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...

A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.