Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency

<div><p>The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited <i>in vitro</i> studies have been performed on MED28, its <i>in vivo</i> function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors <i>Oct4</i> and <i>Nanog</i>. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of <i>Med28</i> in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of <i>Med28</i> results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.</p></div

Loss of Med28 in MEFs results in lack of reprogramming.

<p><b>(A)</b> Western blot analysis shows that Med28 expression is much higher in ES cells compared to MEFs. <b>(B)</b> Western blot shows reduced Med28 expression in <i>Med28</i>^<i>fl/fl</i> MEFs at 6 days post-infection with Ad-Cre compared to Ad-empty control adenovirus. <b>(C)</b> Immunofluorescent staining of <i>Med28</i>^<i>fl/fl</i> MEFs demonstrates significant reduction of Med28 (red) in Ad-Cre-infected cells compared to Ad-empty control. DAPI staining (blue) of cell nuclei is shown. <b>(D)</b> Quantitation of number of iPSC colonies (triplicate experiments) from <i>Med28</i>^<i>fl/fl</i> primary MEFs infected with OKSM and rtTA followed by infection with Ad-empty (OKSM+Ad-empty) or Ad-Cre (OKSM+Ad-Cre) one day after Dox treatment. Note that infection with OKSM+Ad-Cre produced no colonies. Data are presented as mean +/- SD (*<i>p</i><0.05).</p

Cre-mediated heterozygous deletion of Med28 causes differentiation of iPSC.

<p><b>(A)</b><i>Med28</i>^<i>fl/fl</i> iPSCs were infected with Ad-Cre, followed by single cell plating on day 3 and genotyping along with other analyses performed between days 5–8. <b>(B)</b> PCR genotyping shows clones with both deleted (- allele) and non-deleted WT (+ allele) alleles infected with Ad-Cre (+ Cre) compared to uninfected clones (-Cre), respectively. <b>(C)</b> Representative bright field images show noticeable differences in morphology between non-deleted (panel A, -Cre) and deleted (panel B, +Cre) <i>Med28</i>^<i>fl/fl</i> iPSC colonies (scale bar 200μm). Note the differentiated colony morphology of the <i>Med28</i>^<i>fl/fl</i> (panel B, +Cre) cells. <b>(D)</b> Quantitation of real-time PCR for deleted <i>Med28</i>^<i>fl/fl</i> iPSC colonies (+Cre) show ~2-fold decreased expression of <i>Med28</i> as well as significantly lower expression of <i>Oct4</i> and <i>Nanog</i> (n = 4; *, p<0.05) compared to non-deleted <i>Med28</i>^<i>fl/fl</i> iPSC (-Cre) colonies. <b>(E)</b> Immunostaining of deleted <i>Med28</i>^<i>fl/fl</i> colonies (panels E-H) show loss of self-renewal factor Oct4 (red, C and G) and Nanog (green, D and H) compared to non-deleted colonies (panels A-D). Bright field images (A and E) and nuclear DAPI (blue, B and F) are also shown (scale bar 200μm). <b>(F)</b> Quantitation of real—time PCR for differentiation markers reveal increased differentiation markers in deleted (+Cre) <i>Med28</i>^<i>fl/fl</i> iPSC colonies for extra embryonic lineage (<i>Gata4</i>, <i>Hand1</i>, and <i>Cdx2</i>) and decreased germ layer lineage makers nestin (Nes, ectoderm) and T brachyury (T, mesoderm) compared to non-deleted (-Cre) <i>Med28</i>^<i>fl/fl</i> iPSCs colonies (n = 4). Data are presented as mean +/- SD (*<i>p</i><0.05).</p

Targeted disruption of <i>Med28</i>.

<p><b>(A)</b> Schematic representation of the targeted allele and null allele. Targeted <i>Med28</i> allele in mouse ES cells with neo cassette in the reverse orientation (top, Med28(fl-frt), targeted <i>Med28</i> allele without neo cassette after transient transfection with Flp (middle, Med28(fl)) and Med28 null allele with Exon 1 and 2 deletions after breeding with Protamine-Cre mouse (bottom, Med28(-)). Shaded boxes with numbers represent the exons. Open triangle: loxP site; closed triangle: Frt site; B: BspH1 restriction site; S: SpHI restriction site. Numbered arrows represent PCR primers used for genotyping. Short solid bar represents 5’ probe outside the homologous region used for Southern blot analysis. <b>(B)</b> Representative Southern blot analysis used to confirm correct targeting. DNA was digested with either BspHI or SpH1 restrict enzymes and the 5’Probe (shown in <b>A</b>) outside the homologous region was used to detect wildtype (+), floxed (fl) and null (-) alleles at expected sizes for WT (+) allele, 14.6 kb (BspHI) or 15.2 kb (SpHI); floxed allele without neo (fl), 15 kb (BspHI) or 12 kb (SpHI); and null (-) allele, 12.1 kb (BspHI) or 12.7 kb (SpHI). <b>(C)</b> Representative PCR analysis using primers 1 and 2 (shown in A) confirm the presence of 5’loxP site (top panel, 324 bp) and WT allele (top panel, 290 bp). PCR analysis using primers 1 and 3 (shown in <b>A</b>) confirm presence of only the null (-) allele (bottom panel, 310 bp). <b>(D)</b> Histological analysis of 6.5-dpc (E6.5) presumed to be Med28 mutant embryos shows disorganized extraembryonic tissue with no discernible epiblast. Sections from whole decidual swellings were stained with hematoxylin and eosin. Arrow, epiblast; arrowhead, trophoblast giant cell.</p

Impaired ICM outgrowth of <i>Med28</i>^<i>-/-</i> blastocysts.

<p><b>(A)</b> Representative PCR genotyping of E3.5 blastocysts from <i>Med28</i>^<i>+/-</i> intercrosses show WT (+) allele and null (-) allele. Primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140192#pone.0140192.g001" target="_blank">Fig 1C</a> are used for genotyping. <b>(B)</b> Phase contrast microscopy images of embryos from <i>Med28</i>^<i>+/-</i> intercrosses. At embryonic day E3.5, <i>Med28</i>^<i>+/+</i> blastocysts (panel A; a, enlarged inset) are indistinguishable from <i>Med28</i>^<i>-/-</i> blastocysts (panel B; b, enlarged inset). After 3 days in culture (DIV3), unlike <i>Med28</i>^<i>+/+</i> blastocysts that show outgrowth (panel C, arrow), ICMs from <i>Med28</i>^<i>-/-</i> blastocysts fail to show outgrowth (panel D). A total of 14 WT and 12 mutant blastocysts were examined. Scale bar, 100 μm.</p

<i>Med28</i>^<i>-/-</i> ICM is not pluripotent.

<p><b>(A)</b> Semi-quantitative RT-PCR demonstrates reduced expression of pluripotency markers Oct4, Nanog and Sox2 in <i>Med28</i>^<i>-/-</i> (KO) E3.5 blastocysts and cultured blastocysts DIV3 compared to wildtype (WT) controls. Trophoectoderm marker <i>Cdx2</i> expression is reduced in cultured <i>Med28</i>^<i>-/-</i> (KO) blastocysts. <b>(B)</b> Immunofluorescence analysis shows reduced expression of Oct4 (red, top panels) and Nanog (red, bottom panels) in cultured <i>Med28</i>^<i>-/-</i> blastocysts compared to wildtype controls. <b>(C)</b> Semi-quantitative RT-PCR demonstrates increased expression of primitive endoderm markers <i>Gata4</i> and <i>Gata6</i> in DIV3 cultured <i>Med28</i>^<i>-/-</i> (KO) blastocysts, while trophoblast giant cell markers <i>Hand1</i> and <i>Pl1</i> expressions are not up-regulated compared to WT control. <i>Gapdh</i> expression serves as a control (shown in <b>A</b> and <b>C</b>). <b>(D)</b> Confocal microscopy images of immunofluorescence analysis show that Gata4 is expressed in primitive endoderm cells surrounding the ICM in DIV3 cultured <i>Med28</i>^<i>+/+</i> blastocysts (red, top panel) and <i>Med28</i>^<i>-/-</i> blastocysts (red, bottom panel). Nuclear DAPI (blue) and Med28 (green) are also shown (shown in <b>B</b> and <b>D</b>). Scale bar, 100 μm. At least three independent experiments were carried out for all data sets.</p

Kinome changes across human isogenic merlin-wildtype and -deficient AC and SC pairs.

<p><b>(A-B)</b> Top baseline kinome changes across human AC and SC isogenic pairs. Data presented are the mean log2 fold change from 3 experiments, error bars are standard error. <b>(C)</b> Pan-kinome (left) drug induced perturbations relative to DMSO control contain clusters of induced/repressed kinases (right). Each condition is the median log 2 fold change of three replicate experiments relative to vehicle (DMSO) control, with any kinase having >33% non-detection rate removed (grey: kinase not detected in any run). Cell lines treated with HDAC inhibitors (Panobinostat and CUDC-907) cluster most closely.</p

Kinomic response to drug treatment.

<p><b>(A)</b> Ranking the top 10 most-induced and most-suppressed kinases for each of the treatments at 24 hours stresses the similarities in drug response across cell lines, and also the differences between human and mouse cell lines. <b>(B)</b> Drug induced perturbations in HS-01 and Syn5 shown in a kinome tree plot. <b>(C)</b> Kinases that were represented in every cell line were used to generate a MIB-binding response correlation matrix. <b>(D-E)</b> Two of the most-prominent clusters include kinases highly-correlated with PTK2 (FAK1) or RPS6KA1 (p90 RSK) and STK3/4. <b>(F)</b> The most-highly correlated kinases are INSR and IGF1R (corr = 0.91), primarily being induced by GSK458-treatment at 24 hours. <b>(G)</b> The next-most correlated kinase pair across the entire dataset is PTK2 and AAK1 (corr = 0.87). Interestingly, these kinases are preferentially induced in merlin-deficient cell lines.</p

PCA and transcriptomic differences due to merlin deficiency in human AC and SC.

<p><b>(A)</b> Principal components analysis (PCA) of averaged, rank-normalized read counts (overlapping genes only) from RNA-seq of wild type and merlin-deficient human SC (red), mouse SC (orange), human AC (blue, Syn5, Syn1) and human MN cells (blue, Syn6). PC1 explains 61.5% of variance, while PC2 explains 25.5% of variance. <b>(B)</b> Volcano plots showing the significance and log2 fold-change (logFC) due to merlin deficiency for all gene transcripts reliably detected in the RNA-seq analysis. Yellow dots represent genes altered at [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197350#pone.0197350.ref047" target="_blank">47</a>] adjusted significance of P<0.05. The location of the downregulated <i>NF2</i> gene, corresponding with ~7% of normal <i>NF2</i> expression, is labelled for HS01 vs HS11. In the Syn5 vs Syn1 AC comparison, fold-comparisons across the entire gene are not meaningful as there is no significant difference in the level of <i>NF2</i> transcripts in Syn5, but these produce no active merlin due to absence of exon 8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197350#pone.0197350.s014" target="_blank">S5 Fig</a>), which was removed by CRISPR/Cas9. <b>(C)</b> Venn diagrams showing the relatively small degree of overlap between the downregulated (left) and upregulated genes (right) due to merlin deficiency in human AC and SC, respectively.</p

Drug screening outcomes are largely independent of merlin status.

<p><b>(A)</b> Estimated beta coefficients of different assay variables to differences in area under the curve (AUC) as determined by multiple linear modeling. Multiple linear modeling indicates that unlike merlin status, tumor type and treatment with a subset of drugs (CUDC-907, Bortezomib, Panobinostat, GSK2126458, Ganetespib, and Axitinib) are significantly associated with a reduction in cell viability as measured by Simpson AUC. <b>(B)</b> Hierarchical clustering demonstrates similarity of response (Simpson AUC) of all cell lines to tested drugs. Only drugs common to both cell types were included in the analysis. Based on the response of each cell line to the entire panel of drugs, the cell lines appear to cluster by cell type and merlin status.</p