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, 1861 (11), 983-995

Myt1 and Myt1l Transcription Factors Limit Proliferation in GBM Cells by Repressing YAP1 Expression

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Myt1 and Myt1l Transcription Factors Limit Proliferation in GBM Cells by Repressing YAP1 Expression

Tiffany A Melhuish et al. Biochim Biophys Acta Gene Regul Mech.

Abstract

Myelin transcription factor 1 (Myt1) and Myt1l (Myt1-like) are zinc finger transcription factors that regulate neuronal differentiation. Reduced Myt1l expression has been implicated in glioblastoma (GBM), and the related St18 was originally identified as a potential tumor suppressor for breast cancer. We previously analyzed changes in gene expression in a human GBM cell line with re-expression of either Myt1 or Myt1l. This revealed largely overlapping gene expression changes, suggesting similar function in these cells. Here we show that re-expression of Myt1 or Myt1l reduces proliferation in two different GBM cell lines, activates gene expression programs associated with neuronal differentiation, and limits expression of proliferative and epithelial to mesenchymal transition gene-sets. Consistent with this, expression of both MYT1 and MYT1L is lower in more aggressive glioma sub-types. Examination of the gene expression changes in cells expressing Myt1 or Myt1l suggests that both repress expression of the YAP1 transcriptional coactivator, which functions primarily in the Hippo signaling pathway. Expression of YAP1 and its target genes is reduced in Myt-expressing cells, and there is an inverse correlation between YAP1 and MYT1/MYT1L expression in human brain cancer datasets. Proliferation of GBM cell lines is reduced by lowering YAP1 expression and increased with YAP1 over-expression, which overcomes the anti-proliferative effect of Myt1/Myt1l expression. Finally we show that reducing YAP1 expression in a GBM cell line slows the growth of orthotopic tumor xenografts. Together, our data suggest that Myt1 and Myt1l directly repress expression of YAP1, a protein which promotes proliferation and GBM growth.

Keywords: Cancer; Gene expression; Glioma; Proliferation; Repression; Transcription.

Figures

Figure 1.
Figure 1.. Decreased proliferation in Myt1 and Myt1l expressing cells.
A) Expression of Myt1 and Myt1l in U87 cells was analyzed by western blot with a Flag antibody (to the amino-terminal 3×Flag epitope). Blotting for HSP90 is shown below. B) U87 cells were incubated with EdU and the percentage of positive cells in quadruplicate cultures is plotted (mean + sd). C) The percentage of U87 cells in late G2 and M phase (mean + sd, of quadruplicates) was determined by staining with an antibody against pHH3. D) Four separate cultures of each U87 cell line were maintained for four serial passages, and the average cumulative cell number is plotted (left). The right hand panel shows relative cell number (mean + sd) after four passages. E) A172 cells were infected with control or Myt1 or Myt1l expressing lentiviral constructs. Expression of Myt1 and Myt1l in A172 cells was analyzed by Flag western blot, and blotting for HSP90 is shown below. F) Four separate cultures of each A172 line were maintained for four serial passages, and the average cumulative cell number is plotted (left). The right hand panel shows relative cell number (mean + sd) after four passages. p-values were determined by Student’s T-test: * p < 0.05, ** p < 0.01, *** p < 0.001. Panels B, C, D and F show representative experiments, with averages of quadruplicate samples from a single experiment. Panels B, C and F were repeated twice and panel D four times, with similar results. A and E are representative western blots (of three repeats), which were from the cells used in D and F. G) Relative expression of Myt family members in U87 and A172 is shown compared to SKNSH and SW620 as positive controls.
Figure 2.
Figure 2.. MYT1 and MYT1L expression in brain cancer datasets.
A) Relative expression of MYT1 and MYT1L in normal tissue, and three classifications of glioma is shown (data from GSE4290). Expression of MYT1 and MYT1L in the four subtypes of GBM (B) and in the three LGG subtypes (C) is shown (data from TCGA). Expression data is plotted as median with upper and lower quartiles (box) and 5th and 95th percentiles (whiskers). Numbers of samples in each class are indicated below. D) Disease-free survival is plotted for TCGA GBM data (from Betastasis) for patients with high or low MYT1 and MYT1L expression.
Figure 3.
Figure 3.. Regulation of YAP1 by Myt1 and Myt1l.
A) A heat map (z-score per gene) of RNA-seq data from Myt1 or Myt1l expressing and control U87 cells is shown for genes encoding components of the Hippo signaling pathway. B) Comparison of expression of high-confidence YAP1 target genes with changes in expression in U87 RNA-seq data, either +/− 0.5 log2 fold change (lfc). Genes with decreased expression in both Myt1 and Myt1l cells that were tested by qRT-PCR are indicated by asterisks. C) GSEA analysis of U87 RNA-seq data showing enrichment of a conserved YAP1 signature in the control cells. Three of the genes tested in D are near the top of this list (asterisks). D) qRT-PCR expression analysis for YAP1 and putative target genes (from panel B) in control and Myt1 or Myt1l expressing U87 cells. E) Western blot analysis (representative blot of three repeats) for Myt1, Myt1l, and YAP1 in A172 and U87 cells (control or expressing Myt1 or Myt1l). HSP90 loading control is shown below. F) A172 cells expressing Myt1 or Myt1l were analyzed by qRT-PCR as in D. p-values were determined by Student’s Ttest: * p < 0.05, ** p < 0.01. D and F are average and s.d. of triplicate samples from a single experiment.
Figure 4.
Figure 4.. Expression of YAP1 in brain cancers.
YAP1 expression was analyzed as in Figure 2, in normal and glioma from GSE4290 (A), TCGA GBM data (B), and LGG from TCGA (C). Numbers of samples in each class are indicated below. D) Kaplan-Meier analysis of YAP1 in TCGA GBM data (Betastasis) is shown. E) Correlation between zscores for expression of YAP1 and MYT1, MYT1L, or both together was analyzed in the indicated datasets. R-squared values are shown. F) Gene expression changes (Myt1 and Myt1l-expressing versus control cells) were compared to genes with expression that best correlated (either positively or negatively) with MYT1, MYT1L, or YAP1 expression in TCGA LGG, and GBM data-sets. The overlaps are shown as Venn diagrams, with the p-value (Chi squared test on a 2×2 contingency table) for the distribution of the overlap. Red outlines indicate the enriched overlap in each case.
Figure 5.
Figure 5.. GBM cell lines are sensitive to Hippo pathway modulation.
A) U87 cells were treated with increasing concentrations of Verteporfin (0, 2, 4, and 8 μM) after transfection with a YAP-TEAD luciferase reporter (8×GTIIC-luc) and assayed 24 hours later. B) U87 cells were incubated for four days with 0, 2, 4, or 8 μM Verteporfin, and relative cell number determined by counting quadruplicate cultures. C) A172 cells were analyzed as in A and B with 0 or 8 μM Verteporfin. D) U87 cells expressing Myt1 or Myt1l or with the control vector were transiently transfected with the 8×GTIIC-luc reporter with or without a YAP1 expression plasmid, and luciferase activity was determined after 48 hours. E) Cells transfected with the 8×GTIIC-luc reporter and were split at high and low density and assayed for luciferase activity 36 hours later. F) A172 cells expressing Myt1 or Myt1l or with the control vector were transfected with 8×GTIIC-luc with or without a YAP1 plasmid, and luciferase activity was determined after 48 hours. G) U87 cells expressing either Myt1 or Myt1l and control cells were incubated for four days with 0, 2, or 4 μM Verteporfin and relative cell number determined by counting quadruplicate cultures. All luciferase data is presented as mean + sd of triplicate transfections, normalized to a Renilla-luc transfection control. p-values were determined by Student’s T-test: * p < 0.05, ** p < 0.01, *** p < 0.001. Panels A-F show representative experiments, that were repeated twice with similar results. Data in panel G is from a single experiment. Averages shown are of triplicate samples for luciferase assays, and quadruplicates for proliferation assays, from a single representative experiment.
Figure 6.
Figure 6.. Direct regulation of YAP1 by Myts.
A) The human YAP1 upstream region is shown schematically. Above is a screenshot from the USCS genome browser showing H3K27Ac ChIP-seq signal (top), vertebrate conservation (middle) and identity with mouse (lower track). The positions of the MYT consensus sites are shown, relative to the transcriptional start, together with the four luciferase constructs generated. Arrowheads indicate the positions of the PCR primer pairs for ChIP. Below is the region from the mouse Yap1 gene, with two blocks of high sequence identity shown as thicker bars. Consensus MYT sites in mYap1 are marked. B) Binding of Myt1 and Myt1l was analyzed by ChIP-qPCR from U87 cells with either Flag-Myt1, Flag-Myt1l or a control vector. Binding to the proximal and distal regions of the YAP1 promoter was analyzed, together with a negative control region from the CDKN2B gene. Average plus sd of quadruplicate IPs. p-values were determined by Student’s T-test: ** p < 0.01, for comparison to the IP from control cells. C) Luciferase activity of the four constructs shown in A was analyzed in U87 cells expressing Flag-Myt1, Flag-Myt1l or in control cells. p-values were determined by Student’s T-test, with correction for multiple testing: *** p < 0.001, for comparison of WT reporter activity to the control cells, # p < 0.05, ## p < 0.01, ### p < 0.001 for comparison of the mutant reporters to the WT in Myt1 or Myt1l expressing cells. For C, a single representative experiment is shown, from two repeats, with average and s.d. of six replicates. Data in panel B is from a single experiment, with average and s.d. of four replicates.
Figure 7.
Figure 7.. Analysis of proliferation with altered YAP1 expression.
A) U87 or A172 cells were infected with a control virus or a YAP1 expressing lentiviral construct. YAP1 expression was visualized by western blot for the V5 epitope tag. Quadruplicate cultures were counted after four days, and relative cell number is shown. B) YAP1 was knocked down by shRNA lentiviral infection in U87 or A172 cells. Endogenous YAP1 expression is shown by YAP1 western blot together with relative cell number after four days (quadruplicate cultures as in A). C) U87 cells with YAP1 overexpression or knockdown were assayed for growth in soft agar: 10,000 cells per well were plated for sh-lacZ and sh-YAP1, 5,000 cells per well for lacZ and YAP1. Average colony number and images of representative wells are shown. D) U87 cells were co-infected with lentiviral vectors expressing YAP1 (V5 epitope tagged) or lacZ and either the control Flag vector or one with 3×Flag Myt1 or Myt1l. Expression of Myt1, Myt1l and YAP1 was analyzed by western blot for the epitope tags, and HSP90 as a loading control is shown below. Quadruplicate cultures of each of the U87 lines were maintained for two serial passages and cell number after the first passage and cumulative cell number after two passages is shown (mean + sd). p-values were determined by Student’s T test (p < 0.01): # For Myt1 or Myt1 expressing lacZ versus control/lacZ, ^ for YAP1 expressing versus equivalent lacZ line. E) Control or shYAP1 infected U87 cells were injected orthotopically into nude mice and tumors imaged by MRI after three weeks. Average (+ s.d.) tumor volume is shown, together with representative images (red lines outline the tumors). For panels A-C and E, p-values were determined by Student’s T-test: * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001. Panels A, B and D show representative experiments that were repeated twice with similar results. The average and s.d. shown is based on replicates within a single experiment. Data for C and E are from single experiment, with average and s.d. shown based on replicates (6 wells each in C, and 8 mice each in E).

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