Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Podsumowanie

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Streszczenie

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

Wprowadzenie

The ovaries are major players in somatic aging¹, with distinct contributions from specific cell populations. The cellular heterogeneity of the ovary makes it difficult to interpret molecular results from bulk, whole-ovary assays. Understanding the role of specific cell populations in ovarian aging is key to identifying the molecular drivers responsible for fertility and health decline in aged women. Traditionally, the multi-omics assessment of specific ovarian cell types was achieved by techniques such as laser microdissection², single-cell approaches³, or cell sorting⁴. However, microdissection can be expensive and difficult to perform, and cell sorting can alter cellular phenotypic profiles⁵.

A novel approach to assess ovarian cell-type-specific epigenomic and transcriptomic profiles uses the nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse model. The NuTRAP model allows the isolation of cell-type-specific nucleic acids without the need for cell sorting by using the affinity purification methods: translating ribosome affinity purification (TRAP) and isolation of nuclei tagged in specific cell types (INTACT)⁶. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. By crossing the NuTRAP mouse with a cell-type-specific Cre line, the removal of the STOP cassette causes eGFP-tagging of the ribosomal complex and biotin/mCherry-tagging of the nucleus in a Cre-dependent manner⁶. The TRAP and INTACT techniques can then be used to isolate mRNA and nuclear DNA from the cell type of interest and proceed to transcriptomic and epigenomic analyses.

The NuTRAP model has been used in different tissues, such as adipose tissue⁶, brain tissue^7,8,9, and the retina¹⁰, to reveal cell-type-specific epigenomic and transcriptomic changes that may not be detected in whole-tissue homogenate. The benefits of the NuTRAP approach over traditional cell sorting techniques include the following: 1) the prevention of ex vivo activational artifacts⁸, 2) the minimized need for specialized equipment (i.e., cell sorters), and 3) the increased throughput and decreased cost of cell-type-specific analyses. In addition, the ability to isolate cell-type-specific DNA and RNA from a single mouse allows for paired analyses that increase the statistical power. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes^11,12,13, we targeted the NuTRAP expression system to stromal and theca cells using a Cyp17a1-Cre driver. Here, we demonstrate that the induction of the NuTRAP construct is specific to ovarian stromal and theca cells, and sufficient DNA and RNA for sequencing studies are obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with any available Cre line.

For the generation of a cell-type-specific ovarian NuTRAP mouse line, the nuclear tagging and translating ribosome affinity purification (NuTRAP) allele has a floxed STOP codon that controls the expression of BirA, biotin ligase recognition peptide (BLRP)-tagged mCherry/mRANGAP1, and eGFP/L10a. When crossed with a cell-type-specific Cre line, the expression of the NuTRAP cassette labels the nuclear protein mRANGAP1 with biotin/mCherry and ribosomal protein L10a with eGFP in a Cre-dependent manner. This allows for the isolation of nuclei and mRNA from specific cell types without the need for cell sorting. The NuTRAP^flox/flox can be paired with a cell-type-specific Cre relevant to ovarian cell types to assess this.

Protokół

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation (OMRF). Parent mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred and housed under SPF conditions in a HEPA barrier environment on a 14 h/10 h light/dark cycle (lights on at 6:00am) at the OMRF.

NOTE: In this demonstration, we use a Cyp17iCre^+/−(Strain # 028547, The Jackson Laboratory) male paired with a NuTRAP female (Strain # 029899, The Jackson Laboratory). The desired Cyp17-NuTRAP progeny (Cyp17iCre^+/−; NuTRAP^flox/WT) will express the NuTRAP allele under the control of the Cyp17a1 promoter in ovarian stromal cells. For this manuscript preparation, we used 4-month-old female Cyp17-NuTRAP mice (n = 4). DNA was extracted from mouse ear punch samples for genotyping to confirm the inheritance of the Cyp17iCre and NuTRAP transgenes and for the PCR detection of 1) generic Cre, 2) Cyp17iCre, and 3) NuTRAP floxed using the primers listed in the Table of Materials, as previously described^7,8.

1. Mouse ovarian dissection

1. Perform mouse euthanasia according to the animal care guidelines and approval of the institution, followed by ovary collection.
2. Dissect the ovaries to remove any adipose tissue or parts of the oviducts that might be attached to the ovarian tissue prior to placing the ovaries in tubes with buffer. Immediately proceed to TRAP or INTACT techniques.
   NOTE: This protocol has not been optimized for use with frozen tissue. Using one ovary from the same mouse for each technique is recommended for future statistical analysis.

2. Isolation of nuclei from specific ovarian cell types

1. Buffer preparation
   1. Nuclear purification buffer (NPB): To prepare 100 mL of NPB, combine 2 mL of 1 M HEPES (final concentration: 20 mM HEPES), 0.8 mL of 5 M NaCl (40 mM NaCl), 4.5 mL of 2 M KCl (90 mM KCl), 0.4 mL of 0.5 M EDTA (2 mM EDTA), 0.1 mL of 0.5 M EGTA (0.5 mM EGTA), and 92.2 mL of nuclease-free water. Supplement with 1x protease inhibitor on the day of nuclei isolation.
      NOTE: NPB can be prepared without the protease inhibitor in advance and lasts for up to 3 weeks at 4 °C.
   2. Nuclei lysis buffer (complete): Supplement nuclei lysis buffer with 1x protease inhibitor on the day of nuclei isolation.
2. Creation of the nuclei suspension
   NOTE: The samples should be maintained on ice at all times unless otherwise indicated.
   1. Collect one ovary from a Cyp17-NuTRAP mouse (or other relevant Cre-NuTRAP) in 500 µL of nuclei lysis buffer (complete). Chop the ovary into eight parts within the buffer using self-opening micro scissors.
   2. Use wide-bore pipette tips to transfer the minced ovary and 500 µL of lysis buffer into a glass Dounce homogenizer on ice. Homogenize the ovary 10x-20x with the loose pestle A. Add 400 µL of lysis buffer to the Dounce homogenizer, washing pestle A.
   3. Homogenize the ovary with the tight pestle B 10x-20x. Add 1,000 µL of lysis buffer, washing pestle B. Transfer the homogenate (1.9 mL) to a 2 mL round-bottom tube.
   4. Centrifuge at 200 x g for 1.5 min at 4 °C to remove undissociated tissue and blood vessels¹⁴.The supernatant contains the nuclei; discard the pellet of undissociated tissue.
   5. After pre-wetting a 30 µm cell strainer with 100 µL of lysis buffer, and filter the supernatant through the cell strainer into a 15 mL conical tube. Then, transfer the nuclei-containing mixture to a 2 mL round-bottom tube.
   6. Centrifuge the sample at 500 x g for 5 min at 4 °C, and discard the supernatant. The pellet contains nuclei.
   7. Resuspend the nuclear pellet in 250 µL of ice-cold lysis buffer by vortexing briefly at moderate to high speed. Bring the volume up to 1.75 mL by adding 1.5 mL of lysis buffer. Mix gently, and rest the nuclear suspension on ice for 5 min.
   8. Pellet the nuclei by centrifugation at 500 x g for 5 min at 4 °C. Carefully discard the supernatant without disturbing the nuclear pellet. Resuspend the pellet in 200 µL of ice-cold storage buffer, and vortex briefly to completely resuspend the nuclear pellet.
   9. Reserve 10% of the resuspended pellet (20 µL) as an input nuclei sample for downstream analyses.
   10. Take the resuspended nuclei pellet, and make up the volume to 2.0 mL with ice-cold NPB. Proceed to isolate the labeled nuclei using the INTACT affinity-based purification method.
3. Isolation of nuclei tagged in specific cell types (INTACT)
   1. Vortex the streptavidin beads in the vial for 30 s at medium speed to ensure they are fully resuspended. Add 30 µL of resuspended beads for each sample into a 2 mL round-bottom tube (beads for up to 10 samples can be washed in a single tube), add 1 mL of NPB, and resuspend well by pipetting.
   2. Place the tube on the magnetic rack for 1 min to separate the beads. Discard the supernatant, and remove the tube from the magnet. Resuspend the washed magnetic beads in 1 mL of NPB.
   3. Repeat the wash step for a total of three washes. Following the final wash, resuspend the beads in the initial volume of NPB (30 µL per sample).
   4. Add 30 µL of the washed beads to the 2 mL nuclear suspension from step 2.2.10. Resuspend the bead-nuclei mixture well by pipetting and gently inverting the tube.
   5. Place the tubes on a rotating mixer in a cold room or refrigerator for 30 min at low speed. Incubate the input samples without beads in the same conditions.
   6. Place the tubes with the nuclear suspension and beads on the magnet, and separate the biotinylated nuclei bound to streptavidin beads (positive fraction) from the negative fraction of nuclei. Allow the beads to separate on the magnet for 3 min.
   7. Remove the supernatant, pass the negative fraction to a fresh 2.0 mL tube, and reserve on ice. For each positive fraction tube, remove the tube from the magnet, and resuspend the contents in 1 mL of NPB.
   8. Place the tube on the magnet for 1 min, and discard the supernatant. Remove the tube from the magnet, and resuspend the washed bead-nuclei mixture in 1 mL of NPB.
   9. Repeat step 2.3.8 for a total of three washes. After the completion of three washes, resuspend the positive fraction bead-nuclei mixture in 30 µL of NPB.
   10. For each negative fraction tube, centrifuge the nuclear suspension from step 2.3.7 at 500 x g for 7 min at 4 °C. Carefully aspirate and discard the supernatant. Resuspend the negative fraction nuclei pellet in 30 µL of NPB.
   11. Store the nuclei from the input (step 2.2.9), the negative fraction (step 2.3.10), and the positive fraction (step 2.3.9) in a −80 °C freezer until ready for nuclear DNA or RNA isolation.
       NOTE: The nuclei should not be frozen for protocols requiring intact nuclei as input (i.e., assay for transposase-accessible chromatin, chromatin immunoprecipitation).

3. Isolation of mRNA from specific ovarian cell types

1. Preparing the buffers
   1. TRAP buffer base: Prepare 100 mL of TRAP buffer base by combining 78.8 mL of RNase-free water, 5 mL of 1 M Tris-HCl (final concentration: 50 mM Tris [pH 7.5]), 5 mL of 2 M KCl (100 mM KCl), 1.2 mL of 1 M MgCl₂ (12 mM MgCl₂), and 10 mL of 10% NP-40 (1% NP-40). Store at 4 °C for up to 1 month.
   2. High salt buffer base: Prepare 100 mL of high salt buffer base by combining 68.8 mL of RNase-free water, 5 mL of 1 M Tris-HCl (final concentration: 50 mM Tris [pH 7.5]), 15 mL of 2 M KCl (300 mM KCl), 1.2 mL of 1M MgCl₂ (12 mM MgCl₂), and 10 mL of 10% NP-40 (1% NP-40). Store at 4 °C for up to 1 month.
   3. TRAP homogenization buffer (complete): Prepare 1.5 mL per sample on the day of tissue collection. Prepare 10 mL of TRAP homogenization buffer by combining 10 mL of TRAP buffer base (from step 3.1.1) with 10 µL of 100 mg/mL cycloheximide (final concentration: 100 µg/mL cycloheximide), 10 mg of sodium heparin (1 mg/mL sodium heparin), 20 µL of 0.5 M DTT (1 mM DTT), 50 µL of 40 U/µL RNase inhibitor (200 U/mL RNase inhibitor), 200 µL of 0.1 M spermidine (2 mM spermidine), and one EDTA-free protease-inhibitor tablet (1x).
   4. Low salt wash buffer (complete): Make 1.5 mL per sample on the day of tissue collection. To make 10 mL of low salt wash buffer, combine 10 mL of TRAP buffer base (from step 3.1.1) with 10 µL of 100 mg/mL cycloheximide (100 µg/mL cycloheximide) and 20 µL of 0.5 M DTT (1 mM DTT).
   5. High salt wash buffer (complete): Make 1.5 mL per sample on the second day of TRAP isolation. To make 10 mL of high salt wash buffer, combine 10 mL of high salt buffer base (from step 3.1.2) with 10 µL of 100 mg/mL cycloheximide and 20 µL of 0.5 M DTT.
2. Performing translating ribosome affinity purification (TRAP)
   1. Collect the other ovary from a Cyp17-NuTRAP mouse (or other relevant Cre-NuTRAP) and place it in a 1.5 mL RNase-free tube containing 100 µL of ice-cold TRAP homogenization buffer (from step 3.1.3). Chop the ovary into eight parts within the buffer using self-opening micro scissors.
   2. Use wide-bore tips to transfer the ovary and 100 µL of homogenization buffer into a glass Dounce homogenizer. Homogenize 10x-20x with the loose pestle A. Add 400 µL of TRAP homogenization buffer, washing pestle A.
   3. Transfer the homogenate to a 2 mL round-bottom tube. Wash the Dounce homogenizer with an additional 1 mL of TRAP homogenization buffer, and transfer the washed volume to the 2 mL round-bottom tube.
   4. Centrifuge the homogenate at 12,000 x g for 10 min at 4 °C. Transfer the cleared supernatant to a fresh 2 mL round-bottom tube. Discard the pellet.
   5. Transfer 100 µL of the cleared homogenate to a fresh 2 mL round-bottom tube, and reserve as input on ice.
   6. Incubate the remainder of the cleared homogenate (from step 3.2.4) with an anti-GFP antibody (5 µg/mL) for 1 h at 4 °C while rotating end-over-end. Incubate the input sample in the same conditions.
   7. In the last 15 min of the incubation, wash the protein G magnetic beads in the low salt wash buffer using the following steps (3.2.8-3.2.11).
   8. Vortex the vial of protein G magnetic beads for 30 s to completely resuspend. Transfer 50 µL of the protein G magnetic beads for each sample (up to 10 samples can be washed in one tube) into a 2 mL round-bottom tube.
   9. Add 500 µL of low salt wash buffer to the beads, and pipette gently to mix. Place the tube into a magnetic stand for 1 min to collect the beads against the side of the tube. Remove and discard the supernatant.
   10. Remove the tube from the magnetic stand, and add 1 mL of low salt wash buffer to the tube. Pipette gently to mix. Place the tube into a magnetic stand for 1 min to collect the beads against the side of the tube. Remove and discard the supernatant.
   11. Repeat step 3.2.10 for a total of three washes. Following the final wash, remove the tube from the magnetic rack, and resuspend the beads in the initial volume of low salt wash buffer (50 µL per sample).
   12. Transfer the resuspended washed beads (50 µL) to the antigen sample/antibody mixture, and incubate at 4 °C overnight while rotating in an end-over-end mixer. Incubate the input samples rotating end-over-end at 4 °C overnight to maintain equivalent conditions.
   13. After the overnight incubation, remove the tubes from the rotator, and separate the magnetic beads with the target ribosomes/RNA (positive fraction) from the supernatant (negative fraction) using the magnetic stand for 2.5-3 min. Aspirate the negative fraction, place it in a fresh 2 mL round-bottom tube, and set it aside on ice while processing the positive fraction.
   14. Add 500 µL of high salt wash buffer (from step 3.1.5) to the tube with the positive fraction, and gently pipette to mix. Place the tube on a magnetic stand maintained on ice for 1 min to separate the beads. Discard the supernatant, and remove the tube from the magnet.
   15. Repeat step 3.2.14 for a total of three washes. Following the final wash, resuspend the beads in 350 µL of RNA lysis buffer supplemented with 3.5 µL of 2-mercaptoethanol (BME). Incubate the tubes at room temperature while mixing for 10 min at 900 rpm in a digital shaker.
   16. Separate the beads from the solution that now contains the target ribosomes/RNA with a magnetic stand for 1 min at room temperature. Collect the eluted positive fraction (supernatant) in a fresh 1.5 mL tube, and add 350 µL of 100% ethanol. Invert to mix. Proceed to the RNA isolation.
   17. Optional: To isolate RNA from the input sample (step 3.2.5) and negative fraction (step 3.2.13), transfer 100 µL of the input and negative fractions to fresh 1.5 mL tubes. Add 350 µL of RNA lysis buffer supplemented with 3.5 µL of BME to each sample. Invert to mix. Add 250 µL of 100% ethanol to each tube, and invert to mix. Proceed to the RNA isolation.
3. RNA isolation
   1. Transfer 700 µL of the positive fraction and, optionally, the input and/or negative fraction to an RNA spin column. Follow the manufacturer's instructions to isolate RNA from the spin column.

Wyniki

A schematic of the TRAP and INTACT protocols is shown in Figure 1. Here the specificity of the Cyp17-NuTRAP mouse model to ovarian stromal/theca cells is demonstrated by immunofluorescent imaging and RNA-Seq from TRAP-isolated RNA. First, immunofluorescence imaging of the eGFP signal in the ovary and localization of the eGFP signal to the theca and stromal cells were performed. Briefly, 5 µm sections were deparaffinized with a xylene and ethanol gradient. For better eGFP signaling, the ...

Dyskusje

The NuTRAP mouse model⁶ is a powerful transgenic labeling approach for the paired interrogation of the transcriptome and epigenome from specific cell types that can be adapted to any cell type with an available Cre driver. Here, we demonstrate the specificity of the Cyp17-NuTRAP mouse model in targeting ovarian theca and stromal cells. The Cyp17-NuTRAP model can be used to further elucidate the theca and stromal cell-specific epigenetic mechanisms involved in ovarian aging, cancer, and diseas...

Materiały

Name	Company	Catalog Number	Comments
0.1 M Spermidine	Sigma-Aldrich	05292-1ML-F
1 M MgCl2	Thermo Scientific	AM9530G
10% NP-40	Thermo Scientific	85124
100 mg/mL Cycloheximide	Sigma-Aldrich	C4859-1ML
2-mercaptoethanol	Sigma-Aldrich	M3148
30 µm cell strainer	Miltenyi Biotec	130-098-458
All Prep DNA/RNA Mini Kit	Qiagen	80204
anti-GFP antibody	Abcam	Ab290	For TRAP and IHC (Rabbit polyclonal to GFP)
Buffer RLT	Qiagen	79216	RNA Lysis Buffer in protocol
cOmplete, mini, EDTA-free protease inhibitor tablet	Roche	11836170001	For TRAP Homogenization Buffer
Cyp17iCre mouse model	The Jackson Laboratory	28547	B6;SJL-Tg(Cyp17a1-icre)AJako/J
DynaMag-2 magnet	Invitrogen	12321D
Genotyping Primers	IDT	Custom	Generic Cre - Jackson Laboratory protocol 22392, Primers: oIMR1084, oIMR1085, oIMR7338, oIMR7339
			Cyp17iCre - Jackson Laboratory protocol 30847, Primers: 21218, 31704, 31705, 35663
			NuTRAP - Jackson Laboratory protocol 21509, Primers: 21306, 24493, 32625, 32626
Halt Protease Inhibitor cocktail (100X)	Thermo Scientific	1861278	For NPB Buffer
M-280 Streptavidin Dynabeads	Invitrogen	11205D	2.8 µm bead diameter
MixMate	Eppendorf	5353000529
Nuclei Isolation Kit: Nuclei EZ Prep	Sigma-Aldrich	Nuc101	Contains Nuclei Lysis Buffer and Nuclei Storage Buffer
1 M HEPES	Gibco	15630-080
5 M NaCl	Thermo Scientific	AM9760G
2M KCl	Thermo Scientific	AM9640G
0.5 M EDTA	Thermo Scientific	AM9260G
0.5 M EGTA	Fisher Scientific	50-255-956
NuTRAP mouse model	The Jackson Laboratory	29899	B6;129S6-Gt(ROSA)26Sortm2(CAG-NuTRAP)Evdr/J
Pierce DTT No-Weigh Format	Thermo Scientific	A39255
Protein G Dynabeads	ThermoFisher	10004D	For TRAP
RNaseOUT	Invitrogen	10777019
Sodium Heparin	Fisher Scientific	BP2425
Ultrapure 1M Tris-HCl, pH 7.5	Invitrogen	15567-027
VWR Tube Rotator	Fisher Scientific	NC9854190