Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

Transcription of the testis-specific Pgk2 gene is selectively activated in primary spermatocytes to provide a source of phosphoglycerate kinase that is critical to normal motility and fertility of mammalian spermatozoa. We examined dynamic changes in protein-DNA interactions at the Pgk2 gene promoter during murine spermatogenesis in vivo by performing genomic footprinting and chromatin immunoprecipitation assays with enriched populations of murine spermatogenic cells at stages prior to, during, and following transcription of this gene. We found that genes encoding the testis-specific homeodomain factor PBX4 and its coactivator, PREP1, are expressed in patterns that mirror expression of the Pgk2 gene and that these factors become bound to the Pgk2 enhancer in cells in which this gene is actively expressed. We therefore suggest that these factors, along with CREM and SP3, direct stage-and cell type-specific transcription of the Pgk2 gene during spermatogenesis. We propose that binding of PBX4, plus its coactivator PREP1, is a rate-limiting step leading to the initiation of tissue-specific transcription of the Pgk2 gene. This study provides insight into the developmentally dynamic establishment of tissue-specific protein-DNA interactions in vivo. It also allows us to speculate about the events that led to tissue-specific regulation of the Pgk2 gene during mammalian evolution.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Communication between osteoblasts and osteoclasts plays a key role in bone metabolism. We describe here an unexpected role for matrix vesicles (MVs), which bud from boneforming osteoblasts and have a well-established role in initiation of bone mineralization, in osteoclastogenesis. We show that the MV cargo miR-125b accumulates in the bone matrix, with increased accumulation in transgenic (Tg) mice overexpressing miR-125b in osteoblasts. Bone formation and osteoblasts in Tg mice are normal, but the number of bone-resorbing osteoclasts is reduced, leading to higher trabecular bone mass. miR-125b in the bone matrix targets and degrades Prdm1, a transcriptional repressor of anti-osteoclastogenic factors, in osteoclast precursors. Overexpressing miR-125b in osteoblasts abrogates bone loss in different mouse models. Our results show that the MV cargo miR-125b is a regulatory element of osteoblast-osteoclast communication, and that bone matrix provides extracellular storage of miR-125b that is functionally active in bone resorption.