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Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.

Nitzki F, Tolosa EJ, Cuvelier N, Frommhold A, Salinas-Riester G, Johnsen SA, Fernandez-Zapico ME, Hahn H - Oncotarget (2015)

Bottom Line: We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals.Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation.We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University Medical Center, Göttingen, Germany.

ABSTRACT
Mice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch.

No MeSH data available.


Related in: MedlinePlus

Murine Ptch promoter region analyzed in this studyThe upper part shows the upstream genomic region of Ptch, its transcriptome, the three Gli-binding sites (triangles) and the SNP rs29624336 (asterisk). The alternative exons 1a-1e (a–e) and exon 2 (ex 2) are indicated. H3K4me3 occupancy in differentiated C2C12 cells and murine embryonic stem cells (ESC) as downloaded from the European Nucleotide Archive and NCBI Gene Expression Omnibus are indicated below. The CpG islands (A–D), the analyzed PCR amplicons 1–7 (fragment 3 equates the methylation-sensitive DNA fragment described by Ecke et al. [11], all primers are shown in supplemental Table 1) and the CpG content are shown in the lower part. The largest amplicon 5 (391 bp) could only be amplified in the H3K4me3 ChIP experiment and NGS bisulfite sequencing.
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Figure 1: Murine Ptch promoter region analyzed in this studyThe upper part shows the upstream genomic region of Ptch, its transcriptome, the three Gli-binding sites (triangles) and the SNP rs29624336 (asterisk). The alternative exons 1a-1e (a–e) and exon 2 (ex 2) are indicated. H3K4me3 occupancy in differentiated C2C12 cells and murine embryonic stem cells (ESC) as downloaded from the European Nucleotide Archive and NCBI Gene Expression Omnibus are indicated below. The CpG islands (A–D), the analyzed PCR amplicons 1–7 (fragment 3 equates the methylation-sensitive DNA fragment described by Ecke et al. [11], all primers are shown in supplemental Table 1) and the CpG content are shown in the lower part. The largest amplicon 5 (391 bp) could only be amplified in the H3K4me3 ChIP experiment and NGS bisulfite sequencing.

Mentions: We recently showed that RMS of Ptch heterozygous mice consistently overexpress the mutant Ptch allele whereas the expression of the remaining wt Ptch allele is unchanged or even repressed [11, 12 and Supplementary Fig. S1]. Further in silico analysis identified a methylation-sensitive region within the murine Ptch promoter in RMS [11, Fig. 1] that suggests DNA methylation as potential mediator of this phenomenon. We next analyzed methylation of the Ptch promotor by MeDIP using RMS and SM samples from 16 Ptchdel/+(Ptch+/−) [13] and Ptchneo67/+ [14] mice. The total methylation content was calculated as % input. As shown in Fig. 2A, 3 of the 7 amplicons analyzed showed methylation, whereas the remaining amplicons were unmethylated (data not shown). Surprisingly, and in contrast to our previous hypothesis, the methylation content in these fragments (i.e. amplicon 3, 4 and 7 of the Ptch promoter) was reduced in RMS compared to SM (Fig. 2A). Thus, the percentage of methylation in SM in the amplicons 3, 4 and 7 decreased from 32% to 20% (P = 0.0053), from 45% to 22% (P = 0.0112) and from 1.1% to 0.5% (P = 0.0054) in RMS, respectively. Although the decrease was small, especially in fragment 7, the difference between SM and RMS was statistically significant. For positive and negative controls for the MeDIP see supplemental Fig. S2. Next, we used bisulfite next generation sequencing (NGS) to validate the above results. Due to the special requirements of bisulfite specific primers, the amplicons differed slightly from the MeDIP (as indicated in Figs. 2A and 2B). In agreement with the MeDIP results, methylation was only detected in the fragments 3, 4 and 7. Moreover we found a highly significant decrease of methylation at several CpG positions in RMS compared to SM (9/13 CpG positions in fragment 3, 4/4 in fragment 4 and 2/17 in fragment 7). As a control we analyzed two amplicons within the CpG island A (see Fig. 1, Supplemental Table 1) close to the Gli-binding site 1 and we were able to confirm the lack of methylation in this region ([11] and data not shown).


Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.

Nitzki F, Tolosa EJ, Cuvelier N, Frommhold A, Salinas-Riester G, Johnsen SA, Fernandez-Zapico ME, Hahn H - Oncotarget (2015)

Murine Ptch promoter region analyzed in this studyThe upper part shows the upstream genomic region of Ptch, its transcriptome, the three Gli-binding sites (triangles) and the SNP rs29624336 (asterisk). The alternative exons 1a-1e (a–e) and exon 2 (ex 2) are indicated. H3K4me3 occupancy in differentiated C2C12 cells and murine embryonic stem cells (ESC) as downloaded from the European Nucleotide Archive and NCBI Gene Expression Omnibus are indicated below. The CpG islands (A–D), the analyzed PCR amplicons 1–7 (fragment 3 equates the methylation-sensitive DNA fragment described by Ecke et al. [11], all primers are shown in supplemental Table 1) and the CpG content are shown in the lower part. The largest amplicon 5 (391 bp) could only be amplified in the H3K4me3 ChIP experiment and NGS bisulfite sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496206&req=5

Figure 1: Murine Ptch promoter region analyzed in this studyThe upper part shows the upstream genomic region of Ptch, its transcriptome, the three Gli-binding sites (triangles) and the SNP rs29624336 (asterisk). The alternative exons 1a-1e (a–e) and exon 2 (ex 2) are indicated. H3K4me3 occupancy in differentiated C2C12 cells and murine embryonic stem cells (ESC) as downloaded from the European Nucleotide Archive and NCBI Gene Expression Omnibus are indicated below. The CpG islands (A–D), the analyzed PCR amplicons 1–7 (fragment 3 equates the methylation-sensitive DNA fragment described by Ecke et al. [11], all primers are shown in supplemental Table 1) and the CpG content are shown in the lower part. The largest amplicon 5 (391 bp) could only be amplified in the H3K4me3 ChIP experiment and NGS bisulfite sequencing.
Mentions: We recently showed that RMS of Ptch heterozygous mice consistently overexpress the mutant Ptch allele whereas the expression of the remaining wt Ptch allele is unchanged or even repressed [11, 12 and Supplementary Fig. S1]. Further in silico analysis identified a methylation-sensitive region within the murine Ptch promoter in RMS [11, Fig. 1] that suggests DNA methylation as potential mediator of this phenomenon. We next analyzed methylation of the Ptch promotor by MeDIP using RMS and SM samples from 16 Ptchdel/+(Ptch+/−) [13] and Ptchneo67/+ [14] mice. The total methylation content was calculated as % input. As shown in Fig. 2A, 3 of the 7 amplicons analyzed showed methylation, whereas the remaining amplicons were unmethylated (data not shown). Surprisingly, and in contrast to our previous hypothesis, the methylation content in these fragments (i.e. amplicon 3, 4 and 7 of the Ptch promoter) was reduced in RMS compared to SM (Fig. 2A). Thus, the percentage of methylation in SM in the amplicons 3, 4 and 7 decreased from 32% to 20% (P = 0.0053), from 45% to 22% (P = 0.0112) and from 1.1% to 0.5% (P = 0.0054) in RMS, respectively. Although the decrease was small, especially in fragment 7, the difference between SM and RMS was statistically significant. For positive and negative controls for the MeDIP see supplemental Fig. S2. Next, we used bisulfite next generation sequencing (NGS) to validate the above results. Due to the special requirements of bisulfite specific primers, the amplicons differed slightly from the MeDIP (as indicated in Figs. 2A and 2B). In agreement with the MeDIP results, methylation was only detected in the fragments 3, 4 and 7. Moreover we found a highly significant decrease of methylation at several CpG positions in RMS compared to SM (9/13 CpG positions in fragment 3, 4/4 in fragment 4 and 2/17 in fragment 7). As a control we analyzed two amplicons within the CpG island A (see Fig. 1, Supplemental Table 1) close to the Gli-binding site 1 and we were able to confirm the lack of methylation in this region ([11] and data not shown).

Bottom Line: We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals.Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation.We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University Medical Center, Göttingen, Germany.

ABSTRACT
Mice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch.

No MeSH data available.


Related in: MedlinePlus