Name: profiling dna replication timing using zebrafish as an in vivo model system
Uploaded: 2018-04-30T12:30:00
Description: Watch this Scientific Journal Video about Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System at JoVE.com

This work was supported by National Institute of General Medical Sciences of the National Institutes of Health through grants 5P20GM103636-02 (including Flow Cytometry core support) and 1R01GM121703, as well as awards from the Oklahoma Center for Adult Stem Cell Research.

All animals were handled in strict accordance with protocols approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.
1. Setting up adult zebrafish for breeding
<ol>
	<li>Use a large cohort of adult male and female zebrafish of a single strain for breeding. There are small differences in the genetic makeup of zebrafish of even a single strain, use a large cohort to ensure results are representative of the genetic variability of the population and not ...

<ol>
	<li>Rhind, N., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing.">DNA replication timing.</a> Cold Spring Harb Perspect Biol. 5 (8), a010132 (2013).</li><li>Pope, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Rivera-Mulia, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+replication+timing+and+gene+expression+during+lineage+specification+of+human+pluripotent+stem+cells.">Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.</a> Genome Res. 25 (8), 1091-1103 (2015).</li><li>Hiratani, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+reorganization+of+replication+domains+during+embryonic+stem+cell+differentiation.">Global reorganization of replication domains during embryonic stem cell differentiation.</a> PLoS Biol. 6 (10), e245 (2008).</li><li>Hiratani, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+dynamics+of+replication+timing+revealed+by+in+vitro+models+of+mouse+embryogenesis.">Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.</a> Genome Res. 20 (2), 155-169 (2010).</li><li>Koren, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+relationship+of+DNA+replication+timing+to+different+forms+of+human+mutation+and+variation.">Differential relationship of DNA replication timing to different forms of human mutation and variation.</a> Am J Hum Genet. 91 (6), 1033-1040 (2012).</li><li>Ryba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+developmental+control+of+replication-timing+domains+in+pediatric+acute+lymphoblastic+leukemia.">Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.</a> Genome Res. 22 (10), 1833-1844 (2012).</li><li>Sima, J., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+correlations:+replication+timing+and+mutational+landscapes+during+cancer+and+genome+evolution.">Complex correlations: replication timing and mutational landscapes during cancer and genome evolution.</a> Curr Opin Genet Dev. 25, 93-100 (2014).</li><li>Veldman, M. B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Link, B. A., Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+Model+for+Development.">Zebrafish as a Model for Development.</a> Sourcebook of Models for Biomedical Research. , 103-112 (2008).</li><li>Siefert, J. C., Clowdus, E. A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+in+the+early+embryonic+development+of+aquatic+animal+species.">Cell cycle control in the early embryonic development of aquatic animal species.</a> Comp Biochem Physiol C Toxicol Pharmacol. 178, 8-15 (2015).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicol Sci. 86 (1), 6-19 (2005).</li><li>Dooley, K., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+human+disease.">Zebrafish: a model system for the study of human disease.</a> Curr Opin Genet Dev. 10 (3), 252-256 (2000).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> J Clin Invest. 122 (7), 2337-2343 (2012).</li><li>Siefert, J. C., Georgescu, C., Wren, J. D., Koren, A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+during+development+anticipates+transcriptional+programs+and+parallels+enhancer+activation.">DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation.</a> Genome Res. 27 (8), 1406-1416 (2017).</li><li>Koren, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+human+DNA+replication+timing.">Genetic variation in human DNA replication timing.</a> Cell. 159 (5), 1015-1026 (2014).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. 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Zebrafish provide a new and unique in vivo model system to study DNA replication timing. When timed matings are performed as detailed in this experimental protocol, thousands of embryos can be collected in a single day for experiments. These embryos develop synchronously through precisely timed and distinctly characterized stages of development. Zebrafish can be easily and accurately staged by morphology using a stereomicroscope, as zebrafish embryos develop externally and are optically clear. This protocol deta...

For cells to successfully divide, they must first accurately and faithfully replicate their entire genome. Genome duplication occurs in a reproducible pattern, known as the DNA replication timing program1. DNA replication timing is correlated with chromatin organization, epigenetic marks, and gene expression2,3. Changes in replication timing occur throughout development, and are significantly related to transcriptional programs and alterations to chromatin marks and organization4,5. Furthermore, replication timing is correlated with mutational frequencies, and changes in timing are observed in various types of cancer6,7,8. Despite these observations, the mechanisms and determinants of replication timing establishment and regulation are still largely unknown, and the role it plays in development and disease is undetermined. In addition, until recently the genome-wide replication timing changes that occur throughout vertebrate development had only been examined in cell culture models.
Zebrafish, Danio rerio, are well suited to study replication timing in vivo during development, as a single mating pair can yield of hundreds of embryos that develop rapidly with many similarities to mammalian development9,10. Furthermore, throughout zebrafish development, there are changes to the cell cycle, chromatin organization, and transcriptional programs that share relationships with DNA replication timing11. Zebrafish are also an excellent genetic model, as they are particularly amenable to manipulation by transgenesis, mutagenesis, and targeted mutations, and genetic screens have identified many genes required for vertebrate development12. Therefore, zebrafish can be used to identify genes involved in replication timing establishment and maintenance and to observe the effects of deregulating replication timing on vertebrate development. Transgenic lines can also be used to assess replication timing from individual cell types isolated at different developmental timepoints or in disease conditions. Importantly, there are various zebrafish models of human disease that can be used to investigate the role of replication timing in disease formation and progression9,13,14.
Recently, the first replication timing profiles were generated from zebrafish, establishing it as a model system to study replication timing in vivo15. To accomplish this, cells were collected from zebrafish embryos at multiple stages of development and in a cell type isolated from adult zebrafish. Cells were then sorted by FACS (fluorescence-activated cell sorting) based on DNA content to isolate G1 and S phase populations. Using the G1 sample as a copy number control, copy number variations in S phase populations were determined and used to infer relative replication timing16. Changes in replication timing can then be directly compared between different developmental samples and cell types and this was used to determine changes in replication timing that occur in vivo throughout vertebrate development. This method offers several advantages over other genomic methods, chiefly that it does not require labeling with thymidine analogs or immunoprecipitation of DNA4,6.
Here is detailed the protocols to profile genome-wide DNA replication timing at high-resolution in zebrafish. These protocols have been used to determine relationships with genomic and epigenetic features in the zebrafish genome, as well as profiling changes in these relationships that occur throughout development. These protocols are also easily adapted to study changes in replication timing in mutant strains of zebrafish and in disease models. Additionally, these methods provide a foundation that can be expanded upon to study replication timing in specific cell types, by first sorting out the individual cell types from the zebrafish. The zebrafish can serve as an excellent in vivo model system to study replication timing and to ultimately reveal the biological functions of this important epigenetic trait.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>EMD Millipore</td>
			<td>MMX00701</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>10165921001</td>
			<td>protease solution</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>KOPTEC</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9647-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9844</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Santa Cruz</td>
			<td>sc-24950</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform</td>
			<td>Sigma</td>
			<td>P3803-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>J.T.Baker</td>
			<td>3470</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Ambion</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Quanit-iT</td>
			<td>Invitrogen</td>
			<td>Q33130</td>
			<td>Reagents for fluorescence-based DNA quantification</td>
		</tr>
		<tr>
			<td>Covaris AFA microTUBE</td>
			<td>Covaris</td>
			<td>520045</td>
			<td>specialized tube for sonication</td>
		</tr>
		<tr>
			<td>Covaris E220 Sonicator</td>
			<td>Covaris</td>
			<td>E220</td>
			<td>focused ultrasonicator</td>
		</tr>
		<tr>
			<td>Agilent 4200 Tapestation</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>automated electrophoresis machine</td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td>Reagents for automated electrophoresis machine</td>
		</tr>
		<tr>
			<td>NEBNext Ultra DNA Library Prep Kit for Illumina</td>
			<td>NEB</td>
			<td>Cat#E7370L</td>
			<td>DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 1)</td>
			<td>NEB</td>
			<td>Cat#E7335S</td>
			<td>multiplex oligos for DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 2)</td>
			<td>NEB</td>
			<td>Cat#E7500S</td>
			<td>additional multiplex oligos for DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Library Quant Kit for Illumina</td>
			<td>NEB</td>
			<td>E7630L</td>
			<td>quantification kit for library preparation</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>magnetic beads</td>
		</tr>
		<tr>
			<td>Illumina HiSeq 2500</td>
			<td>Illumina</td>
			<td>SY&#8211;401&#8211;2501</td>
			<td>next generation DNA sequencing platform</td>
		</tr>
		<tr>
			<td>40 &#181;m Falcon Nylon Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Disposable Petri Dish 100 x 25 mm</td>
			<td>VWR</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 mL Syringe for Nichiryo Model 8100</td>
			<td>VWR</td>
			<td>89078-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click Tubes, 1.7 mL, Natural Color</td>
			<td>Denville Scientific</td>
			<td>C2170 (1001002)</td>
			<td>Dnase/Rnase free</td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Bottles</td>
			<td>VWR</td>
			<td>16650-022</td>
			<td>Low-Density Polyethylene, Wide Mouth</td>
		</tr>
		<tr>
			<td>Strainer</td>
			<td>VWR</td>
			<td>470092-440</td>
			<td>6.9 cm, fine mesh</td>
		</tr>
		<tr>
			<td>Corssing tank</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td>individual breeding tank</td>
		</tr>
		<tr>
			<td>iSpawn</td>
			<td>Techniplast</td>
			<td>N/A</td>
			<td>large breeding tank</td>
		</tr>
		<tr>
			<td>FACSAria II</td>
			<td>BD biosciences</td>
			<td>N/A</td>
			<td>cell sorting machine</td>
		</tr>
		<tr>
			<td>Wild M5a steromicroscope</td>
			<td>Wild Heerbrugg</td>
			<td>N/A</td>
			<td>dissecting microscope</td>
		</tr>
		<tr>
			<td>Qubit 3 Fluorometer</td>
			<td>Thermo Scientific</td>
			<td>Q33216</td>
			<td>quantitative fluorescence-based method for determining DNA concentration</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>version 2017a</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab Statistics Toolbox</td>
			<td>Mathworks</td>
			<td>version 11.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab Curve Fitting Toolbox</td>
			<td>Mathworks</td>
			<td>version 3.5.5</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling dna replication timing using zebrafish as an in vivo model system

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Using published replication timing data, representative replication timing profiles and quality control measures are provided15. The initial steps of processing involve aligning the sequencing data to the genome, calculating read length and genome coverage statistics, and filtering low quality, unpaired, and PCR duplicate reads. Read statistics for a typical zebrafish sequencing sample are shown in Figure 2. After filtering, read count...

Watch this Scientific Journal Video about Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System at JoVE.com

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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