Methyltransferase-based single-promoter analysis assay

Introduction

Nucleosomal positioning [reviewed in (1-3)] plays a pivotal role in the regulation of transcriptional initiation. Transcriptional co-activator complexes interact with nucleosomes (4) to induce nucleosomal rearrangements. Nucleosomes often have to unfold completely (5) or be disassembled (6) at the transcription start site, to allow for transcriptional initiation (7, 8).

 

Most of the studies done on the nucleosomal rearrangements of histones use conventional footprinting techniques, which rely on nuclease digestion and primer extension. However, promoters are molecular ‘modules’, which are controlled as individual entities. When analyzed by conventional methodologies this modularity is destroyed. Our lab has modified a previously described footprinting strategy (9,10), which now allows us to study the chromatin structure of individual molecules. MSPA (methylation-sensitive promoter analysis) allows for the study of unmethylated CpG islands by treatment of nuclei with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by genomic bisulfite sequencing of individual progeny DNA molecules (Fig. 1) (11-13) (see comment 1). This gives single molecule resolution over the promoter and allows for the physical linkage between binding sites on individual promoter molecules to be maintained.

Our lab has successfully used this method to study the difference in nucleosomal positioning in the p16 promoters in two human cell lines (11), to identify transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress (12), and to study the changes in nucleosome occupancy silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells (13).

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Procedure

Treatment of Nuclei with M.SssI

Nuclei are first purified from cells. M.SssI is then added to the purified nuclei. The M.SssI then enters the nuclei and methylates the chromatin (comment 2).

Nuclei extraction

1. Actively growing cells are trypsinized and washed once with cold phosphate buffer saline (PBS). It is recommended to start with at least 10⁷ cells, however, this procedure has been done successfully with 200,000 cells (variations below).
2. Cells are then resuspended in 1 ml of ice cold RSB Buffer and kept on ice for 10 min.

The following steps are all done at 4°C:

1. Following the 10 min incubation, 0.1 ml of 10% Nonidet P-40 (NP-40) detergent is added and the cells are homogenized with 15 strokes of the tight pestle of a Dounce homogenizer. Homogenized cells are then put in eppendorf tube and centrifuged for 5 min at 5000 rpm at 4°C. The supernatant is discarded.
2. Nuclei are then resuspended in 1 ml of RSB Buffer. At this time a small aliquot can be checked for intact nuclei and complete lysis of the cellular membrane using a microscope. Samples are then centrifuged for 5 min at 5000 rpm at 4°C. The supernatant is discarded.
3. Nuclei are then washed again with either RSB Buffer or with 1x M.SssI buffer. (It should be noted that epithelial nuclei tend to lyse during centrifugation if washed with 1X M.SssI buffer, however fibroblast nuclei stay intact with the 1X M.SssI buffer wash) (comment 3). Samples are then centrifuged for 5 min at 5000 rpm at 4°C. The supernatant is discarded.
4. The nuclei are then resuspended in 1X M.SssI buffer so that there are 10⁶ nuclei per 74.25µl.

M.SssI treatments

M.SssI treatments of nuclei are done immediately after nuclei are prepared.

M.SssI is purchased from New England Biolabs. A 10X M.SssI buffer and AdoMet (SAM) is included. (Note: We actually buy fresh M.SssI every time we perform the assay to ensure that the footprints we see are due to protection of the DNA by nucleosomes and not by the inefficiency of an old enzyme. At a minimum fresh AdoMet should be used.)

1. For M.SssI treatment of 10⁶ nuclei add the following to an eppendorf tube:

   10X M.SssI buffer	15 µl
   32 mM SAM	0.75 µl
   1 M Sucrose	45 µl
   Nuclei 106 (6 µg DNA)	74.25 µl
   M.SssI (NEB)	15 µl (60 U) + (comment 3)
   H2 O	to make volume up to 150 µl

   Incubate at 37°C for 15 mins
2. Reactions are stopped by the addition of an equal volume of stop solution (150 µl).
3. Samples are then incubated with 200 µg/ml proteinase K at 55°C for 16 h.
4. DNA is purified by phenol/chloroform extraction and ethanol precipitation.
5. The DNA is then subjected to genomic bisulfite sequencing which was first reported by Frommer et al (14) and described in detail below.