Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Summary

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Abstract

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)¹. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT². Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Introduction

Telomere protects chromosomal ends from aberrant fusion and degradation. Mammalian telomere is composed of G-rich hexameric repeats, TTAGGG, and shelterin complexes. The telomere repeat sequence of the nematode is similar to those of mammals (TTAGGC). Most eukaryotes utilize telomerase to add telomere repeats to their chromosomal ends. However, 10 – 15% of cancer cells utilize telomerase independent mechanism, known as Alternative Lengthening of Telomeres (ALT)³. Previously, we reported that telomere repeats and its associated sequences, named as TALT, were amplified in the telomeres of telomerase mutant lines that survived critical sterility².

Telomere length was measured by quantitative PCR or by Southern blot, which provides average length of total telomeres^4,5,6,7. Read count of telomere repeat in whole genome sequencing data is also an indicator of total telomere contents⁸. Although Single TElomere Length Analysis (STELA) could provide the length of a single telomere, it cannot provide spatial information of telomeres⁹. While POT-1::mCherry reporter protein provides the spatial information of telomeres in vivo, it cannot represent lengths of double-stranded telomeres, as POT-1 is a single-strand telomere binding protein¹⁰.

While aforementioned methods provide the averaged information of repetitive sequences, fluorescence in situ hybridization (FISH) allows to observe the amount and spatial pattern of individual sequences of interest on a chromosomal scale. Instead of purification of DNA, tissues or cells are fixed to preserve the native spatial information in FISH. Thus, FISH is a both quantitative and qualitative tool for observation of individual repeat sequences, such as telomere repeats.

This protocol provides an efficient method for simultaneous detection of both telomere and other repeats based on improvements from previously described methods ^11,12. C. elegans larvae or adults are multicellular organism with highly differentiated cells. The heterogeneity of cells impedes on the quantitative analysis of a large number of telomere spots. To maximize the number of cells analyzed, embryos are isolated and spread on the polylysine-coated slides for FISH. In addition, this protocol can also be combined with immunofluorescence.

As a proof that the protocol works, we show that it is possible to observe and quantify two different repetitive sequences. DNA probe against TALT1 was generated with simple PCR incorporating digoxigenin-dUTP. Then this TALT1 probe and fluorescence-labeled telomere PNA probe were hybridized simultaneously. Subsequently, digoxigenin was detected by canonical immunofluorescence methods. We present here the representative images where TALT1 colocalized with the telomere in trt-1 survivors.

Protocol

1. Labeling Probes with Digoxigenin-dUTP by PCR Perform PCR labeling with 10x dNTP mix containing digoxigenin-dUTP as previously described13. Purify PCR product with spin-column purification according to manufacturer's instruction. If the probe is shorter than 200 bp, remove free digoxigenin-dUTP with spin-column chromatography from the reaction mixture rather than spin-column purification. 2. Preparing Polylysine Coated Slide…

Representative Results

It was previously reported that ALT survivor can emerge from telomerase-deficient mutant, trt-1(ok410), in low frequency by replicating internally localized 'Template of ALT' (TALT) sequences for telomere maintenance2. Using PNA probe, we were able to visualize telomeres in the dissected gonads (Figure 2A). The faint telomere signal was detected both in trt-1(ok410) and ALT survivor. The fuzzy signal was overlapped only with DAPI, sugg…

Discussion

The main advantage of our protocol is the simplicity of the procedure without noticeable damage to the morphology of cellular structure. Several steps were optimized for C. elegans FISH in this protocol. The critical steps for successful FISH include labeling of probes, fixation of embryos and penetration. Digoxigenin-dUTP labeling method provides an easy-to-use labeling method by PCR or nick-translation. To label long target sequence, nick-translation is preferred. In this case, the probes should be digested wi…

Materials

PNA probe	PANAGENE	custom order
Anti-Digoxigenin-Fluorescein, Fab fragments	Roche	11207741910	use 1:200 diluted in PBST
Digoxigenin-dUTP	Roche	11573152910
Bovine serum albumin	SIGMA-ALDRICH	A-7906
Paraformaldehyde	SIGMA-ALDRICH	P-6148	prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.
Vectashield	Vector Laboratories	H-1200
Hybridizaiton solution			3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA
Hybridizaiton wash solution			2X SSC, 50% formamide
Formamide	BIONEER	C-9012	toxic
Methanol	Carlo Erba
Acetone	Carlo Erba
Heparin	SIGMA-ALDRICH	H3393	make 10 mg/ml for stock solution
Dextran sulfate	SIGMA-ALDRICH	67578
10X PBS			For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO
PBST			1X PBS, 0.1% tween-20
Polysorbate 20	SIGMA-ALDRICH	P-2287	Commercial name is Tween-20
Poly-L-Lysine solution (0.1 % w/v)	SIGMA-ALDRICH	P-8920	prepare fresh 0.01 % w/v solution before use
M9			3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L
Bleaching solution			20% sodium hypochlorite, 0.5 M KOH
Antibody buffer			1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)
Blocking solution			Antibody buffer with 5% bovine serum albumin (BSA)
illustra Microspin G-50	GE healthcare	27-53310-01
20X SSC			To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0
2X SSCT			2X SSC, 0.1 % tween-20
10x digoxigenin-dUTP mix			1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP
PCR purification columns	Cosmo genetech	CMR0112
Glass cleaner / ULTRA CLEAN	Dukssan pure chemicals	8AV721
Multi-well glass slide	MP biomedicals	96041205
Nematode growth media			to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.
Levamisole	SIGMA-ALDRICH	196142
Razor	Feather	blade No. 11
Rnase A	Enzynomics
BSA	SIGMA-ALDRICH	A7906
Equipments
Confocal microsope	Zeiss	LSM 510	EC Plan-Neofluar 100x was used as objective lens.
Dry block / aluminum block	Labtech	LBH-T03	Set temperature to 80℃
Humid chamber			Plastic box filled with paper towel soaked in DW
Image Analysis Software	Dr. Peter Landsdorp	TFL-telo	http://www.flintbox.com/public/project/502