Fine mapping of chromatin structure in Drosophila melanogaster embryos using micrococcal nuclease

Abstract

The structure of chromatin in eukaryotes exerts significant influences on many DNA related processes, including transcription, replication, recombination and repair. A useful tool for mapping chromatin structure is micrococcal nuclease (MNase), which induces double-strand breaks within nucleosome linker regions, and with more extensive digestion, single-strand nicks within the nucleosome itself. Many studies, carried out largely with microbes and cell cultures, have used MN ase to determine the positions of nucleosomes within a region of DNA to identify dynamic changes induced during gene regulation. To measure similar processes in a developmental context, we turned to a tractable model system, the Drosophila embryo. Here we describe a protocol that enables MN ase mapping of the enhancer chromatin structure in the embryo, and show how it can be used to identify structural changes on a cis-regulatory element targeted by the Knirps repressor.

Introduction

In eukaryotes, DNA is packaged into chromatin, which imposes a natural barrier for various DNA-related processes including transcription and replication.¹ Eukaryotic cells deploy multiple mechanisms to alter the positions of nucleosomes in order to enhance the accessibility of DNA.² In the case of transcription, regulatory factors recruit chromatin remodeling complexes such as SWI/SNF and ISWI to remove, transfer or slide a nucleosome along the DNA template.³ Alternatively, histone modifying enzymes can also be recruited by transcription factors to acetylate, methylate, phosphorylate, monoubiquitinate, sumoylate or ADP-ribosylate histones, or carry out reverse reactions.² Such modifications can directly affect inter- and intranucleosomal interactions, or recruit downstream effectors to modulate the structure of nucleosomes.^4,5 Thus, measurement of chromatin structure and changes associated with transcriptional activation and repression is essential for understanding of gene regulatory mechanisms.

Chromatin immunoprecipitation (ChIP) is used to analyze histone occupancy and modifications, and can pinpoint whether a specific histone protein or modification is associated with a particular piece of DNA; however the method does not provide direct information on overall chromatin structure.⁶ Nuclease digestion does provide such structural information; DNase I has been used to map hypersensitive sites created by the loss or remodeling of nucleosomes, providing an indication of an active regulatory function.⁷ Alternatively, fine structure mapping can be obtained by the use of micrococcal nuclease (MNase). This nuclease can induce double-stranded breaks within the nucleosome linker region, providing an indication of whether a region of interest is protected within a nucleosome.⁸ In standard MNase experiments, chromatin accessibility is determined in a qualitative manner by Southern blot using radioactive-labeled probes against the DNA of interest.⁸ To obtain a more quantitative picture of nucleosome structure, MNase digested chromatin can be analyzed by quantitative PCR using overlapping primers. This technique has been used to determine chromatin structure in yeast, as well as tissue-culture cells.^9–11 Current studies are also analyzing chromatin from multicellular organisms, but no protocols are available for application of MNase mapping in embryos. Here, we describe a protocol we developed and optimized for use with the Drosophila blastoderm embryo, providing a method to study chromatin structure in a developmental setting.

Collection of Drosophila Embryos and Formaldehyde Crosslinking

To ensure reproducibility, it is critical to standardize the amount of starting materials. For each MNase experiment, we start with approximately 500 mg of 2–4 hr old embryos collected from ten laying bottles. Embryos are rinsed free of yeast paste and any adults by washing through coarse nylon mesh into a large collection basket, dechorinated by bleaching for 2.5 min, rinsed in stream of tap water for 1 min and blotted dry from below using Kimwipes.

Embryos are then transferred into a 50 ml Corning tube and crosslinked by vigorously shaking for 30 min in 10 ml crosslinking buffer (3% formaldehyde in 50 mM HEPES, pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl) and 30 ml heptane. Heptane is added to the crosslinking mixture to increase the permeability of the embryos. Embryos are centrifuged at 1,000 rpm (167 g) for 1 min in a clinical centrifuge. The top organic layer and the crosslinking buffer are carefully removed so as not to lose too many embryos, and the reaction is quenched by addition of 25 ml crosslinking stop buffer (0.125 M glycine in PBS (137 mM NaCl, 4.3 mM Na₂HPO₄, 1.4 mM NaH₂PO₄)-Triton-X-100 0.01%). After gently mixing at room temperature for 30 min, the mixture is centrifuged at 1,000 rpm (167 g) for 1 min, and the supernatant is discarded. At this point, the embryos can be flash frozen by immersing the Corning tube in liquid nitrogen for later processing, or the material can be directly processed as described below.

Preparation of Nuclei and MNase Digestion

Embryos are washed three times in 10 ml PBS-Triton-X-100 (0.01%) with gentle rocking for 5 min. The crosslinked embryos are resuspended in 5 ml homogenization buffer (10 mM HEPES, pH 7.6, 0.3 M sucrose, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT, 1 pellet of protease inhibitor (cOmplete mini, Roche, Catalog No. 04 693 124 001) per 10 ml) per 0.5 gram of embryos and disrupted in a Dounce homogenizer 10 times with a loose pestle, and 15 times with a tight pestle. To collect the nuclei, the lysate is transferred to a 15 ml Corning tube and centrifuged at 1,000 rpm (167 g) for 10 min at 4°C in a clinical centrifuge. The supernatant is removed and the pellet is resuspended in 1 ml MNase digestion buffer (10 mM Tris-HCl, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine). After transferring to a 1.7 ml microcentrifuge tube, the material is centrifuged at 14,000 rpm (16,000 g) for 10 min at 4°C. The supernatant is removed and discarded, and the pellet is flash frozen until ready for use.

Nuclei from 500 mg embryos are suspended in 1 ml of MNase digestion buffer in a microcentrifuge tube. Half of the volume is reserved to serve as the undigested control. 100 U MNase (USB, Catalog No. 70196Y) is added to the rest of the sample and incubated at 37°C for 30 min, long enough to digest most of the chromosomal DNA into mononucleosomal DNA. The sample is chilled on ice for 10 min and then EDTA is added to the final concentration of 10 mM to quench the digestion. The digested nuclei are collected by centrifuging at 14,000 rpm (16,000 g) for 10 min at 4°C in a microcentrifuge.

Dissolving the Nuclei and Reversing Crosslinking and DNA Purification

Digested nuclei are washed twice in 1 ml sonication buffer (10 mM HEPES pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate). For each wash, the pellet is completely resuspended, and then recollected by centrifuging at 14,000 rpm (16,000 g) for 10 min at 4°C using a microcentrifuge. Nuclei are then suspended by addition of 500 µl sonication buffer and sonicating three times at output 4, 60% duty cycle and 20 pulses with a Branson sonicator, using a microtip. The material is then centrifuged at 14,000 rpm (16,000 g) for 20 min to pellet the nuclear membrane; the supernatant contains the mononucleosomes.

The crosslinking of both the digested sample and the undigested control is reversed by addition of SDS to a final concentration of 1%, and NaCl is added to a final concentration of 0.2 M. The samples are heated at 65°C overnight to reverse the crosslinking. RNA within the samples is removed by incubating tubes at 37°C for 30 min with 10 µg RNAse (Roche, Catalog No. 10109142001) per reaction. To remove protein, the solution is adjusted to 10 mM EDTA, 40 mM Tris-Cl pH 6.5, and 20 µg Proteinase K (Roche, Catalog No. 03115836001) is added to the reaction. Samples are incubated at 42°C for 2 hours. DNA is purified by extraction once with buffered phenol-chloroform. To precipitate DNA, 400 µl of supernatant is transferred into a microcentrifuge tube (avoid sucking up the interface), and incubated with 1 µl of GlycoBlue (Ambion, AM9515), 44 µl NaOAc 0.3 M, and 444 µl of room temperature isopropanol for at least 30 min. DNA is then collected by centrifugation at 14,000 rpm (16,000 g) for 15 min at room temperature, washed once with 70% EtOH, and dried in a Speedvac for 10 min at 65°C or in air overnight. The pellet is dissolved in 100 µl water; typically 1 µl of this solution is sufficient for real-time PCR analysis.

Analysis of Digested Nucleosomes

To obtain a detailed map of chromatin structure, the resulting materials (both the digested and undigested) are analyzed by real-time PCR with an Applied Biosystem 7500 thermocycler using Power Syber Master Mix (Catalog No. 4367659). For a detailed mapping of nucleosome structure, primer pairs that generate short-overlapping PCR products are normally used.¹⁰ If the purpose of the experiment is to identify changes in nucleosome density, less densely spaced, non-overlapping primer pairs can be used. Primers were designed using Primer Express software (Applied Biosystems) with lengths of 100–150 bp and Tms 57–60°C. Nucleosome density of a given region is defined by the ratio of digested DNA versus undigested control DNA.

If a nucleosome is reproducibly positioned on a length of DNA in all cells, the DNA will be protected from MNase digestion and an amplicon from a primer pair located totally within this nucleosome will yield a digested/undigested ratio of 1. On the other hand, if part of the amplicon is not protected by a nucleosome, MNase digestion will reduce the amount of intact genomic DNA for this amplification, yielding a digested/undigested DNA ratio of as little as 0. Thus, a positioned nucleosome array will generate peaks and valleys, with the valleys corresponding to linker regions between nucleosomes. If only a fraction of cells contain a positioned nucleosome, the ratio of digested/undigested DNA will lie between 0–1.

Analysis of Chromatin Structure at Early Drosophila Zygotic Genes using MNase Mapping

During early Drosophila development, zygotic gap, pair-rule and segment polarity genes are expressed in a spatially dynamic manner representing a well-studied transcriptional cascade.¹² A set of transcriptional repressors encoded by knirps, Kruppel and giant gap genes dictate critical positional information in the blastoderm embryos, but little is known of these proteins' biochemical activities. We applied this MNase mapping method to assess the effect of the short-range repressor Knirps on the chromatin structure of a cis-regulatory region located 9.5 kbp downstream of the hunchback (hb) transcription start site, and compared it to the chromatin structure of adam, a non-target locus. Knirps protein was uniformly expressed in 2–4 hr blastoderm embryos to repress target genes using a heatshock driver.¹³

We observed a significant increase in the resistance to MNase digestion across the 400 bp hb enhancer, whereas the digestion pattern remained unchanged at the nonspecific adam gene, as well as an intergenic regions tested on the 3rd chromosome (Fig. 1 and data not shown). We have observed similar increases in nuclease resistance at specific even-skipped enhancers targeted by Knirps (Li LM and Arnosti DN, submitted). These results suggest that chromatin structure is significantly altered during transcriptional repression by this factor, and that such effects can be observed using the whole Drosophila embryos. This protocol is suitable for local analysis of individual regulatory elements controlled by gap gene repressors, and may provide useful insight into chromatin alterations mapped by more global approaches.¹⁴

Figure 1.

Chromatin sensitivity to MNase digestion in the Drosophila embryo, before and after induction of Knirps repressor. (A) Chromatin accessibility of adam promoter region before (solid line) and after (dashed line) induction of Knirps repressor. (B) Increase in resistance to MNase digestion at hb enhancer located at +9.5 kbp after repression of the gene by Knirps. Data represent averages of 3 biological replicates; error bars indicate standard errors; points in the graph represent the centers of the amplicons used in real-time PCR analysis.