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...

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss-and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.