Abstract

   TRIM28, a multi-domain protein, is crucial in the development of mouse
   embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal
   potential. As the epigenetic factor modulating chromatin structure,
   TRIM28 regulates the expression of numerous genes and is associated
   with progression and poor prognosis in many types of cancer. Because of
   many similarities between highly dedifferentiated cancer cells and
   normal pluripotent stem cells, we applied human induced pluripotent
   stem cells (hiPSC) as a model for stemness studies. For the first time
   in hiPSC, we analyzed the function of individual TRIM28 domains. Here
   we demonstrate the essential role of a really interesting new gene
   (RING) domain and plant homeodomain (PHD) in regulating pluripotency
   maintenance and self-renewal capacity of hiPSC. Our data indicate that
   mutation within the RING or PHD domain leads to the loss of stem cell
   phenotypes and downregulation of the FGF signaling. Moreover,
   impairment of RING or PHD domain results in decreased proliferation and
   impedes embryoid body formation. In opposition to previous data
   indicating the impact of phosphorylation on TRIM28 function, our data
   suggest that TRIM28 phosphorylation does not significantly affect the
   pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with
   disrupted RING and PHD functions display downregulation of genes
   associated with tumor metastasis, which are considered important
   targets in cancer treatment. Our data suggest the potential use of RING
   and PHD domains of TRIM28 as targets in cancer therapy.

   Keywords: TRIM28, KAP1, iPS, stem cells, stemness, pluripotency,
   self-renewal, epigenetics, differentiation, cancer

1. Introduction

   Due to self-renewal and pluripotency maintenance properties, induced
   pluripotent stem cells (iPSC) exhibit several features that are also
   characteristic for cancer cells, e.g., the similar expression profile
   of many genes or activity of signaling pathways regulating self-renewal
   [[34]1,[35]2,[36]3]. Epigenetic and transcriptional dysregulations in
   tumor cells disturb many signaling pathways also responsible for
   maintaining the phenotype of normal stem cells, leading to progressive
   dedifferentiation and acquisition of stemness features [[37]2,[38]3].
   The stemness score is the lowest in somatic cells, increased in primary
   tumors, and reaches the highest level in tumor metastases. This
   indicates that tumor progression usually involves the process of
   oncogenic dedifferentiation [[39]3]. Because of many similarities, iPSC
   can contribute to developing strategies for inhibiting the oncogenic
   switch of primary tumor cells to more malignant cells found in
   metastases.

   A number of different mechanisms determine the ability of stem cells to
   maintain their phenotype, and many of them play important roles in
   embryonic, somatic, induced, and cancer stem cells
   [[40]2,[41]3,[42]4,[43]5,[44]6]. These mechanisms include numerous
   signaling pathways and the core pluripotency network comprised of
   OCT3/4, SOX2, and NANOG. The conversion between stemness and
   differentiation also depends on more complex epigenetic and
   non-epigenetic gene expression regulation.

   We and others showed that tripartite motif-containing protein 28
   (TRIM28) is implicated in the epigenetic control of genes responsible
   for maintaining stemness and self-renewal [[45]7,[46]8,[47]9,[48]10].
   TRIM28, also known as KAP1 (KRAB (Krüppel associated box)-associated
   protein 1)), or TIF1β (transcription intermediary factor 1-beta), is a
   multi-domain protein, modulating the structure of chromatin
   [[49]11,[50]12]. It is highly expressed in embryonic stem cells (ESC)
   and is crucial in embryogenesis and gastrulation in mice
   [[51]13,[52]14]. Trim28 is indispensable for the reprogramming of mouse
   fibroblasts into iPSC [[53]15] and in stemness maintenance in mouse and
   human PSC [[54]8,[55]16,[56]17,[57]18], as well as in human cancer
   cells [[58]9,[59]19]. Several studies indicated a positive correlation
   between TRIM28 upregulation and poor cancer prognosis in specific tumor
   types [[60]20,[61]21,[62]22,[63]23,[64]24,[65]25].

   Each of the TRIM28 protein domains performs a separate, strictly
   defined function (Graphical Abstract, [66]Figure S1, Table S1). At the
   N-terminus, TRIM28 contains the tripartite motif (TRIM) responsible for
   protein–protein interaction and oligomerization. The TRIM motif
   consists of three sub-units: the really interesting new gene (RING)
   finger, two B-box zinc fingers, and the coiled-coil (CC) motif,
   altogether described as RING-B-box-Coiled-Coil (RBCC) domain [[67]11].
   The RBCC domain interacts with the KRAB domain of epigenetic
   repressors—KRAB zinc-finger proteins (KRAB-ZFPs) [[68]11]. In the
   central part, TRIM28 contains the heterochromatin protein 1-binding
   domain (HP1BD) [[69]26]. HP1BD recruits HP1 protein to activate
   heterochromatization. The C-terminus of TRIM28 contains the plant
   homeodomain (PHD) and bromodomain (BROMO) functional unit [[70]27]. PHD
   mediates the sumoylation of adjacent BROMO [[71]28,[72]29]. This
   induces local chromatin concentration by recruiting SET domain
   bifurcated 1 histone lysine methyltransferase (SETDB1) specific to H3K9
   and the nucleosome remodeling and deacetylation (NuRD) complex
   [[73]30]. It is considered that PHD, BROMO, and the PxVxL motif present
   in the HP1BD domain [[74]31] work in cooperation to initiate
   heterochromatin formation, which is characterized by low histone
   acetylation, increased H3K9me3, and binding of HP1 [[75]30].

   Due to the numerous common features between highly dedifferentiated
   cancer cells and normal pluripotent stem cells, and, above all,
   similarities in stemness and self-renewal maintenance, we applied human
   iPSC as a model for stemness studies. Here we show that cells lacking
   TRIM28 lose the expression of pluripotency markers, as well as the
   ability to self-renew, and they start to differentiate. Moreover, we
   identified two TRIM28 domains, RING and PHD, responsible for iPSC’s
   self-renewal capacity and pluripotency maintenance. Mutation of RING or
   PHD resulted in the downregulation of pluripotency markers and
   proliferation, self-renewal inhibition, and restriction in embryoid
   bodies (EBs) formation. Finally, we found that mutation in the RING or
   PHD domain caused the downregulation of genes frequently associated
   with metastasis or poor prognosis in cancer patients. Altogether, our
   observations indicate that TRIM28 maintains the state of pluripotency
   and self-renewal by its transcriptional co-repressor activity via RING
   and PHD.

2. Materials and Methods

2.1. Organisms

   Immunodeficient NUDE mice, NOD.CB17-Prkdc^scid/NCrCrl
   (RRID:IMSR_CRL:394, Charles River Laboratories, Wilmington, MA, USA)
   were kept in the animal rooms of the Institute of Human Genetics,
   Polish Academy of Sciences, Poznan, Poland. Cages were equipped with
   HEPA filters and internal air circulation. The mice were provided with
   a 12-h day/night cycle and unlimited access to water and food. The
   temperature in the breeding rooms was regulated automatically.

2.2. Cell Culture

   PHDF cells were isolated from the skin fragments obtained during
   mastectomy surgeries performed at the Greater Poland Cancer Centre,
   Poznań, Poland. Each time, informed patient consent participation in
   “The Cancer Genome Atlas (TCGA)” project was obtained to use biological
   material for scientific research. Patient identities were not known to
   researchers. The skin fragment was cut into smaller sizes and incubated
   overnight at 37 °C in a digestion medium consisting of DMEM High
   Glucose (#L0102, Biowest, Nuaillé, France), 3 mg/mL collagenase IV
   (#17104019, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1%
   penicillin-streptomycin (#P4333, Sigma-Aldrich, St. Louis, MO, USA).
   After incubation, the enzyme was inactivated with a culture medium:
   DMEM High Glucose (#L0102, Biowest, Nuaillé, France) with 20% FBS
   (#S181H, Biowest, Nuaillé, France) and 1% penicillin-streptomycin
   (#P4333, Sigma-Aldrich, St. Louis, MO, USA). Cells were transferred to
   100 mm plates and incubated under standard culture conditions (37 °C,
   5% CO[2]) for 72 h. Cells were then washed with DPBS (#L0615, Biowest,
   Nuaillé, France) to remove tissue debris and cultured in 20% FBS medium
   to achieve approximately 90% confluence. After the first passage, the
   cells were maintained in a complete culture medium: DMEM High Glucose
   (#L0102, Biowest, Nuaillé, France), 10% FBS (#S181H, Biowest, Nuaillé,
   France) and 1% penicillin-streptomycin (#P4333, Sigma-Aldrich, St.
   Louis, MO, USA).

   HEK 293T cells (RRID:CVCL_0063, Cat# CRL-3216, ATCC, Manassas, VA, USA)
   were maintained in a complete culture medium: DMEM High Glucose
   (#L0102, Biowest, Nuaillé, France) with 10% FBS (#S181H, Biowest,
   Nuaillé, France) and 1% penicillin-streptomycin (#P4333, Sigma-Aldrich,
   St. Louis, MO, USA), under standard culture conditions. After reaching
   about 80–90% confluence, cells were passaged by trypsinization
   (#25-053-CI, Corning, Corning, NY, USA).

   Generated iPSC and ESC (hES BG01V, RRID:CVCL_9727, Cat# SCRC-2002,
   ATCC, Manassas, VA, USA) were maintained in feeder-dependent culture
   under hypoxic conditions (37 °C, 5% CO[2], 5% O[2]). Cells were
   cultured in plates coated with BD Matrigel™ Matrix, Basement Membrane,
   GFR (#354230, BD Biosciences, San Jose, CA, USA) and inactivated MEF
   cells (Cat#PMEF-CF, Millipore Merck KGaA, Darmstadt, Germany KGaA,
   Darmstadt, Germany) at 100% confluency, in iPSC medium: DMEM/F12
   (#DF-042-B, Millipore Merck KGaA, Darmstadt, Germany KGaA, Darmstadt,
   Germany), 20% KSR (#10828028, Gibco, Thermo Fisher Scientific, Waltham,
   MA, USA), 1× NEAA (#M7145, Merck KGaA, Darmstadt, Germany), 1×
   β-mercaptoethanol (#ES-007-E, Merck KGaA, Darmstadt, Germany), 0.5%
   penicillin-streptomycin (#P4333, Sigma-Aldrich, St. Louis, MO, USA) and
   10 ng/mL FGF (#PHG0264, Gibco, Thermo Fisher Scientific, Waltham, MA,
   USA). The medium was changed daily. Colonies were passaged after
   reaching about 70% confluence by incubation with 0.1% collagenase IV
   (#17104019, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in
   DMEM/F12 (#DF-042-B, Millipore Merck KGaA, Darmstadt, Germany KGaA,
   Darmstadt, Germany), for 2–5 min at 37 °C. After incubation, the enzyme
   was removed, the cells were gently washed with DPBS (#L0615, Biowest,
   Nuaillé, France), and a fresh iPSC medium was added. IPSC colonies were
   gently detached from the culture dish with a soft silicone scraper,
   carefully broken up in suspension into smaller aggregates with an
   automatic pipette, and transferred to a new Matrigel and MEF-coated
   culture vessel.

   IPSC ND41658 (RRID:CVCL_Y836, Cat# ND41658, Coriell, Camden, NJ, USA)
   were cultured in feeder-free conditions in plates coated with BD
   Matrigel™ Matrix, Basement Membrane, GFR (#354230, BD Biosciences, San
   Jose, CA, USA). Cells were maintained in an Essential 8 Flex Medium Kit
   (#A2858501, Thermo Fisher Scientific, Waltham, MA, USA) under hypoxic
   conditions (37 °C, 5% O[2], 5% CO[2]). The medium was changed every
   other day. Colonies were passaged after reaching about 60% confluence
   by incubation with a 0.48 mM EDTA (#E6511, Sigma-Aldrich, St. Louis,
   MO, USA) in DPBS (#L0615, Biowest, Nuaillé, France) for 3 min at 37 °C.
   After incubation, the solution was removed and residues inactivated
   with a culture medium. The colonies were gently detached from the plate
   using a soft silicone scraper, carefully broken up in suspension into
   smaller aggregates by pipetting, and transferred to new Matrigel-coated
   culture vessel.

2.3. Cloning Procedures

   The pStemcca-TetO plasmid vector was constructed as previously
   described [[76]15]. pLV-HK plasmid was a kind gift, generated in Didier
   Trono Lab, on the backbone of a pLV-tTRKRAB vector (RRID:
   Addgene_12249, Addgene, Watertown, MA, USA).

   All generated vectors for the TRIM28 domains study were constructed on
   the 2nd generation lentiviral transfer plasmid pWPXL
   (RRID:Addgene_12257, Addgene, Watertown, MA, USA) backbone. Generation
   of 11 pWPXL vectors required complex multi-step cloning procedures.
   Detailed information on cloning steps is available from the Lead
   Contact. The hairpin targeted 5′-GCACTAGCTGTGAGGATAA-3′ of the
   endogenous TRIM28 (457–476 nt). All exogenous variants of TRIM28
   contained a mutation introducing hairpin resistance
   5′-GCACcAGtTGcGAaGAcAA-3′ (silent mutations marked with lowercase
   letters). Fragments of TRIM28 phospho-mutants and phospho-mimetics were
   synthesized by GeneArt (Invitrogen, Carlsbad, CA, USA). Fragments of
   TRIM28 structural mutants were obtained by directed mutagenesis
   (GeneArt Site-Directed Mutagenesis System, #A13282, Invitrogen,
   Carlsbad, CA, USA). All pWPXL- plasmids were sequenced (Genomed), and
   results were analyzed with Chromas 2.6.6 (RRID:SCR_000598, Technelysium
   Pty Ltd., [77]https://technelysium.com.au/wp/chromas/, accessed on 27
   June 2021). Mutagenesis and sequencing primers are listed in [78]Table
   S2.

   All plasmid vectors were transformed into E. coli JM109 (#P9751,
   Promega, Madison, WI, USA Research) and isolated with JETSTAR 2.0
   Plasmid Maxiprep Kit (#220100, Gentaur, Kampenhout, Belgium).

2.4. Lentiviral Vectors Production, Purification, and Titration

   Lentiviral vectors were produced by transient transfection of HEK 293T
   cells (ATCC, Manassas, VA, USA) by calcium phosphate precipitation.
   Briefly, HEK 293T (2.5 × 10^6 cells) were seeded on 100 mm plates in a
   complete culture medium. The next day 20 μg of transfer plasmid, 15 μg
   of psPAX2 packaging plasmid (RRID:Addgene_12260, Addgene, Watertown,
   MA, USA), and 6 μg of pMD2G plasmid coding for virus envelope proteins
   G (RRID:Addgene_12259, Addgene, Watertown, MA, USA) were mixed with 50
   μL 2.5 M CaCl[2] (#C3881, Sigma-Aldrich, St. Louis, MO, USA), and
   sterile H[2]O to a volume of 500 μL. The mixture was added dropwise to
   500 μL of 2X HBS buffer: 150 mM NaCl (#S3014, Sigma-Aldrich, St. Louis,
   MO, USA), 20 mM HEPES (#A3724, AppliChem, PanReac, Darmstadt, Germany)
   in H[2]O, and pH 7.05, with simultaneous aerating with an automatic
   pipettor. The volumes given are sufficient for one 100 mm plate. The
   transfection mixture was incubated for 5 min at RT and added to the
   cells. Cells were incubated for 6 h under standard culture conditions.
   Then, the medium was changed to 6 mL of fresh medium. The supernatant
   containing the virus particles was collected 48 h after transfection
   and centrifuged at 3000 rpm, 5 min, RT.

   Virus-containing supernatant was purified and concentrated by
   ultracentrifugation in Ultra-Cone Polyallomer Centrifuge Tubes (Seton
   Scientific, Petaluma, CA, USA), in Sorvall Discovery 100S
   Ultracentrifuge (Kendro Laboratory Products, Asheville, NC, USA).
   Supernatants (20 mL) were carefully added dropwise on a 4 mL layer of
   20% saccharose (#107651, Merck KGaA, Darmstadt, Germany) in distilled
   H[2]O. Tubes were centrifuged at 26,000 rpm, for 1.5 h at 4 °C. The
   supernatant was decanted, and the pellet (barely visible/invisible) was
   suspended in 2% BSA (#A9418, Sigma-Aldrich, St. Louis, MO, USA) in DPBS
   (#L0615, Biowest, Nuaillé, France) and incubated for 20 min at RT. The
   precipitate dissolved in the buffer was intensively pipetted,
   aliquoted, and stored at −80 °C.

   Viral titers were determined by real-time PCR. HEK 293T cells were
   seeded at 20,000 cells/well of a 6-well plate in a complete culture
   medium. After 24 h, cells were transduced with each lentivirus
   produced, in volumes of 2, 5, and 10 μL, in duplicates. The flow
   cytometry-titered pWPXL lentiviral vector expressing EGFP was used as
   the reference. The medium was replaced 24 h after transduction with a
   fresh one, and the cells were cultured for another 5 days with the
   medium changed every other day. Cells were harvested by trypsinization
   (#25-053-CI, Corning, Corning, NY, USA), and DNA was isolated with
   Quick-DNA Miniprep (#D3025, Zymo Research, Irvine, CA, USA). All
   procedures were performed according to the manufacturer’s protocol. We
   used primers amplifying WPRE (present in integrating vector fragment)
   and GAPDH sequence. Primer sequences are listed in [79]Table S2.
   Amplicons were detected with TaqMan hydrolysis probes (#04683633001,
   Roche, Basel, Switzerland). The number of infectious particles was
   calculated as described in the protocol [[80]32].

2.5. Reprogramming of Human Fibroblasts towards iPSC

   PHDF cells, in an early (2–3) passage, were seeded 10,000 cells/well of
   a 6-well plate in a complete culture medium. After 24 h and 48 h, cells
   were transduced with Stemcca-TetO lentiviral vector (50 IU/well). On
   day 7 after transduction, cells were passaged into 6-well plates,
   coated with BD Matrigel™ Matrix, Basement Membrane, GFR (#354230, BD
   Biosciences, San Jose, CA, USA), and MEF cells (#PMEF-CF, Millipore
   Merck KGaA, Darmstadt, Germany), approx. 4000/well. Cells were cultured
   in an iPSC medium as described in Cell culture, p. 3. The medium was
   changed every other day. Twenty-one days after transduction, the
   clusters of cells with stem cell-like morphology were manually
   transferred to freshly prepared wells of 6-well plates coated with
   Matrigel and MEF cells at 100% confluency. From this point, the iPSC
   medium was changed daily. After 2 passages, iPSC were transduced with
   LVE-HK lentiviral vector (hygromycin resistance), 10 IU/well, to
   silence the expression of exogenous reprogramming agents. IPSC were
   selected for 7 days with 50 μg/mL Hygromycin B (#H3274, Sigma-Aldrich,
   St. Louis, MO, USA).

2.6. Generation of TRIM28-Depleted or Mutated iPSC Populations

   Silencing of TRIM28 with siRNA in iPSC (obtained by reprogramming) was
   performed in 5 iPSC lines generated from different fibroblast donors,
   in duplicate. The results collected in this paper are representative
   results from the iPSC 26.6. iPSC were treated with a mix of two
   equimolar (50 nM) siRNA particles ([81]Table S2) and Lipofectamine
   RNAiMAX Transfection Reagent (#13778-075, Invitrogen, Carlsbad, CA,
   USA), according to the manufacturer’s protocol. SiRNA BLOCK-iT™
   Fluorescent Oligo (#2013, Invitrogen, Carlsbad, CA, USA) was used as a
   control in iPSC-CTRL. Transfection was performed every 72 h, for 21
   days. TRIM28 expression was analyzed by real-time PCR and
   immunofluorescence staining.

   Transduction of iPSC ND41658 with LV-WPXL carrying exogenous TRIM28
   variants was performed in suspension, in an Essential 8 Flex Medium Kit
   (#A2858501, Thermo Fisher Scientific, Waltham, MA, USA), with 10 µM
   Polybrene (#H9268, Sigma-Aldrich, St. Louis, MO, USA). Cells were
   transduced in 3 biological replicates (MOI = 20). IPSC were selected
   for 7 days with 0.3 mg/mL Puromycin (#P8833, Sigma-Aldrich, St. Louis,
   MO, USA).

2.7. Immunofluorescence Staining

   Cells (seeded on 24-well plates) were washed 3 × 5 min with DPBS
   (#L0615, Biowest, Nuaillé, France) and fixed with a 4% formalin
   (#HT501128, Sigma-Aldrich, St. Louis, MO, USA) in DPBS, 10 min, at RT.
   Cells were washed 3 × 5 min with DPBS. The cell membrane was
   permeabilized (10 min, RT) with a 0.15% Triton X-100 (#T8787,
   Sigma-Aldrich, St. Louis, MO, USA) in DPBS and washed 3 × 5 min with
   DPBS. Cells were then incubated 30 min, RT with 1 mL blocking buffer:
   1% BSA (#A9418, Sigma-Aldrich, St. Louis, MO, USA) and 0.1% Tween 20
   (#P9416, Sigma-Aldrich, St. Louis, MO, USA) in DPBS. Cells were
   incubated with 200 μL primary antibodies in DPBS with 1% BSA,
   overnight, at 4 °C. Generated iPSC and hESC were stained every 7 days
   after transduction, for 3 weeks: anti-OCT3/4 (rabbit) 1:200; anti-NANOG
   1:50; anti-SSEA4 1:50; anti-TRA-1-60 1:50; anti-TRA-1-81 1:50;
   anti-SSEA1 1:100. To siRNA-treated iPSC: anti-TRIM28 (rabbit) 1:50,
   anti-OCT3/4 (mouse) 1:200; anti-NANOG 1:50; anti-ATP5H 1:200; anti-PGK1
   1:400; anti-PKM2 1:40; anti-HK1 1:250; anti-HK2 1:200. LV-treated
   ND41658 iPSC were stained after the 2nd, 6th, or 10th passage.:
   anti-TRIM28 (mouse) 1:200; anti-FLAG 1:800; anti-OCT3/4 (mouse) 1:200;
   anti-SOX2 1:200; anti-NANOG 1:50.

   The next day, the cells were washed 3 × 5 min with DPBS and incubated 1
   h in the dark, RT, with 200 μL fluorescently labeled secondary
   antibodies in DPBS with 1% BSA, in a ratio of 1:1000. After washing the
   cells three times with DPBS, the cell nuclei were stained with a DAPI
   (#32670, Sigma-Aldrich, St. Louis, MO, USA) in distilled water
   (1:10,000) for 5 min in the dark at RT. After washing the cells three
   times with DPBS, the cells were analyzed with Leica DMI3000B
   fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and
   Leica Application Suite (RRID:SCR_016555, Leica Microsystems, Wetzlar,
   Germany). Primary and secondary antibodies are listed in [82]Table S3.

2.8. cDNA Samples Preparation

   Total RNA was isolated from 2 biological replicates (for siRNA-treated
   iPSC) or 3 biological replicates (for LV-treated ND41658 iPSC) using
   TRI Reagent (#T9424, Sigma-Aldrich, St. Louis, MO, USA). Reverse
   transcription was performed with an EvoScript Universal cDNA Master
   (#07912439001, Roche, Basel, Switzerland), according to the
   manufacturer’s protocol. One μg of total cellular RNA was used for each
   reaction. Obtained cDNA was diluted 10-fold in sterile DEPC water and
   used as an RT-PCR and real-time PCR template.

2.9. Real-Time PCR Quantification

   Samples were amplified on a LightCycler^®480 instrument (Roche, Basel,
   Switzerland). The gene expression level was quantified with 2^−ΔΔCT
   method, relative to the control sample. Primer sequences are listed in
   [83]Table S2.

   siRNA-treated iPSC were analyzed every 7 days after the first siRNA
   transfection for 3 weeks. Each reaction contained 2 μL cDNA, 1×
   LightCycler 480 Probes Master (#04887301001, Roche, Basel,
   Switzerland), 100 nM hydrolysis probes (Universal Probe Library), and
   200 nM of primers for TRIM28, OCT3/4, NANOG, CDX2, MSX1, FSP1, PAX6,
   SOX1, GAPDH. Quantitative values for individual samples were normalized
   to GAPDH. Statistical results were calculated in GraphPad Prism6
   (RRID:SCR_002798, GraphPad Software), using t-student and ANOVA tests.

   LV-treated ND41658 iPSC were analyzed 6 or 10 passages after
   transduction. Each reaction contained 2 μL of cDNA template, 1× SYBR
   Green I Master Mix (#04707516001, Roche, Basel, Switzerland), and 200
   nM of primers for NANOG, LIN28A, BMP7, FGFR, PI3K, CDH1, ID1, TWIST2,
   PTCH1, SMO, NOTCH1, MAML1, WNT1, EPCAM, DKK1, DNMT3A, DNMT3B, TERT,
   EZH2, Nestin, TUJ, MAP2, GFAP, SMA, Brachyury, GATA4, GATA6, SOX17,
   B2M, HPRT1, and RPLP0. The specificity of the amplified PCR product was
   assessed by melting curve analysis. Differentiated iPSC consisted of
   many types of cells, so quantitative determined values for individual
   samples were normalized to three reference genes: B2M, HPRT1, and
   RPLP0. Statistical results were calculated in GraphPad Prism6
   (RRID:SCR_002798, GraphPad Software). One-way ANOVA variance analysis
   with a post-hoc Dunnett’s test was conducted.

2.10. Flow Cytometry

   Cells were stained in 3 biological replicates, 10 passages after
   lentiviral transduction. Cell pellets were suspended in 100 μL DPBS
   (#L0615, Biowest, Nuaillé, France) with 1% BSA (#A9418, Sigma-Aldrich,
   St. Louis, MO, USA) and a primary antibody. The antibodies are listed
   in [84]Table S3. Antibodies were administered in the following
   proportions: anti SSEA-4 1:50; anti TRA-1-60 1:50; anti TRA-1-81 1:50;
   anti IgG 1:50; anti IgM 1:50. Cells were incubated for 30 min at 4 °C
   and then washed 3 times with DPBS. Cell pellets were suspended in 100
   μL 1% BSA in DPBS with biotin-conjugated secondary antibodies and
   incubated for 30 min at RT (IgG and IgM, 1:200). Cells were washed 3
   times with DPBS, and pellets were suspended in 100 μL 1% BSA in DPBS
   and streptavidin APC conjugate (#17-4317-82, eBioscience, San Diego,
   CA, USA) and incubated at RT in the dark for 15 min. Cells were washed
   3 times with DPBS, and pellets were suspended in 150 μL of 1× BD
   CellFIX fixing agent (#340181, BD Biosciences, San Jose, CA, USA).
   Cells were counted on a Becton Dickinson flow cytometer (BD
   Biosciences, San Jose, CA, USA FACSAria). The results were analyzed
   with the FlowJo_V10 (RRID:SCR_008520, FLOWJO, LLC Data Analysis
   Software).

2.11. RT-PCR

   RT-PCRs were performed in 3 technical replicates with 2 μL of cDNA
   template, 500 nM primers (for REX1, NODAL, DNMT3B, OCT3/4, GABRB3,
   NANOG, Stemcca-TetO transgene or GAPDH), and ReadyMix™ Taq PCR Reaction
   Mix (#P4600, Sigma-Aldrich, St. Louis, MO, USA) according to the
   manufacturer’s protocols. Primer sequences are listed in [85]Table S2.

2.12. Proliferation Assay

   The proliferation ratio was determined in 3 biological replicates, with
   Cell Proliferation ELISA, BrdU, and colorimetric (#11 647 229 001,
   Roche, Basel, Switzerland), according to the manufacturer’s protocol.
   Briefly, 5000 cells were seeded in a 96-well plate in 100 µL/well and
   incubated at hypoxic conditions at 37 °C for 48 h. BrdU labeling was
   performed for 2 h. Cells were incubated with the anti-BrdU solution for
   90 min and then with a substrate solution for 10 min. The absorbance
   was measured at 370 nm and calculated by subtracting the blank control
   absorbance value. Statistical results were calculated in GraphPad
   Prism6 (RRID:SCR_002798, GraphPad Software). One-way ANOVA variance
   analysis with a post-hoc Dunnett’s test was conducted.

2.13. Spontaneous In Vitro Differentiation Potential Assessment (Embryoid
Bodies Formation)

   Analysis was performed on 3 biological replicates of the iPSC 6
   passages after lentiviral transduction. All variants of iPSC were
   harvested and washed twice with DPBS to remove Matrigel residues. Cells
   were then counted and seeded at 5000 cells/well on a 96 well,
   non-adherent, U-shaped plate in Essential 6™ Medium (#A1516401, Gibco,
   Thermo Fisher Scientific, Waltham, MA, USA) in standard culture
   conditions. Images and videos were taken with the Incucyte SX1
   Live-Cell Analysis System (#4788, Sartorius, Göttingen, Germany), and
   the spheres’ area was calculated with ImageJ (RRID:SCR_003070).
   Statistical results were calculated in GraphPad Prism6
   (RRID:SCR_002798, GraphPad Software) with a Kruskal–Wallis test
   followed by a Dunns’ test.

2.14. Western Blot

   Cells were washed with DPBS (#L0615, Biowest, Nuaillé, France) and
   lysed in 200 μL of RIPA buffer (#J63306, Alfa Aesar, Ward Hill, MA,
   USA) at 4 °C, for 30 min. Cell lysates were centrifuged at 13,000 rpm
   at 4 °C for 30 min, and the supernatant was collected. Protein
   concentration was determined by BCA reaction with a Pierce™ BCA Protein
   Assay Kit (#23225, Thermo Fisher Scientific, Waltham, MA, USA),
   according to the manufacturer’s protocol. Further, 10 μg of protein was
   mixed with Laemmli Sample Buffer (#1610747, Bio-Rad Laboratories,
   Hercules, CA, USA) and RIPA to a final volume of 15 uL, and denatured
   at 95 °C, for 5 min. Electrophoresis was run in Tris/Glycine/SDS buffer
   (#1610772, Bio-Rad Laboratories, Hercules, CA, USA) on a Mini-PROTEAN
   TGX Precast Gel (#4561086, Bio-Rad Laboratories, Hercules, CA, USA),
   with Precision Plus Protein Kaleidoscope Pre-stained Protein Standards
   (#1610375, Bio-Rad Laboratories, Hercules, CA, USA). Proteins were
   transferred on a PVDF membrane, Trans-Blot Turbo Transfer Pack
   (#1704156, Bio-Rad Laboratories, Hercules, CA, USA). The membrane was
   blocked for 30 min in 5% milk in TBST buffer: 0.01 M TRIS (#T1503,
   Sigma-Aldrich, St. Louis, MO, USA), 0.15 M NaCl (#S3014, Sigma-Aldrich,
   St. Louis, MO, USA) and 0.1% Tween 20 (#P9416, Sigma-Aldrich, St.
   Louis, MO, USA). Membrane fragments were incubated with primary
   antibodies in 5 mL 5% milk in TBST buffer, at 4 °C, overnight.
   Membranes were washed 3 times with 10 mL TBST buffer, for 10 min, and
   incubated with secondary HRP-conjugated antibodies in 5 mL 5% milk in
   TBST buffer, followed by triple washing with 10 mL TBST buffer.
   Antibodies were visualized with a WesternBright Quantum HRP substrate
   (#K-12042, Advansta, San Jose, CA, USA). Antibodies are listed in
   [86]Table S3.

2.15. Teratoma Formation

   iPSC were harvested with 0.1% collagenase IV (#17104019, Gibco, Thermo
   Fisher Scientific, Waltham, MA, USA), and 2 × 10^6 of cells were
   resuspended in 50 μL iPSC medium. Before injection, 50 μL of BD
   Matrigel™ Matrix Basement Membrane GFR (#354230, BD Biosciences, San
   Jose, CA, USA) was added to the cell suspension at 4 °C, and the
   mixture was injected subcutaneously into the lower flank of
   immunodeficient NOD SCID mice NOD.CB17-Prkdc^scid/NCrCrl (Charles River
   Laboratories, Wilmington, MA, USA). All animal experiments were
   performed following institutional guidelines. After 7–9 weeks, tumors
   were resected, measured, and subjected to RNA isolation and
   immunohistochemical staining. Paraffin sections of formalin-fixed
   teratomas were stained with hematoxylin and eosin (H + E) and
   antibodies specific for markers of three germ layers: endoderm
   cytokeratins, ectoderm GFAP, and mesoderm desmin. Analysis was
   performed in the Department of Tumor Pathology, Greater Poland Cancer
   Centre in Poznań.

2.16. Karyotyping of Generated iPSC Lines

   Karyotype analyses were performed by the Cytogenetic Laboratory, Cancer
   Centre-Maria Sklodowska–Curie Institute in Warsaw, according to the
   standard protocol for G-banding.

2.17. Bisulfite Sequencing for Promoter Methylation Analysis

   DNA from 8 iPSC clones and 4 PHDF lines was isolated and
   bisulfite-treated as described [[87]8]. DNA from 8 iPSC clones and 4
   PHDF cell lines was isolated with a Quick-DNA Miniprep Kit (#D3025,
   Zymo Research, Irvine, CA, USA). Then, 1.5 µg DNA was bisulfite
   converted with EZ DNA Methylation Kit (#D5001, Zymo Research, Irvine,
   CA, USA), according to the manufacturer’s protocol. OCT3/4 and NANOG
   promoter was amplified with 500 nM specific primers (listed in
   [88]Table S2) and the GoTaq Green Master Mix (#M7122, Promega, Madison,
   WI, USA). PCR was performed with the following thermal profile: 95 °C/2
   min, 42 cycles of 95 °C/30 s + 61 °C/30 s + 72 °C/30 s, and a final
   extension at 72 °C for 7 min. Amplified fragments of OCT3/4 and NANOG
   promoters were ligated to pGEM-T Easy vector (#A1360, Promega, Madison,
   WI, USA) with a LigaFast Rapid DNA Ligation System (#M8221, Promega,
   Madison, WI, USA) and cloned into E. coli DH5a (#T3007, Zymo Research,
   Irvine, CA, USA). The plasmids from individual clones were purified
   with a Zyppy™ Plasmid Miniprep Kit (#D4036, Zymo Research, Irvine, CA,
   USA), sequenced (Genomed, Warsaw, Poland), and analyzed with the BISMA
   application (RRID:SCR_000688, Jacobs University Bremen; Germany)
   [[89]33]. The percentage of methylation (MtI) was calculated according
   to the formula: MtI% = Cm/(Cm + Cnm).

2.18. Proteomic Profiling–Reverse Phase Protein Array (RPPA)

   Analysis was performed on 1 biological replicate. Pellets of PHDF cells
   and derived iPSC were washed with DPBS (#L0615, Biowest, Nuaillé,
   France) and lysed in 100 μL of RIPA buffer (#J63306, Alfa Aesar, Ward
   Hill, MA, USA). Cell lysates were centrifuged at 13,000 rpm, at 4 °C
   for 30 min, and supernatant was collected. Protein concentration was
   determined by BCA reaction with a Pierce™ BCA Protein Assay Kit
   (#23225, Thermo Fisher Scientific, Waltham, MA, USA), according to the
   manufacturer’s protocol. Each sample (40 μL) was mixed with 4X SDS
   sample buffer: 40% Glycerol (#A1123, AppliChem, PanReac, Darmstadt,
   Germany), 8% SDS (#L4509, Sigma-Aldrich, St. Louis, MO, USA), 0.25 M
   Tris-HCl (#10812846001, Roche, Basel, Switzerland), pH 6.8, and 10%
   β-mercaptoethanol (#ES-007-E, Merck KGaA, Darmstadt, Germany), and
   boiled for 5 min. Samples were stored in −80 °C. Samples were analyzed
   in the RPPA Core Facility, The University of Texas, MD Anderson Cancer
   Center (Houston, TX, USA). Each sample was diluted in five 2-fold
   serial dilutions in 1% SDS lysis buffer. Serially diluted lysates were
   arrayed on nitrocellulose-coated ONCYTE^® Film slides (Grace Bio-Labs,
   Bend, OR, USA) by Aushon 2470 Arrayer (Aushon BioSystems, Billerica,
   MA, USA) in 11 × 11 format. Each slide was probed with a validated
   primary antibody plus a biotin-conjugated secondary antibody. A total
   of 305 unique antibodies were used. The signal obtained was amplified
   using a Dako Cytomation–catalyzed system (Agilent Technologies, Santa
   Clara, CA, USA) and visualized by DAB colorimetric reaction. The slides
   were scanned, analyzed, and quantified using MicroVigene
   (RRID:SCR_002820, VigeneTech Inc., Carlisle, MA, USA) software to
   generate spot intensity. Each dilution curve was fitted with a logistic
   model SuperCurve Fitting (Department of Bioinformatics and
   Computational Biology in MD Anderson Cancer Center, Houston, TX, USA)
   utilizing the R environment (RRID:SCR_001905, CRAN family,
   [90]http://www.r-project.org/, accessed on 27 June 2021). The fitted
   curve was plotted with the signal intensities on the y-axis and the
   log2-concentration of proteins on the x-axis. The protein
   concentrations of each set of slides were then normalized by median
   polish, corrected across samples by the linear expression values. A
   correction was performed using the median expression level of all
   antibody experiments to calculate a loading correction factor for each
   sample.

2.19. Principal Component Analysis

   Principal components were calculated using the ClustVis web tool
   [[91]34] (RRID:SCR_017133, University of Tartu, Tartu, Estonia). The
   imputation and Singular Value Decomposition (SVD) were performed
   iteratively until estimates of missing values converge was performed.
   As an input, linear normalized RPPA data were used for all tested
   samples. The first two principal components (PC1, PC2) were plotted.

2.20. Differential Expression Analysis

   Proteins from RPPA data were filtered based on the adjusted p-value <
   0.05 and presented in volcano plots. All significantly differentially
   expressed proteins were clustered with the Morpheus tool [[92]35]
   (RRID:SCR_014975, Dresden University of Technology, Dresden, Germany)
   and visualized as heatmaps. The distance was calculated with one minus
   Pearson’s correlation coefficient metric.

2.21. Gene Set Enrichment Analysis (GSEA)

   GSEA ([93]http://www.broad.mit.edu/gsea/index.html access date: 26
   March 2020) was used to detect the coordinated expression of a priori
   defined groups of genes within the tested samples. Gene sets are
   available from the Molecular Signatures Database (MSigDB,
   RRID:SCR_016863, Broad Institute, Cambridge, MA, USA,
   [94]http://software.broadinstitute.org/gsea/msigdb/index.jsp access
   date: 26 March 2020). Briefly, GSEA generated an enrichment score (ES)
   reflective of the degree to which a gene set is overrepresented at the
   extremes (top or bottom) of the entire list of RPPA data. Genes are
   ranked according to expression difference (signal/noise ratio) between
   the tested group of samples: PHDF-WT/PHDF-CTRL and iPSC-WT/iPSC-CTRL,
   where CTRL cells are treated with a control siRNA. The ES calculation
   and estimation of the p-value, together with the normalized enrichment
   score (NES) and FDR calculations, have been previously described in
   detail [[95]36]. A total of 305 markers (previously ranked based on
   their log2FC between analyzed groups) were imported for GSEA. The GSEA
   was run according to the default parameters: each probe set was
   collapsed into a single gene vector (identified by its HUGO gene
   symbol), permutation number = 1000; permutation type = “gene-sets.” The
   FDR was used to correct for multiple comparisons and gene set sizes.

2.22. Over-Representation Enrichment Analysis (ORA)

   The ORA [[96]37] was performed with a WEB-based Gene SeT AnaLysis
   Toolkit (WebGestalt; [97]http://www.webgestalt.org/ access date: 28
   March 2020) with the “pathway” database. Protein names were transferred
   into gene symbols, and the reference gene list was set at “genome
   protein-coding”. Upregulated and down-regulated markers were considered
   separately.

3. Results

3.1. Generating Human iPSC with Doxycycline-Inducible System Results in
Repression of Transgene Expression in Established Clones

   IPSC were generated by reprogramming primary human dermal fibroblasts
   (PHDFs) with Yamanaka factors: OCT3/4, SOX2, KLF4, and c-MYC (OSKM)
   [[98]38], delivered in Stemcca-TetO lentivirus, under the control of
   the doxycycline-inducible system ([99]Figure S2A). PHDF cell lines were
   established from the healthy skin margins surrounding breast cancers
   excised during mastectomy. Application of the inducible system allowed
   to switch off OSKM expression in obtained iPSC colonies by transducing
   them on day 21 with LV-HK lentivirus. LV-HK vector carried the
   expression of the KRAB tet-repression domain, which, in the absence of
   doxycycline, inhibits EF-1α promoter ([100]Figure S2A). Re-induction of
   transgene expression would require the presence of doxycycline in
   culture media.

   Obtained iPSC displayed typical [[101]38] round colony morphology with
   a clear, regular peripheral outline ([102]Figure S2B). Colonies
   expressed intra- and extra-cellular pluripotency markers (positive for
   OCT3/4, NANOG, SSEA-4, TRA-1-60, and TRA-1-81, and negative to
   differentiation marker SSEA-1), comparable to the expression profile of
   hESC ([103]Figure S2C). The CpG methylation status in the promoter
   regions of OCT3/4 and NANOG was evaluated by bisulfite sequencing. The
   analyzed promoters were unmethylated in iPSC compared to parental PHDF
   cells, indicating the activity of these promoters in iPSC ([104]Figure
   S2D). Moreover, we confirmed a similar expression profile of
   pluripotency markers (REX1, NODAL, DNMT3B, OCT3/4, GABRB3, NANOG) in
   hESC and obtained iPSC ([105]Figure S2E). Chromosomal G-band analysis
   showed normal karyotypes with no chromosomal aberrations in generated
   lines ([106]Figure S2F). Immunohistochemical and H + E staining of
   iPSC-derived teratoma sections proved iPSC potential to differentiate
   into ecto-, endo- and mesoderm ([107]Figure S2G). We also confirmed no
   transgene expression from the integrated Stemcca-TetO vector in
   teratomas ([108]Figure S2H).

3.2. Silencing of Endogenous TRIM28 Induces Downregulation of Pluripotency
Markers and Differentiation of Human iPSC

   To evaluate the role of TRIM28 in stemness, we silenced endogenous
   TRIM28 expression with small interfering RNA (siRNA) in two generated
   iPSC lines. IPSC treated with siRNA with no target sequence served as a
   control (siCTRL) of the experiment. Upon TRIM28 silencing, the stemness
   and differentiation status was examined every 7 days for 3 weeks
   ([109]Figure 1A). The silencing efficiency was confirmed on
   transcriptional and protein level by qRT-PCR and immunofluorescence
   staining ([110]Figure 1B). The significant downregulation of TRIM28 in
   the CTRL sample at day 21 might result from that control siRNA, and
   transfection reagents can influence mRNA and protein levels [[111]39].
   Upon TRIM28 silencing, we observed decreased expression of
   extracellular pluripotency markers, SSEA-4, TRA-1-60, TRA-1-81
   ([112]Figure 1C), and progressive loss of intracellular pluripotency
   markers OCT3/4 and NANOG ([113]Figure 1D). Finally,
   differentiation-associated markers (CDX2, MSX1, FSP1, PAX6, SOX1) were
   upregulated in siTRIM28 cells on the transcript level ([114]Figure 1E).
   Our data indicate that TRIM28 knock-down facilitates differentiation of
   iPSC.

Figure 1.

   [115]Figure 1
   [116]Open in a new tab

   Silencing of endogenous TRIM28 induces downregulation of pluripotency
   markers and differentiation of human iPSC. (A) Endogenous TRIM28 was
   silenced in human iPSC with siRNA. Stemness and differentiation status
   were analyzed every 7 days for 3 weeks. (B) Silencing of TRIM28 at
   transcriptional and protein level confirmed by qRT-PCR (left) and
   immunofluorescence staining (right; green—TRIM28, blue—DAPI). Scale
   bar: 100 μm. Representative images are shown in the figures. (C) Flow
   cytometry analysis revealed decreased expression of extracellular
   pluripotency markers SSEA-4, TRA-1-60, and TRA-1-81 in siTRIM28 iPSC
   (red) vs. CTRL iPSC (black). Isotype control is marked in gray. (D) The
   progressive loss of intracellular pluripotency markers (NANOG, OCT3/4)
   expression in siTRIM28 iPSC confirmed with qRT-PCR (left) and
   immunofluorescence staining (right). Cells were stained at day 21.
   Scale bar: 100 μm. (E) qRT-PCR of differentiation-associated markers
   shows their upregulation during spontaneous differentiation of siTRIM28
   iPSC. (B–E) Graphs represent mean (SD), n = 3. Statistical analysis was
   performed with t-student and ANOVA tests; p ≤ 0.05 (<0.0001 ****;
   0.0001–0.001 ***; 0.001–0.01 **; 0.01–0.05 *; ≥0.05 not significant).

3.3. iPSC with Silenced TRIM28 Display Metabolic Changes, and Their Proteomic
Profile Differs from the Control iPSC

   Two weeks after silencing TRIM28, we evaluated metabolic changes by
   immunofluorescence staining ([117]Figure 2A). During reprogramming of
   somatic cells into iPSC, the metabolic profile shifts from oxidative
   phosphorylation (OXPHOS) to glycolysis [[118]40,[119]41,[120]42]. Upon
   cellular differentiation, the metabolic profile shifts back to OXPHOS,
   and mitochondrial activity is restored. Silencing of TRIM28 resulted in
   high upregulation of mitochondrial ATP synthase subunit D (ATP5PD),
   which indicates OXPHOS metabolism in differentiated cells. Evident
   downregulation of glycolysis-related markers, including
   phosphoglycerate kinase (PGK), pyruvate kinase (PKM2), and hexokinase 1
   (HK1), compared to wild type (WT) cells, also confirmed inhibition of
   glycolysis processes as a result of differentiation. The expression of
   HK2 did not change significantly upon TRIM28 silencing.

Figure 2.

   [121]Figure 2
   [122]Open in a new tab

   IPSC with silenced TRIM28 display metabolic changes and their proteomic
   profile differs significantly from the control iPSC. (A) Representative
   images of immunofluorescence staining showing metabolic changes from
   glycolysis (PGK1, PKM2—red; HK1, HK2—green) to oxidative
   phosphorylation (ATP5H—green) in differentiating iPSC-siTRIM28
   (TRIM28—green/red; DAPI—blue). Scale bar 100 μm. Graphs represent the
   quantitative analysis of MFI (SD), n = 3. Cells were analyzed on day 14
   upon TRIM28 silencing. (B) Principal Component Analysis (PCA) of RPPA
   data from 3 timepoints upon iPSC differentiation, n = 1. Numbers in
   parentheses represent the percentage of total variance explained by the
   first and the second PC. Prediction ellipses are such that with a
   probability 0.95, a new observation from the same group will fall
   inside the ellipse. Dots represent samples: purple—iPSC-siTRIM28,
   yellow—iPSC-WT, grey—iPSC-CTRL. (C) Protein level changes between
   iPSC-siTRIM28 cells and reference iPSC (WT and CTRL). The number of
   significantly downregulated (blue) or upregulated (red) proteins, with
   p-val < 0.05, is presented on the volcano plot, n = 1. (D) Heatmap of
   185 differentially expressed markers in WT/CTRL and siTRIM28 cells,
   based on RPPA data (p-val < 0.05). The relative protein level is
   presented as z-score. Blue—minimal value, red—maximal value in each
   column, n = 1. (E,F) Gene Ontology Biological Processes significantly
   enriched in the list of upregulated markers in iPSC-siTRIM28 (E) and
   reference iPSC (F), as determined with Overrepresentation Enrichment
   Analysis (ORA) using WebGestalt on-line tool. Numbers in parentheses
   indicate the number of markers in individual processes. Top 25 terms
   with FDR < 0.05% are presented.

   iPSC with silenced TRIM28 were subjected to RPPA analysis, and their
   proteomic profile was compared with WT and siCTRL iPSC. Raw data are
   openly available in GEO at [123]www.ncbi.nlm.nih.gov/geo access date: 3
   July 2020, reference number: [124]GSE153726. Principal Component
   Analysis (PCA) showed that the samples from each group clustered
   together, and the groups were clearly segregated ([125]Figure 2B). We
   determined 185 differentially expressed proteins, of which 64 were
   significantly downregulated and 121 upregulated between iPSC siTRIM28
   and reference iPSC (WT and siCTRL) ([126]Figure 2C). Differentially
   expressed proteins showed clustering of markers from iPSC_WT,
   iPSC_siCTRL, and iPSC_siTRIM28 samples ([127]Figure 2D). Pathway
   enrichment analysis using Gene Ontology datasets showed significant
   upregulation of pathways related to apoptosis, differentiation,
   cellular response to DNA damage stimulus, and cell cycle regulation in
   iPSC-siTRIM28, relative to reference iPSC ([128]Figure 2E). In
   contrast, reference iPSC demonstrated enrichment of the processes
   involved in the cellular response to organonitrogen and nitrogen
   compounds and processes related to the regulation of phosphorylation
   and cell proliferation ([129]Figure 2F). The list of markers assigned
   to individual processes is presented in [130]Figure S3. Among the
   markers upregulated upon TRIM28 silencing, we found some tumor
   suppressor genes, e.g., MSH2, CHEK2, ANXA, or CAV1 ([131]Figure S3A).
   One of the downregulated markers in iPSC-siTRIM28 (upregulated in
   reference iPSC) was TRIM28, and a few protooncogenes, e.g., EIF4E,
   BRAF, ARAF ([132]Figure S3B). These results indicate the role of TRIM28
   in the regulation of several signaling pathways implicated in
   maintaining self-renewal and stemness of iPSC, and probably of highly
   dedifferentiated metastatic tumor cells as well.

3.4. Selection of Mutation Sites Impairing the Function of TRIM28 Protein
Domains

   To determine the TRIM28-dependent mechanisms responsible for the
   self-renewal and pluripotency maintenance, we selected eight different
   mutation sites within TRIM28. Mutations impaired the functions of its
   particular domains by their effect on phosphorylation of the domain
   structure ([133]Figure 3A,B). All mutations introduced into the TRIM28
   sequence and predicted effects on protein activity are summarized in
   [134]Table 1.

Figure 3.

   [135]Figure 3
   [136]Open in a new tab

   The constructed lentiviral system enables efficient transgene
   expression and silencing of endogenous TRIM28. (A) Localization of
   structural (S) or phosphorylation (p) mutations chosen to affect the
   function of each TRIM28 domain. (B) Prediction of the functional impact
   of selected mutations on TRIM28 activity performed with Mutation
   Assessor algorithms. Table source: [137]http://mutationassessor.org/
   access date: 17 February 2019. MSA—annotated in multiple sequence
   alignment (MSA) browser. PDB—annotated in 3D structure browser. FI
   score—functional impact combined score. VC score—variant conservation
   score. VS score—variant specificity score. Gaps in MSA—portion of gaps
   in variant position in MSA, MSA height—number of diverse sequences in
   multiple sequence alignment. (C) Lentiviral system generated to silence
   endogenous TRIM28 by shRNA (shTRIM28) and express exogenous,
   shRNA-resistant (shTRIM-res), and FLAG-tagged TRIM28 sequences. (D)
   Silencing of endogenous TRIM28 and shRNA specificity to endogenous
   TRIM28 sequence evaluated by immunofluorescence staining (green—TRIM28,
   blue—DAPI). Scale bar: 100 μm. (E) Silencing of endogenous TRIM28 and
   expression of exogenous FLAG-tagged proteins confirmed by Western Blot.
   (F) Efficient transgene expression assessed by immunofluorescence
   staining against FLAG-tag attached to N-terminus of exogenous TRIM28
   protein (red—FLAG, blue—DAPI). Scale bar: 100 μm.

Table 1.

   Selected TRIM28 mutation sites with predicted effects on protein
   activity.
   Mutation Domain Predicted Influence on TRIM28 Activity Reference
   C91A
   (structural mutant) RING Destabilization of RING domain structure
   [[138]28,[139]45,[140]46,[141]47,[142]48]
   Inhibition of interaction with KRAB domain of KRAB-ZNFs
   Impairment of E3 ubiquitin ligase function
   Restriction of TRIM28 transcription co-repressor function
   S473A
   (phospho-mutant) HP1BD Inhibition of TRIM28 Ser473 phosphorylation
   [[143]31,[144]43]
   HP1BD interaction with HP1
   TRIM28 functions as transcription co-repressor
   S473D
   (phospho-mimetic) HP1BD Imitation of permanent TRIM28 Ser473
   phosphorylation [[145]31,[146]43]
   Inhibition of HP1 binding by HP1BD
   Restriction of TRIM28 transcription co-repressor function
   3YF
   (phospho-mutant) HP1BD Inhibition of triple tyrosine phosphorylation
   [[147]19]
   HP1BD interaction with HP1
   TRIM28 functions as a transcription repressor
   C628R
   (structural mutant) PHD Destabilization of PHD domain structure
   [[148]30,[149]49]
   Impairment of E3 SUMO ligase function
   Inhibition of NuRD complex and SETDB1 methyltransferase binding
   Restriction of TRIM28 transcription repressor function
   N773G
   (structural mutant) BROMO Destabilization of bromodomain structure -
   Low influence on TRIM28 activity
   S824A
   (phospho-mutant) BROMO Inhibition of TRIM28 Ser824 phosphorylation
   [[150]18,[151]44]
   Increased TRIM28 BROMO sumoylation
   Induction of differentiation
   S824D
   (phospho-mimetic) BROMO Imitation of permanent TRIM28 Ser824
   phosphorylation [[152]18,[153]44]
   Decreased TRIM28 BROMO sumoylation
   Inhibition of spontaneous differentiation
   Induction of SOX2 and NANOG expression
   [154]Open in a new tab

   Five of the TRIM28 mutation sites were chosen based on the literature
   reports related to the key amino acids of the TRIM28 protein undergoing
   phosphorylation that affect particular domains’ activity.
   Phosphorylation on Ser474, which is located directly before HP1BD,
   lowers the ability to bind HP1 protein and inhibits TRIM28
   transcription repressor activity [[155]31,[156]43]. TRIM28
   phosphorylation on three tyrosines, Y449F/Y458F/Y517F (3YF), flanking
   HP1BD, also reduces the HP1 binding ability, preventing silencing of
   gene expression [[157]19]. Phosphorylation on Ser824 affects the
   activity of BROMO by decreasing TRIM28 sumoylation and is associated
   with relaxed chromatin. It blocks the differentiation of mouse
   pluripotent cells and induces the expression of SOX2 and NANOG
   [[158]18,[159]44].

   Structural mutants were selected using Mutation Assessor algorithms
   (RRID:SCR_005762, Computational Biology Center, Memorial Sloan
   Kettering Cancer Center) [[160]50]. The algorithm analyzes protein
   family multiple sequence alignments (MSA) of homologous sequences,
   exploits sequence homologs 3D structures, and generates conservation
   scores to predict functional specificity. The functional impact score
   of mutation is calculated based on the evolutionary conservation in a
   protein family and, separately, in every subfamily [[161]50].

   The C91A mutation in the RING domain results in the abolition of E3
   ubiquitin ligase function and inhibition of binding to transcription
   factors containing the KRAB domain, leading to the loss of the
   transcription repressor function [[162]45,[163]46,[164]47,[165]48].
   Structural C628R mutation in PHD inhibits its endogenous E3 SUMO ligase
   function and hindrance of BROMO sumoylation [[166]28]. It impedes the
   interaction with the NuRD complex and SETDB1 methyltransferase and
   restricts the function of TRIM28 as a transcription repressor
   [[167]30,[168]49]. The N773G structural mutation, located in BROMO, was
   selected as a structural control mutation that has a low impact on the
   impairment of protein function.

3.5. Mutations in RING and PHD Domains Are Classified as Mutations with a
High Impact on TRIM28 Function

   Predictive algorithms indicated C91A and C628R to be mutations having a
   high impact on the function of the TRIM28 protein ([169]Figure 3B).
   Many previous reports demonstrated common TRIM28 phosphorylation sites
   (S473, 3YF, S824) as crucial for TRIM28 function. Experiments were
   performed on mouse ESC [[170]18], HEK-293 cells
   [[171]31,[172]43,[173]44], breast cancer cell line MCF-7 [[174]44], or
   cervical cancer cell line HeLa [[175]19,[176]31,[177]43]. In contrast,
   our predictive analysis indicated that phosphorylation site mutations
   have a low or neutral functional impact.

3.6. Constructed Lentiviral System Enables Efficient Transgene Expression and
Silencing of Endogenous TRIM28

   To obtain more homogenous populations and reduce the factors that might
   influence the subtle phenotype changes, we decided to perform further
   experiments on commercial feeder-free human iPSC line ND41658*H (NINDS
   Human Genetics DNA and Cell Line Repository, Coriell, Camden, NJ, USA).
   We also wanted to evaluate whether previously observed differentiation
   due to TRIM28 silencing will be observed on other iPSC lines.

   To generate populations expressing exogenous TRIM28 or its mutated
   variants, iPSC were transduced with lentiviral vectors carrying mutated
   exogenous TRIM28 sequence and shRNA sequence silencing endogenous
   TRIM28 (mut-shTRIM28) ([178]Figure 3C). We also generated the empty
   vector control population (CTRL) only with silenced endogenous TRIM28
   expression (shTRIM28) and population expressing exogenous non-mutated
   TRIM28 and hairpin silencing endogenous TRIM28 (RESCUE). Exogenous
   mutated (S473A, S473D, 3YF, S824A, S824D, C91A, C628R, N773G) and
   non-mutated (RESCUE) TRIM28 sequences were resistant to shRNA
   (shTRIM28-res) and were tagged at the N-terminus with FLAG sequence.

   First, we evaluated the shRNA effect on endo- and exogenous TRIM28
   expression by immunofluorescence staining ([179]Figure 3D). At the
   initial stages (2nd passage), we observed some colonies expressing
   TRIM28 in cells transduced with the shTRIM28 vector, probably due to
   ongoing and not yet complete puromycin selection. After the 6th
   passage, we re-assessed TRIM28 silencing, and this time TRIM28
   expression was decreased in all studied colonies. The control RESCUE
   population expressed TRIM28 among all the colonies, confirming
   resistance of exogenous sequence to shRNA.

   Analysis of anti-FLAG antibodies proved the functionality of applied
   lentiviral vectors. We detected transgene expression in all populations
   of iPSC with exogenous variants of TRIM28, but the intensity varied
   within transduced populations ([180]Figure 3E) and cells in colonies
   ([181]Figure 3F). Heterogenous transgene expression might result from
   transducing iPSC as small aggregates, not as a single cell suspension,
   as well as from non-clonal selection.

3.7. Mutations of RING and PHD Domains Influence Human iPSC Morphology and
Pluripotency Markers Expression

   First, morphological changes appeared right after the 1st passage in
   the variant with the silenced endogenous TRIM28 transcript (shTRIM28).
   After the 2nd passage, similar changes were observed in iPSC with
   mutations within RING (C91A) and within PHD (C628R) ([182]Figure 4A).
   These populations demonstrated the loss of morphology typical for
   pluripotent cells [[183]38]. Non-modified cell colonies (WT) and cells
   transduced with control vectors (CTRL and RESCUE) showed no changes in
   the characteristic uniform morphology. Among other modified variants of
   iPSC, no morphology changes indicating differentiation were noted.
   Occasionally, differentiating single cells were left among the
   undifferentiated colonies to not interfere with the differences arising
   between individual variant populations.

Figure 4.

   [184]Figure 4
   [185]Open in a new tab

   Mutations of RING and PHD domains influence iPSC morphology and
   pluripotency marker expression. (A) Morphology of iPSC colonies was
   visually inspected every day for 10 passages after LV transduction.
   Pictures show representative morphology after the 2nd, 6th, and 10th
   passage. Scale bar: 200 µm. (B,C) Loss of endogenous pluripotency
   markers expression in differentiating populations (shTRIM28, C91A,
   C628R) evaluated by immunofluorescence staining against OCT3/4, SOX2,
   and NANOG (red—pluripotency markers; blue—DAPI). Scale bar: 100 µm.
   Graphs represent the quantitative analysis of mean MFI (SD), n = 3.
   Statistical analysis performed with one-way ANOVA and a post-hoc
   Dunnett’s test; p ≤ 0.05 (<0.0001 ****; 0.0001–0.001 ***; 0.01–0.05 *;
   ≥0.05 not significant).

   Silencing of endogenous TRIM28 (shTRIM28) resulted in downregulation of
   OCT3/4 expression compared to other analyzed populations ([186]Figure
   4B). On the other hand, a downregulated level of NANOG was demonstrated
   in the shTRIM28 population and, to a smaller extent, in populations
   with mutated RING (C91A) or PHD domains (C628R). In differentiating
   populations (shTRIM28, C19A, and C628R), the SOX2 level was also
   reduced. Also, in some cells of the S824D population, SOX2 was
   decreased, but no other signs of cell differentiation were noted, and
   no changes in colony morphology after the 10th passage were found. The
   remaining phospho-mutants and phospho-mimetics did not affect the
   expression of the analyzed pluripotency markers.

3.8. Dysfunction of RING and PHD Domains Results in Decreased Proliferation
and Inhibition of Embryoid Bodies Formation

   We also evaluated the influence of TRIM28 mutations on proliferation
   and in vitro spontaneous differentiation potential ([187]Figure 5). As
   expected, differentiating populations (shTRIM28, C19A, and C628R)
   displayed a decreased proliferation compared to WT or CTRL/RESCUE iPSC
   ([188]Figure 5A). However, analyzed phospho-mutants and
   phospho-mimetics did not significantly affect proliferation.

Figure 5.

   [189]Figure 5
   [190]Open in a new tab

   Dysfunction of RING and PHD domains results in decreased proliferation
   and inhibition of embryoid body formation. (A) Changes in iPSC
   proliferation calculated by spectrophotometry analysis of BrdU
   incorporation on the 6th passage after LV transduction (n = 3). (B) EBs
   area was calculated from images taken by Incucyte for 8 days after
   induction of EBs formation. Each bar represents the mean area in µm
   (SD), n = 3. Statistical analysis performed with Kruskal–Wallis test; p
   ≤ 0.05 (0.0001–0.001 ***; 0.001–0.01 **; 0.01–0.05 *; ≥ 0.05 not
   significant). (C) Example images taken by Incucyte during EBs formation
   (n = 3). Scale bar: 400 µm. See also [191]Videos S1–S12. (D) QRT-PCR
   analysis of differentiation markers in iPSC populations 6 passages
   after LV transduction. Graphs represent markers related to ectoderm
   (Nestin, MAP2), mesoderm (SMA, Brachyury), endoderm (GATA6, SOX17).
   Each bar represents the mean mRNA expression level (SD), n = 3, SOX17:
   n = 1. If no amplification occurred, the expression level was
   calculated as 0. Statistical analysis performed with one-way ANOVA and
   a post-hoc Dunnett’s test; p ≤ 0.05 (0.0001–0.001 ***; 0.001–0.01 **;
   0.01–0.05 *; ≥0.05 not significant).

   Differentiation potential was analyzed by EBs formation. EBs were
   formed by forced aggregation in a non-adherent 96-well plate and
   monitored with an Incucyte SX1 Live-Cell Analysis System (#4788,
   Sartorius, Göttingen, Germany). We observed smaller or no spheres
   derived from shTRIM28, C91A, and C628R compared to the rest of the
   populations ([192]Figure 5B,C, [193]Videos S1–S12). We assumed this
   effect was caused by differentiation induction after impairment of the
   RING and PHD function. Therefore we evaluated differentiation markers
   expression in the populations used to obtain EBs ([194]Figure 5D).
   Unfortunately, due to late or no amplification in many samples, SD is
   very high and disabled drawing conclusions. However, we observed a
   clear trend in the expression of MAP2 (ectoderm) in shTRIM28 and SMA
   (mesoderm) in shTRIM28 and C628R cells. MAP2 is a neuronal marker, yet
   its expression can be found in differentiating EBs, as soon as 16 days
   upon EBs formation, which might explain its presence in differentiating
   shTRIM28 population [[195]51]. We also captured the amplification of
   SOX17 (endoderm), but only in one out of three biological replicates.
   Due to high deviations, the graph for SOX17 expression represents only
   one replicate. The result is not statistically significant, but it is
   interesting enough to mention it in this study.

   Until this point, our results suggested that TRIM28 phosphorylation
   does not significantly affect the mechanisms contributing to
   pluripotency and self-renewal maintenance in human iPSC. However, in
   the 3YF population, we noticed that the expression level of some
   differentiation markers ([196]Figure 5D) was lower than in WT cells.
   This may support findings suggesting the inhibition of triple tyrosine
   phosphorylation impact on HP1BD interaction with HP1, which enables
   transcription repression by TRIM28 [[197]19,[198]52,[199]53].

   Still, our results imply that mutations of Ser473 or Ser824 do not
   affect TRIM28 function in human iPSC. These data stand in opposition to
   previous reports indicating the impact of Ser473 and Ser824
   phosphorylation on TRIM28 function [[200]18,[201]31,[202]43,[203]44].
   What is important, mentioned reports did not include the research on
   human iPSC. Nevertheless, the phosphorylation-dependent regulation of
   genes responsible for sustaining the undifferentiated state cannot be
   completely ruled out. Obtained data, however, indicates a much stronger
   influence of structural mutations on the mechanisms supporting
   pluripotency. Thus, only the populations with mutants in RING (C91A) or
   PHD (C628R) and shTRIM28 were subjected to further experiments.

3.9. Impairment of RING and PHD Functions Results in Dysregulation of Stem
Cell-Associated Signaling Pathways

   The differentiating shTRIM28, C19A, and C628R populations exhibited
   downregulation of extracellular pluripotency markers TRA-1-60 and
   TRA-1-81 ([204]Figure 6A). The decreased expression of surface markers
   is more evident in cells with silenced TRIM28 (shTRIM28) than in cells
   with a structural defect of RING or PHD domain. The SSEA-4 level was
   only slightly reduced in C19A and C628R mutants. However, the
   literature data indicate that the most rapid changes occur in the
   expression of TRA-1-81 and TRA-1-60 antigens, and the expression of
   SSEA-4 decreases much slower during differentiation of hESC [[205]54].
   QRT-PCR analysis of differentiating populations also showed significant
   downregulation of pluripotency markers NANOG and LIN28A ([206]Figure
   6B).

Figure 6.

   [207]Figure 6
   [208]Open in a new tab

   Impairment of RING and PHD functions results in dysregulation of stem
   cell-associated signaling pathways. (A) Extracellular pluripotency
   marker changes evaluated by flow cytometry. Fluorescence intensity for
   analyzed populations was compared to CTRL population and isotype
   control (n = 3). Representative histograms are shown for each
   population. (B–H) QRT-PCR analysis of genes dysregulated in
   differentiating populations. Graphs represent markers related to
   pluripotency (B), epigenetic regulation (C), self-renewal (D), Hedgehog
   pathway (E), WNT pathway (F), NOTCH pathway (G), migration, and
   metastasis (H). Each bar represents the mean mRNA expression level
   (SD), n = 3. Statistical analysis performed with one-way ANOVA and a
   post-hoc Dunnett’s test; p ≤ 0.05 (0.0001–0.001 ***; 0.001–0.01 **;
   0.01–0.05 *; ≥0.05 not significant).

   We next analyzed the expression of various markers implicated in the
   maintenance of self-renewal and stemness ([209]Figure 6C–H). These
   genes are important for the functioning of both pluripotent and cancer
   cells. First, we investigated the expression of the genes engaged in
   chromatin modification ([210]Figure 6C). Analyzed populations displayed
   downregulation of DNA methyltransferases (DNMT) 3A and 3B, which
   contributes to pluripotency maintenance and inhibition of
   differentiation [[211]55,[212]56]. Many reports indicate rapid
   repression of TERT within a few days upon differentiation
   [[213]57,[214]58]. However, according to our observations, TRIM28 RING
   and PHD domain dysfunctions do not seem to affect TERT expression
   level. EZH2, a set-domain containing histone methyltransferase specific
   to H3K27 [[215]59], was upregulated only in the PHD-mutated population.
   However, this shift cannot be compared to the expression level in
   control or silenced TRIM28 populations, and the results remain
   inconclusive.

   Self-renewal regulation also depends on several signaling pathways,
   such as Wingless (Wnt) [[216]60], Hedgehog (Hh) [[217]61],
   phosphoinositide 3-kinase (Pi3K)/Akt kinase [[218]5], or
   mitogen-activated protein kinases (MAPK) [[219]62]. Therefore, we
   analyzed the expression of critical genes involved in these pathways.
   We observed that the downregulation of FGF receptor (FGFR), PI3K, and
   BMP7 mRNA levels correlated with the impairment of RING and PHD
   function and the silencing of TRIM28 ([220]Figure 6D). The expression
   of key genes involved in the Hh pathway, receptor patched 1 (PTCH1) and
   smoothened (SMO), was also remarkably reduced in iPSC populations with
   silenced TRIM28, and in cells with RING and PHD mutants, indicating the
   inhibition of Hh signal transduction ([221]Figure 6E). Structural
   mutations of TRIM28 resulted in a significant decrease in the
   expression of EPCAM, pluripotency, and proliferation marker in mouse
   and human stem cells [[222]63,[223]64,[224]65] ([225]Figure 6F).
   Populations with silenced TRIM28 and the population with PHD mutant
   showed a slight increase in WNT expression and downstream WNT signaling
   inhibitor DKK1 [[226]66]. Silencing of TRIM28 and defects within the
   PHD contributed to a double increase in NOTCH1 ([227]Figure 6G). The
   expression of MAML1, which acts as a transcriptional coactivator for
   NOTCH signaling [[228]67,[229]68], did not alter under the influence of
   TRIM28 dysfunction. However, due to high deviations, the results for
   WNT, DKK1, NOTCH1, and MAML1 expression were not considered
   statistically significant.

   Finally, we examined the expression of genes that are known to be
   associated with migration and metastasis of cancer cells, and we
   observed a significant reduction in E-cadherin (CDH1), an inhibitor of
   differentiation 1 (ID1) and TWIST2 ([230]Figure 6H). We assumed that
   CDH1 shift is directly related to the loss of typical compact colony
   morphology, regularly maintained in an undifferentiated state by
   cell-cell contact via E-cadherin [[231]69,[232]70]. Altogether, our
   results demonstrated that TRIM28 affects the expression of the genes
   implicated in the pathways common for stem cells and cancer cells.

4. Discussion

   The TRIM28 protein was shown to have a crucial impact on self-renewal
   ability and maintenance of pluripotency in mouse and human ESC
   [[233]8,[234]16,[235]17,[236]18,[237]71,[238]72], as well as in human
   cancer cells [[239]9,[240]19,[241]43]. In this study, we confirmed that
   TRIM28 preserves stemness and self-renewal in human iPSC. In addition,
   for the first time in human iPSC, we analyzed the function of
   individual TRIM28 domains. We identified RING and PHD as the principal
   domains responsible for these TRIM28 properties.

   Impairment of RING or PHD activity, as well as TRIM28 silencing, had a
   very rapid influence on iPSC populations. IPSC dissociated into
   individual cells, lost a typical compact colony structure, and showed a
   decrease in NANOG and SOX2 protein level. In contrast to previous
   studies suggesting the impact of Ser473 and Ser824 phosphorylation on
   TRIM28 function, examined phospho-mutants and phospho-mimetics did not
   affect the self-renewal in human iPSC
   [[242]18,[243]31,[244]43,[245]44]. Nevertheless, the inhibition of
   triple tyrosine phosphorylation (Y449F/Y458F/Y517F) slightly reduced
   the occasional differentiation of the 3YF population. This observation
   may support reports indicating 3YF mutation enables HP1BD interaction
   with HP1, resulting in transcription repression by TRIM28
   [[246]19,[247]52,[248]53]. Therefore, TRIM28 phosphorylation-dependent
   regulation of genes responsible for sustaining the undifferentiated
   state cannot be excluded. However, our data indicate a much stronger
   influence of structural mutations on the mechanisms supporting
   pluripotency.

   Both mutations with observed effects on stemness properties (C91A,
   C628R) were predicted to have a high functional impact in our initial
   analysis with the Mutation Assessor algorithms. Indeed, our results
   underline the importance of both substituted amino acids for early
   developmental processes associated with the maintenance of self-renewal
   and pluripotency. Therefore, only the iPSC populations exhibiting
   phenotype changes were selected for further study.

   Silencing TRIM28 and impairment of RING or PHD domains activity led to
   downregulation of intra- and extra-cellular pluripotency markers. It
   also resulted in downregulation of methyltransferases DNMT3A and
   DNMT3B, associated with the ongoing switch in gene expression profile,
   progressive cell differentiation, and loss of the parental phenotype
   [[249]55,[250]56]. Our observations regarding RING domain function in
   stemness maintenance support the results indicating the engagement of
   KRAB-ZFPs in pluripotency maintenance. Silencing particular KRAB-ZFPs
   was shown to induce differentiation of pluripotent stem cells by
   epigenetic repression of crucial differentiation genes [[251]8]. This
   may conclude that the RING domain, which is responsible for interaction
   with the KRAB domain of ZFPs, maintains self-renewal and stemness in
   human pluripotent stem cells through mediating KRAB-ZFPs repression
   function. On the other hand, we report that stemness maintenance in
   human iPSC is also determined by the activity of PHD, which is
   responsible for sumoylation, and therefore activation of the BROMO
   domain. Cooperation of PHD and BROMO domains results in chromatin
   remodeling, histone deacetylation, enhanced methylation of H3K9, and
   finally binding HP1 to tri-methylated H3K9 and silencing gene
   expression [[252]30,[253]73]. Our data indicate that TRIM28, to support
   pluripotency and self-renewal mechanisms, requires functional RBCC and
   PHD domains that are responsible for the activity of TRIM28 as a
   transcriptional co-repressor. Gene repression mediated by TRIM28 is
   also compromised by phosphorylation of S824 TRIM28 residue. In
   opposition to previous data indicating the important role of this
   post-translational modification in maintaining pluripotency in murine
   cells [[254]18], S824A phospho-mutant was shown to be insufficient to
   initiate differentiation in human iPSC in our study.

   In mouse ESC, Trim28, along with Cnot3 co-repressor, were identified as
   factors necessary to maintain self-renewal capacity [[255]71]. They
   were shown to bind to numerous gene promoters, creating a unique module
   distinct from the main module of the core pluripotency network, formed
   by OCT3/4, SOX2, and NANOG. The sequences regulated by TRIM28 included
   genes involved in the cell cycle, cell death, and tumorigenesis
   [[256]71]. Here, we show that during differentiation of iPSC due to
   TRIM28 depletion, there is a significant shift in cellular signaling.
   We demonstrate that silencing TRIM28 leads to upregulation of processes
   related to the regulation of apoptosis, differentiation into a
   multicellular organism, positive regulation of the developmental
   process, and regulation of the cell cycle, which is also confirmed by
   earlier evidence [[257]9,[258]74,[259]75].

   As previously reported, TRIM28 overexpression correlates with poor
   prognosis in many cancer types
   [[260]20,[261]21,[262]22,[263]23,[264]24,[265]25]. Furthermore, there
   is growing evidence that cancer stemness is associated with the
   expression of OCT3/4, SOX2, c-MYC, and other genes involved in the
   self-renewal regulation of malignant cancer cells, as well as normal
   stem cells [[266]3]. Numerous studies have been performed to
   characterize tumor cells in terms of their stemness score and
   similarity to normal stem cells. Genes upregulated in ESC, for instance
   genes regulated by NANOG, OCT3/4, SOX2, and c-MYC, are often
   overexpressed in undifferentiated tumors compared to differentiated
   ones [[267]76,[268]77,[269]78]. Collectively, with many reports
   presenting TRIM28 contribution to cancer, these findings suggest that
   regulatory networks controlling self-renewal in stem cells may also be
   active in some types of cancer and may constitute new cancer cell
   therapy.

   Our results also indicate that the dysfunction of RING or PHD domains
   decreases proliferation and EBs formation of hiPSC, which might be very
   important considering TRIM28 correlation with some tumors. For this
   reason, we also analyzed the expression of various markers involved in
   the maintenance of self-renewal, stemness, and the functioning of
   pluripotent stem cells, as well as of highly dedifferentiated cancer
   cells.

   In human ESC, the pivotal factor in maintaining the state of
   pluripotency is FGF, which activates PI3K/Akt and MAPK/Erk signaling
   cascades [[270]62]. Our results reveal a significant reduction in FGFR2
   and PI3K expression in all differentiating iPSC populations. PI3K/Akt
   signaling maintains pluripotency by regulating OCT4, SOX2, and NANOG
   [[271]79,[272]80]. Zhou et al. demonstrated that in hESC inhibition of
   mTOR, which is a PI3K effector, this results in a decrease in OCT4,
   SOX2, and NANOG expression [[273]79]. On the contrary, our results
   indicate that the most significant changes in the expression of core
   pluripotency transcription factors occurring within the timescale of
   our experiment are limited to NANOG. Thus, our data may suggest that
   the differentiation effect was caused not by the primary inhibition of
   FGF/PI3K signaling but by the lower NANOG expression caused by
   disrupted TRIM28 activity.

   Recently Do et al. determined that Trim28 prevents the degradation of
   Oct4, and Trim28 overexpression stabilizes Oct4 in mouse ESC [[274]81].
   Furthermore, they found the Trim28 CC domain to be responsible for
   interaction with Oct4. This supports our findings, indicating that only
   silencing of TRIM28 resulted in downregulation of OCT4 expression, and
   none of the introduced mutations were able to induce such an effect.
   Data presented by Do et al. may also support reports regarding TRIM28
   overexpression correlation with poor prognosis in certain cancer types
   [[275]20,[276]21,[277]22,[278]23,[279]24,[280]25].

   We also demonstrate that dysfunction of the RING or PHD domains and
   TRIM28 silencing leads to the downregulation of the Hedgehog pathway
   and EPCAM, implicated in stemness maintenance in iPSC. These factors
   are also therapeutic targets for cancer. In adults, the mutation or
   deregulation of the Hedgehog pathway plays a key role in both
   proliferation and differentiation, leading to tumorigenesis or
   accelerated tumor growth in many different tissues
   [[281]4,[282]82,[283]83]. EPCAM is frequently overexpressed in tumor
   cells [[284]84], while its suppression is considered a new approach for
   the treatment of colon cancer [[285]85]. Furthermore, we show that
   TRIM28 significantly impacts the expression of metastasis-related
   genes, TWIST2 and ID-1. Many reports indicate the involvement of TRIM28
   in the induction of EMT through regulation of TWIST
   [[286]9,[287]86,[288]87]. ID-1 contributes to many cellular processes,
   including cell growth, aging, differentiation, apoptosis, angiogenesis,
   and neoplastic transformation [[289]88,[290]89,[291]90]. Recent studies
   suggest that ID-1 knock-down in endothelial cells derived from angioma
   inhibits proliferation and induces apoptosis by inhibiting
   PI3K/Akt/mTOR signaling [[292]90]. Our data also indicate a significant
   reduction in ID-1 expression, as well as a decrease in PI3K activity in
   cells with impaired TRIM28 functions, which supports previous findings.

   Although both RING and PHD domains contribute to the self-renewal and
   stemness of human iPSC, they may use different interactions and
   mechanisms to repress gene expression. We found that WNT1, DKK1, and
   NOTCH1 showed a trend of increased expression in the population with
   mutated PHD compared to RESCUE and RING mutants. The expression level
   of WNT1, DKK1, and NOTCH1 in the PHD mutant population is similar to
   the expression in cells with silenced TRIM28. In stem cells, the
   activation of Wnt signaling can induce the expression of Notch pathway
   components [[293]91]. This may suggest that in contrast to the RING
   domain, PHD takes part in Wnt/Notch signaling.

5. Conclusions

   In conclusion, our study provides new insights into the role of TRIM28
   protein domains in the regulation of pluripotency processes and
   self-renewal mechanisms in human induced pluripotent stem cells. Of the
   numerous biological functions of TRIM28 protein, our results indicate
   the activity of RING and PHD domains in the transcriptional repression
   to be one of the main molecular mechanisms responsible for maintaining
   self-renewal and pluripotency. In addition, we demonstrate that in
   iPSC, TRIM28 influences the expression of the genes involved in the
   cell cycle, self-renewal, cell death, mobility, and other gene
   characteristic for cancer cells. Therefore, regulatory networks
   dependent on TRIM28 signaling that control self-renewal in stem cells
   may also be active in some types of cancer cells. As such, TRIM28 RING
   and PHD domains may be considered new targets for cancer therapy, e.g.,
   by designing a drug inhibitor complementary to selected domains. The
   application of an oncolytic virus expressing a short peptide, binding
   to crucial TRIM28 amino acids and blocking domain interactions, could
   also prove useful.

Acknowledgments