Additional file 2: of Transcription factor-associated combinatorial epigenetic pattern reveals higher transcriptional activity of TCF7L2-regulated intragenic enhancers

Gene List associated with TF-states. (XLSX 330 kb

Additional file 1: of Integrative analysis reveals functional and regulatory roles of H3K79me2 in mediating alternative splicing

Supplemental Tables S1–S4. (PDF 88 kb

Additional file 2: of Integrative analysis reveals functional and regulatory roles of H3K79me2 in mediating alternative splicing

Supplemental Figures S1–S11. (PDF 2762 kb

CNVs in MEFs, mouse kidney-derived cells and mouse tissue.

<p>(A) Summary of CNVs detected at chromosome loci not known to be fragile, in mouse kidney-derived cells, kidney-derived cloned cells, and pre- and post-senescence MEF cell lines derived from <i>+/+</i> and <i>-/</i>- mice, and <i>-/</i>- tail tissue, using the Affymetrix Mouse Diversity Genotype Array; CNV data for MEF cultures was previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080730#B14" target="_blank">14</a>] and is included here for comparison to the kidney culture DNAs. Blue columns indicate allele loss (fold-change range, 0.26 to 0.61), and red columns indicate gain (fold-change range, 1.51 to 1.91). For more detail, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080730#pone.0080730.s006" target="_blank">Table S1</a>. (B) CNVs at chromosome fragile loci. CNVs at fragile loci detected in the same mouse cell lines and tissues. Blue columns indicate allele loss (fold-change range, -0.5 to -0.83) and red columns indicate gain at specific fragile chromosome loci (fold-change range, 1.50 to 2.51). </p

Gene expression changes in +/+ and -/- kidney cells at late <i>vs</i> early passage.

<p>mRNA expression in kidney cells at early and late passages was examined by RT² Profiler PCR Array (Mouse Cellular Senescence PCR Array, Qiagen) and fold change was calculated. Dark blue in the columns indicates >10-fold decrease of gene expression at late passage, medium blue indicates >5-fold decrease and light blue indicates >2-fold decrease. White columns indicate less than 2-fold change. Light pink indicates >2-fold increase of gene expression at late passage, medium pink indicates >5-fold increase and red indicates >10-fold increase in expression. Gene names are given in blue or red for those genes that show significant differences in expression. Gene names marked in bold indicate possibly interconnected signal pathways that are altered in <i>-/</i>- kidney cells.</p

Mouse kidney cells exhibit progressive changes during <i>in</i><i>vitro</i> culture.

<p>(A) Immunoblots for p53, p21, Survivin, Mcl1, Fhit and GAPDH expression in the mouse kidney cells with progressive culture. In <i>+/+</i> cells, Fhit expression is decreased gradually with passage progression, but capacity for apoptosis is likely maintained at late passage as shown by expression of p21. <i>-/</i>- cells undergo loss of apoptotic response with passage progression, dramatic increase of p53 in parallel with abrupt loss of p21 expression and dramatic increase of Mcl1 expression. (B) Representative micrographs of fixed, crystal biored stained +/+ and -/- kidney cell clones exhibiting morphological diversity, particularly in the -/- clones (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080730#pone.0080730.s001" target="_blank">Figure S1</a>).</p

Decreased sensitivity to apoptosis-related proteins in late passage <i>-/</i>- MEF cells.

<p>(A) Clonogenicity of <i>+/+</i> and <i>-/</i>- MEFs at P30. The asterisks represent significant differences (*<i>P</i> <0.05) between +/+ and -/- MEFs. (B) Clonogenicity of MEF+/+ 3 and MEF-/- 5 cells after treatment with 20 µM DMBA for 24 h (P31). Bar graphs represent the means, and error bars the standard deviation from at least 3 independent experiments; (*<i>P</i> <0.05). (C, D) Western blots for Caspase 3, PARP, Survivin, Mcl1, p53, p21 and GAPDH expression in MEF+/+ 3 and -/- 5 cells before and 6 and 24 h after 20 µM DMBA treatment at P31. Note that although the +/+ and -/- MEFs show similar expression patterns for cleaved Caspase 3 and cleaved PARP after DMBA treatment, the MEF-/-5 cells likely survive DMBA treatment as shown in (B) because Survivin and Mcl1 are up-regulated in these cells by 24 h DMBA treatment, in contrast to the loss of these proteins at 24 h after DMBA in the MEF+/+3 cells. </p

Gene expression changes in MEF cells.

<p>mRNA expression in MEF cells at early and late passages was also examined using the RT² Profiler PCR Array and fold change was calculated. Dark blue in the columns indicates >10-fold decrease of gene expression at late relative to early passage, medium blue indicates >5-fold decrease and light blue indicates >2-fold decrease. White columns indicate less than 2-fold change. Light pink indicates >2-fold increase of gene expression at late passage, medium pink indicates >5-fold increase and red indicates >10-fold increase in expression. Gene names are given in blue or red for those genes that show statistically significant differences in expression in the -/- <i>vs</i> +/+ cells in at least two of the three -/- <i>vs</i> +/+ MEF cell lines. Note the intra- and inter-strain variation in expression changes among the three +/+ and -/- MEF cell lines and the few changes in common with the kidney cell lines (E2f1, up in -/- of both cell types, Ccnd1 up in two of three -/-MEFs as well as -/- kidney); also Atm and Chek2 up in -/- lines, perhaps indicating ongoing DNA damage.</p

Restoration of p21 expression in late passage Fhit^-/- cells by WT p53 overexpression.

<p>(A) Western blot analysis of p53, p21 and vinculin in <i>Fhit </i>^<i>-/-</i> mouse kidney cells transfected with either control vector (EV), WT or A135P mutant <i>Trp53</i> expression vector. The end lane shows expression of p21 in early passage <i>Fhit </i>^<i>-/-</i> kidney cells treated with DMBA for 24 h. (B) Western blot analysis of p21, Fhit and GAPDH in <i>Fhit</i>^<i>-/-</i> mouse kidney cells, at tissue culture passages shown across tops of gels, transfected with control (EV) or WT <i>FHIT</i> expression vector. The end lane contains lysate from early passage <i>Fhit</i>^<i>-/-</i> mouse kidney cell treated 24 h with DMBA for a p21 positive control. </p

Expression of apoptosis-related proteins in <i>-/</i>- kidney cells with and without exposure to DMBA.

<p>(A) Clonogenicity of <i>+/+</i> and <i>-/</i>- kidney-derived cells with passage progression. Note the remarkable increase in colony number in -/- cells post P12 while the colony number for +/+ cells shows a steady rise to P12 and then decreases. *<i>P</i> <0.05; **<i>P</i> =0.069. (B) Clonogenicity of +/+ and -/- kidney cells after a 24 h DMBA (20 µM) treatment (at P10 and P21). *<i>P</i> <0.05. (C) Expression of apoptosis-associated proteins in the kidney cells before and 6 and 24 h after DMBA exposure, detected by immunoblot (at P10 and P22) (left panel). Expression of apoptosis-related proteins in -/- DMBA survivors (right panel): Note that cleaved Caspase 3 and cleaved PARP, both apoptosis markers, are markedly decreased in the DMBA-surviving cells whereas they were very similarly activated in the +/+ and -/- cells following initial DMBA exposure. In (A, B) bar graphs represent means and error bars standard deviations from at least 3 independent experiments.</p