The mediator complex is known to serve as an intermediary between transcription factors and RNA polymerase II (pol II) for the transmission of intracellular signals. The mediator complex can be divided into four distinct modules: a head module, a middle module, and a tail module, which function as the main components, and the CDK8 kinase module, which is transiently associated with the complex [1
]. After the recruitment to DNA through the mediator-transcription complex, the mediator complex controls a variety of processes that are critical for transcription, including the reorganization of chromatin architecture and the regulation of pol II-mediated preinitiation, initiation, and elongation [3
] in transcription. When considering that the essential role of the mediator complex in transcriptional control, genetic variation, or change in the expression of the mediator complex subunits that affect the pol II activity could lead to a variety of diseases. For instance, mutations in MED12, MED17, and MED23 are associated with X-linked mental retardation syndrome, infantile cerebral atrophy, and intellectual disability, respectively [2
]. Mutational analysis of the mediator complex subunits can provide valuable insights that could be useful in the development of novel drugs against critical human diseases [8
Mammalian mediator complex subunit 28 (MED28) belongs to the “head” module of the mediator complex and it was first identified as an endothelial-derived gene 1 using tumor-conditioned media of endothelial cells [9
]. MED28 is highly expressed in breast, colon, and prostate cancers, and MED28 induces cancer cell proliferation via the mitogen-activated protein kinase kinase-1 (MAP2K1) signaling pathway [10
]. Recently, we reported that the interplay between MED28 and ZNF224, a Krüppel-associated-box-containing zinc finger protein transcriptional cofactor, was associated with cancer cell proliferation upon initiation of the DNA damage response [13
]. A study in knockout mice has shown that the genetic deletion of MED28
results in peri-implantation embryonic lethality by reducing the expression of OCT4 and NANOG, which are pluripotency transcription factors [14
]. Although the expression level of MED28 is closely associated with cell proliferation, the regulatory mechanism that is involved in enhancing MED28 expression is unknown.
Chromosome segregation is the most critical event in the cell cycle, and chromosome mis-segregation can be observed by the direct examination of chromosome movements. A high degree of mis-segregation is called chromosomal instability, and the persistent mis-segregation of chromosomes at a high rate causes aneuploidy in tumors with chromosome numbers in the range of 40–60 [15
]. Aneuploidy is caused by various factors, including chemicals, environmental toxins, and DNA replication errors, and it induces increased proliferation with an abnormal cell cycle [16
]. It is known that the alteration of the cell cycle by aneuploidy can change the intracellular or extracellular environments, thereby inducing resistance to chemotherapeutic drugs [18
Although the molecular mechanisms underlying MED28-mediated oncogenesis are unknown, previous studies have suggested that MED28 can increase cancer cell proliferation, and phenotypes that are related to the dysregulation of MED28 have been demonstrated in breast cancer cells [11
]. In this study, we aimed to identify and characterize the transcription factors that increase MED28 expression and investigated the involvement of MED28 in cell cycle regulation.
Recent studies indicate that the overexpression of MED28
induces cancer cell proliferation and increases the colony-forming ability of cancer cells upon DNA damage [11
]. In this study, we first identified four transcription factors, namely E2F-1, NRF-1, ETS-1, and C/EBPβ, which increase the expression level of MED28
. E2F-1 is reported to be an oncogene that is involved in DNA replication and the G1–S phase transition, and hence affects cell cycle progression. [25
]. The overexpression and gene amplification of E2F-1 in a variety of cancers are reported. E2F-1 transgenic mice develop cancers in the vagina, skin, forestomach, and odontogenic epithelium [26
]. Moreover, the overexpression of E2F-1 has been reported to have a prognostic value in breast cancer [26
]. In addition, the target genes of E2F-1 are known to be required in cell cycle progression and they are mainly involved in DNA synthesis [27
]. The screening of MED28 binding proteins in MED28
-overexpressing cells using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) showed that a putative binding protein for MED28 is similarly associated with DNA replication (results not shown). However, this association needs further elucidation. In addition, ETS-1 is mainly expressed in triple-negative breast cancer, which is closely associated with poor survival. Furthermore, ETS-1 induces cancer cell invasion and regulates epithelial-to-mesenchymal transition (EMT), thereby leading to drug resistance and neovascularization [28
Of note, an in vitro drug resistance model that is based on a breast cancer cell line has been reported to manifest enhanced expression of MED28, which regulates epithelial–mesenchymal transition in human breast cancer [29
]. C/EBPβ is associated with metastatic breast cancer and an overall poor prognosis [30
]. In addition, the high expression of NRF-1 is closely correlated with poor survival in the luminal A (ER+
) subtype of breast cancer [32
]. Taken together, these observations strongly suggest that the increase in MED28 expression by E2F-1, ETS-1, C/EBPβ, and NRF-1 is associated with cancer progression and poor prognosis.
As E2F-1 is an oncogene that stimulates DNA replication and the G1–S phase transition [25
], and MED28 is one of its target genes, we hypothesized that the pattern of MED28 expression would change during cell cycle progression. As illustrated in Figure 2
, the expression level of MED28 gradually increased from the early G1 phase until the early S phase and decreased from mid-S phase to mid-G2 phase, and then increased again from the late G2 to M phase. Decreased expression of MED28 was observed as cells exited mitosis. These findings clearly show that MED28 is involved in cell cycle regulation, at least in part.
Cyclin-dependent kinases (CDKs), a family of serine/threonine kinases, control the cell cycle by forming an active heterodimeric complex with cyclins. CDK2 forms a complex with cyclin E for the G1–S transition and with cyclin A for S phase progression [33
]. Therefore, we examined whether MED28 contains a motif that is phosphorylated by CDK, being represented by the sequence (S/T)PX(R/K). We found that MED28 had two consensus motifs that were recognized by CDK (Supplementary Figure S3A
). An in vitro binding assay using CDKs confirmed that MED28 can bind to CDK2 (Supplementary Figure S3B,C
). In addition, the post-translational modification analysis revealed that residues S103 and T134 of MED28 were phosphorylated in HeLa cells (results are not shown). Retinoblastoma protein (pRb) inhibits the transcriptional activity of E2F-1 by forming the pRb/E2F-1 complex. The active CDK2–cyclin complex hyperphosphorylates pRb, which releases E2F-1 and enables E2F-1 to function as an active transcription factor [34
]. Our study showed that MED28 expression is cell cycle dependent and increased by E2F-1, and that CDK2 may mediate the phosphorylation of MED28. Thus, CDK2 and E2F-1 might regulate the increase in MED28 levels at the G1–S transition. However, the underlying molecular mechanism needs further elucidation.
Defects at the cell cycle checkpoints can be induced by alterations of transcriptional or post-translational regulation via tumor suppressor genes or oncogenes [35
]. In addition, tumor cells that progress with abnormal chromosome segregation during mitosis acquire aneuploidy. Given that mitotic checkpoints are important for precise chromosome segregation, aberrant cell division that is due to abnormal mitotic checkpoints often induces aneuploidy in cancer cells [35
]. In this study, we found that the overexpression of MED28 shortened the mitosis by increasing chromosome condensation, segregation, cytokinesis, and decondensation, which resulted in a more abnormal nuclear phenotype (nuclear budding and the presence of a micronucleus) and aneuploidy (Figure 3
, Figure 4
and Figure 5
). This phenotype was further confirmed by the FACS analyses, as depicted in Figure 4
and Supplementary Figure S2
. In addition, the overexpression of MED28 decreased cell populations at the G1–S transition. The mechanism of MED28-driven shortening of the S phase and the subsequent induction of chromosome instability and aneuploidy need further elucidation. We could not elucidate the molecular mechanism behind the abnormal cell cycle and aneuploidy induced by MED28 overexpression in this study. However, cancer genomics data, where MED28
was found to be amplified in various cancers, including breast cancer, ovarian cancer, pancreatic cancer, sarcoma, lung adenoma, prostate cancer, and melanoma, clearly indicate that the overexpression of MED28 is closely related to cancer progression (Supplementary Figure S4A
). In addition, the analyses of RNA-seq data from various cancers suggest that the gene amplification of MED28
closely correlates with an increase in MED28 mRNA expression (Supplementary Figure S4B
Overall, we have identified and characterized the transcription factors that increase MED28 expression. In addition, we found that the regulation of MED28 expression is cell cycle dependent, and MED28 overexpression induces abnormal cell cycle progression, genomic instability, and aneuploidy. Therefore, our results suggest that MED28 plays a critical role in tumorigenesis by inducing genomic instability.
4. Materials and Methods
4.1. Cell Culture and Plasmid Construction
HeLa, HEK293, and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, GE Healthcare Life Sciences, Marlborough, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, GE Healthcare Life Sciences, UT, USA), and 1% (v/v) penicillin/streptomycin solution (PS; Hyclone, GE Healthcare Life Sciences, UT, USA) in a humidified CO2 incubator. Full-length MED28, CDKs, ETS-1, E2F-1, NRF-1, and C/EBPβ cDNAs were PCR-amplified and subcloned into vectors pcDNA3-Flag, pcDNA3-Myc (Invitrogen, Carlsbad, CA, USA), pEXPR-Iba-105 (Iba Lifescience, Goettingen, Germany), or pEGFP-C1 (Clontech, Mountain View, CA, USA) vectors.
4.2. Mammalian Cell Transfection
Plasmid transfection was performed while using a mixture of 150 mM NaCl and polyethylenimine (PEI; Polysciences, Inc., Warrington, PA, USA) or Lipofectamine™ 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocols.
4.3. The Luciferase Reporter Assay
promoter region was analyzed in UCSC Genome Browser (http://genome.ucsc.edu/
). The MED28
promoter region was cloned into the pGL3-basic luciferase reporter vector. The pGL3 basic vector or pGL3 basic vector containing the MED28
promoter (200 ng) with a Renilla
vector (100 ng) was transfected into the HEK293 cells. To examine the effects of transcription factors, the HEK293 cells were cotransfected with pGL3-MED28 promoter (200 ng), with Renilla
vector (100 ng), and one of the transcription factor constructs (each 200 ng, ETS-1, E2F-1, NRF-1, and C/EBPβ) using PEI. After 24 h incubation, the cells were lysed with passive lysis buffer, and a Dual Luciferase Reporter Assay System measured luciferase activity (Promega, Madison, WI, USA) and quantitated using GloMax (Promega, WI, USA) according to the manufacturer’s instructions.
4.4. The ChIP Assay
After the transfection of the empty vector or the ETS-1, E2F-1, NRF-1, or C/EBPβ plasmid (5 μg) into HEK293 cells, the cells were fixed with 1% (w
) formaldehyde for 10 min at 25 °C. ChIP was performed, as described previously [7
]. The cells were incubated in 200 µL of sonication buffer and sonicated for 70 s (10 s pulse and 60 s rest). DNA was sheared to lengths between 200 and 1000 bp. The sonication mixture was incubated with mock immunoglobulin G (Santa Cruz Biotechnology, CA, USA), anti-E2F-1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-NRF-1 (Santa Cruz Biotechnology, CA, USA), anti-ETS-1 (Santa Cruz Biotechnology, CA, USA), or anti-C/EBP antibody (Santa Cruz Biotechnology, CA, USA), followed by immunoprecipitation using Protein A and G agarose bead mixture (Invitrogen, Paisley, UK). The protein–DNA complexes were separated from the beads by elution buffer (50 mM Tris HCl, pH 8.0, 1 mM EDTA, 1% [w
] sodium dodecyl sulfate (SDS), 50 mM NaHCO3
), and then heated at 65 °C for 5 h with protease K to reverse the formaldehyde crosslinks. Finally, the DNA was purified with the PCR Clean-up Kit (Promega, WI, USA). ChIPed DNA was used as a template for PCR (Forward: 5′-GCCTACTACACCAGCC-3′, Reverse: 5′-CATGTTTGGAATGGCG-3′).
4.5. Cell Cycle Analysis
HeLa cells were arrested at the G1–S or G2–M transition by a double thymidine block (2 mM) or thymidine-nocodazole block (thymidine 2 mM, nocodazole 100 ng/mL), respectively. The synchronized cells were harvested, fixed in 70% (v/v) ice-cold ethanol, washed with ice-cold phosphate-buffered saline (PBS), and then stained with 40 μg/mL propidium iodide (PI) in the presence of 50 μg/mL RNase A for 30 min at 25 °C. All of the DNA samples (1 × 104 cells/sample) were analyzed with a Cube 6 system (Sysmex Partec, Sysmex, GmbH, Germany) and in the FCS Express 4 Flow Research Edition (De Novo Software™, Glendale, CA, USA) software. To generate G2–M-synchronized cell populations, the HeLa cells were synchronized by treatment with thymidine (2 mM) to generate a population enriched in cells in the postmitotic and G1 phases of the cell cycle, and the cells were then released from the arrest for 9 h with a fresh cell culture medium. The cells were then treated with nocodazole (100 ng/mL) for 16 h, followed by a release from the arrest with a fresh cell culture medium. The cells were harvested at various time points after synchronization and then analyzed for DNA content by flow cytometry, as described above.
4.6. Immunoblot Analysis, Strep-Tactin-Based Protein Purification, Antibodies, and Chemicals
The cells were lysed with lysis buffer (1% [v/v] Triton X-100, 50 mM HEPES, pH 7.6, 150 mM NaCl, 10% [v/v] glycerol, 10 mM NaF, 1 mM Na3OV4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA) supplemented with a protease inhibitor cocktail (PIC; Thermo Fisher Scientific, Waltham, MA, USA), and the proteins were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA), followed by antibody-based detection using an Enhanced Chemiluminescence Eetection system (Santa Cruz Biotechnology, TX, USA). For streptavidin-based protein purification, the HEK283 cells were transfected with the pEXPR-IBA103-MED28 plasmid in the presence of Myc-CDK1, Myc-CDK2, Myc-CDK3, or Myc-CDK4 for 24 h and then lysed with lysis buffer (1% [v/v] Triton X-100, 50 mM HEPES, pH 7.6, 150 mM NaCl, 10% [v/v] glycerol, 10 mM NaF, 1 mM Na3OV4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA) containing the protease inhibitor cocktail at 4 °C for 30 min. Subsequently, Strep-Tactin Sepharose beads (Iba Lifesciences, Gottingen, Germany) were added and rotated at 4 °C for 2 h. The beads were collected by centrifugation and then washed three times in lysis buffer. The samples were boiled in SDS-PAGE sample buffer at 100 °C for 10 min and resolved by SDS-PAGE on a 12.5% gel and analyzed by immunoblotting. The antibodies used in this study included M2 anti-Flag (Sigma Aldrich, St. Louis, MO, USA), anti-Myc (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), anti-HA (1:1000; Santa Cruz Biotechnology, TX, USA), anti-MED28 (1:10,000; laboratory-made), and anti-γ-tubulin (1:20,000; laboratory-made).
4.7. A Glutathione S Transferase (GST) Pull-Down Assay
GST pull-down assay was performed, as described previously [9
]. Briefly, the pGEX4T-1 vector or pGEX4T-1 vector containing MED28 was transformed into the BL21(DE3) Escherichia coli
strain, and the expression of the GST or GST-MED28 protein was induced while using 0.1 mM IPTG at 30 °C for 5 h. The cells were harvested and then lysed in PBS containing 0.1% (v
) Triton X-100 with sonication. The cell extracts containing GST or GST-MED28 were bound to glutathione-Sepharose beads (GE healthcare, Pittsburgh, PA, USA), and the beads were washed five times with PBS containing 0.1% (v
) Triton X-100 to remove the nonspecifically bound proteins. Beads containing GST or GST-MED28 were incubated with HEK293 cell lysates containing Myc-CDK1 or Myc-CDK2 for 2 h at 4 °C, washed three times with PBS containing 0.1% (v
) Triton X-100, and then boiled in SDS-PAGE sample buffer at 100 °C for 10 min. The samples were subjected to SDS-PAGE for immunoblot.
4.8. Live Cell Imaging and Fluorescence Microscopy
The HeLa cells were transfected with the pcDNA3 vector or pcDNA-mycMED28 and selected with G418 (200 μg/mL) for 10 days. For the estimation of mitosis duration and whole cell cycle analysis with time-lapse photomicroscopy, HeLa cells that were transfected with the pcDNA3 vector or pcDNA-Myc-MED28 were seeded (1 × 104 cells/well) in a four-well glass dish (Thermo Scientific™ Nunc™Lab-Tek II Chambered Coverglass, MA, USA) and incubated overnight in standard culture conditions. To visualize chromosomes, the cells were incubated with 1 μg/mL Hoechst 33342 (Thermo Scientific™ Hoechst® 33342, MA, USA) for 30 min. Fluorescence images were acquired every 5 min for 24 h while using a Nikon eclipse Ti camera (Tokyo, Japan) with a 40× dry Plan-Apochromat objective. Images were captured with an iXonEM +897 Electron Multiplying charge-coupled device camera (Teledyne Princeton Instruments, Trenton, NJ, USA) and analyzed in the Nikon Imaging Software (NIS)-elements advanced research (AR) (Nikon, Tokyo, Japan). For immunocytochemistry, the cells were fixed in 4% formaldehyde in PBS for 15 min at 25 °C and permeabilized with 0.2% (v/v) Triton X-100 (Sigma Aldrich, MO, USA) in PBS for 10 min at 25 °C. The fixed cells were preincubated in blocking solution (3% (w/v) bovine serum albumin in PBS), followed by incubation with primary antibodies at 4 °C overnight. After incubation with primary antibodies, the cells were washed three times via shaking in PBS and probed with fluorescein (Cy3, Alexa 488)-conjugated anti-mouse or anti-rabbit secondary antibodies. After three washes with PBS, DAPI (1 μg/mL, Invitrogen, CA, USA) was used for DNA counterstaining. The cells were then washed three times with PBS and subsequently incubated in a mounting solution (Biomeda, CA, USA). The samples were examined under a fluorescence (Axio Imager M1, Carl Zeiss, Oberkochen, Germany) or confocal microscope (LSM710 or LSM510, Carl Zeiss, Oberkochen, Germany).
4.9. Statistical Analysis
The results are expressed as mean ± SD or mean ± SEM. A student’s t test was performed to determine the statistical significance of all the results obtained. p < 0.05 was considered to be statistically significant.