A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells

Abstract

Although there is now evidence that the expression of centromeric (CT) and pericentric (PCT) sequences are key players in major genomic functions, their transcriptional status in human cells is still poorly known. The main reason for this lack of data is the complexity and high level of polymorphism of these repeated sequences, which hampers straightforward analyses by available transcriptomic approaches. Here a transcriptomic macro-array dedicated to the analysis of CT and PCT expression is developed and validated in heat-shocked (HS) HeLa cells. For the first time, the expression status of CT and PCT sequences is analyzed in a series of normal and cancer human cells and tissues demonstrating that they are repressed in all normal tissues except in the testis, where PCT transcripts are found. Moreover, PCT sequences are specifically expressed in HS cells in a Heat-Shock Factor 1 (HSF1)-dependent fashion, and we show here that another independent pathway, involving DNA hypo-methylation, can also trigger their expression. Interestingly, CT and PCT were found illegitimately expressed in somatic cancer samples, whereas PCT were repressed in testis cancer, suggesting that the expression of CT and PCT sequences may represent a good indicator of epigenetic deregulations occurring in response to environmental changes or in cell transformation.

Figure 1.

Kinetics of PCT sequence expression during heat-shock response in HeLa cells, detected by RepChip. (a) Expression of sense PCT transcripts in the course of the heat-shock response. The expression of PCT sequences was evaluated in NHS HeLa cells, in HeLa cells over a continuous heat-shock exposure of 30–60 min, as well as in HeLa cells submitted to a 1 h of continuous heat-shock followed by a recovery period of 1–6 h. A peak of accumulation of hsp and PCT specific sense transcripts was observed after 1 h of recovery following heat-shock. No heat-shock dependent variation of expression was observed for control genes. (b) Sense and antisense PCT sequences expression in NHS and HS cells. Raw values of signal intensities corresponding to sense and antisense PCT sequences expression in NHS HeLa cells and in HeLa cells submitted to a 1 h of heat-shock (HS) are displayed as box plots. The P-values were calculated using a paired T-test. The induction rates of sense and antisense PCT sequences upon stress are respectively 44.9- and 11.6-fold indicating that PCT transcripts in sense and antisense orientations are more abundant in HS cells than in NHS cells. (c) Impact of HSF1 on PCT sequence expression. PCT sequence expression induction was determined in HSF1 expressing (WT) and non-expressing (KD) HeLa cells. The values of signal intensities corresponding to sense and antisense PCT sequences expression in HS versus NHS HeLa cells are displayed as ratios (HS/NHS). Heat-induced accumulation of sense and antisense PCT transcripts and of hsp transcripts is severely impaired in KD HeLa cells. (d) Heat-shock is accompanied by a nuclear redistribution of HP1ß. In NHS cells, HP1ß displays a non-homogeneous nuclear distribution and is enriched in pericentric heterochromatic regions (white arrows). In HS cells [assessed by the presence of HSF1 nSBS (white arrows)], HP1ß displays a homogeneous nuclear distribution. Bar: 5 µm.

Figure 2.

PCT sequence expression upon stress is a common feature of normal and cancer cell lines. The amount of sense and antisense PCT transcripts was analyzed in HeLa, HCT116, A431 and IMR90 cell lines submitted or not to a 1 h heat-shock with or without a period of recovery of 3 h following heat-shock. For each condition and cell type, the relative levels of expression of PCT sequences and of genes were normalized using the corresponding values in NHS cells. Heat-induced accumulation of PCT transcripts in both orientations is observed in all cell lines with marked differences in the amplitude and kinetics of accumulation. REC: recovery. The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively, and the top and bottom circles the maximum and minimum values, respectively.

Figure 3.

Comparative analysis of PCT sequences expression in HeLa cells and testis. The intensity values obtained for control genes, HS genes and PCT sequences expression in testis, in NHS and HS HeLa cells are presented as box plots. For each condition, the relative levels of expression of PCT sequences and of control genes were normalized using the corresponding values in NHS HeLa cells. In contrast to HS HeLa cells, where PCT transcripts are detected in both orientations, PCT sequences in testis are mostly expressed in an antisense orientation. The accumulation of PCT transcripts in testis does not seem to involve the heat-shock signaling pathway since the intensity values corresponding to the heat-shock genes remained at a lower level than in HS HeLa cells. The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively and the top and bottom circles the maximum and minimum values, respectively.

Figure 4.

Variations in CT and PCT sequences expression in normal and tumor tissues. (a) Expression of PCT transcripts in testis, liver, ovary and lung normal and tumor tissues. For each tissue, the RNA fractions from normal and tumor tissues originating from the same patient were analyzed. Signal intensities corresponding to control and hsp genes and to PCT sequences in a sense and antisense orientations are represented as box plots. An accumulation of antisense PCT sequences is observed in normal testis. PCT transcripts in both orientations are abundant in tumor lung tissue. The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively, and the top and bottom circles the maximum and minimum values, respectively. (b) hsp genes expression in testis and lung normal and tumor tissues. Hsp genes expression were analysed by qPCR. No variation of hsp genes expression was detected between normal and tumor testis tissues. No induction of hsp genes expression was observed in tumor lung tissue compared to normal lung tissue.

Figure 5.

Variations in CT and PCT sequences expression in a series of normal and tumor lung tissues. (a) Analysis of the expression of CT and PCT in four normal (N) and corresponding tumor lung tissues (T). The signal intensities corresponding to control and hsp genes and to CT and PCT sequences are represented as box plots. An accumulation of sense and antisense CT and PCT sequences in both orientations is observed in tumor lung tissues. N: normal, T: tumoral, #: patient identification. The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively, and the top and bottom circles the maximum and minimum values, respectively. (b) Global loss of H3K27me3 specific labeling in lung tumors. Immunohistochemistry analysis of lung cancer tissues from Patients 1, 2, 3 and 4 was performed on frozen sections using anti-H3K27me3 (immunoperoxidase labeling ×200). Nuclear staining of trimethylated H3K27 is observed in non-tumor cells while a decrease of H3K27me3 labeling is observed in tumor cells as compared to normal cells: in one case (Patient 1) the tumor limit is indicated by a dotted line between tumor cells (left) and adjacent normal cells (right). For each patient analyzed, the percentage of H3K27me3 positive cells respectively found in normal (N) and tumor tissues (T) is given in the table.

Figure 6.

Epigenetic control of PCT sequences expression. (a) Drug-induced DNA demethylation can induce PCT sequences expression. Comparative analysis of PCT sequences expression was performed in NHS HeLa cells treated with Trichostatin A (TSA), butyrate (BUT) or 5-Azacytidin (Aza-C). Expression of PCT sequences was also analyzed in HCT116 cells deficient for dnmt3b (3bKO) and for both dnmt1 and dnmt3b (DKO) genes. The relative levels of expression of PCT sequences and of control genes were normalized using the corresponding values in NHS HeLa cells (left) or in normal HCT116 cells (right). PCT sequences expression is only detected in Aza-C treated cells (in both orientations). The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively, and the top and bottom circles the maximum and minimum values, respectively. (b) PCT sequences are hypomethylated in Aza-C treated cells. MeDIP analyses of PCT and CT sequences were performed in Aza-C treated cells. The hypomethylated status of CT and PCT sequences in HCT116 dnmt mutated cells, described in ref. (14) is not represented herein. The histograms indicate the relative enrichment in 5-methyl-cytosine of target sequences after normalization to a reference promoteur (GAPDH promoter). IP-IgG: immunoprecipitation with non-specific IgG, IP-5mC: immunoprecipitation with antibody specific for 5-methyl-cytosine. (c) The expression of PCT sequences in Aza-C treated cells is not correlated to the accumulation of hsp specific transcripts. Hsp gene (hsp 90, hsp40 and hsp70) expression was evaluated by qPCR in Aza-C treated cells and in control HeLa cells (HeLa). Results were normalized, thanks to three control genes (GAPDH, U6 and histone H4). No induction of hsp genes was detected in these cells. (d) The expression of PCT sequences in Aza-C treated cells is HSF1 independent. The status of HSF1 activation in NHS and HS HeLa cells, Aza-C treated HeLa cells was determined by western blot analysis (right) and immunofluorescence with an antibody against HSF1 (left). Active HSF1 which displays a lower electrophoretic mobility was only detected in HS cells, but not in NHS Aza-C treated cells. Similarly, nuclear foci formed of active HSF1 and which form primarily form at the 9q12 locus were only present in HeLa HS cells. (e) PCT sequences expression upon stress is not associated with DNA demethylation. MeDIP analysis of PCT sequences was performed in NHS and HS HeLa cells. The histograms indicate the relative enrichment of PCT sequence after normalization to histone H4 promoter. No variation of DNA methylation of PCT sequences was observed between NHS and HS cells. IP-IgG: immunoprecipitation with non-specific IgG, IP-5mC: immunoprecipitation with antibody specific for 5-methyl-cytosine.

Figure 7.

The knock down of Dicer (dcr) does not induce PCT sequences expression. (a) siRNA efficiency to generate a knock down of Dicer expression was validated by RT-PCR (left) and western blot analysis (right) on siRNA treated (Si-dcr) and control (Ctl) cells. U6 snRNA expression was used as a control in RT-PCT analysis while anti HSF1 was used as a control in western blot analysis. Dicer mRNA and Dicer protein rates were greatly decreased in Si-dcr cells. (b) Impact of Dicer knock-down on PCT sequences expression. Signal intensities corresponding to control genes, hsp genes and PCT sequences expression in control (Ctl) and Si-dcr HeLa cells are displayed as box plots. No significant difference in the expression of any of the sequences was observed between Ctl and Si-dcr treated cells. The thick line represents the median, the cross the mean, the top and bottom boundaries of the boxes corresponding to the 75 and 25 percentiles, respectively, and the top and bottom circles the maximum and minimum values, respectively.