Overexpression of mutantPtchin rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy

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.

Measuring quantitative effects of methylation on transcription factor–DNA binding affinity

“…This essentially forms an autoregulatory feedback to modulate the protein homeostasis of GLI1 inside the nucleus. Although the GLI1 consensus site contains no CpGs, expression of PTCH1 and binding by GLI1 have been shown to be sensitive to methylation of the promoter region in certain cancers (38)(39)(40). Using Spec-seq and libraries with a 7-bplong region 3′ of the known consensus, we find an extended consensus sequence of CGTCGCA that may interact with zinc fingers 2 and 3 (Fig.…”

Antagonism between Hedgehog and Wnt signaling pathways regulates tumorigenicity (Review)

“…An example of this is the inactivation of PTCH or oncogenic activation of SMO, which have been demonstrated to be common features in a high proportion of tumors. To date, this mode of Hh signaling is considered the most important for tumor development (36)(37)(38)(39)(40). The second mode of aberrant activation is autocrine: The Hh ligand is secreted by tumor cells and also affects the tumor cells themselves (41,42).…”