A Method to Study de novo Formation of Chromatin Domains

Podsumowanie

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

Streszczenie

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

Wprowadzenie

Eukaryotic genomes are highly organized and changes in the chromatin accessibility directly controls gene transcription¹. The genome contains distinct types of chromatin domains, which correlate with transcriptional activity and replication timing^2,3. These chromatin domains range in size from a few kilobases (kb) to more than 100 kb and are characterized by an enrichment in distinct histone modifications⁴. The central questions are: how are these domains formed and how are they propagated?

One of the most well-characterized chromatin domains is fostered through the activity of the Polycomb repressive complex 2 (PRC2). PRC2 is a multi-subunit complex composed of a subset of the Polycomb Group (PcG) of proteins^5,6, and catalyzes the mono-, di- and trimethylation of lysine 27 of histone H3 (H3K27me1/me2/me3)^7,8,9,10. H3K27me2/me3 are associated with a repressive chromatin state, but the function of H3K27me1 is unclear^6,11. One of the core components of PRC2, embryonic ectoderm development (EED), binds to the end product of PRC2 catalysis, H3K27me3, through its aromatic cage and this feature results in the allosteric stimulation of PRC2^12,13. The PRC2 enzymatic activity is crucial for preserving cellular identity during development as the inappropriate expression of certain developmental genes that are contraindicated for a specific lineage, would be detrimental^5,6. Hence, unraveling the mechanisms by which PRC2 fosters the formation of repressive chromatin domains in mammals is of fundamental importance to understanding cellular identity.

All of the past experimental systems designed to investigate chromatin domain formation including PRC2-mediated chromatin domains, were performed under steady-state conditions, which are unable to track the unfolding events of chromatin domain formation in cells. Here, we present a detailed protocol to generate an inducible cellular system which monitors the initial recruitment and propagation of chromatin domains. Specifically, we focus on tracking the formation of PRC2-mediated repressive chromatin domains that comprise H3K27me2/3. This system that can capture the mechanistic details of chromatin domain formation, could be adapted to incorporate other chromatin domains, such as the widely studied domains comprising either H2AK119ub or H3K9me. In combination with genomics and imaging technologies, this approach has the potential to successfully address various, key questions in chromatin biology.

Protokół

Generation of inducible EED rescue mESCs

1. Cell culture

1. Use feeder-free C57BL/6 mouse embryonic stem cells (mESCs) possessing a stably integrated CreERT2 transgene, which can translocate to the nucleus upon administration of 4-Hydroxytamoxifen (4-OHT)¹³.
2. Grow mESCs in conventional ESC medium^14,15, supplemented with 1000 U/mL LIF, 1 μM ERK inhibitor PD0325901 and 3 μM GSK3 inhibitor CHIR99021. For conventional ESC medium, use knockout DMEM containing 15% fetal bovine serum (FBS), 2 mM L-glutamine, 1X penicillin/streptomycin and 0.1 mM 2-mercaptoethanol.
3. Use plates coated with 0.1% gelatin solution for culturing mESCs.

2. Generation of clonal EED knockout (KO) mESCs

1. Design guide RNAs (gRNAs) to delete Exon 10 and Exon 11 of endogenous copy of EED in mESCs using the CRISPR design tool in Benchling¹⁶. EED-KO-gRNA-1 targets the intron immediately upstream of exon 10 and EED-KO-gRNA-2 targets the intron immediately downstream of Exon 11. The simultaneous application of these gRNAs deletes both Exon 10 and Exon 11 by non-homologous end joining (NHEJ).
2. Clone gRNAs into pSpCas9(BB)-2A-GFP (PX458) using instructions in Ran et al., 2013¹⁷.
3. In a 6-well plate format, transfect 2 x 10⁵ mESCs with 1 μg of each EED-KO-gRNA-1 and EED-KO-gRNA-2 (see Table of Materials) using transfection reagent by following the manufacturer's instructions. Change the media 24 h after transfection.
4. Two days after the transfection, isolate GFP positive cells using Fluorescence-activated cell sorting (FACS). Expect the transfection efficiency to vary around 10-30 %. Sort around 5 x 10⁵ cells to capture sufficient GFP positive cells for plating.
5. Plate the isolated GFP positive mESCs into 15 cm plates pre-coated with 0.1% gelatin (10-20 x10³ cells per plate) for colony picking.
6. Grow the cells in ESC medium for about one week until single colonies are visible. Change the media every 2 days.
7. Pick a minimum of 48 colonies using 20 μL micropipette tip. Scrape over the colony while aspirating into the micropipette tip. Do not break up the colony into single cells. Transfer the colony into an accutase-containing (20 μL) 96 well plate.
8. Incubate the colonies for 10 min at 37 °C until all cells are dissociated.
9. Add 200 μL of ESC media into each well using multichannel pipette.
10. Mix well and plate the cells into two separate 96 well plates using multichannel pipette.
11. Use one of the 96 well plates for genotyping and keep the other growing until genotyping is concluded. Use DNA extraction solution to extract DNA from the 96 well plate by following the manufacturer's instructions.
12. Use genotyping primers Gnt_EED-KO-up and Gnt_EED-KO_down, which span the deleted site and perform genotyping PCR with Taq DNA polymerase following the manufacturer’s instructions.
    NOTE: Other types of DNA polymerases can also be used for genotyping, however, Taq DNA polymerase offers convenience as the PCR reaction can directly be loaded on a gel when its colored reaction buffer is used.
    1. Observe a DNA product of lower molecular weight in cells with a homozygous deletion relative to the wild-type (WT) case.
       NOTE: To generate PRC2 null cells, homozygous deletion of exons 10 and 11 is necessary to destabilize and degrade EED, an essential subunit of core PRC2¹³. The CRISPR targeting efficiency is around 10 %.
13. Validate the deletion of EED exon 10 and 11 and the loss of EED protein by Sanger sequencing and Western blotting, respectively.
14. Confirm the depletion of EED and H3K27me2/me3 from chromatin in EED KO cells by ChIP-seq using antibodies against H3K27me2/me3 and EED. Use the protocol given in Oksuz et al., 2017¹⁵ for ChIP-seq experiments.

3. Engineering the EED knockout mESCs to harbor Cre-ERT2 based inducible EED expression

1. Design gRNA (EED-gRNA-inducible) to introduce a cut within the intron following exon 9 of EED using the CRISPR design tool in Benchling¹⁶ (see Table of Materials).
2. Clone gRNAs into pSpCas9(BB)-2A-GFP (PX458) using instructions in Ran et al., 2013¹⁷.
3. Design a donor template DNA that comprises 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.
   1. Flank the cassette with a splice-acceptor and a polyadenylation sequence nested between heterologous inverted loxP sites (lox66 and lox71)¹⁸.
   2. Include at least 500 bp of homology arms from each end. Split the donor template into 2 segments of gBlocks gene fragments (see gBlock-1, gBlock-2-WT "https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI" and gBlock-2-cage-mutant "https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM") and assemble them into PCR Blunt vector using Gibson cloning following the manufacturer's instructions.
      NOTE: gblock-2 contains an aromatic residue (Y365) within the cage of EED that is important for interaction with H3K27me3¹². gBlock-2-Wt contains wild type residue, whereas gblock-2-cage-mutant contains the cage-mutant of EED (Y365A), incapable of binding to H3K27me3. Inducible WT EED rescue is denoted as i-WT-r and inducible cage-mutant EED rescue is denoted as i-MT-r for convenience.
4. In a 6-well plate format, transfect 2 x 10⁵ mESCs with 1 μg of the gRNA (EED-gRNA-inducible) and 1 μg of the Donor templates (i-WT-r or i-MT-r) using transfection reagent and isolate GFP positive cells using FACS.
5. Follow steps 2.3-2.11 to isolate individual colonies ready for genotyping for successful targeting.
6. Use genotyping primers Inducible_Genotype-FW-1 and Inducible_Genotype-REV-1, which span the inserted cassette and perform genotyping PCR using Taq DNA polymerase.
   NOTE: The targeting efficiency for CRISPR is around 10 %.
   1. Confirm correct integration of the cassette by PCR using Inducible_Genotype-FW-2 and Inducible_Genotype-REV-2 primers, which are outside of the homology arms.
      NOTE: Homozygous integration of the cassette is necessary to accomplish efficient rescue of EED. Note that cells with homozygous integration will produce a single large DNA product as compared to WT EED, which will produce a short product.
7. Confirm the integration of the cassette by Sanger sequencing.
8. Confirm flipping of the cassette and the expression of EED and other PRC2 core components (e.g., EZH2 and SUZ12) upon 4-OHT administration by Western blotting.
9. Confirm the expression of T2A-GFP and percentage of flip by flow cytometry. Note that expression of GFP is indicative of flipping of the cassette and the expression of EED.

4. Following nucleation and spreading of PRC2 activity on chromatin

1. Confirm that i-WT-r and i-MT-r mESCs do not have leaky expression of GFP by flow cytometry. In the case of leaky expression of GFP, isolate GFP-negative cells by FACS before starting the experiment.
2. Expand the i-WT-r and i-MT-r mESCs into five 15 cm plates (5x 10⁶ cells per plate).
3. Induce expression of WT or cage-mutant EED by administration of 0.5 μM 4-OHT for 0 h, 12 h, 24 h, 36 h and 8 days (one 15 cm plate per condition). Change the media after 12 h for treatments longer than 12 h. Isolate the successfully recombined cells by FACS using GFP. Adjust the time of the treatments such that all of the conditions are collected at once.
4. Perform ChIP-seq for H3K27me2, H3K27me3 to investigate their temporal deposition to chromatin in response to re-expression of WT or cage-mutant EED. Use ChIP-seq protocol including library preparation detailed in Oksuz et al., 2017¹⁵.
5. Use spike-in control in each sample to allow for quantitative comparison among different time points¹⁹.
   1. For spike-in control, use chromatin from Drosophila melanogaster (in a 1:50 ratio to the mESC-derived chromatin) as well as Drosophila specific H2Av antibody (1 μL of H2Av antibody per 4 μg of Drosophila chromatin according to manufacturer’s instructions) in each sample.
   2. Prepare the chromatin from Drosophila in a manner similar to that from mESCs using the protocol detailed in Oksuz et al., 2017¹⁵.
6. Map the sequence reads for ChIP-seq to mm10 genome with Bowtie 2 using default parameters²⁰.
7. Normalize the mouse ChIP-seq reads to spike-in Drosophila read counts²¹. Calculate the spike-in normalization factor using the following formula: 1 x 10⁶/unique Drosophila read counts. Expect to get around 1 x 10⁶ Drosophila read counts and 20 x 10⁶ mouse read counts per experiment.
   1. Do not include the Drosophila chromatin when sequencing the input samples.
8. Use genomecov tool from bedtools to convert bam file into bedgraph²². Next, convert the bedgraph into bigwig file using bedGraphToBigWig tool from USCS^23,24. See the following script:
   gen="path to mm10 genome"
   chr="path to mm10 chromosome sizes"
   inp_bam="path to input bam file "
   multiply="calculate the spike-in normalization factor"
   bedtools genomecov -bg -scale $multiply -ibam $inp_bam -g $gen > output.bedGraph
   sort -k1,1 -k2,2n output.bedGraph > output_sorted.bedGraph
   bedGraphToBigWig output_sorted.bedGraph $chr output.bw
9. Visualize the ChIP-seq read densities by uploading the bigwig files on the USCS genome browser²⁴.

5. Monitoring emergence and growth of the H3K27me3 foci in the mESCs nuclei

1. Plate 1x 10⁴ mESCs into 8-well chamber slides pre-coated with 0.1% gelatin.
2. Next day, start performing consecutive 4-OHT (0.5 μM) inductions to collect the cells expressing EED for indicated time points (0 h, 12 h, 24 h and 36 h). Change the media after 12 h for treatments longer than 12 h.
3. Fix the mESCs with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature (RT) for 10 min.
4. Permeabilize the cells with PBS/0.25% Triton X-100 at RT for 30 min.
5. Block the cells with PBS/5% donkey serum/0.1% Triton X-100 (blocking buffer) at RT for 30 min.
6. Dilute H3K27me3 primary antibody 1:500 in the blocking buffer and add onto the cells.
7. Incubate the cells with primary antibody overnight at 4 °C.
8. Next day, perform 3 washes with PBS/0.1% Triton X-100.
9. Dilute secondary antibody (Alexa fluor 595) 1:1000 in the blocking buffer and add onto cells.
   1. Perform all incubations in dark in the following steps as secondary antibodies conjugated with Alexa Fluor are light sensitive.
10. Incubate the secondary antibody for 2 h at RT.
11. Perform 3 washes with PBS/0.1% Triton X-100.
12. Dilute DAPI (1 mg/mL) 1:4000 with PBS/0.1% Triton X-100 and add onto cells.
13. Incubate the cells with DAPI for 10 min at RT.
14. Mount the cells with Aqua mount.
15. Image the cells with confocal microscopy at 63X magnification.
16. Process and pseudo-color the images using a distribution of ImageJ, Fiji²⁵. 

Wyniki

A general scheme of the conditional rescue system
Figure 1 shows the targeting scheme to conditionally rescue EED KO cells with either WT or cage-mutant (Y365A) EED that is expressed from the endogenous EED locus. After knocking out EED, a core subunit of PRC2 that is essential for its stability and enzymatic activity, a cassette within the intron following exon 9 of EED is introduced (Figure 1). The cassette consists of the remaining 3...

Dyskusje

A powerful approach towards understanding the mechanistic details during the formation of a given chromatin domain, is to first disrupt the domain and then track its reconstruction in progress within cells. The process can be paused at any time during the reconstruction to analyze in detail the events in progress. Previous studies on chromatin domains were unable to resolve such events as they were performed under steady-state conditions (e.g., comparing wild-type and gene knockout). Here, we outline a system to assess t...

Materiały

Name	Company	Catalog Number	Comments
(Z)-4-Hydroxytamoxifen (5 mg)	Sigma	H7904-5MG	For induction of EED expression
16% Paraformaldehyde aqueous solution (10x10 ml)	Electron Microscopy Sciences	15710	For immunofluorescence
2-mercaptoethanol	LifeTechnologies	21985-023	For mESCs culture
2% Gelatin Solution	Sigma	G1393-100ml	For mESCs culture
Accutase 500 ML	Innovative Cell Tech/FISHER	AT 104-500	For mESCs culture
Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson immunoresaerch	711-585-152	For immunofluorescence
Aqua-Mount Mounting Medium	FISHER/VWR	41799-008	For immunofluorescence
CHAMBER SLD TC PRMA 8-CHM 16 PK	Fisher Sci	177445PK	For immunofluorescence
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) - 10 mg	Life Tech	D1306	For immunofluorescence
ERK inhibitor, PD0325901	Stemgent	04-0006	For mESCs culture
ESGRO Recombinant Mouse LIF Protein	Millipore/Fisher	ESG1107	For mESCs culture
FBS Stem Cell Qualified	Atlanta	S10250	For mESCs culture
Gibson Assembly Master Mix	NEB	E2611L	For Donor template cloning
GSK3 inhibitor, CHIR99021	Stemgent	04-0004	For mESCs culture
H3K27me2 (D18C8) rabbit mAB	Cell Signaling	9728S	Antibody for ChIP-seq
H3K27me3	Cell Signaling	9733S	Antibody for ChIP-seq
Histone H2Av antibody (pAb)	Active motif	39715	Spike-in control for ChIP-seq
Knockout DMEM	Invitrogen	10829-018	For mESCs culture
L-glutamine	Sigma	G7513	For mESCs culture
Lipofectamine 2000	LifeTech	11668019	For transfection
MangoTaq DNA Polymerase	Bioline	BIO-21079	For Genotyping PCR
Normal donkey serum (10 mL)	Jackson ImmunoResearch	017-000-121	For immunofluorescence
Penicillin-Streptomycin	Sigma/Roche	P0781	For mESCs culture
pSpCas9(BB)-2A-GFP (PX458)	Addgene	48138	For gRNA cloning
QuickExtract DNA Extraction Solution	Lucigen	QE0905T	For Genotyping PCR
Triton X-100	Sigma	T8787-250ML
Zero Blunt PCR Cloning Kit	Thermo Fisher	K270020	For Donor template cloning
Primers/gBlocks
EED-KO-gRNA-1			Sequence: ctctggctactgtcaactag. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.
EED-KO-gRNA-2			Sequence: TAGGCTATGACGCAGCTCAG. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.
EED-gRNA-inducible			Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.
i-WT-r Donor			https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI. gRNA and Donor to generate i-WT-r system in the EED-KO background.
EED-gRNA-inducible			Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.
i-MT-r Donor			https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM. gRNA and Donor to generate i-MT-r system in the EED-KO background.
Genotyping Primers
Gnt-EED-KO-FW-1			Sequence: ctgtaggctgccatctgtga. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.
Gnt-EED-KO-REV-1			Sequence: agccagggctacacagagaa. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.
Inducible_Genotype-FW-1			Sequence: tgcagtgaaacaaatttggaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.
Inducible_Genotype-REV-1			Sequence: gagaggggtggcactgtaaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.
Inducible_Genotype-FW-2			Sequence: ccccctctttctccttttct. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.
Inducible_Genotype-REV-2			Sequence: atgcctgggtgaatgaaaaa. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.