Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days. INTRODUCTIONInsight into genomic organization is key to understanding gene regulation in mammals. However, owing to technical limitations, we still have little idea about how the mammalian genome is structured in vivo at the scale at which long-range physical interactions between genes and dispersed regulatory elements most often take place (1-10 3 kbp). The recent development of the ''Tagging and recovery of associated proteins'' 1 and 3C (see ref.2) assays allowed the very first glimpse into this crucial level of organization of the genome 3-5 . However, the RNA-TRAP technique, which is based on the targeting of peroxidase activity to nascent transcripts, is restricted to physical interactions occurring with actively transcribed genes, while 3C assays potentially allow identification of physical interactions between any chromatin segments. 3C technology is particularly suited to identify chromatin loops formed in genomic regions of up to several hundreds of kilobases in size. 5C technology 6,7 offers a robust high-throughput alternative for this analysis, based on large-scale sequencing or microarray analysis. 5C is however more laborious to set up. To identify DNA segments that interact over distances larger than several hundreds of kilobases, we recommend using 4C technology [8][9][10][11] , which allows for an unbiased genome-wide screen for DNA elements that interact with a genomic site of choice.The principle of 3C technology 2 (Fig. 1) is based on formaldehyde crosslinking of interacting chromatin segments, followed by restriction digestion and intramolecular ligation of crosslinked fragments. Ligation products are subsequently analyzed by PCR using primers specific for the restriction fragments of interest. The mere detection of a ligation product between two segmen...

The pleomorphic adenoma gene 1 ( Plag1 ) proto-oncogene encodes a transcription factor and is implicated in human tumorigenesis via ectopic overexpression. No information is available about its developmental role. To address this, a Plag1 -/-mouse strain was generated and it appears that Plag1 -deficient mice are viable. No anatomical differences are obvious at birth, except that the weight of Plag1 -/-mice is significantly lower in comparison to control litter mates. This early growth retardation is maintained throughout adult life with proportionally smaller organs except for the disproportionally small seminal vesicles and ventral prostate; however, plasma testosterone levels in males were not affected. Furthermore, fertility of both male and female Plag1 -/-is reduced. Northern blot analysis revealed that Plag1 is developmentally regulated with high overall fetal expression levels, which drop after birth. Furthermore, Plag1 is differentially expressed and is readily detectable in the reproductive organs and pituitary. Expression of growth regulatory Igf2, a known target gene of Plag1 in tumorigenesis, was not affected in Plag1 -/-embryos and pups. The general morphology and histology of the size-reduced pituitaries was not affected. Our results establish that Plag1 disruption in mouse differentially affects pre-and postnatal growth and development of organs, with reproductive repercussions.

Pleomorphic adenoma gene 1 (PLAG1) proto-oncogene overexpression is implicated in various human neoplasias, including salivary gland pleomorphic adenomas. To further assess the oncogenic capacity of PLAG1, two independent PLAG1 transgenic mouse strains were established, PTMS1 and PTMS2, in which activation of PLAG1 overexpression is Cre mediated. Crossbreeding of PTMS1 or PTMS2 mice with MMTV-Cre transgenic mice was done to target PLAG1 overexpression to salivary and mammary glands, in the P1-Mcre/ P2-Mcre offspring. With a prevalence of 100% and 6%, respectively, P1-Mcre and P2-Mcre mice developed salivary gland tumors displaying various pleomorphic adenoma features. Moreover, histopathologic analysis of salivary glands of 1-week-old P1-Mcre mice pointed at early tumoral stages in epithelial structures. Malignant characteristics in the salivary gland tumors and frequent lung metastases were found in older tumor-bearing mice. PLAG1 overexpression was shown in all tumors, including early tumoral stages. The tumors revealed an up-regulation of the expression of two distinct, imprinted gene clusters (i.e., Igf2/H19 and Dlk1/Gtl2). With a latency period of about 1 year, 8% of the P2-Mcre mice developed mammary gland tumors displaying similar histopathologic features as the salivary gland tumors. In conclusion, our results establish the strong and apparently direct in vivo tumorigenic capacity of PLAG1 and indicate that the transgenic mice constitute a valuable model for pleomorphic salivary gland tumorigenesis and potentially for other glands as well. (Cancer Res 2005; 65(11): 4544-53)