Introduction
Cohesin is a ubiquitously expressed multiprotein complex best known for its involvement in sister chromatid cohesin, but it also plays important roles in chromosome organization, gene expression, and DNA repair (
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5SMC complexes: from DNA to chromosomes.
). In vertebrate cells, cohesin is a ring-like structure encircling chromatin composed of four core subunits: SMC1A, SMC3, RAD21, and either STAG1 or STAG2. Several additional subunits serve to regulate the core complex, including NIPBL, MAU2, WAPL, PDS5A, PDS5B, and sororin. The cohesin complex is highly conserved in both prokaryotic and eukaryotic unicellular organisms, as well as in metazoans. Somatic mutations of cohesin are present in a wide range of human cancers, and inherited mutations result in developmental disorders known as cohesinopathies (
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Mutational inactivation of STAG2 causes aneuploidy in human cancer.
,
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Cohesin mutations in human cancer.
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Etiology and pathogenesis of the cohesinopathies.
).
Current models of cohesin function focus on the ability of the cohesin ring to encircle DNA in a cell cycle and developmentally-regulated fashion. This entrapment of DNA by cohesin is thought to result in several important mechanistic outcomes: (i) encirclement of homologous regions of sister chromatids (in trans) enforces sister chromatid cohesion until cohesin is degraded at the metaphase to anaphase transition, and (ii) encirclement of otherwise distant regions of contiguous DNA (in cis) enables the formation of loop structures important in regulating gene expression and chromosomal organization. The mechanistic implications of the cohesin ring model drive most current research into cohesin function and into the consequences of its inactivation in human disease.
In an effort to further our understanding of cohesin function using an unbiased approach, we set out to generate a complete cohesin interactome in human cells. To do this, gene editing was used to create a panel of 11 isogenic human cell lines in which each of the genes encoding the 11 known components of cohesin were modified with the addition of a dual epitope tag in either the C or N terminus of the encoded protein. Mass spectrometry was then performed on dual-affinity purifications to generate an endogenous human cohesin interactome. This effort, together with subsequent confirmation of the interactions and functional validation, revealed that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs)
2The abbreviations used are:
RBP
RNA-binding protein
snRNP
small nuclear ribonucleoprotein
SBP
streptavidin-binding peptide
HBSS
Hanks' buffered saline solution
FDR
false-discovery rate
AAV
adeno-associated virus.
required for mitotic progression, identifying splicing factors and RBPs as a novel class of functionally significant cohesin-interacting proteins.
Discussion
In this study we used gene editing to introduce dual epitope tags into the endogenous allele of each of 11 known components of cohesin in human cells, and we performed dual-affinity purification and MS to define the endogenous cohesin interactome. We found the following: (i) cohesin interacts with a panoply of splicing factors and RBPs in chromatin; (ii) the interactions between splicing factors and cohesin and between splicing factors and chromatin are cell cycle-regulated, and (iii) cohesin and interacting splicing factors are similarly required for mitotic progression.
The data presented here build on prior studies that identified intriguing intersections between cohesin and splicing factors. In 2005, McCracken
et al. (
30- McCracken S.
- Longman D.
- Marcon E.
- Moens P.
- Downey M.
- Nickerson J.A.
- Jessberger R.
- Wilde A.
- Caceres J.F.
- Emili A.
- Blencowe B.J.
Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin.
) reported an evolutionarily conserved, robust physical interaction between cohesin and the SRRM1 splicing co-activator. Then, in 2014, four independent groups simultaneously reported that depletion of splicing factors, including many of those we reported here interact with cohesin, results in attenuation of sister chromatid cohesion that phenocopies depletion of cohesin itself (
21- Sundaramoorthy S.
- Vázquez-Novelle M.D.
- Lekomtsev S.
- Howell M.
- Petronczki M.
Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesin.
22- van der Lelij P.
- Stocsits R.R.
- Ladurner R.
- Petzold G.
- Kreidl E.
- Koch B.
- Schmitz J.
- Neumann B.
- Ellenberg J.
- Peters J.M.
SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs.
,
23- Watrin E.
- Demidova M.
- Watrin T.
- Hu Z.
- Prigent C.
Sororin pre-mRNA splicing is required for proper sister chromatid cohesin in human cells.
24- Oka Y.
- Varmark H.
- Vitting-Seerup K.
- Beli P.
- Waage J.
- Hakobyan A.
- Mistrik M.
- Choudhary C.
- Rohde M.
- Bekker-Jensen S.
- Mailand N.
UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells.
). They further demonstrated that depletion of splicing factors results in aberrant splicing of the cohesin subunit sororin, resulting in its functional inactivation. However, the fact that these splicing factors interact physically with cohesin provides another possible mechanism for the observation that depletion of splicing factors results in loss of sister chromatid cohesion.
The molecular details of the physical interaction between cohesin and splicing factors/RBPs are of substantial interest to us. In this study, we show that the interactions occur in chromatin and require neither RNA nor DNA. We also demonstrate that RAD21, but no other individual component of cohesin, is required for the interaction with splicing factors and RBPs in HCT116 and HeLa cells. RAD21 is well-known to be an especially important component of cohesin, as it is required for the opening and closing of the cohesin ring around chromatin and is degraded by Separase to drive the metaphase to anaphase transition. Further studies are required to determine how these known functions for RAD21 interface mechanistically with the new function we uncover here, bridging the interaction between cohesin and interacting splicing factors/RBPs.
We are also interested in determining which, if any, individual splicing factors and RBPs are required for the interaction between cohesin and splicing factors/RBPs. However, because our MS data identified >30 such interacting splicing factors/RBPs, and none were overwhelmingly more abundant than others in the affinity purifications, we are at present unable to generate or test hypotheses regarding the identity of splicing factors required for maintenance of the complex.
We are intrigued by the possibility that splicing factors and RBPs are required for the loading and/or proper positioning of cohesin on chromatin. Our initial experiments testing this hypothesis demonstrated that depletion of splicing factors/RBPs does not alter the total amount of cohesin that interacts with chromatin in asynchronous proliferating HeLa cells (data not shown). These results are consistent with the findings of Sundaramoorthy
et al. and van der Lelij
et al. (
21- Sundaramoorthy S.
- Vázquez-Novelle M.D.
- Lekomtsev S.
- Howell M.
- Petronczki M.
Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesin.
,
22- van der Lelij P.
- Stocsits R.R.
- Ladurner R.
- Petzold G.
- Kreidl E.
- Koch B.
- Schmitz J.
- Neumann B.
- Ellenberg J.
- Peters J.M.
SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs.
), who demonstrated that splicing factors alter the interaction of cohesin with chromatin only in G
2 cells. Further experiments using synchronized cells and more specific readouts such as cohesin ChIP-seq may prove informative.
Upon initially making the discovery reported here, we were surprised to find a large number of published studies that report discrete cell cycle phenotypes of splicing factor inactivation in a wide range of organisms, from
Saccharomyces cerevisiae to metazoans (
31- Kittler R.
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- et al.
Genome-scale RNAi profiling of cell division in human tissue culture cells.
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- Sieckmann F.
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- Kabbe R.
- Wünsche A.
- et al.
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
,
33- Huen M.S.
- Sy S.M.
- Leung K.M.
- Ching Y.P.
- Tipoe G.L.
- Man C.
- Dong S.
- Chen J.
SON is a spliceosome-associated factor required for mitotic progression.
,
34- Hofmann J.C.
- Tegha-Dunghu J.
- Dräger S.
- Will C.L.
- Lührmann R.
- Gruss O.J.
The Prp19 complex directly functions in mitotic spindle assembly.
,
35- Hofmann J.C.
- Husedzinovic A.
- Gruss O.J.
The function of spliceosome components in open mitosis.
36- Karamysheva Z.
- Díaz-Martínez L.A.
- Warrington R.
- Yu H.
Graded requirement for the spliceosome in cell cycle progression.
). In most of these studies the authors propose that inactivation of splicing factors results in aberrant splicing of key cell cycle regulatory genes, resulting in cell cycle arrest and/or aberrant progression through the cell cycle. Such a splicing-dependent mechanism is consistent with current models of splicing factor function. However, inactivation of splicing factors is predicted to result in aberrant splicing of thousands of cellular mRNAs, a subset of which would be expected to be essential for viability. Therefore, why inactivation of splicing factors results in discrete and specific cell cycle abnormalities rather than rapid toxicity and cell death is a mystery.
We believe the findings reported here have the potential to provide insight into the mechanisms through which inactivation of splicing factors results in cell cycle arrest. Important next steps will include determining whether the requirement for splicing factors in mitotic progression depends on their interactions with cohesin and/or their splicing activities. However, such studies will require the development of techniques for rapid inactivation of cohesin-interacting splicing factors and the identification of splicing factor/RBP mutants that separate their splicing functions from their ability to interact with cohesin and to regulate mitotic progression.
This study also demonstrates the power of applying gene-editing approaches to the discovery of endogenous interactomes in human cells. To date, the addition of epitope tags to endogenous genes in human cells has been performed on an individual gene basis. This is, to our knowledge, the first application of endogenous tagging to an entire human protein complex or pathway.
In conclusion, here we demonstrate the existence of a robust physical interaction between endogenous cohesin and endogenous splicing factors/RBPs in the chromatin of human cells. Further studies are required to reveal the details of the biomolecular interactions within the complex and its importance in the various critical functions of cohesin and splicing factors in cell biology and human disease.
Experimental procedures
Cell lines
HCT116, HeLa, and RPE-hTERT cells were obtained from the ATCC. HeLa-H2B-GFP cells were obtained from Millipore. Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2.
Gene editing
AAV-based gene editing was used to modify an endogenous allele of each of the genes encoding the 11 known subunits of cohesin. The biological and technical principles underlying AAV-based gene editing are described in detail in Refs.
9- Khan I.F.
- Hirata R.K.
- Russell D.W.
AAV-mediated gene targeting methods for human cells.
,
10- Kim J.S.
- Bonifant C.
- Bunz F.
- Lane W.S.
- Waldman T.
Epitope tagging of endogenous genes in diverse human cell lines.
11- Gaj T.
- Epstein B.E.
- Schaffer D.V.
Genome engineering using adeno-associated virus: basic and clinical research applications.
.
Gene-editing vectors were designed as shown in
Fig. 1A and
Fig. S1. Homology arms were synthesized by Genscript and cloned sequentially into pAAV-SEPT, an AAV-based gene-editing acceptor vector we previously reported in which polylinkers for the cloning of left and right homology arms flank a promoterless splice–acceptor–IRES–neo
R gene (
10- Kim J.S.
- Bonifant C.
- Bunz F.
- Lane W.S.
- Waldman T.
Epitope tagging of endogenous genes in diverse human cell lines.
). Next, transient stocks of AAV-2 virions were created by co-transfection of 293T cells with gene-editing vectors together with pAAV-RC (Stratagene) and pHELPER (Stratagene) using X-tremeGENE 9 (Roche Applied Science). Two days after transfection, the medium was aspirated, and cell monolayers were scraped into 1 ml of PBS and subjected to four cycles of freeze/thaw. The lysate was then clarified by centrifugation at 12,000 rpm for 10 min in a benchtop microcentrifuge to remove cell debris, and the virus-containing supernatant was aliquoted and stored at −80 °C.
Virus was then used to infect HCT116 cells in T25 tissue culture flask, and 24 h later, cells were passaged at limiting dilution into 96-well plates in the presence of 1.0 mg/ml G418. After 14 days, individual G418-resistant clones were expanded, and genomic DNA was prepared in a 96-well format using the QIAamp 96 DNA blood kit (Qiagen). Clones were tested for homologous integration of the targeting vector using a primer pair specific for the targeted allele. Efficiencies of gene editing are shown in
Table S1. Homologous integration of the dual epitope tag was then confirmed by DNA sequencing. Gene-edited clones were then infected with Cre-expressing adenovirus overnight and plated at limiting dilution into 96-well plates. Single colonies were expanded and tested for G418 sensitivity to identify clones in which the now gene-edited allele had been restored to its otherwise natural configuration. All homology arm and primer sequences are available from the authors upon request.
Protein preparations
Nuclear extracts for dual-affinity purification followed by GeLC-MS/MS were prepared using a modification of Dignam's nondetergent lysis method (
37- Dignam J.D.
- Lebovitz R.M.
- Roeder R.G.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
,
38- Jung S.Y.
- Malovannaya A.
- Wei J.
- O'Malley B.W.
- Qin J.
Proteomic analysis of steady-state nuclear hormone receptor co-activator complexes.
). Whole-cell protein lysates for immunoprecipitation and Western blotting were prepared in either Nonidet P-40 lysis buffer (50 m
m Tris-HCl, pH 7.5, 150 m
m NaCl, 1% Nonidet P-40) or RIPA buffer (50 m
m Tris-HCl, pH 7.5, 150 m
m NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Chromatin-associated proteins and total soluble proteins were prepared as described (
39- Busslinger G.A.
- Stocsits R.R.
- van der Lelij P.
- Axelsson E.
- Tedeschi A.
- Galjart N.
- Peters J.M.
Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl.
). Protein concentrations were determined using the bicinchoninic assay (Pierce).
Dual-affinity purification
Nuclear extracts were prepared from ∼109 cells (∼60 confluent 15-cm dishes) for parental HCT116 cells and each of the 11 cohesin epitope-tagged derivatives. Next, either 30 mg of nuclear extract (HCT116, SMC1A, SMC3, RAD21, STAG1, STAG2, WAPL, and PDS5A) or 100 mg of nuclear extract (HCT116, PDS5B, sororin, NIPBL, and MAU2) was incubated with FLAG M2 beads (Sigma) rotating at 4 °C for 1 h. Beads were then transferred to Poly-Prep® chromatography columns (Bio-Rad) and washed with 30× bed volume Tris-buffered saline (TBS) containing 150 mm NaCl. Proteins were then eluted with 5× bed volume 100 ng/μl 1× FLAG peptide (Sigma). FLAG eluents were then applied to Streptavidin Plus UltraLink resin (Pierce) rotating at 4 °C for 1 h. Beads were then transferred to Poly-Prep® chromatography columns and washed with 30× bed volume TBS containing 150 mm NaCl. Proteins were then eluted with 2× bed volume 1 mm d-biotin. Proteins were then concentrated by TCA precipitation, dissolved in LDS Sample Buffer (Thermo Fisher Scientific), and subjected to GeLC-MS/MS.
GeLC-MS/MS
Dual-affinity purified proteins derived from either 20 mg of nuclear extract (HCT116, SMC1A, SMC3, RAD21, STAG1, STAG2, WAPL, and PDS5A) or 75 mg of nuclear extract (HCT116, PDS5B, sororin, NIPBL, and MAU2) were separated by SDS-PAGE. Gels were fixed in 50% methanol, 7% acetic acid for 1 h at room temperature, washed with water, stained with Coomassie Brilliant Blue (Thermo Fisher Scientific) for 2 h, and destained with water overnight. Each lane was then excised in seven pieces. Gel slices were washed in 50% acetonitrile and rehydrated with a 50 mm ammonia bicarbonate/trypsin solution and digested at 37 °C overnight. Peptides were then extracted with a series of elutions, dried in a speed vac, and solubilized in 0.1% formic acid in water for analysis by tandem MS.
LC-MS/MS was performed on an LTQ Orbitrap Elite (Thermo Fisher Scientific) equipped with Waters (Milford, MA) NanoAcquity HPLC pump. Peptides were separated onto a 100-μm inner diameter microcapillary trapping column packed first with ∼5 cm of C18 Reprosil resin (5 μm, 100 Å, Dr. Maisch GmbH, Germany) followed by an analytical column with ∼20 cm of Reprosil resin (1.8 μm, 200 Å, Dr. Maisch GmbH, Germany). Separation was achieved by applying a gradient from 5 to 27% acetonitrile in 0.1% formic acid over 90 min at 200 nl/min. Electrospray ionization was enabled by applying a voltage of 1.8 kV using a homemade electrode junction at the end of the microcapillary column and sprayed from fused silica pico tips (New Objective, MA). The LTQ Orbitrap Elite was operated in the data-dependent mode for the MS methods. The MS survey scan was performed in the Orbitrap in the range of 395–1800 m/z at a resolution of 6 × 104, followed by the selection of the 20 most intense ions (TOP20) for CID-MS2 fragmentation in the ion trap using a precursor isolation width window of 2 m/z, AGC setting of 10,000, and a maximum ion accumulation of 200 ms. Singly charged ion species were not subjected to CID fragmentation. Normalized collision energy was set to 35 V and an activation time of 10 ms; AGC was set to 50,000, and the maximum ion time was 200 ms. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60 s.
Raw data were submitted for analysis in Proteome Discoverer 2.1.0.81 (Thermo Fisher Scientific) software. Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against a protein sequence database, including all entries from the Human UniProt database (SwissProt 16,768, 2016) and other known contaminants such as human keratins and common lab contaminants. SEQUEST HT searches were performed using a 20-ppm precursor ion tolerance and requiring each peptide's N/C terminus to adhere to trypsin protease specificity while allowing up to two missed cleavages. Cysteine carbamidomethyl (+57.021) was set as static modifications, whereas methionine oxidation (+15.99492 Da) was set as variable modification. MS2 spectra assignment FDR of 1% on protein level was achieved by applying the target-decoy database search. Filtering was performed using Percolator (64-bit version). For quantification, a 0.02 m/z window centered on the theoretical m/z value of each of the six reporter ions and the intensity of the signal closest to the theoretical m/z value was recorded. Reporter ion intensities were exported in the result file of Proteome Discoverer 2.1 search engine as Excel tables.
STRING
Proteins identified by MS and listed in the 11 tabs of
Table S2 were analyzed using the STRING database (
http://string-db.org; version 10.0)
3Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
(
17- Szklarczyk D.
- Morris J.H.
- Cook H.
- Kuhn M.
- Wyder S.
- Simonovic M.
- Santos A.
- Doncheva N.T.
- Roth A.
- Bork P.
- Jensen L.J.
- von Mering C.
The STRING database in 2017: quality-controlled protein–protein association networks, made broadly accessible.
) to construct a protein–protein interaction network. The network (
Fig. 1C) was constructed using the highest confidence level setting, and unconnected nodes were removed.
Immunoprecipitation
For SMC1A immunoprecipitation, protein lysates were incubated with 3 μg of SMC1A antibody (clone H-6, Santa Cruz Biotechnology sc-393171) or IgG control antibody in TBS containing 0.1% Nonidet P-40 for 1 h rotating at 4 °C. Protein A/G-agarose beads (Santa Cruz Biotechnology) were then added, and the lysates were further incubated for 1 h rotating at 4 °C. Beads were then washed three times with TBS (either 150 or 350 mm NaCl) containing 0.1% Nonidet P-40. Proteins were then eluted into 2× LDS sample buffer at room temperature for 20 min. DTT was then added to a final concentration of 50 mm. Samples were boiled for 5 min and separated by SDS-PAGE.
For FLAG immunoprecipitation, FLAG M2 beads (Sigma) were washed once with TBS (150 mm NaCl) and then incubated with protein lysates for 1 h rotating at 4 °C. Beads were then washed three times in TBS, and bound proteins were eluted with 100 ng/μl 1× FLAG peptide (Sigma). Eluents were then concentrated by TCA precipitation, resuspended in sample buffer, and separated by SDS-PAGE.
Silver staining
Silver staining was performed with the SilverQuest staining kit (Thermo Fisher Scientific), following the manufacturer's instructions.
Western blotting
Protein lysates, affinity purifications, and immunoprecipitations were dissolved in LDS sample buffer, boiled for 5 min, and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes, which were then probed with a 1:1000 dilution of primary antibodies overnight rotating at 4 °C. After incubation with horseradish peroxidase–conjugated secondary antibodies (Cell Signaling) rotating for 1 h at room temperature, membranes were developed with SuperSignal West Pico PLUS chemiluminescent substrate (Pierce) and imaged using a myECL imager (Pierce).
Immunofluorescence
HeLa cells were cultured on coverslips, fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% Triton X-100 in PBS at room temperature for 15 min. Fixed and permeabilized cells were then blocked in 5% BSA and 0.25% Triton X-100 in PBS for 1 h at room temperature and then incubated with primary antibodies (1:200) in wash buffer (1% BSA and 0.25% Triton X-100 in PBS) overnight at 4 °C. Cells were then washed three times with wash buffer, then incubated with fluorescent secondary antibodies (Alexa Fluor 488 and Alexa Fluor 647, Thermo Fisher Scientific) in antibody dilution buffer for 1 h at room temperature, and then washed three times with wash buffer. The slides were mounted with antifade mountant with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific) and imaged with a Leica TCS SP8 confocal laser-scanning microscope fitted with ×63/1.40 plan apochromat oil immersion objective. Images were processed and analyzed using ImageJ software (
40- Schneider C.A.
- Rasband W.S.
- Eliceiri K.W.
NIH Image to ImageJ: 25 years of image analysis.
).
Antibodies
Primary antibodies for immunoblotting were FLAG (M2) and tubulin (DM1α) from Sigma; sororin (ab192237) from Abcam; STAG2 (sc-81852), WAPL (sc-365189), HSPA8 (sc-7298), MAU2 (sc-243420), and NIPBL (sc-374625) from Santa Cruz Biotechnology; cyclin B1 (12231), cyclin E1 (20808), glyceraldehyde-3-phosphate dehydrogenase (2118), histone H3 (14269), and phospho-H3 (53348) from Cell Signaling Technologies; and ADAR1 (A303-884), DDX47 (A302-977), EFTUD2 (A300-957), HNRNPH (A300-511), PDCD11 (A303-804), PDS5A (A300-088), PDS5B (A300-538), PRPF31 (A303-919), RAD21 (A300-080), RBM10 (A301-006), RBM15 (A300-821), SF3B1 (A300-997), SF3B3 (A302-508), SMC1A (A300-055), SMC3 (A300-060), SNRNP200 (A303-453), and STAG1 (A302-579) from Bethyl Laboratories.
Primary antibodies for immunofluorescence were STAG2 (sc-81852) and SMC1A (sc-393171) from Santa Cruz Biotechnology, and HNRNPH (A300-511), EFTUD2 (A300-957), and SF3B1 (A300-997) from Bethyl Laboratories.
Double thymidine block for cell synchronization
HeLa cells (2 × 106) were plated in 10-cm2 dishes. After 24 h, cells were treated with 2 mm thymidine (Sigma) for 24 h to arrest cells in S phase. Cells were then released by washing in Hanks’ buffered saline solution (HBSS) followed by an 8-h incubation in regular media. Cells were then treated again with 2 mm thymidine (Sigma) for 16 h to generate a synchronized population of cells arrested in S phase. Cells were then washed in HBSS, incubated in regular media to release them from the cell cycle arrest, and harvested at different time points for flow cytometry and protein preparation.
Drug treatments
Nocodazole (Sigma) was used at a final concentration of 100 ng/ml. RO-3306 (Sigma) was used at a final concentration of 10 μm. Hydroxyurea (Sigma) was used at a final concentration of 0.5 mm. BI 2536 (Selleckchem) was used at final concentrations of 50 and 100 nm.
Flow cytometry
Cells were fixed in 70% ethanol and stained in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 50 μg/ml RNase, and 50 μg/ml propidium iodide. DNA content was measured on a FACSort flow cytometer (BD Biosciences), and data were analyzed using ModFit software (Verity Software House). At least 20,000 cells were analyzed for each sample.
CRISPR
A guide RNA targeting the first coding exon of PDS5A (GTGGCGTCGTGAGTGCCGAC) was designed using the Feng Zhang lab’s CRISPR guide RNA design tool (crispr.mit.edu) and cloned into lentiCRISPR version 2 (a gift from Feng Zhang, Addgene no. 52961). Lentivirus was packaged using pMD2.G (Addgene no. 12259) and psPAX2 (Addgene no. 12260) and was used to infect HCT116-SMC1A–tagged cells. Individual puromycin
R colonies were obtained by limiting dilution and tested by Western blotting to identify clones in which expression of PDS5A had been lost. DNA sequencing was performed to confirm the presence of homozygous truncating mutations in the first exon of PDS5A, and the cells used for the experiments are shown in
Fig. 5A.
siRNA
Cells were transfected with Silencer Select siRNAs (Ambion) using RNAiMax (Thermo Fisher Scientific) according to the manufacturer's instructions. Catalogue numbers for individual siRNAs and their depletion efficiencies are shown in
Figs. 5D and
8A.
Live-cell imaging
Histone H2B-GFP–expressing HeLa cells (Millipore) were plated on μ-Slide 8-well glass bottom plate (Ibidi), transfected with siRNAs, and cultured for 36 h. The chamber slide was placed in a Leica TCS SP8 confocal laser-scanning microscope fitted with environmental chamber and stage-top CO2/humidity incubator held at 37 °C and 5% CO2 (Life Imaging Services). For each siRNA treatment, four positions were recorded every 5 min over a 36-h imaging session. Data acquisition was performed with Leica LAS X control software.
Article info
Publication history
Published online: April 22, 2019
Received in revised form:
April 18,
2019
Received:
February 1,
2019
Edited by Joel M. Gottesfeld
Footnotes
This work was supported by National Institutes of Health Grant R01CA169345 (to T. W.), Alex's Lemonade Stand (to T. W.), the Hyundai Hope on Wheels Foundation (to T. W.), and National Institutes of Health Common Fund 4D Nucleome Project Grant U54DK107979 (to W. S. N.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
This article contains Figs. S1–S4, Tables S1–S6, and supporting Movies S1 and S2.
Copyright
© 2019 Kim et al.