Randomly inserted and targeted Hox/ reporter fusions transcriptionally silenced in Polycomb mutants

Abstract

Polycomb-group (Pc-G) proteins ensure late maintenance of transcriptional repression outside the expression domain of target genes in flies and vertebrates. They act in complexes, presumably by modulating chromatin structure. In Drosophila, they have been found to be associated with transcriptionally inactive loci but seem to be present in association with actively transcribed promoters as well, a feature which is not yet understood. In the mouse, mutations in several Pc-G genes result in an often subtle, local derepression of only a subset of the Hox genes rostral to their expression domains. We report here that Hox͞reporter fusion genes, either randomly integrated as transgenes or as insertions within endogenous loci, are transcriptionally silenced in two mouse Pc-G-null mutants, Mel18 and rae28. Transcriptional silencing of Hox͞reporter transgenes in Pc-G mutants was accompanied by increased DNA methylation in the promoter region. Gene silencing was observed at early developmental stages, long before Pc-G and trithorax-group proteins exert their function in maintenance of the Hox patterns. Although all five Hox genes tested as Hox͞reporter fusions were silenced in the Pc-G mutants, transcription of the endogenous loci was mildly decreased in a subset of these Hox genes, and Hoxb1 was the most strongly affected. We discuss the possibilities that the observed negative effect of Pc-G mutations on Hox and Hox͞reporter expression may reflect a positive involvement of the Pc-G epigenetic repressors in initial Hox gene transcription and that this requirement is exacerbated by the reporter insertion.

Mammalian Polyhomeotic Homologues Phc2 and Phc1 Act in Synergy To Mediate Polycomb Repression of Hox Genes

“…Therefore, Phc2 gene products were shown to act in synergy to repress the expression of Hoxb4 and -b8 rostral to the expression domain and also to maintain Hoxb8 expression within its expression domain. Very similar changes have been reported in the Rnf110-Bmi1 double mutant (12). In addition, since the derepression of Hoxb6 in Rnf110-Bmi1 double homozygotes has been shown to occur progressively between 8.5 and 9.5 dpc (3), Representative results show the association of the Phc2 protein with the second exonic region of Cdkn2a, using the adam34 coding region as a negative control.…”

“…It is possible that these results tie in with the recent discovery that class II PcG complexes are associated with a positive function. Null mutations in the Rnf110 and Phc1 loci have been shown to decrease the transcription level of endogenous Hoxb1 and to severely impair the transcription from Hox-LacZ reporters and knockin loci (12). In addition, a positive action of PcG complexes could explain the fall in gene expression levels within Hoxb8 expression domains, which has been reported to be around 9.5 dpc in Phc2-Phc1 and Bmi1-Rnf110 and double mutants (3,12).…”

“…Null mutations in the Rnf110 and Phc1 loci have been shown to decrease the transcription level of endogenous Hoxb1 and to severely impair the transcription from Hox-LacZ reporters and knockin loci (12). In addition, a positive action of PcG complexes could explain the fall in gene expression levels within Hoxb8 expression domains, which has been reported to be around 9.5 dpc in Phc2-Phc1 and Bmi1-Rnf110 and double mutants (3,12). Importantly, the transcriptional silencing of Hox-reporter loci in Rnf110 and Phc1 mutants is accompanied by DNA methylation of the promoter region (12).…”

“…6B). Therefore, based on these results, it appears that Phc1 and Phc2 are involved in maintaining the transcriptional status of Hoxb8 rather than in its early induction, as observed in Rnf110-Bmi1 double homozygotes (3,12). Following this, we looked at the genetic interactions between the Phc2 and Rnf110 mutations.…”

Developmental regulation of the Hox genes during axial morphogenesis in the mouse

“…In addition to their role in the epigenetic maintenance of transcriptional states of their target genes, PcG and trxG protein complexes probably regulate the transcription of their targets in Drosophila (Breiling et al, 2001;Saurin et al, 2001), as well as in early mouse embryos (de Graaff et al, 2003). A recent study (Milne et al, 2002) elegantly showed that the binding of SET domain methyl transferase activity to the proximal promoter of human HOXC8 in cultured fibroblasts was crucially required for transcription of the gene.…”