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Arid4b Alters Cell Cycle and Cell Death Dynamics During Mouse Embryonic Stem Cell Differentiation

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Arid4b Alters Cell Cycle and Cell Death Dynamics During Mouse Embryonic Stem Cell Differentiation Turkish Journal of Biology Turk J Biol (2021) 45: 56-64 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-2009-6 Arid4b alters cell cycle and cell death dynamics during mouse embryonic stem cell differentiation Gözde GÜVEN, Nihal TERZİ ÇİZMECİOĞLU* Department of Biological Sciences, Faculty of Arts and Sciences, Middle East Technical University, Ankara, Turkey Received: 02.09.2020 Accepted/Published Online: 24.11.2020 Final Version: 09.02.2021 Abstract: Cell division and death play an important role in embryonic development. Cell specialization is accompanied with slow proliferation and quiescence. Cell death is important for morphogenesis. Gene expression changes during differentiation is coordinated by lineage-specific transcription factors and chromatin factors. It is not yet fully understood how alterations in gene expression and cell cycle/death mechanisms are connected. We previously identified a chromatin protein Arid4b as a critical factor for meso/ endoderm differentiation of mouse embryonic stem cells (mESCs). The differentiation defect of Arid4b-deficient mESCs might be due to misregulation of cell proliferation or death. Here, we identified a role for Arid4b in cell cycle rewiring at the onset of differentiation. Arid4b-deficient differentiating cells have less proliferative capacity and their cell cycle profile is more similar to mESC stage than the differentiating wild-type cells. We found no evidence of increased DNA damage or checkpoint activation. Our investigation of cell death mechanisms found no contribution from autophagy but revealed a slight increase in Caspase-3 activation implying early apoptosis in Arid4b-deficient differentiating cells. Taken together, our data suggest Arid4b regulates cell cycle alterations during exit from pluripotency. Future studies will be instrumental in understanding whether these changes directly contribute to Arid4b-dependent differentiation control. Key words: Embryonic stem cells, chromatin, cell cycle, cell death, endoderm differentiation 1. Introduction and cooccupy genes that are important for pluripotency Embryonic stem cells (ESC) are pluripotent and self- (Saunders et al., 2017). We previously identified Arid4b renewing cells that originate from the inner cell mass as a critical factor for mouse ESC differentiation towards (ICM) of the developing blastocyst. Owing to their limitless meso/endoderm (Terzi Cizmecioglu et al., 2020). Arid4b- proliferation capacity and in vitro differentiation potential, deficient mESCs failed to upregulate meso/endoderm they are widely used as a model for molecular studies of specific gene expression program and exhibited altered early mammalian development (Niwa, 2007; Young, H3K27 trimethylation and acetylation in lineage specific 2011). The ESC state is transcriptionally maintained by genes. pluripotency transcription factors (TFs) and chromatin ESC differentiation and cell cycle are closely linked. factors (Masui et al., 2007). Working together, they Previous studies show that G1 cyclin CDKs directly enable stable expression of the pluripotency-related gene regulate Oct4, Sox2 and Nanog by phosphorylation. expression network and suppress lineage specific genes Inhibition of Cyclin D directs ESCs to differentiation (Liu (Orkin and Hochedlinger, 2011). et al., 2017). On the other hand, in ESCs, Cyclin D is highly Sin3a is a multisubunit corepressor complex that active because of inactivation of hyperphosphorylated Rb contains histone deacetylases, Hdac1, and Hdac2 by CDK2-cyclin E (Pauklin and Vallier, 2013). After the (Kadamb et al., 2013). Arid4b is a nonenzymatic member exit from pluripotency and the onset of differentiation, of the Sin3a corepressor complex that belongs to the cell cycle duration is lengthened, and G1-S checkpoint Arid family DNA binding proteins. Along with the weak is properly established. It was also found that G1 DNA-binding ability, it has chromatin reader domains cyclins regulate phosphorylation and the stability of that might be important for complex recruitment to the pluripotency TFs at the ESC stage (Liu et al., 2017). chromatin (Fleischer, Yun and Ayer, 2003). In ESCs, Sin3a Previous studies also showed a role for G1-phase restricted was previously found to physically interact with Nanog expression of TGFβ pathway TFs Smad2 and Smad3 and * Correspondence: [email protected] 56 This work is licensed under a Creative Commons Attribution 4.0 International License. GÜVEN and TERZİ ÇİZMECİOĞLU / Turk J Biol meso/endoderm differentiation (Pauklin and Vallier, 2013; Scientific, Waltham, MA, USA), monothioglycerol (1.5x10-4 Soufi and Dalton, 2016). M final) (Sigma- Aldrich Corp., St. Louis, MO, USA) and We hypothesized that the differentiation defect 4% FBS (Thermo Fisher Scientific, Waltham, MA, USA). observed in Arid4b-deficient mESCs might be due to The media was supplemented with 3 µM CHIR99021 alterations in cell proliferation or cell death. We observed (Selleckchem), 1 µM PD0325901 (Selleckchem), and 1% that during differentiation, arid4bΔ cells reach lower cell LIF (Millipore). When cells were 70%-80% confluent, cells numbers when compared to WT cells. We set out to test were split by using TrypLE express enzyme (1×) (Thermo whether Arid4b alters cell cycle or cell death dynamics Fisher Scientific, Waltham, MA, USA). during mESC differentiation. During ESC and endoderm differentiation protocols, cell growth and viability were routinely assessed by trypan 2. Materials and methods blue staining, followed with counting using Countess 2.1. Cell culture II automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA). 2.1.1. mESC growth CJ9 (WT) and mESCs that Arid4b genes deleted by the 2.1.2. Endoderm differentiation CRISPR-Cas9 system (arid4bΔ) were obtained from When CJ9 and Arid4bΔ cells reached 70%-80% confluency, the laboratory of Prof. Stuart Orkin (Boston Children they were counted the cells with trypan blue staining 5 Hospital). CJ9 and arid4bΔ mESCs were expanded in the and seeded in 10 cm petri dishes as 7.5 × 10 cells each, high glucose DMEM (Gibco, Thermo Fisher Scientific, how and cells were grown in serum-free differentiation Waltham, MA, USA), including 15% FCS (Gibco, Thermo medium (SFD medium) that contains 75% IMDM (Gibco, Fisher Scientific, Waltham, MA, USA), 1% Pen-strep Thermo Fisher Scientific, Waltham, MA, USA), 25% Ham’s (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), F-12 Nutrient Mix, 1% GlutaMAX Supplement (Gibco, 1% Nucleoside mix (80 mg Adenine, 24 mg thymidine, Thermo Fisher Scientific, Waltham, MA, USA), 0.5% BSA 85 mg Guanosine, 73 mg Uracil, and 73 mg cytosine), (Sigma-Aldrich Corp., St. Louis, MO, USA), 1% 50 × B-27 1% Glutamax (200mM, Gibco, Thermo Fisher Scientific, supplement without RA (Gibco, Thermo Fisher, Waltham, Waltham, MA, USA), NEAA (nonessential amino acids) MA, USA), 0.5% 100× N-2 supplement (Gibco, Thermo (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), Fisher, Waltham, MA, USA), 1% Glutamax (200 mM, 10-4 M beta-mercaptoethanol on the irradiated mouse Gibco, Thermo Fisher Scientific, Waltham, MA, USA), embryonic fibroblast (MEF) and cultured at 37°C and 5% 10 µl/mL ascorbic acid (Sigma-Aldrich Corp., St. Louis, MO, USA), and 3 µl/mL from diluted MTG (13 µL / 1000 CO2. When cells were 70%-80% confluent, cells were split by using 0.25% Trypsin-EDTA (Sigma-Aldrich Corp., µL) (Sigma-Aldrich Corp., St. Louis, MO, USA). The cells St. Louis, MO, USA). Previous studies suggested that the were incubated at 37°C at 5% CO2. On the second day of ESCs which are grown in 2i containing defined medium differentiation, embryoid bodies (EBs) were collected in more homogenously expressed transcription factors that 50 mL falcon tubes and centrifuged at 1000 rpm for 5 min are necessary for ESC pluripotency. We optimized the at 4°C. They were resuspended in Accutase (Millipore) at ESC maintenance in 2i+LIF containing defined medium 37°C water bath until become a single cell. Accutase was suggested by the protocol (Mulas et al., 2019). We quenched with IMDM, and the cells were centrifuged at confirmed that WT mESCs grown in 2i+LIF conditions 1500 rpm for 5 min at 4°C. They were counted and seeded 5 can be differentiated to endoderm in similar kinetics as 5.0 × 10 cells into each 6 cm petri dishes. SFD medium was the traditional LIF containing serum medium conditions. supplied with 50 µg/mL Activin A (75 ng/mL final) (R&D; We also validated that mESCs in 2i+LIF medium are 338-AC). The EBs were collected by the same method and pluripotent and do not demonstrate expression of prepared for flow cytometry and western blot analysis on neuroectoderm marker Sox1 (not shown). The defined the fifth day of differentiation. Cells were collected and media contains 50% Neurobasal (Gibco, Thermo Fisher washed with ice-cold 0.1% BSA-PBS solution and PBS Scientific, Waltham, MA, USA), 50% DMEM-F12 (Gibco, solutions three times for flow cytometry and western blot, Thermo Fisher Scientific, Waltham, MA, USA), 0.5% N-2 respectively. The cells that were washed with PBS were Supplement (100X) (Thermo Fisher Scientific, Waltham, snap-freeze with dry-ice. MA, USA), 1% 50× B-27 Supplement (Thermo Fisher 2.2. Flow cytometry Scientific, Waltham, MA, USA), 0.5% BSA (Sigma- Aldrich CJ9 and arid4bΔ mESCs were collected and washed tree Corp., St. Louis, MO, USA), 1% GlutaMAX Supplement times with 0.1% BSA-PBS solution (FACS buffer). CJ9 cells (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% have been separated equally as unstained, single stained Penicillin-Streptomycin
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