (Istituto Nazionale di Genetica Molecolare &#34;Romeo ed Enrica Invernizzi&#34; (INGM), Milan, Italy), in particular C. Cordiglieri, for assistance during 3D multicolor DNA FISH images acquisition. This work has been supported by the following grants to B.B.: EPIGEN Italian flagship program, Association Fran&#231;aise contre les Myopathies (AFM-Telethon, grant nr 18754) and Giovani Ricercatori, Italian Ministry of Health (GR-2011-02349383). This work has been supported by the following grant to F.M.: Fondazione Cariplo (Bando Giovani, grant nr 2018-0321).

1. DNA probe preparation and labelling procedures with nick translation
<ol>
	<li>Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH2O, check by electrophoresis on an agarose gel and quantify.</li>
	<li>Perform nick translation on 1.5&#8722;2 &#181;g of DNA from step 1.1 in a final volume of 50 &#181;L by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1.<br ...

<ol>
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The current method describes a step by step protocol to perform 3D multicolor DNA FISH on a wide range of human primary cells. Although DNA FISH is a technology in wide use, 3D multicolor DNA FISH on preserved 3D interphase nuclei is still difficult to perform in many laboratories, mainly due to the characteristics of the samples used23,24.
Probe nick translation is a fundamental step for successful 3D multicolor DNA FISH; many differe...

Higher eukaryotes need to systematically condense and compact a huge amount of genetic information in the minute 3D space of the nucleus1,2,3,4. Today, we know that the genome is spatially ordered in compartments and topologically associated domains5 and that the multiple levels of DNA folding generate contacts between different genomic regions that may involve chromatin loop formation6,7. The 3D dynamic looping of chromatin can influence many different biological processes such as transcription8,9, differentiation and development10,11, DNA repair12,13, while its perturbations are involved in various diseases14,15,16 and developmental defects15,17,18.
Many approaches have been developed to study the 3D genome organization. Chromosome conformation capture-based technologies (C-technologies, 3C, 4C, 5C, Hi-C and derivatives) have been developed to study genome organization in fixed cells3,4,19,20. Such approaches are based on the ability to capture the contact frequencies between genomic loci in physical proximity. C-technologies, depending on their complexity, catch the global 3D genome organization and nuclear topology of a cell population3,4,19,20. Nevertheless, 3D interactions are dynamic in time and space, highly variable between individual cells consisting of multiplex interactions, and are extensively heterogenous21,22.
3D multicolor DNA fluorescence in situ hybridization (FISH) is a technique that allows the visualization of specific genomic loci at a single cell level, enabling direct investigation of the 3D nuclear architecture in a complementary manner to C-technologies. It represents a technology currently used to unambiguously validate C-results. 3D multicolor DNA FISH uses fluorescently labeled probes complementary to the genomic loci of interests. The use of different fluorophores and suitable microscopy equipment allow contemporary visualization of multiple targets within the nuclear space23,24. In recent years, FISH has been combined with technological advances in microscopy to obtain the visualization of fine-scale structures at high resolution25,26 or with CRISPR-Cas approaches for the visualization of the nucleic acids in live imaging27,28. Despite wide adoption, the 3D multicolor DNA FISH approach is still considered difficult in many laboratories because the biological material used must be adapted.
Here, we provide a comprehensive protocol for 3D multicolor DNA FISH (from cell/probe preparation to data analysis) applicable to a wide range of human primary cells, enabling the visualization of multiple genomic loci and preserving the 3D structure of nuclei. In order to study nuclear architecture, the 3D structure of nuclei must be preserved. For this reason, contrasting from other existing protocols29,30,31, we avoid the use of an alcohol gradient and the storage of the coverslips in alcohol that can affect chromatin structure32. The method is adapted from preserved 3D DNA FISH protocols24,33 to be applied to a wide range of human primary cells, both isolated ex vivo or cultured in vitro. There are permeabilization and deproteinization parameters for different nuclear morphology and cytological characteristics (e.g., different degrees of nuclear compaction, cytoskeleton abundance)34. These parameters are often generally described in other protocols24,33, without providing a clear discrimination of the procedure within different cell types. Furthermore, we developed a specific tool named NuCL&#949;D (nuclear contacts locator in 3D)16, providing principles for data analysis that will improve the 3D proximity between different loci and their nuclear topological distribution within the nuclear space in an automated way.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin 488</td>
			<td>DBA</td>
			<td>DI7488</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin 11 d-UTP</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0081</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverlsips</td>
			<td>Marienfeld</td>
			<td>117500</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 d-UTP</td>
			<td>GE Healthcare</td>
			<td>PA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D21490</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acids single strand from salmon testes</td>
			<td>Sigma</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate (powder)</td>
			<td>Santa Cruz</td>
			<td>sc-203917A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin 11 d-UTP</td>
			<td>Roche</td>
			<td>11093088910</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21969-035 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>18010-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>AMPD1</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (C-G-A-T)</td>
			<td>Euroclone</td>
			<td>BL0423A/C/G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644.2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Hyclone</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30109</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutammine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Human</td>
			<td>Sigma</td>
			<td>I9278-5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate-buffered saline)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennycillin/Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Biorad</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>PhasePrep BAC DNA Kit</td>
			<td>Sigma</td>
			<td>NA0100-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Gel Extraction &#38; PCR Purification Combo Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K220001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2288</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbercement</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>631-0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidina Alexa fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>S21374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate</td>
			<td>Sigma</td>
			<td>1110379026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P9416-100mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

The method of 3D multicolor DNA FISH described in this article allows contemporary visualization of different genomic loci within preserved 3D nuclei (Figure 1B). This protocol permits the measurement of distances between alleles, and different genomic loci in order to evaluate their spatial proximity, and to assess their location within the nuclear space (e.g., loci distance from the centroid or the periphery of the nuclei)16. However, there are many...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

3D multicolor DNA FISH enables the visualization of multiple genomic loci within preserved nuclei to ambiguously define their reciprocal interaction and localization at single cell level. 3D multicolor DNA FISH allows the direct investigation of the nuclear architecture. In general, it works in conjunction with chromosome capture-based technologies making the technique an available tool for C data validation within single cells.Demonstrating the procedure will be Federica Marasca. She's a postdoc in my laboratory. Begin by incubating the nick translation mix in a thermal mixer at 16 degrees Celsius according to the length of the starting DNA material.At the end of the incubation, check the size of the probes on a 2.2%agarose gel. For each DNA FISH experiment, precipitate the appropriate quantity of probe according to the starting DNA material from which the probes were produced in 150 microliters of double distilled water, 20 micrograms of unlabeled salmon sperm DNA, 3.5 micrograms of species specific Cot-1 DNA, three volumes of 100%ethanol, and a 1/10th volume of three molar sodium acetate for one hour at minus 80 degrees Celsius. At the end of the incubation, centrifuge the sample at maximum speed for one hour at four degrees Celsius.After discarding the supernatant, wash the pellet two times with 500 microliters of 70%ethanol. After the second wash, resuspend the pellet in two microliters of 100%formamide and shake the probe for 30 minutes at 40 degrees Celsius before adding an equal volume of 4X saline sodium citrate in 20%dextran sulfate. For the fixation pre-treatment and permeabilization of small cells with small nuclei and a low amount of cytoplasm, first add two times 10 to the sixth cells in 200 microliters of PBS directly onto glass coverslips in individual wells of a 24-well plate and allow the cells to settle for 30 minutes at room temperature.At the end of the incubation, quickly replace the PBS with 300 microliters of freshly prepared 4%paraformaldehyde supplemented with 0.1%Tween 20. After 10 minutes, wash the fixed cells three times in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature. After the last wash, permeabilize the T cells with 300 microliters of 0.5%TPBS for 10 minutes at room temperature before treating the cells with 250 microliters per well of RNAse cocktail diluted 1:100 in PBS for one hour at 37 degrees Celsius.At the end of the incubation, rinse the cells in 300 microliters of PBS and add 300 microliters of 20%glycerol in PBS to each well with an overnight incubation at four degrees Celsius. The next morning, place the glass on dry ice for 15 to 30 seconds to freeze the cells before gradually thawing the samples at room temperature and soaking them in 300 microliters of 20%glycerol in PBS for 30 seconds. After freeze/thawing the cells three more times as just demonstrated, wash the samples in 300 microliters of 0.5%TPBS for five minutes at room temperature, followed by two washes in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature.After the last wash, treat the cells with 300 microliters of 0.1 normal hydrochloric acid for 12 minutes at room temperature, followed by two rinses in 300 microliters of saline sodium citrate. Then fix the samples overnight in 300 microliters of 50%formamide in 2X saline sodium citrate at room temperature. For the fixation, pre-treatment and permeabilization of large cells with a high amount of cytoplasm, fix, pre-treat and permeabilize the cells similarly to as demonstrated for human primary T cells and treat the samples with 300 microliters of 0.0025%pepsin in 0.01 normal hydrochloric acid for a few seconds up to five minutes.Observe the cells under an optical microscope and stop the reaction with two five-minute washes in 300 microliters of 50 millimolar magnesium chloride as soon as the nuclei are free from the cytoplasm but the nucleoli are still visible and intact. For 3D multicolor DNA FISH, denature the hybridization probes at 80 degrees Celsius for five minutes and immediately place the probes on ice. Load the probes onto a clean microscope slide and place one coverslip of cells onto each drop of probe.Seal the coverslip edges with rubber cement. When the cement has dried completely, denature the samples on a 75 degrees Celsius heating block. After four minutes, hydridize the samples at 37 degrees Celsius overnight in a metallic box floating in a water bath.The next morning, peel off the rubber cement and with the slides immersed in 2X saline sodium citrate carefully lift off the coverslips. Place each coverslip into individual wells of a six-well plate containing two milliliters of fresh saline sodium citrate per well for two five-minute washes at 37 degrees Celsius with shaking at 90 revolutions per minute. At the end of the incubation, rinse the coverslips briefly in two milliliters of 0.2%Tween 20 in 4X saline sodium citrate.For 3D multicolor DNA FISH detection, transfer the coverslips into individual wells of a new 24-well plate and block any nonspecific binding in 300 microliters of blocking buffer for 20 minutes at 37 degrees Celsius and 20 revolutions per minute. At the end of the incubation, treat the samples with the appropriate concentration of anti-digoxigenin and/or streptavidin diluted in blocking buffer for 35 minutes in a dark and wet chamber at 37 degrees Celsius. At the end of the incubation, transfer the samples to individual wells of a six-well plate and wash the samples three times in two milliliters of 0.2%Tween 20 and 4X saline sodium citrate for five minutes per wash with shaking at 90 revolutions per minute.After the last wash, equilibrate the samples in two milliliters of PBS before transferring the coverslips to a new 24-well plate for post-fixing in 300 microliters of 2%formaldehyde in PBS per well for two minutes at room temperature. Wash the fixed cells five times briefly in 300 microliters of PBS, followed by staining with DAPI diluted at one nanogram per milliliter in 300 microliters of PBS for five minutes at room temperature. Wash the coverslips five more times in PBS and mount the samples with an appropriate mounting medium.Then acquire 3D images with a microscope system. 3D multicolor DNA FISH allows the contemporary visualization of different genomic loci within preserved 3D nuclei. For DNA probe preparation, a probe size of less than 200 base pairs ensures a successful 3D multicolor DNA FISH procedure.Suboptimal DNA FISH probes produced by nick translation can be partially or over digested. Over digested probes will result in a nonspecific signal due to a loss of specificity in the hybridization and a consequent increase of the background. For human primary T lymphocytes and small cells with small nuclei and a low amount of cytoplasm, a 12-minute 0.1 normal hydrochloric acid treatment is recommended to promote nuclei accessibility to the DNA probes and to preserve the nuclear integrity.Cytoplasmic pepsin digestion is not needed to obtain a DNA FISH good signal in small cells. For human primary myoblasts and cells that have large nuclei and abundant cytoplasm, however, the pepsinization step is fundamental. A short and suboptimal cytoskeleton pepsinization will hamper the entry of the probe into the nuclei ending in the absence of a DNA FISH signal.However, if the cells are over pepsinized, the nuclei will not remain intact losing their 3D structure. A successful 3D multicolor DNA FISH will result in the presence of signal within the nuclei. I suggest working with fresh biological material, testing probes before performing the 3D multicolor DNA FISH, preparing fresh solutions and being precise with incubation times and the temperatures.Remember that formamide and HCL are hazardous substances and should always be used in a chemical fume hood with appropriate disposable materials.

3d-multicolor-dna-fish-tool-to-study-nuclear-architecture-human

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

A Pathway Association Study Tool for GWAS Analyses of Metabolic Pathway Information

Recently, a new implementation of a previously described method for interpreting genome-wide association study (GWAS) data using metabolic pathway analysis has been developed and released. The Pathway Association Study Tool (PAST) was developed to address concerns with user-friendliness and slow-running analyses. This new user-friendly tool has been released on Bioconductor and Github. In testing, PAST ran analyses in less than one hour that previously required twenty-four or more hours. In this article, we present the protocol for using either the Shiny application or the R console to run PAST.

By running the Pathway Association Study Tool (PAST), either through the Shiny application or through the R console, researchers can gain a deeper understanding of the biological meaning of their genome-wide association study (GWAS) results by investigating the metabolic pathways involved.