A Method to Study de novo Formation of Chromatin Domains

We outline a system to assess the dynamic formation of chromatin domains. We utilize this system to follow the recruitment and spreading of PRC2-mediated chromatin domains in cells. The process can be paused at any time to analyze the events in progress.

This system could be geared to monitor dynamic changes in any given cellular process and hence is not limited to tracking chromatin domain formation. Start by designing guide RNAs to delete exon 10 and 11 of an endogenous copy of embryonic ectoderm development, or EED, in mouse embryonic stem cells using CRISPR Design Tool. Clone the guide RNAs into the CRISPR/Cas9 plasmid according to manuscript directions.

In a six-well plate format, transfect 200, 000 mouse embryonic stem cells, or mESCs, with one microgram of each guide RNA using transfection reagents according to manufacturer's instructions. Change the media 24 hours after transfection. Two days after the transfection, isolate GFP-positive cells using FACS.

Transfection efficiency ranges from about 10 to 30%so sort around 500, 000 cells to capture sufficient GFP-positive cells for plating. Plate the isolated GFP-positive cells into 15-centimeter plates that are pre-coated with 0.1%gelatin for colony picking. Grow the cells in ESC medium for about one week until single colonies are visible, making sure to change the media every two days.

Pick a minimum of 48 colonies by using a 20-microliter pipette tip to scrape over the colony while aspirating. Without breaking up the colony into single cells, transfer it to an Accutase-containing 96-well plate. Incubate the colonies at 37 degrees Celsius for 10 minutes to dissociate the cells.

Then add 200 microliters of ESC media to each well with a multichannel pipette. Mix well and plate the cells into two separate 96-well plates. Use one of the plates for genotyping, and keep the other growing until genotyping is concluded.

Extract DNA from the plate using commercial reagents and following manufacturer's directions. Use genotyping primers that span the deleted site to perform genotyping PCR with Taq DNA polymerase. Observe a DNA product of lower molecular weight in cells with a homozygous deletion relative to the wild-type cells.

Design guide RNA to introduce a cut within the intron following exon 9 of EED using a CRISPR Design Tool, and clone it into the CRISPR/Cas9 plasmid according to manuscript directions. Design a donor template DNA that includes the EED cDNA sequence after exon 9 and a C-terminal Flag-HA tag upstream of a T2A-GFP sequence, all in reverse orientation with respect to the endogenous gene sequence. Flank the cassette with a splice-acceptor and a polyadenylation sequence nested between heterologous inverted loxP sites, and include at least 500 base pairs of homology arms from each end.

Split the donor template into two segments of gBlocks Gene Fragments, and assemble them into a PCR Blunt vector using Gibson cloning following manufacturer's instructions. In a six-well plate format, transfect 200, 000 mESCs with one microgram of the guide RNA and one microgram of the donor templates. Then isolate GFP-positive cells using FACS.

Isolate individual colonies for genotyping according to manuscript directions. Use flow cytometry to confirm that the mESCs do not have leaky expression of GFP, and, if they do, isolate GFP-negative cells by FACS before starting the experiment. Expand the cells into five 15-centimeter plates, and induce expression of wild-type or cage-mutant EED by administering 5-micromolar 4-hydroxytamoxifen for five different time durations ranging from zero hours to eight days.

Change the media after 12 hours for treatments lasting longer than that. Isolate the successfully recombined cells by FACS using GFP, and perform ChIP-seq for H3K27me2 and H3K27me3 to investigate their temporal deposition to chromatin in response to re-expression of wild-type or cage-mutant EED. To validate the inducible wild-type rescue and inducible mutant rescue systems, western blot was performed on whole extracts after induction of wild-type or cage-mutant EED with 4-hydroxytamoxifen treatment.

Flow cytometry analysis was used to confirm the efficiency of T2A-GFP expression in i-WT-r cells before and after the 4-hydroxytamoxifen treatment, which demonstrates the successful flip of the inverted cassette. ChIP-seq of H3K27me3 was performed after re-expression of wild-type or cage-mutant EED. The emergence of H3K27me3 is observed at 12 hours after wild-type EED expression at discrete nucleation sites and then at 24 hours at regions distant from the initial nucleation sites.

ChIP-seq was also used to track the temporal deposition of H3K27me2, which appears to precede H3K27me3 deposition. The H3K27me3 foci and their growth have also been visualized with fluorescence microscopy. After 12 hours of wild-type EED expression, there is evidence of H3K27me3 foci formation.

These foci increase in number and size by 24 hours and eventually spread to large regions of the nucleus by 36 hours. The accurate design of DNA constructs is very important to generate the desired inducible rescue system. This method can provide a temporal and special control of mutations of proteins in mice.

In this way, one could determine the effect of mutations in a specific tissue or at a specific stage of development. We are currently using this system to monitor dynamic establishment of another chromatin domain, Polycomb repressive complex 1.