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Reproduction, Fertility and Development, 2019, 31, 1497–1506 https://doi.org/10.1071/RD19030

Nuclear trafficking dynamics of Bromodomain-containing protein 7 (BRD7), a switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex subunit, in porcine oocytes and cleavage-stage embryos

Jennifer S. CrodianA,*, Bethany M. WeldonA,*, Yu-Chun TsengA, Birgit CabotA and Ryan Cabot A,B

ADepartment of Animal Sciences, Purdue University, 270 South Russell Street, West Lafayette, IN, 47907, USA. BCorresponding author. Email: [email protected]

Abstract. In the work presented here, we investigated how bromodomain-containing protein 7 (BRD7), a subunit associated with switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes, is trafficked between cellular compartments during embryo development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4, or member 2; SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci; BRD7 is one of these additional subunits. We hypothesised that BRD7 is exported from the nuclei of porcine oocytes and embryos in a Chromosome Region Maintenance 1 (CRM1)-dependent manner and imported into the nuclei using the karyopherin a/b1 heterodimer. Porcine oocytes and embryos were treated with inhibitors of CRM1-mediated nuclear export and karyopherin a/b1-mediated nuclear import to test this hypothesis. An RNA interference assay and a dominant negative overexpression assay were also performed to determine if karyopherin a7 serves a specific role in BRD7 trafficking. Our findings indicate that BRD7 shuttles between nuclear and cytoplasmic compartments during cleavage development. The shuttling of BRD7 indicates that it serves a unique role in remodelling chromatin during this developmental window.

Additional keywords: epigenetic, importin, karyopherin, trafficking, SWI/SNF.

Received 22 January 2019, accepted 30 March 2019, published online 13 May 2019

Introduction during cleavage development and is required for successful Dynamic changes in chromatin structure, which can impact embryo development (Li et al. 1992). developmental competence, occur during the first week of Non-covalent chromatin modifications involve repositioning mammalian embryo development. These early changes include and restructuring nucleosomes (Bartholomew 2014). Chromatin remodelling of epigenetic marks, which include heritable remodelling machinery utilises the energy released from ATP covalent and non-covalent modifications to DNA that impact hydrolysis to reposition nucleosomes to facilitate activation or gene expression. Changes in chromatin structure that impact repression of transcription. The switch/sucrose non-fermentable transcription must be tightly regulated to ensure successful (SWI/SNF) chromatin remodelling complexes are one group of embryo development. ATP-dependent chromatin remodelling complexes; they are Epigenetic marks accumulated during germ cell develop- utilised extensively during embryonic development in mamma- ment are remodelled during the cleavage stage to prepare for lian embryos (Bultman et al. 2006). further development (Sasaki and Matsui 2008). Both covalent The large, multi-subunit SWI/SNF complexes contain a and non-covalent chromatin modifications take place during catalytic subunit, either SWI/SNF related, Matrix associated, early embryo development. An example of a covalent epigenetic Actin dependent Regulator of Chromatin, subfamily A, member modification is the methylation of either DNA or histone 4 (SMARCA4; also known as Brahma-related gene 1, BRG1) or proteins. DNA can be methylated at the cytosine-5 position. SWI/SNF related, Matrix associated, Actin dependent Regula- DNA methylation is dramatically remodelled on a global level tor of Chromatin, subfamily A, member 2 (SMARCA2; also

*These authors contributed equally to this paper.

Journal Compilation Ó CSIRO 2019 Open Access CC BY-NC-ND www.publish.csiro.au/journals/rfd 1498 Reproduction, Fertility and Development J. S. Crodian et al. known as Brahma, BRM); these catalytic subunits have ATPase nuclear export signal (NES) out of the nucleus. The NESs activity (Clapier and Cairns 2009). A functional SWI/SNF recognised by CRM1 are part of the primary amino acid chromatin remodelling complex contains a collection of sequence in proteins and are typically short (,10 amino acids) BRG1-associated factors (BAFs) in addition to the catalytic and rich in leucine residues (Fukuda et al. 1997). CRM1- subunit. The specific combination of BAFs that associate with mediated nuclear export occurs in GV-stage porcine oocytes the catalytic subunit creates unique SWI/SNF complexes that and throughout cleavage development, but CRM1-mediated act at discrete loci within the genome (Clapier and Cairns 2009). export is not required for development until the 4-cell stage of SWI/SNF chromatin remodelling complexes participate in a development (Cabot et al. 2002). myriad of cellular processes. It has been shown that SWI/SNF- One of the most documented transport pathways is nuclear mediated chromatin remodelling is necessary for mammalian import mediated by the karyopherin a/b1 heterodimer (also embryo development. When SMARCA4 is knocked out in referred to as importin a/b; Go¨rlich et al. 1996). Karyopherin a murine embryos, they fail to develop past the blastocyst stage functions as an adaptor and binds to proteins that contain a (Bultman et al. 2000). When SMARCA2 is knocked out in nuclear localisation signal (NLS). Classical NLSs typically embryos, the embryos develop to full term but display a mild consist of short stretches of basic amino acids, typically lysine overgrowth phenotype as adults (Reyes et al. 1998). SMARCA4 residues. These NLSs exist in either monopartite or bipartite also appears to be required for zygotic genome activation and arrangements. The former is exemplified by the NLSs found trophectoderm development (Bultman et al. 2006; Wang et al. in the SV40 large T antigen (PKKKRKV), whereas the latter is 2010). In addition to these two catalytic subunits, SWI/SNF characterised by two small clusters of basic amino acids complexes contain a core group of subunits that include separated by 10 amino acids. The NLS from the protein BAF155, BAF170 and SMARCB1 (SNF5). These BAFs are nucleoplasmin (PAATKKAGQAKKK) is an example of a also required for development (Euskirchen et al. 2012). bipartite NLS (Dingwall and Laskey 1991). Once karyopherin Each BAF subunit has a unique role within a given SWI/SNF a binds to the NLS region of the cargo, it forms a trimeric chromatin remodelling complex. The subunits that make up the complex with karyopherin b1. The karyopherin a/b1–cargo functional core of the SWI/SNF complex are essential for full trimeric complex then moves through the nuclear pore complex remodelling activity; downregulation of these subunits results (NPC) by interactions with the NPC proteins. Once the trimeric in defects in transcription (Phelan et al. 1999). The AT-rich complex enters the nucleus, karyopherin b1 binds to the guano- interacting domain (ARID) family of BAF subunits are utilised sine triphosphate (GTP)-bound form of the GTPase Ran; the for DNA binding (Wilsker et al. 2005) and are needed for binding of Ran-GTP causes the complex to disassociate appropriate cell cycle control (Nagl et al. 2005). Bromodomain- (Ribbeck et al. 1998). The karyopherin a and b1 proteins are containing domain 7 (BRD7) is a polybromo-associated factor exported back to the cytoplasm where they will bind another (PBAF) that is found in both undifferentiated embryonic stem cargo containing an NLS signal. cells and differentiated cells (Kaeser et al. 2008). In embryonic Because intracellular proteins are trafficked to cellular stem cells, BRD7 and ARID1A work to control the expression of compartments by various transport receptors, we wanted to SWI/SNF target genes (Kaeser et al. 2008). BRD7 is required determine if CRM1-mediated nuclear export and karyopherin for successful embryo development; studies involving a BRD7 a/b1-mediated import were involved in transporting BRD7 knockout mouse model revealed that BRD7-null embryos die by within porcine oocytes and cleavage-stage embryos. We mid-gestation (Kim et al. 2016). hypothesised that BRD7 is exported from the nuclei of porcine Previous work from our group has shown that BRD7 under- oocytes and embryos in a CRM1-dependent manner and goes a change in intracellular distribution during progression in imported using the karyopherin a/b1 heterodimer. To test this development from immature, germinal vesicle (GV)-stage hypothesis, GV-stage porcine oocytes and 4-cell-stage porcine oocyte to blastocyst-stage embryo (Cabot et al. 2017). Approxi- embryos were incubated with leptomycin B (LMB), an inhibitor mately 50% of GV-stage oocytes presented a predominately of CRM1-mediated nuclear export and with ivermectin (IVER), cytoplasmic localisation of BRD7, with the other 50% posses- an inhibitor of karyopherin a/b1-mediated import. In addition, sing a nuclear enrichment of BRD7. Pronuclear-stage embryos we selectively blocked karyopherin a/b1-mediated import displayed similar findings with half of the pronuclear-stage mediated by a specific karyopherin a subtype, KPNA7, which embryos examined presenting BRD7 restricted to the cyto- has been shown to be exclusively expressed in mammalian plasm, whereas the other 50% of oocytes presented BRD7 in oocytes and early cleavage-stage embryos. both nuclear and cytoplasmic compartments. BRD7 was found to be predominantly cytoplasmic in embryos at later cleavage Materials and methods stages (4-cell and blastocyst stages of development). The vari- able pattern of BRD7 intracellular localisation suggests that All chemicals were obtained from Sigma-Aldrich unless stated BRD7 may shuttle dynamically between nuclear and cyto- otherwise. plasmic compartments, allowing BRD7-containing SWI/SNF complexes to exist transiently. Porcine oocyte collection Movement of intracellular proteins between the nucleus Ovaries from prepubertal gilts were collected from a local meat and cytoplasm is mediated by a host of transport factors. processing facility, rinsed in a saline solution containing Chromosome Region Maintenance 1 (CRM1) is a nuclear 75 mgmL 1 penicillin and 50 mgmL 1 streptomycin at 378Cand export factor that functions to carry proteins that possess a transported in an insulated container to the laboratory for BRD7 in porcine embryos Reproduction, Fertility and Development 1499 processing. Antral ovarian follicles were manually aspirated with Leptomycin B culture experiments a sterile 18-gauge needle and 10-mL syringe; follicular fluid Leptomycin B (LMB) was added to in vitro maturation medium 8 was pooled and held at 39 C. Cumulus–oocyte complexes (7 nM LMB final concentration in ethanol; LMB treatment) (COCs) were allowed to settle by gravity, follicular fluid was and denuded GV-stage oocytes were incubated for 12 h. As a discarded and COCs were resuspended in a 4-(2-hydroxyethyl)- control, ethanol was added to in vitro maturation medium (1 mL 1-piperazineethanesulphonic acid (HEPES)-buffered medium ethanol per 500 mL maturation medium; 0.2% ethanol, ETOH containing 0.01% polyvinyl alcohol (PVA; Abeydeera et al. treatment) and denuded GV-stage oocytes were incubated for 1998). Cumulus–oocyte complexes with multiple layers of 12 h. Following the 12-h incubation, all cells were fixed in 3.7% intact cumulus cells were selected for experiments. Cumulus paraformaldehyde for 1 h, subjected to an immunocytochemical cells were removed from selected COCs immediately after staining protocol and evaluated using confocal microscopy selection for immunocytochemical staining assays involving (Nikon A1R-MP; Nikon). GV-stage oocytes. For experiments involving porcine embryos, oocytes were matured and fertilised in vitro; embryos were placed in embryo In vitro maturation culture medium for 48 h. After 48 h, 4-cell-stage embryos were For experiments involving RNA microinjection (green fluo- moved to embryo culture medium contained either 7 nm LMB rescent protein, GFP, or dominant negative karyopherin a7, (LMB treatment) or 0.2% ethanol (ETOH treatment) for 12 h. KPNA7) and KPNA7 small interfering RNA (siRNA) injection Following treatment, 4-cell-stage embryos were fixed in 3.7% into porcine embryos, oocytes were first matured in groups of paraformaldehyde, subjected to an immunocytochemical 50–100 COCs in 500 mL of Medium 199 containing 0.14% staining protocol and evaluated using confocal microscopy. PVA, 10 ng mL1 epidermal growth factor, 0.57 mM cysteine, To determine the quality of the embryos produced in each 0.5 IU mL1 FSH, 0.5 IU mL1 LH, 20 ng mL1 leukaemia biological replicate, a group of LMB and ETOH embryos was inhibitory factor (LIF; MilliporeSigma), 20 ng mL1 insulin- also cultured to Day 6 after gamete co-incubation. Replicates in like growth factor 1 (IGF1; Prospec Protein Specialists) and which control (ETOH-treated) embryos failed to yield 40 ng mL1 fibroblast growth factor 2 (FGF2; PeproTech) as blastocyst-stage embryos by Day 6 after fertilisation were previously described (Yuan et al. 2017). The COCs were excluded from our analysis. matured for 42–44 h in a humidified incubator (100% humidity) at 398C with 5% CO2 in air. Cumulus cells were removed from Ivermectin culture experiments matured COCs by vortexing in 0.1% hyaluronidase in a HEPES- Porcine GV-stage oocytes and 4-cell-stage embryos were buffered medium containing 0.01% PVA for 4 min. For cultured with 500 mM ivermectin (Alfa Aesar) to determine if experiments involving embryos incubated with leptomycin B the karyopherin a/b1 import pathway controlled the trafficking and ivermectin, groups of 50–100 COCs were placed in 500 mL of BRD7 at these developmental stages. GV-stage oocytes were of Medium 199 containing 0.14% PVA, 10 ng mL 1 epidermal added to maturation medium that contained either 500 mM growth factor, 0.57 mM cysteine, 0.5 IU mL 1 FSH and ivermectin in dimethyl sulphoxide (DMSO; IVER treatment) or 0.5 IU mL 1 LH as previously described (Abeydeera et al. a control group that contained 4.0% DMSO (DMSO treatment). 1998). The COCs were matured for 42–44 h in a humidified Following 12 h of incubation, all cells were fixed in 3.7% incubator (100% humidity) at 398C with 5% CO2 in air. paraformaldehyde for 1 h, subjected to an immunocytochemical Cumulus cells were removed from matured COCs by vortexing staining protocol and evaluated using confocal microscopy. For in 0.1% hyaluronidase in a HEPES-buffered medium containing experiments involving porcine embryos, oocytes were matured 0.01% PVA for 4 min. and fertilised in vitro; immediately after gamete co-incubation the embryos were moved to embryo culture medium for 48 h. In vitro fertilisation and embryo culture After 48 h, 4-cell-stage embryos were moved to embryo culture medium containing either 500 mM ivermectin (IVER) or 4.0% Denuded oocytes were transferred to a modified Tris-buffered dimethyl sulfoxide (DMSO). After 12 h of incubation, 4-cell- medium (mTBM) and fertilised as previously described stage embryos were fixed in 3.7% paraformaldehyde, subjected (Abeydeera and Day 1997). Fresh semen was collected from a to an immunocytochemical staining protocol and evaluated mature boar with proven fertility housed at Purdue’s Animal using confocal microscopy. To determine the quality of the Science Research and Educational Centre, extended with a embryos produced in each biological replicate, a group of IVER commercial semen extender (EnduraGuard Plus; Mofa Global) and DMSO embryos was also cultured to Day 6 after gamete co- and stored at 17.58C until use for in vitro fertilisation. The incubation. Replicates in which control (DMSO) embryos failed spermatozoa were washed three times in Dulbecco’s phosphate- to yield blastocyst-stage embryos by Day 6 after fertilisation buffered saline (PBS) and then resuspended in mTBM. The were excluded from our analysis. sperm suspension was added to droplets containing 15–30 mature oocytes such that the final concentration of spermatozoa during gamete co-incubation was 5 105 spermatozoa mL1. Ivermectin dose titration The gametes were allowed to co-incubate for 5 h and were then Porcine oocytes were incubated in varying concentrations transferred to porcine zygotic medium 3 (PZM3), an embryo (25–500 mM) of ivermectin, an inhibitor of karyopherin culture medium (Yoshioka et al. 2002) supplemented with a/b1-mediated import, to determine the effective concentration 3mgmL1 bovine serum albumin (BSA). of ivermectin necessary to block nuclear import using a reporter 1500 Reproduction, Fertility and Development J. S. Crodian et al. protein injection assay. Briefly, Alexa 488-labelled glutathione RNA interference-mediated knockdown of KPNA7 S-transferase (GST)-NLS was injected into GV-stage porcine Interfering RNAs were used to knockdown the KPNA7 oocytes that were incubated for 4 h in ivermectin (or DMSO, transcript as previously described (Wang et al. 2012). Briefly, control). Injected oocytes were cultured in oocyte maturation custom-made Stealth siRNAs targeting porcine KPNA7 medium for 24 h after microinjection in the presence of the (50-CAUGAAUGCUUAACGCCCUUAACAA and 50-UUG- respective concentrations of ivermectin. Following culture, UUAAGGGCGUUAAGCAUUCAUG) and control siRNAs oocytes were fixed in 3.7% paraformaldehyde for 1 h and sub- (50-CAUCGUAAAUUCCGCAUUCAAGCAA and 50-UUG- sequently washed three times in PBS containing 0.1% Tween-20 CUUGAAUGCGGAAUUUACGAUG) were synthesised by a (PBST); 15 min for each wash. Cells were permeabilised by commercial vendor (Invitrogen). Duplexed RNAs were diluted incubation in PBS containing 1% Triton X-100 for 1 h; oocytes in RNase-free water to a concentration of 20 mM; aliquots were were washed three times in PBST and then stained with Hoechst frozen at 208C. Aliquots of interfering RNAs were diluted to m 1 33342 (5 gmL ) for 20 min. Cells were then mounted on 1 mM in RNase-free water immediately before microinjection. slides with 20 mL Vectashield (Vector Laboratories, Inc.) and examined by a confocal microscope (Nikon A1R-MP). Microinjection Spermatozoa were removed from presumptive zygotes by GST-NLS production vortexing embryos in 0.1% hyaluronidase in HEPES-buffered A recombinant fusion protein consisting of glutathione medium for 4 min after gamete co-incubation. For mRNA S-transferase (GST) and the nuclear localisation signal (NLS) injections, presumptive zygotes were then divided into three from the SV40 T-antigen (GST-NLS) was produced in BL21 treatments: DN-KPNA7 mRNA injection, GFP mRNA injection cells as described previously (Cabot and Prather 2003). Briefly, and non-injected controls. Injection pipettes were loaded with transfected bacteria were grown in liquid culture medium at 5 mL of mRNA; a Femtojet microinjector (Eppendorf) was used 378C to an optical density between 0.5 and 0.8 at 600 nm; protein to perform microinjection. Embryos that lysed immediately expression was induced with 0.2 mM isopropyl b-D-1-thioga- after microinjection were discarded and excluded from analysis. lactopyranoside (IPTG) for 12 h at 188C. Liquid cultures were After injection, surviving embryos were placed in PZM3 cooled to 48C following induction; bacteria were pelleted by embryo culture medium and cultured for 46 h. Cleaved embryos centrifugation at 10 000g at 48C for 15 min. Bacterial pellets in all treatment groups were fixed in 3.7% paraformaldehyde were resuspended in 20 mL PBS containing 0.3% Triton X-100 and processed to detect BRD7 immunocytochemically. and protease inhibitors (Complete Protease Inhibitor Cocktail; For siRNA experiments, presumptive zygotes were assigned Roche) and lysed by sonication. Bacterial lysates were cen- to one of three treatments: KPNA7 siRNA injected, nonsense trifuged at 12 000g for 15 min at 48C; supernatants were then siRNA injected and non-injected controls. Injection pipettes incubated with a glutathione–agarose column at 48C for 2 h. The were loaded with 5 mLof1mM siRNA; a Femtojet microinjector column was then washed four times with PBS containing 1% was used to perform microinjection. Embryos that lysed imme- Triton X-100, proteins were eluted with 10 mM reduced gluta- diately after microinjection were discarded and excluded from thione and eluted proteins were labelled with Alexa488, quan- analysis. After injection, surviving embryos were placed in tified with the Bradford assay and stored at 808C until use. PZM3 embryo culture medium and cultured for 46 h. Cleaved embryos in all treatment groups were fixed in 3.7% paraformal- Dominant negative KPNA7 dehyde and processed to detect BRD7 immunocytochemically. A dominant negative KPNA7 (DN-KPNA7) construct was generated by deleting the 50 end of the porcine KPNA7 open Immunocytochemical analysis reading frame (Wang et al. 2012; National Center for Bio- Oocytes and embryos were processed according to an estab- technology Information (NCBI) reference sequence lished immunocytochemical staining protocol (Cabot et al. NM_001163411.1), which encodes the import b binding (IBB) 2017). Briefly, oocytes and embryos were fixed in 3.7% para- domain. A forward oligonucleotide primer (50-CACCAG- formaldehyde for 1 h at 48C. After fixation the embryos and GAAAATGCTGTCCCAGGAgGAGG) and reverse oligonu- oocytes were washed three times in PBS containing 0.1% cleotide primer (50- GCCAGCGTCTTCTTCCTCACTGAAGT) Tween-20 for 30 min at 48C. Oocytes and embryos were stored were used to amplify the remaining open reading frame of in 500 mL PBST and stored for up to 3 days at 48C until further KPNA7 downstream of the IBB domain by polymerase chain processing. All incubations were carried out at 48C. Fixed reaction (PCR). The PCR product was cloned into the Gateway- oocytes and embryos were permeabilised with 1% Triton X-100 enabled entry vector, pENTR/s.d./D-TOPO (Invitrogen), in PBS for 1 h. Embryos and oocytes were then placed in sequenced to verify its identity and recombined into the Gateway blocking solution consisting of 0.1 M glycine, 1% goat serum, pcDNA-pDEST53 destination vector (Invitrogen) to yield an 0.01% Triton X-100, 1% powdered nonfat dry milk, 0.5% BSA N-terminal GFP fusion protein. This vector was linearised by and 0.02% sodium azide in PBS (Prather and Schatten 1992). restriction enzyme digestion with StuI and used as the template Oocytes and embryos were probed with a commercially avail- for in vitro mRNA synthesis using the mMessage mMachine able antibody directed against BRD7 (Abcam) diluted 1 : 500 in kit (Thermo-Fisher) following the manufacturer’s directions. PBS containing 0.1% Tween-20 for 16 h then washed three Aliquots of purified in vitro-transcribed mRNA (750 mgmL1) times in PBS containing 0.1% Tween-20 (30 min for each wash). were stored at 808C until use. Embryos and oocytes were stained with secondary antibody BRD7 in porcine embryos Reproduction, Fertility and Development 1501

(fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG), (n ¼ 9/9, n ¼ 13/13 for LMB and ETOH treatments respectively). which was diluted 1 : 500 in PBS containing 0.1% Tween-20 for The nuclear intensity of the ETOH treatment group was 16 h, then washed three times in PBS containing 0.1% Tween-20 1.17 0.37 ADU mm1 and the average nuclear intensity of (30 min for each wash). For the dominant negative KPNA7 the LMB treatment group was 1.09 0.26 ADU mm1. Repre- ectopic expression studies the secondary antibody used was sentative images are shown in Fig. 1. tetramethylrhodamine (TRITC)-conjugated anti-rabbit IgG. Embryos and oocytes were stained with 5 mgmL 1 Hoechst Effect of LMB on BRD7 localisation in 4-cell-stage porcine 33342 in PBS for 15 min, mounted on slides using Vectashield embryos mounting medium (Vector Laboratories, Inc.) and examined The distribution of BRD7 was significantly different between using confocal microscopy (Nikon A1R-MP; Nikon). LMB and ETOH treatment groups. Embryos in the ETOH treatment displayed two distribution patterns: either even dis- Quantification of intracellular distribution of BRD7 signal in tribution of BRD7 between the nucleus and cytoplasm (n ¼ 8/15) oocytes and embryos or a predominantly cytoplasmic localisation (n ¼ 7/15). Embryos Images were taken on a confocal microscope equipped with a in the LMB treatment group possessed a dramatic nuclear camera, with a max fluorescence below 4000 optical units. localisation of BRD7 (n ¼ 7/7). The average nuclear intensity Photos were taken of an optical section of oocytes and embryos in the ETOH treatment group was 1.17 0.29 ADU mm1 and at 461 nm and 495 nm in order to capture Hoechst 33342 and the average nuclear intensity in the LMB treatment group was FITC staining respectively. Analytical imaging software (Nikon 10.72 2.82 ADU mm1 (LMB vs ETOH, P , 0.05). Repre- Elements; Nikon Corporation) was used to analyse the images sentative images are shown in Fig. 1. taken at the confocal microscope for the sum intensity of fluo- rescence of nuclear localisation in comparison to cytoplasmic Effect of ivermectin on BRD7 localisation in porcine oocytes localisation by using regions of interest (ROI) around each and embryos respective area. The nuclear ROI was determined based on Multiple putative nuclear localisation signals (NLSs) were Hoechst staining that showed the edges of the nucleus. The identified within BRD7 (UniProt accession number I3 L640) cytoplasmic ROI was determined based on the edge of the using an online NLS prediction platform (http://nls-mapper.iab. oocyte or blastomere. The nuclear-to-cytoplasmic ratio was keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed 23 April calculated by dividing the sum intensity by the ROI area for the 2019; Table 1). A dose titration experiment was performed to nuclear and cytoplasmic regions. The intensity was measured in identify the effective dose of ivermectin necessary to block terms of optical density using the Nikon Elements software and karyopherin a/b1-mediated nuclear import. Our results show recorded as analogue to digital units (ADU). that a concentration of 500 mM ivermectin was able to block nuclear accumulation of a fluorescently labelled reporter protein Statistical analyses in 93% of GV-stage oocytes (Table 2). The ratio of nuclear-to-cytoplasmic intensity was calculated on No significant differences were found between IVER- and each individual oocyte or embryo in the ivermectin and lepto- DMSO-treated oocytes. BRD7 was found to have an even mycin B culture experiments. A higher ratio indicated an distribution between nuclear and cytoplasmic compartments in increased nuclear localisation. The measurements of intracel- the majority of oocytes examined (n ¼ 21/33, n ¼ 20/25 for lular localisation of BRD7 were analysed by two-way ANOVA DMSO and IVER treatments respectively). The remaining using R-Studio (R-Studio). Significance was determined by a oocytes showed a predominantly cytoplasmic localisation two-sample t-test. A P value less than 0.05 was considered to be (n ¼ 12/33, n ¼ 5/25 for DMSO and IVER treatments respec- significant. A Chi-square test was used to detect differences tively). The nuclear intensity of the DMSO treatment group pertaining to the intracellular distribution of BRD7 in both the was 0.95 0.55 ADU mm1 and the average nuclear intensity KPNA7 RNA interference and dominant negative KPNA7 of the IVER treatment group was 0.88 0.39 ADU mm1. overexpression assays. No significant differences between localisation patterns of BRD7 were detected in 4-cell-stage embryos in the respective Results DMSO or IVER treatment groups. BRD7 was found to have a Effect of LMB on BRD7 localisation in porcine oocytes ubiquitous staining throughout the nucleus and cytoplasm in blastomeres of all embryos examined (n ¼ 7/7, n ¼ 7/7 for Two putative nuclear export signals (NESs) were found within DMSO and IVER treatments respectively). The nuclear inten- porcine BRD7 (UniProt accession number I3 L640) using an sity of the DMSO treatment group was 3.25 1.07 ADU mm 1 online NES prediction platform sponsored by The Netherlands and the average nuclear intensity of the IVER treatment group Cancer Institute (http://research.nki.nl/fornerodlab/NES-Finder. was 3.20 1.60 ADU mm 1. Representative images are shown htm, accessed 23 April 2019). The first presumptive NES begins in Fig. 1. at amino acid 193 (N’-IEELKDNFKL) and the second NES begins at amino acid 237 (N’-IQSLKQSIDF). No significant differences between localisation patterns of BRD7 were detected RNA interference-mediated knockdown of KPNA7 restricts in GV-stage oocytes in the respective LMB and ETOH treatment BRD7 nuclear localisation groups. BRD7 was found to have an even distribution between We found a significant difference in the BRD7 intracellular nuclear and cytoplasmic compartments in all oocytes examined localisation patterns in porcine embryos 46 h after treatment with 1502 Reproduction, Fertility and Development J. S. Crodian et al.

DNA BRD7 DNA BRD7

ETOH

A1 A2 C1 C2

LMB

B1 B2 D1 D2

DMSO

E1 E2 G1 G2

IVER

F1 F2 H1 H2

Fig. 1. Nuclear trafficking pathways that facilitate transport of BRD7 in porcine oocytes and 4-cell-stage embryos. Representative images of a (a) GV-stage oocyte and (c) 4-cell-stage embryo from the ETOH control groups, a (e) GV-stage oocyte and (g) 4-cell-stage embryo from the DMSO control groups, a (b) GV-stage oocyte and (d ) 4-cell-stage embryo from the LMB groups and a ( f ) GV-stage oocyte and (h) 4-cell-stage embryo from the IVER control groups. DNA staining is shown in panels labelled 1 (a1, b1, etc.) and while BRD7 intracellular localisation is shown in panels labelled 2 (a2, b2, etc.).

Table 1. Predicted nuclear localisation signals (NLSs) in porcine BRD7

NLS Sequence Position Type

MGKKHKKHKSDKHLY 1 Monopartite HKDRKRKKRKKGEK 58 Monopartite EKGRKRRRVKE 77 Monopartite GKKHKKHKSDKHLYEEYVEKPLKLVL 2 Bipartite KDRKRKKRKKGEKQVPGEEKGRKRRRVKEDKKK 59 Bipartite IEELKDNFKLMCTNAMIYNKPETIYYKAAK 193 Bipartite KILSQERIQSLKQSIDFMADLQKSRKQK 230 Bipartite

Table 2. Effective dose of ivermectin necessary to block karyopherin a/b1-mediated import in porcine oocytes

Treatment Number of oocytes Number (%) of oocytes injected with GST-NLS with nuclear GST-NLS

DMSO 74 71 (96%) 25 mM ivermectin 90 74 (82%) 100 mM ivermectin 55 36 (65%) 250 mM ivermectin 90 17 (19%) 500 mM ivermectin 55 4 (7.3%) BRD7 in porcine embryos Reproduction, Fertility and Development 1503

DNA BRD7 with equal distribution between the nuclear and cytoplasmic compartments (n ¼ 3/8 GFP, n ¼ 16/18 non-injected) or with greater intensity within the nucleus (n ¼ 5/8 GFP, n ¼ 2/18 non-injected). BRD7 staining was enriched in the cytoplasm in KPNA7 siRNA embryos expressing DN-KPNA7 (n ¼ 6/17), evenly distributed between the nucleus and cytoplasm (n ¼ 9/17) or enriched in the nucleus (n ¼ 2/17; DN-KPNA7 vs GFP and non-injected, P , 0.05). Representative images are shown in Fig. 3. A1 A2

Discussion SWI/SNF chromatin remodelling complexes play an essential Control siRNA role during early embryogenesis, impacting both zygotic genome activation and trophectoderm differentiation (Bultman et al. 2006). The collection of BAF subunits that associate with one B1 B2 another determines the activity of a given SWI/SNF chromatin remodelling complex. Determining the mechanisms that regulate the intracellular localisation of discrete BAF subunits will help us to understand which SWI/SNF complexes can exist in the Non-injected nucleus at specific time points during early embryogenesis. Because the intracellular localisation of BRD7 was found to C1 C2 change over the course of development from immature oocyte to cleavage-stage embryo, we wanted to determine which nuclear trafficking pathways played a role in regulating the intracellular localisation of BRD7. We identified classical NLSs recognised by the karyopherin a/b1 heterodimer (Table 1) and NESs recognised by CRM1 within porcine BRD7; Zhou et al. Њ 2 antibody (2011) had also reported that BRD7 contained an LMB- only sensitive NES. As these two pathways have been shown to be D1 D2 operational in porcine oocytes and embryos (Cabot et al. 2002; Cabot and Prather 2003) we hypothesised that they were involved in trafficking BRD7. Fig. 2. RNA interference-mediated knockdown of KPNA7 leads to It was interesting to find that LMB treatment led to nuclear changes in BRD7 intracellular localisation. Representative images of a accumulation of BRD7 in 4-cell-stage embryos but not porcine embryo from (a) the KPNA7 siRNA treatment, (b) the control GV-stage oocytes, as CRM1-mediated nuclear export has been siRNA treatment, (c) the non-injected treatment and (d) a porcine embryo for shown to operate at both of these developmental stages (Cabot which immunocytochemical staining with BRD7 primary antibody was et al. 2002). While CRM1-mediated export can occur in porcine omitted (secondary antibody incubation only). The location of nuclei (DNA oocytes, this activity was ascertained by using a microinjection staining with Hoechst 33342) is shown in panels labelled 1 (a1, a2, etc.) and assay involving fluorescently labelled, recombinant protein the intracellular localisation of BRD7 (FITC-labelled secondary antibody) is (Cabot et al. 2002). The kinetics of this transport system have shown in panels labelled 2 (a2, b2, etc.). Scale bars ¼ 50 mm. not been established in porcine cells, therefore it is possible that the activity of CRM1-mediated export differs between the GV- stage oocyte and the 4-cell-stage embryo. It is also possible for KPNA7 interfering RNAs. While the majority of embryos in the multiple intracellular proteins to associate with one another and ¼ control siRNA treatment (n 16/22) and non-injected embryo mask nuclear localisation signals. In addition, post-translational ¼ treatment (n 11/15) revealed an even distribution of BRD7 modifications that impact protein folding may occlude localisa- between nuclear and cytoplasmic compartments, the majority of tion signals at discrete stages of development. Other members of ¼ embryos in the KPNA7 siRNA treatment (n 24/28) showed a the karyopherin b family of transport receptors also mediate reduced amount of BRD7 in the nucleus as compared with the intracellular trafficking. Karyopherin b2, for example, binds to cytoplasm (KPNA7 siRNA vs control siRNA and non-injected, the M9 domain (found in the primary amino acid sequence of , P 0.05). Representative images are shown in Fig. 2. intracellular cargos) and transports proteins in a bidirectional manner across the nuclear envelope (Bonifaci et al. 1997). Expression of a dominant negative KPNA7 restricts BRD7 Although we did not detect an M9 domain within BRD7, it is nuclear localisation plausible that BRD7 utilises additional transport receptors to We found a significant difference in the BRD7 intracellular translocate between the nuclear and cytoplasmic compartments. localisation patterns in porcine embryos 46 h after injection Aside from the possibilities outlined above, we speculate that with mRNA encoding DN-KPNA7. All embryos in the GFP and the lack of nuclear accumulation of BRD7 in response to LMB non-injected treatment groups revealed detectable BRD7 either treatment in porcine oocytes was due to attenuated BRD7 1504 Reproduction, Fertility and Development J. S. Crodian et al.

DNA GFP BRD7

DN-KPNA7

A1 A2 A3

GFP

B1 B2 B3

Non-injected

C1 C2 C3

GFP 2ЊAb only

D1 D2 D3

Non-injected 2ЊAb only

E1 E2 E3

Fig. 3. Ectopic overexpression of dominant negative KPNA7 leads to changes in BRD7 intracellular localisation. Representative images of a porcine embryo from (a) the dominant negative KPNA7 treatment, (b) the GFP treatment, (c) the non-injected treatment, (d) the GFP treatment for which immunocytochemical staining with BRD7 primary antibody was omitted (secondary antibody incuba- tion only) and (e) the non-injected treatment for which immunocytochemical staining with BRD7 primary antibody was omitted (secondary antibody incubation only). The location of nuclei (DNA staining with Hoechst 33342) are shown in panels labelled 1 (a1, b1, etc.), the intracellular localisation of GFP is shown in panels labelled 2 (a2, b2, etc.) and the intracellular localisation of BRD7 (TRITC- labelled secondary antibody) is shown in panels labelled 3 (a3, b3, etc.). Scale bars ¼ 50 mm. nuclear import at this stage of development. If BRD7 is not nuclear import. Ivermectin has been shown to be an effective trafficked into the nucleus, then inhibiting its export would show inhibitor of karyopherin a/b1-mediated nuclear import no change in intracellular distribution. We first attempted to (Wagstaff et al. 2012). Although Wagstaff et al. (2012) deter- address this question with an ivermectin-based assay to block mined that a 25 mM concentration of ivermectin was sufficient to BRD7 in porcine embryos Reproduction, Fertility and Development 1505 block karyopherin a/b1-mediated nuclear import in HeLa cells, SWI/SNF complexes also serve a role in regulating the low the dose titration performed in this report (Table 2) revealed that level of transcription before the 4-cell stage. We hypothesise an ivermectin concentration of 500 mM of ivermectin was that BRD7 participates in chromatin remodelling events during necessary to inhibit karyopherin a/b1-mediated import. the 4-cell stage of development that are distinct from those that The fact that no change in BRD7 intracellular localisation occur in the GV-stage oocyte. was observed following ivermectin treatment in either GV-stage Porcine embryos used in the nuclear export assay we reported oocytes or 4-cell-stage embryos was somewhat surprising. It here were cultured in the presence of LMB for 12 h. This is possible that ivermectin is simply not 100% effective in incubation time does not allow us to ascertain how rapidly (or blocking karyopherin a/b1-mediated nuclear import in porcine frequently) a given SWI/SNF subunit transits between the oocytes and embryos at the concentration used in our study. nucleus and cytoplasm. There are some differences in localisa- Alternatively, although ivermectin can block karyopherin tion patterns between what we have reported here and what was a/b1-mediated nuclear import, it is possible that ivermectin previously published by (Cabot et al. 2017). For instance, we preferentially disrupts trafficking of only a subset of karyo- previous reported that BRD7 shows a cytoplasmic localisation pherin a subtypes. Karyopherin a subtypes are known to be in the majority of embryos at the 4-cell stage of development, differentially expressed in various cell types and discrete whereas 47% of our control embryos had a cytoplasmic locali- developmental stages (Wang et al. 2012). We therefore chose sation of BRD7 and 53% revealed an even distribution between to target a specific karyopherin a-subtype by two complemen- the nucleus and cytoplasm. This subtle difference is likely due to tary methods to further test the hypothesis that karyopherin the different age of the 4-cell-stage embryos in the two studies. a/b1-mediated nuclear import regulates the intracellular loca- Four-cell-stage embryos were fixed 48 h after gamete mixing in lisation of BRD7. We focussed on karyopherin a7 (KPNA7), as the former study (Cabot et al. 2017); 4-cell-stage embryos in the KPNA7 appears to be an oocyte- and early embryo-specific present study were collected 48 h after gamete mixing and then karyopherin a subtype that is required for embryogenesis incubated for 12 h before fixation. This added incubation puts (Tejomurtula et al. 2009; Wang et al. 2012). embryos much closer to the onset of mitosis. It is plausible that The dominant negative KPNA7 mutant that we generated the shuttling of BRD7 may serve a role in directing specific lacks the IBB domain, which is required for the interaction SWI/SNF complexes to genomic loci at discrete time points between karyopherin a and karyopherin b1, and retains the during the cell cycle or in remodelling chromatin to mediate armadillo (ARM) repeats, which are responsible for binding aspects of zygotic genome activation. NLS-bearing cargo (Lott and Cingolani 2011). Overexpression SWI/SNF chromatin remodelling complexes are known to of the dominant negative KPNA7 should be able to bind its serve critical roles in the events of zygotic genome activation respective cargo in the cytoplasm, but be unable to transport the and trophectoderm formation. The findings in this work add to cargo to the nucleus. While our results clearly show that BRD7 our understanding of how discrete SWI/SNF complex subunits is redistributed in 4-cell-stage embryos as a result of the are partitioned between the nucleus and cytoplasm in cleavage- presence of dominant negative KPNA7, it is possible that the stage embryos. Understanding the mechanism by which these redistribution may be an indirect effect of ectopic protein over- protein subunits are partitioned within cells of the cleavage- expression. We used a previously reported RNA interference stage embryo will help us determine the types of SWI/SNF approach to reduce KPNA7 levels in porcine embryos (Wang complexes that exist at discrete time points during development. et al. 2012). As shown in Figs 2 and 3, both approaches to Future investigations that examine how BRD7 gains entry to modulate KPNA7 levels resulted in a redistribution of BRD7. the nucleus and how transcript profiles change when the These experiments suggest a role for KPNA7 in BRD7 import, intracellular localisation or the levels of BRD7 are altered but these experiments do not exclude the possibility that other will help us to determine the networks that are regulated by karyopherin a subtypes also participate in BRD7 import. SWI/SNF chromatin remodelling. This new knowledge will We have previously shown that BRD7 is largely restricted to improve our understanding of mammalian embryogenesis and the cytoplasm at the 4-cell stage of development (Cabot et al. provide a means to develop new and novel approaches to 2017). The new data presented here suggest that the steady-state improve current conditions for handling embryos in vitro. localisation of BRD7 at the 4-cell stage is cytoplasmic because the rate of CRM1-mediated nuclear export exceeds the rate of Conflicts of interest karyopherin a/b1-mediated nuclear import. This apparent shut- The authors declare no conflicts of interest. tling of BRD7 between the nucleus and cytoplasm at this stage of development was surprising and may reflect a unique role for BRD7 during the 4-cell stage of development. Acknowledgements Zygotic genome activation occurs during the 4-cell stage in The authors acknowledge the use of the facilities of the Bindley Bioscience the porcine embryo (Anderson et al. 1999). The shuttling of Center, a core facility of the NIH-funded Indiana Clinical and Translational BRD7 at this stage may reflect a role that BRD7-containing Sciences Institute. We would also like to thank Indiana Packers Corporation for supplying the porcine ovaries used in this study, as well as all of SWI/SNF complexes serve in modifying chromatin structure in the support from our laboratory members, Hayly M. Goebel, Marta preparation for the global increase in transcription observed at Kwiatkowska and Charlie C. Zhang. We also want to thank our statistical the 4-cell stage. Previously published data have shown that consultant, Qi Lu, and the Purdue Statistical Consulting Service for assis- CRM1-mediated nuclear export occurs before the 4-cell stage tance in analysing the data presented in this manuscript. This project was (Cabot et al. 2002). It is possible that BRD7-containing supported, in part, by the Eunice Kennedy Shriver National Institute of 1506 Reproduction, Fertility and Development J. S. Crodian et al.

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