Transfection and colony isolation for creating AID cell lines

Stable transfection was achieved as previously described [33]. Briefly, we generated conditional human HCT116 mutants by homology-directed repair (HDR)-mediated gene tagging using CRISPR-Cas9. Around 60–70% confluent cells expressing OsTIR1 were plated at 3 × 105 cells in a 6-well plate and cultured for 24 h at 37°C. On the next day of transfection, 4 mL of 200 ng/mL CRISPR plasmid, 3 mL of 200 ng/mL donor plasmid, 90 mL of OptiMEM I Reduced Serum Medium (Gibco, #31985070), and 8 mL of FuGENE 6 Transfection Reagent (Promega, #E2691) were mixed and incubated at room temperature for 15 min before being applied to the cells. For antibiotic selection and colony formation, Hygromycin B Gold (Gibco, #10,687010), 100 mg/mL was used. For colony isolation, single colonies were picked under a stereo microscope and transferred to a 96-well plate containing 10 mL of trypsin/ EDTA and neutralized with 200 mL of media. After 2 weeks of culture, the cells were transferred to a 24-well plate. After a few days of culture, genomic DNA was isolated. Briefly, the cells were harvested, lysed with SDS buffer (100 mM NaCl, 50 mM Tris–Cl pH 8.1, 5mM EDTA, and 1% wt/vol SDS), and treated with proteinase k (New England Biolabs, #P8102) (0.6 mg/mL) at 55 °C for 2 h. Then, the lysis solutions were treated with PCl (phenol/ chloroform/ isoamyl alcohol) and used in EtOH precipitation. Genomic DNA pellets were washed with 70% EtOH and resuspended in RNase-containing water. The PCR reaction was set up using 0.5 U of Taq DNA Polymerase (G Biosciences, #786–447) (1 × PCR Buffer), 0.5 mM primers, and 1 mL of genomic DNA from the HCT116 CMV-OsTIR1 parental cells to a 20-mL total volume reaction mixture. PCR was performed using the following conditions: 30 cycles of 98 °C for 2 min, 55 °C for 30 s, and 68 °C for 0.5 min/kb. PCR products were examined for biallelic insertion using agarose gel electrophoresis. Initially, progenitor cells were produced by integrating OsTIR1 (CMV-OsTIR1) into the safe-harbor AAVS1 locus. We then co-transfected the donor template plasmid with Cas9 and sgRNAs that targeted the STOP codon of the LMNB1 or LMNB2 genes in HCT116 cells expressing OsTIR1. To generate cells with mAID in both genes, we simultaneously transfected the two donor templates with two sgRNA that target both genes. Cells expressing mClover for each protein target (LMNB1, LMNB2, or LMNB1 & B2) were sorted and grown as single-cell colonies. We PCR-screened 179 colonies in total to identify clones with homozygous LAMINB1-mAID (5 + /40 colonies) and LAMIN B2-mAID (5 + /55 colonies) as well as clones that had homozygous mAID in both genes (3 + /84 colonies).

Primers and sgRNA

Appropriate primers were designed to check the insertion by PCR. For creating stable cell lines, primer sets detected both the wild type (WT) (1–1.5 kb) and inserted alleles (1–1.5 kb plus the size of the insertion), and another primer set to detect only the inserted allele. The first primer set was designed outside of the homology arms. Primers and sgRNA sequences used to create all AID cell lines are listed in Additional file 1.

Plasmids for AID cell lines

The donor construct contained the AID domain fused to mClover and an intervening T2A site with a hygromycin resistance marker. The construct was flanked by 50-base pair homology arms corresponding to the last exon region of the sgRNA recognition sequence and Cas9 cleavage site. To identify a CRISPR–Cas9 targeting site, we chose an appropriate sequence within 50-bp upstream or downstream from the stop codon. The following target finder sites were used to construct the CRISPR–Cas9 plasmid: IDT custom Alt-R guide design and WEG CRISPR finder. Construction of the CRISPR–Cas9 plasmid and donor plasmids have been previously described [33]. The AAVS1 T2 CRISPR in pX330 plasmid (Addgene plasmid # 72833; http://n2t.net/addgene:72833; RRID: Addgene_72833) is based on pX330-U6-Chimeric_BB-CBh-hSpCas9 from Dr. Feng Zhang (Addgene #42230) (Cong et al., Science, 2013) and was a gift from Masato Kanemaki. The AAVS1 target sequence is described in Mali et al. (Mali et al., Science, 2013). pMK232 (CMV-OsTIR1-PURO) was a gift from Masato Kanemaki (Addgene plasmid # 72834; http://n2t.net/addgene:72834; RRID: Addgene 72834). pMK364 (CMV-OsTIR1-loxP-PURO-loxP) was a gift from Masato Kanemaki (Addgene plasmid # 121184; http://n2t.net/addgene:121184; RRID: Addgene_121184). pMK290 (mAID-mClover-Hygro) was a gift from Masato Kanemaki (Addgene plasmid # 72828; http://n2t.net/addgene:72828; RRID: Addgene_72828).

Auxin treatment

For auxin treatment, HCT116LMN(B1&B2)−AID cells were plated at 50,000 cells per well of a 6-well plate (Cellvis, P12-1.5H-N). Cells were given at least 24 h to re-adhere prior to treatment. To induce expression of OsTIR1, 2 μg/ml of doxycycline (Fisher Scientific, #10592–13-9) was added to cells 24 h prior to auxin treatment. For live-cell flow cytometry, western blots, RT-qPCR, RNA-seq, and in situ Hi-C experiments, 1000 μM 3-Indoleacetic acid (IAA, Sigma-Aldrich, #12886) was solubilized in 100% EtOH before each treatment as a fresh solution. For live-cell confocal microscopy, fixed-cell flow cytometry, fixed-cell immunofluorescence, CRISPR-Sirius fluorescent imaging, and Dual-PWS experiments, 1000 μM Indole-3-acetic acid sodium salt (IAA, Sigma-Aldrich, #6505–45-9) was solubilized in RNase-free water (Fisher Scientific, #10–977-015) before each treatment as a fresh solution and added to HCT116LMN(B1&B2)−AID cells. Optimal auxin treatment time was determined based on the results from western blot, immunofluorescence, and flow cytometry experiments (Fig. 1D).

GSK343 treatment

For GSK343 treatment, HCT116 cells were plated at 50,000 cells per well of a 6-well plate (Cellvis, P12-1.5H-N). Cells were given at least 24 h to re-adhere before treatment. GSK343 (Millipore Sigma, #SML0766) was dissolved in DMSO to create a 10 mM stock solution. This was further diluted in complete cell media to a final treatment concentration of 10 µm.

Fixed-cell flow cytometry (FACS) analysis

Flow cytometry analysis for HCT116LMNB1−AID, HCT116LMNB2−AID, and HCT116LMN(B1&B2)−AID cells for AID system verification and nuclear morphology experiments was performed on the Amnis ImageStreamXTM, located at the University of Virginia Flow Cytometry Core Facility in Charlottesville, VA. To assess the degree of apoptosis induced by auxin treatment, we used the Annexin V APC Kit (Cayman Chemical, #601410) and followed the manufacturer’s protocol. Flow cytometry analysis for HCT116LMN(B1&B2)−AID cells to determine proper auxin treatment concentration was performed on a BD LSRFortessa Cell Analyzer FACSymphony S6 SORP system, located at the Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility at Northwestern University in Evanston, IL. For all FACS analysis, the same protocol was used. After 24 h of doxycycline treatment followed by auxin treatment, cells were harvested and fixed. Briefly, cells were washed with DPBS (Gibco, #14190–144), trypsinized (Gibco, #25200–056), neutralized with media, and then centrifuged at 500 × g for 5 min. Cells were then resuspended in 500 μL of 4% PFA and DPBS and fixed for 10 min at room temperature, followed by centrifugation and resuspension in cold FACS buffer (DPBS with 1% of BSA and 2mM EDTA added) at 4°C until analysis could be performed the following day. For propidium iodine (PI) staining, cells were harvested, and cell pellets were washed with DPBS. Then, DPBS was removed and ice-cold 70% EtOH was added drop by drop to the cell pellets for fixation. Cells were fixed for 3 h at 4°C. After fixation, EtOH was removed, and cells were washed twice with DPBS. PI staining solution (20 µL/ sample; BioLegend, #421301) including RNase A (0.5 µL/ sample; Thermo Fisher Scientific, #EN0531) was added to the cells and incubated with the cells for 30 min protected from light. Staining solution was subsequently removed, and cells were washed twice using 2% FBS containing DPBS. Finally, cells were filtered through 70 µM filters. All flow cytometry data were analyzed using FlowJo software.

Live-cell imaging for growth kinetics

The Incucyte Live cell imaging system (Sartorius) was used to measure growth kinetics of the cells. Cells were seeded to 96-well plates and next day, as soon as the cells were treated with doxycycline, the system was set to collect images every 2 h in phase and green channels (phase: proliferation of the cells, green: degradation of lamins). Twenty-four hours later, cells were treated with IAA and the system collected images for 72 h.

Protein detection and antibodies

HCT116LMNB1−AID, HCT116LMNB2−AID, and HCT116LMN(B1&B2)−AID cells were lysed using Radio Immuno Precipitation Assay (RIPA) buffer (Sigma-Aldrich, #R0278) with protease inhibitor added (Sigma-Aldrich, #P8340). Cell lysates were quantified with a standard Bradford assay using the Protein Assay Dye Concentrate (BioRad, #500–0006) and BSA as a control. Heat denatured protein samples were resolved on a 4–12% bis–tris gradient gel, transferred to a PVDF membrane using the Life Technologies Invitrogen iBlot Dry Transfer System (Thermo Fisher Scientific, IB1001) (20 V for 7 min), and blocked in 5% nonfat dried milk (BioRad, #120–6404) in 1 × TBST. Whole-cell lysates were blotted against the following primary antibodies: Lamin B1 (Cell Signaling, #13435, dilution 1:1000), Lamin B2 (Cell Signaling, #12255, dilution 1:1000), and alpha-tubulin (Thermo Fischer Scientific, #62204, dilution 1:2000). The following secondary antibodies were used: anti-rabbit IgG HRP (Promega, #W4018). Blots were incubated with the primary antibody overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h at room temperature. To develop blots for protein detection, chemiluminescent substrates were used (Thermo Fischer Scientific, #32106). To quantify the western blot bands, we used the iBright Analysis Software from Thermo Fisher Scientific to define bands as regions of interest. By measuring the mean grey intensity values, the final relative quantification values were calculated as the ratio of each protein band relative to the lane’s loading control for all three replicates. Uncropped blots are included in Additional file 3.

Quantitative real-time PCR and RNA isolation

Total RNA from transfected cells was harvested using the RNeasy Plus Mini Kit (Qiagen, #74134) following the manufacturer’s protocol. One milligram of RNA was converted to cDNA using the Applied Biosystems High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, #4387406). The GAPDH or HPRT-1 gene was used as an internal control for analysis. RT-qPCR was performed on a StepOnePlus Applied Biosystems instrument with SYBR Green. RNA quantity was measured using the Nanodrop 2000 Spectrophotometer at 260nm.

Fixed-cell immunofluorescence

HCT116LMN(B1)−AID cells, HCT116LMN(B2)−AID cells, or HCT116LMN(B1&B2)−AID cells at a low passage (< P10) were plated at 100,000 cells per well of a 6-well glass-bottom plate (Cellvis, #P06-1.5H-N). Following auxin treatment, cells were washed twice with 1 × Phosphate Buffered Saline (PBS) (Gibco, #10010031). Cells were fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, #15,710) for 10 min at room temperature, followed by washing with PBS 3 times for 5 min each. Cells were permeabilized using 0.2% TritonX-100 (10%) (Sigma-Aldrich, #93443) in 1 × PBS, followed by another wash with 1 × PBS for 3 times for 5 min each. Cells were blocked using 3% BSA (Sigma-Aldrich, #A7906) in PBST (Tween-20 in 1 × PBS) (Sigma-Aldrich, #P9416) at room temperature. The following primary antibodies were added overnight at 4 °C: anti-lamin B1 (Abcam, #ab16048, dilution 1:1000), anti-lamin B2 (Abcam, #ab155319, dilution 1:1000), anti-lamin A (Abcam, #ab8980, dilution 1:1000), anti-H3K27ac (Abcam, #ab177178, dilution 1:7000), and anti-H3K27me3 (Abcam, #ab6002, dilution 1:200). Cells were washed with 1 × PBS 3 times for 5 min each. Cells were washed with 1 × PBS 3 times for 5 min each. The following secondary antibodies were added for 1 h at room temperature: Goat anti-Rabbit IgG (H + L) Alexa Fluor 568 (Abcam, #ab175471, dilution 1:1000), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Thermo Fisher Scientific, #A32728, dilution 1:200), and Invitrogen Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, #A-21245). Cells were washed with 1 × PBS 3 times for 5 min each. Finally, cells were stained with DAPI (Thermo Fisher Scientific, #62248, diluted to 0.5 μg/mL in 1 × PBS) for 10 min at room temperature. Prior to imaging, cells were washed with 1 × PBS twice for 5 min each.

Preparation of Hi-C Libraries for in situ Hi-C

In situ Hi-C was performed as previously described [2]. Briefly, 2–5 million cells at 80% confluence were detached and pelleted by centrifugation at 300 × G for 5 min. Cells were resuspended in fresh medium at a concentration of 1E6 cells per 1 mL media. In a fume hood, 1% formaldehyde was used to crosslink cells, with 10 min of incubation at room temperature with mixing. 2.5M glycine solution was added to a final concentration of 0.2M to quench the reaction, and the reaction was incubated at room temperature for 5 min with gentle rocking. Samples were centrifuged at 300 × g for 5 min at 4°C. Cells were resuspended in 1mL of ice-cold PBS and spun at 300 × g for 5 min at 4°C. Cell pellets were flash-frozen in liquid nitrogen and either stored at − 80°C or used immediately for lysis and restriction digest. Nuclei were permeabilized. Two hundred fifty microliters of Hi-C lysis buffer (10mM Tris–HCl pH 8.0, 10mM NaCl, 0.2% Igepal CA930 (Sigma-Aldrich, #I3021)) with 50 μL of protease inhibitors (Sigma-Aldrich, #P8340) was added to each crosslinked pellet of cells. After incubation on ice for > 15 min, samples were centrifuged at 2500 × g for 5 min and washed with 500 μL of ice-cold Hi-C lysis buffer. Pelleted nuclei were resuspended in 50 μL of 0.5% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, #436143) and incubated at 62°C for 10 min. Next, 145 μL of water (Fischer Scientific, #10–977-015) and 25 μL of 10% Triton X-100 (Sigma-Aldrich, #93443) were added to quench the SDS. Samples were mixed and incubated at 37°C for 15 min. DNA was digested with 100U of MboI restriction enzyme (New England Biolabs, #R0147) and 25 μL of 10X NEBuffer 2 (New England Biolabs, #B7002S). Chromatin was digested overnight at 37°C with rotation. Samples were incubated at 62°C for 20 min to inactivate MboI, and then cooled to room temperature. The ends of restriction fragments were labeled using biotinylated nucleotides (Thermo Fisher Scientific, #19524016) and ligated in a small volume (~ 900 μL) using 10X NEB T4 DNA ligase buffer (New England Biolabs, #B0202) and DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs, #M0202) after 1 h of incubation at 37°C. Samples were mixed and incubated at room temperature for 4 h prior to reversal of crosslinks. We added 50 μL of 20 mg/mL proteinase K (New England Biolabs, #P8102) and 120 μL of 10% SDS and incubated samples at 55°C for 30 min. Next, 130 μL of 5M sodium chloride was added and samples were incubated at 68°C overnight. Ligated DNA was purified and sheared to a length of ~ 400 bp, as previously described [2] using a LE220-plus Focused-ultrasonicator (Covaris, #500569) and AMPure XP beads (Beckman Coulter, #A63881). DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, #Q33230) and undiluted DNA was run on a 2% agarose gel to verify successful size selection. Point ligation junctions were pulled down with 10 m/mL Dynabeads MyOne Steptavidin T1 beads (Thermo Fisher Scientific, #65601) and prepared for Illumina sequencing using Illumina primers and protocol (Illumina, 2007) as previously described [2]. Paired-end sequencing was performed using the Illumina HiSeq 2000 OR 2500 platform. A no-ligation control was also used.

Hi-C data processing and analysis

Juicebox was used to visualize Hi-C contact maps [79]. All Hi-C data reported were produced using Illumina paired-end sequencing. We followed the Hi-C data processing pipeline that has previously been described [2]. This pipeline uses the Burrows-Wheeler single end aligner (BWA) [80] to map each read end separately to the hg19 reference genome, removes reads that map to the same fragment, removes duplicate or near-duplicate reads, and filters the remaining reads based on the mapping quality score. All analysis (i.e., aggregate peak analysis) and annotations (i.e., annotation of domains, assigning loci to subcompartments, and peaks) were performed as previously described [2, 81]. All contact matrices were KR-normalized with Juicer. Domains were annotated using TopDom.

Chromosome paint hybridization

Cells were trypsinized, resuspended in appropriate growth media and then plated onto glass coverslips and allowed to adhere for approximately 16 h prior to fixation. Cells were rinsed once in PBS prior to fixation in 4% PFA for 10 min at room temperature. Cells were then washed 3 times in PBS, followed by incubation in PBS with 0.01% Triton X-100 at room temperature 3 times for 3 min, then incubation in 0.5% Triton X-100/1 × PBS at room temperature for 15 min. Cells were then incubated in 20% glycerol/ PBS at room temperature for 3 h. Cells were washed in PBS 3 times for 10 min each and then incubated in 0.1 N HCl for 5 min at room temperature. The cells were incubated in 2 × SSC twice for 3 min each before being placed in 50% formamide (Electron Microscopy Sciences)/2 × SSC at room temperature for approximately 18 h. After the addition of chromosome paints (Metasystems) to the coverslips, slides were heated to 75°C for 2 min before being placed at 37° for approximately 72 h for hybridization. After hybridization, coverslips were washed in 2 × SSC washes at 37° three times for 5 min each, followed by washes in 0.1 × SSC at 60° three times for 5 min each. The coverslips were then washed in 4 × SSC/0.2% Tween-20 three times for 3 min and mounted on microscope slides using Diamond antifade with DAPI (Invitrogen). The following chromosome paints were used in this study: Chr 1: D-0301–050-OR, Chr 2: D-0302–050-OR, Chr 18: D-0318–050-OR, and Chr 19: D-0319–050-OR.

Dual-PWS imaging

Briefly, PWS measures the spectral interference signal resulting from internal light scattering originating from nuclear chromatin. This is related to variations in the refractive index distribution (Σ) (extracted by calculating the standard deviation of the spectral interference at each pixel), characterized by the chromatin packing scaling (D). D was calculated using maps of Σ, as previously described [39, 49, 51, 52]. Measurements were normalized by the reflectance of the glass medium interface (i.e., to an independent reference measurement acquired in a region lacking cells on the dish). This allows us to obtain the interference signal directly related to refractive index (RI) fluctuations within the cell. Although it is a diffraction-limited imaging modality, PWS can measure chromatin density variations because the RI is proportional to the local density of macromolecules (e.g., DNA, RNA, proteins). Therefore, the standard deviation of the RI (Σ) is proportional to nanoscale density variations and can be used to characterize packing scaling behavior of chromatin domains with length scale sensitivity around 20–200 nm, depending on sample thickness and height. Changes in D resulting from each condition are quantified by averaging over nearly 2000 cells, taken across 3 technical replicates. Live-cell PWS measurements obtained using a commercial inverted microscope (Leica, DMIRB) using a Hamamatsu Image-EM charge-coupled device (CCD) camera (C9100-13) coupled to a liquid crystal tunable filter (LCTF, CRi VariSpec) to acquire monochromatic, spectrally resolved images ranging from 500 to 700 nm at 2-nm intervals as previously described [46, 49, 50]. Broadband illumination is provided by a broad-spectrum white light LED source (Xcite-120 LED, Excelitas). The system is equipped with a long pass filter (Semrock BLP01-405R-25) and a × 63 oil immersion objective (Leica HCX PL APO). Cells were imaged under physiological conditions (37°C and 5% CO2) using a stage top incubator (In vivo Scientific; Stage Top Systems). All cells were given at least 24 h to re-adhere before treatment (for treated cells) and imaging.

Dynamic PWS measurements

Dynamic PWS measurements were obtained as previously described [49]. Briefly, dynamics measurements (\(_^\), fractional moving mass (\(_}} )\), and diffusion) are collected by acquiring multiple backscattered wide-field images at a single wavelength (550 nm) over time (acquisition time), to produce a three-dimensional image cube, where \(_^\) is temporal interference and t is time. Diffusion is extracted by calculating the decay rate of the autocorrelation of the temporal interference as previously described [49]. The fractional moving mass is calculated by normalizing the variance of \(_^\) at each pixel. Using the equations and parameters supplied and explained in detail in the supplementary information of our recent publication [49], the fractional moving mass is obtained by using the following equation to normalize \(_^\) by\(_\), the density of a typical macromolecular cluster:

$$_^\left(\frac_}^^_}\right)_}_}\right)}^_}_ - _}\right)}^= _}}_\varphi = _\varphi = _}}$$

With this normalization, \(_^\) is equivalent to \(_}}\), which measures the mass moving within the sample. This value is calculated from the product of the mass of the typical moving cluster (\(_)\) and the volume fraction of mobile mass (\(\varphi\)). \(_\) is obtained by \(_= }}__\), where \(}}_\) is the volume of the typical moving macromolecular cluster. To calculate this normalization, we approximate \(_\) = 1.43 as the refractive index (RI) of a nucleosome, \(_\) = 1.37 as the RI of a nucleus, \(_\) = 1.518 as the refractive index of the immersion oil, and \(_\) = 0.55 g cm−3 as the dry density of a nucleosome. Additionally, \(k\) = 1.57E5 cm−1 is the scalar wavenumber of the illumination light, and \(\Gamma\) is a Fresnel intensity coefficient for normal incidence. \(_\) = 1.49 is the numerical aperture (NA) of collection and \(_\) = 0.52 is the NA of illumination. As stated previously [49], \(_^\) is sensitive to instrument parameters such as the depth of field and substrate refractive index. These dependencies are removed through normalization with the proper pre-factor calculated above for obtaining biological measurements. It should also be noted that backscattered intensity is prone to errors along the transverse direction [49]. Due to these variations, these parameters are more accurate when calculating the expected value over each pixel.

Regional PWS analysis

We used PWS to calculate D values via measuring the variations in spectral light interference resulting from light scattering due to heterogeneities in chromatin density as previously described [51]. The same cells used to analyze average chromatin packing scaling in whole nuclear regions were used for regional chromatin packing scaling analysis. For the periphery characterization, individual nuclei were segmented into 6 ribbons of 260 nm width each using MATLAB. The remaining region at the center of the nucleus was classified as the center. We calculated the average D for each pixel, followed by averaging all these values to estimate the average D in each region.

Confocal imaging

For CRISPR-Sirius, when MCP-Halo was added to cells for fluorescent imaging, HaloTag-JF646 was added to the cells at 10 μM 24 h before imaging and incubated overnight at 37°C and 5% CO2. On the day of imaging, live cells were washed three times with DPBS (Gibco, #14190–144) and further incubated with phenol-red free media (Cytiva, #SH30270.01). The optical instrument was built on a commercial inverted microscope (Eclipse Ti-U with the perfect focus system, Nikon). Images of live cells were collected using a × 100 objective and sent to an electron-multiplying CCD (iXon Ultra 888, Andor). A 637-nm laser (Obis, Coherent) was co-illuminated through a × 100/ 1.49 NA (numerical aperture) oil objective lens (SR APO TIRF, Nikon) with an average power at the sample of 3 to 10 kW/cm3. The microscope stage incubation chamber was maintained at 37 °C and supplemented with 5% CO2. For single image acquisition, at least 50 frames were taken at 30 ms exposure time and a gain of 150. Z-stack images were acquired at 0.024 µm per step for a total of 401–701 frames depending on the size of the nuclei being imaged [46, 49, 50]. Images of fixed-cells previously transfected with CRISPR-Sirius plasmids were imaged using the Nikon SoRa Spinning Disk confocal microscope equipped with a Hamamatsu ORCA-Fusion Digital CMOS camera. Images were collected using a × 60/1.42 NA oil immersion objective mounted with a × 2.8 magnifier. mClover was excited with a 488-nm laser, HaloTag-JF646 was excited with a 640-nm laser, and DAPI was excited with a 405-nm laser. Imaging data were acquired by Nikon acquisition software.

SMLM sample preparation and imaging

The primary antibody rabbit anti-H3K9me3 (Abcam, #ab176916) was aliquoted and stored at − 80 °C. The secondary antibody goat anti-rabbit AF647 (Thermo Fisher Scientific, #A-21245) was stored at 4 °C. The cells were plated on No. 1 borosilicate bottom eight-well Lab-Tek Chambered cover glass with at a seeding density of 1.25 × 104. After 48 h, the cells were fixed in 3% paraformaldehyde in PBS for 10 min, and then subsequently washed with PBS once for five min. Thereafter, the samples were quenched with freshly prepared 0.1% sodium borohydride in PBS for 7 min and rinsed with PBS three times at room temperature. The fixed samples were permeabilized with a blocking buffer (3% bovine serum albumin (BSA), 0.5% Triton X-100 in PBS) for 20 min and then incubated with rabbit anti-H3K9me3 in blocking buffer for 1–2 h at room temperature and rinsed with a washing buffer (0.2% BSA, 0.1% Triton X-100 in PBS) three times. The fixed samples were further incubated with the corresponding goat secondary antibody–dye conjugates, anti-rabbit AF647, for 40 min, washed thoroughly with PBS three times at room temperature and stored at 4 °C.

SMLM image analysis

To segment nuclei, nuclear, nuclear periphery, and nuclear interior masks were generated using built-in OpenCV Python package methods to perform combination erosion and dilation operations on the reconstructed H3K9me3 image. To identify the nuclear periphery and what was considered the nuclear lamina, the cv2.findContours method was used to first identify the contour around each nucleus. Contours were then dilated n = 5 times with a 5 × 5 one’s kernel to approximate 100 nm distance from the identified edge. Contours then underwent a bitwise and multiplication with the nuclear mask to keep only the dilated portion of the contour that lies within the original nuclear mask. Nuclear interior masks were identified as the subtraction of the larger nuclear mask by the dilated contour. To measure Normalized STORM Intensity (NSI), reconstructed images were generated using the built-in ThunderSTORM Fiji plugin average shifted histograms algorithms where pixel intensity in resultant image is a proxy for count of localized events within that bin (bin size = 26 nm per pixel). NSI is the quotient of the sum of all pixel intensities within a specific regional mask (e.g., nuclear periphery) normalized by the area of that mask (number of pixels) and the total nuclear intensity normalized by nuclear area. NSI reports the proportion of signal intensity, which is a proxy for the number of events within a given area, relative to the entire nuclear signal. In the equation below, Amask is the nuclear regional mask area, si is the ith pixel of that regional mask, Anuc is area of total nuclear mask, and sj is jth pixel of that total nuclear mask.

$$NSI= \frac_}\sum_^_}_}\sum_^_}$$

Lentivirus packaging

HEK293T cells were used to produce lentiviral particles using FuGENE HD Transfection Reagent (Promega, #E2311) following the manufacturer’s protocol. Briefly, 1 day before transfection, HEK293T cells at low passage (< P10) were plated at 100,000 cells per well of a 12-well plate (Cellvis, P12-1.5H-N). At the time of transfection, cells reached a confluency of 70–80%. For lentivirus packaging, a master mix of DNA was prepared in reduced serum media (OptiMEM, Gibco, #31985–070). This master mix contained the lentiviral packaging plasmid pCMV-VSV-G (a gift from Bob Weinberg, Addgene plasmid # 8454) and pCMV-dR8.2 (a gift from Bob Weinberg, Addgene plasmid # 8455). For packaging each virus, the following amounts of each plasmid were mixed: 0.5 μg transfer vector + 0.45 μg pCMV-dR8.2 + 0.05 μg pCMV-VSV-G. Media was changed 24 h post-transfection to fresh DMEM. Lentiviral particles were harvested 60 h after transfection. The viral supernatants were filtered using a 33-mm-diameter sterile syringe filter with a 0.45-µm pore size hydrophilic PVDF membrane (Millipore Sigma, SLHVR33RS) and added to HEK293T cells. The virus was immediately used or stored at − 80 °C. Polybrene (8 μg/mL; Sigma-Aldrich) was supplemented to enhance transduction efficiency.

CRISPR-Sirius labeling

Plasmids were obtained from Addgene as bacterial stabs and streaked onto LB-ampicillin plates. Upon overnight growth and single-colony selection, a single colony was inoculated into LB-ampicillin liquid culture overnight. Plasmid DNA isolation was performed using QIAprep Spin Miniprep kit (Qiagen, # 27104) following the manufacturer’s protocol. pHAGE-TO-dCas9-P2A-HSA (Addgene plasmid # 121936; http://n2t.net/addgene:121936; RRID: Addgene_121936), pHAGE-EFS-MCP-HALOnls (Addgene plasmid # 121937; http://n2t.net/addgene:121937; RRID: Addgene_121937), and pPUR-hU6-sgRNA-Sirius-8XMS2 (Addgene plasmid # 121942; http://n2t.net/addgene:121942; RRID: Addgene_121942) were gifts from Thoru Pederson. Target sequences for CRISPR-Sirius labeling are listed in Additional file 1.

CRISPR-Sirius transduction

For live-cell CRISPR-Sirius, HCT116LMN(B1&B2)−AID cells were transfected with CRISPR-dCas9 and donor plasmids using FuGENE HD Transfection Reagent (Promega, #E2311) following the manufacturer’s protocol. Briefly, cells at low passage (< P10) were plated at 100,000 cells per well of a 6-well glass-bottom plate (Cellvis, P06-1.5H-N). Twenty-four hours after plating, 50 μL dCas9, 50 μL MCP-HALOnls, and 100 μL sgRNA lentiviral particles were added to each well. Twenty-four hours after transduction, lentiviral particles were removed by replacing media.

CRISPR-Sirius transfection

For additional quantification of foci and distances of foci to the nuclear periphery, HCT116LMN(B1&B2)−AID cells were co-transfected with 200 ng MCP-HaloTag, 400 ng of dCas9 plasmid DNA, and 2 µg of plasmid DNA for the desired guide RNAs using Lipofectamine LTX and Plus Reagent. Cells were incubated for 24 h prior to overnight staining with HaloTag-JF646 before fixation and imaging.

Fluorescence in situ hybridization (FISH)

Cells were trypsinized, resuspended in appropriate growth media, and then plated onto glass coverslips and allowed to adhere for approximately 16 h prior to fixation. Cells were rinsed once in PBS prior to fixation in 4% PFA for 10 min at room temperature. Cells were then washed 3 times in PBS, followed by incubation in PBS with 0.01% Triton X-100 at room temperature 3 times for 3 min, then incubation in 0.5% Triton X-100/1 × PBS at room temperature for 15 min. Cells were then incubated in 20% glycerol/PBS at room temperature for 3 h. Cells were washed in PBS 3 times for 10 min each and then incubated in 0.1 N HCl for 5 min at room temperature. The cells were incubated in 2 × SSC twice for 3 min each before being placed in 50% formamide (Electron Microscopy Sciences)/2 × SSC at room temperature for approximately 18 h. After the addition of FISH probes (Empire Genomics) to the coverslips, slides were heated to 75°C for 2 min before being placed at 37° for approximately 24 h for hybridization. After hybridization, coverslips were washed in 2 × SSC washes at 37° three times for 5 min each, followed by washes in 0.1 × SSC at 60° three times for 5 min each. The coverslips were then washed in 4 × SSC/0.2% Tween-20 three times for 3 min and mounted on microscope slides using either Diamond antifade with DAPI (Invitrogen) or 5 µm of DRAQ5 Fluorescent Probe Solution (Thermo Fisher Scientific, #62251). The following gene-specific FISH probes were used in this study: CEMIP-20-AQ (Chr15:80,779,370–80,951,771), SLCO3A1-20-RE (Chr15:91,853,708–92,172,435), and CYP1A1-20-AQ (Chr15:74,719,542–74,725,528).

Data and image analysis

We used GraphPad Prism 10.1.1 or Excel for statistical analysis and for making all boxplots. Flow cytometric analysis (FACS) data were analyzed using FlowJo software version 10.6.1. To localize the fluorescent puncta, we used ImageJ software to first generate max projections of the Z-stack images/50 frame single-layer acquisition. Max projected images were then background subtracted using the standard rolling ball algorithm with a radius of 12.0. Processed images were then input into the ThunderSTORM ImageJ plugin with a peak intensity threshold coefficient of 5.0 to locate the fluorescent puncta associated with the CRISPR-Sirius and FISH foci. Puncta coordinates were saved in Microsoft Excel as a.csv file to be input to the Python algorithm. The processed image was then fed into a Python computer vision algorithm that segmented out the nucleus and determined the coordinates for the nuclear periphery as well as the centroid of the nucleus. To quantify the spatial distance to the nuclear center or between foci, only pairs of foci lying in the same focal plane were analyzed. Distances were measured from each fluorescent puncta to the nearest nuclear periphery coordinate as well as the centroid of the segmented nucleus. To account for differences in cell area, the distance from each focus to the centroid was divided by the radius, assuming a circular area. This was further verified by dividing the distance by the nuclear area. To detect foci numbers, maximum intensity projection of Z-series images was performed. For chromosome paint experiments, images were analyzed through imaging processing pipelines utilizing standard tools on Cell Profiler [82] and Fiji [83].

Coefficient of variation analysis

To assess chromatin compaction through the Coefficient of Variation (CoV) analysis, DAPI-stained cells (see section Fixed-cell immunofluorescence) treated with Auxin (see section “Auxin treatment”) were imaged on a Nikon SoRa Spinning Disk confocal microscope (see section “Confocal imaging”). Following a published workflow [53], we used ImageJ to create masks of each nucleus. The coefficient of variation of individual nuclei was calculated in MATLAB, with CoV = σ/μ, where σ represents the standard deviation of the intensity values and μ representing the mean value of intensity of the nucleus.

RNA-Seq library preparation

Total RNA extraction was performed on samples from colon carcinoma epithelial HCT116 cells and HCT116LMN(B1&B2)−AID cells using the RNeasy Plus Mini Kit (Qiagen, #74134) following the manufacturer’s protocol. The conditions for these samples were control, 12-h auxin, 48-h auxin, and 48-h auxin with 6 days of removal by changing cell culture media. These samples were collected with three biological replicates per condition. RNA-Seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, #E7760), according to the company’s instruction. Library quality was measured using the Qubit 2.0 and Bioanalyzer.

RNA-Seq data analysis

Bulk mRNA sequencing was conducted in The Genome Analysis and Technology Core in Charlottesville, VA, at the University of Virginia. Paired-end reads were acquired using NextSeq 2000 (75bp) system on high-throughput mode. Reads were aligned to the hg19 genome using HISAT2 and quantified using StringTie. Read counts were normalized and compared for differential gene expression using the DESeq2 package in R. Heatmaps were generated using the pheatmap package. Other plots were generated using the ggplot2 package. We used Metascape (https://metascape.org/gp/index.html#/main/step1) to perform pathway enrichment analysis. The publicly available hg19 DamID track was downloaded from the 4D Nucleome data repository (data.4dnucleome.org) [45, 84]. The tracks are obtained from two independent biological replicates. We used bedtools to compare gene coordinates with the DamID LAD coordinates. We specifically used the “reldist,” “closest,” and “coverage” options of bedtools.