CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allelespecific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.The transcriptional regulator CCCTC-binding factor (CTCF) is a highly conserved 11-zinc-finger nuclear protein that controls the expression of a number of genes via chromatin insulation or enhancer blocking (for reviews, see references 5, 8, 23, and 28). CTCF silences genes by binding to sites within promoters, silencers, and insulators through the use of different combinations of zinc fingers (20). More than 15,000 CTCFbinding sites have been identified throughout the genome (16).The role of CTCF as an insulator regulating the imprinting of Igf2 and H19 has been extensively studied. Igf2 and H19 imprinting is directed by epigenetic modifications in the differentially methylated region (DMR) of the imprinting control region (ICR) located between these two adjacent genes (1,9,19,21,29,30). The binding of CTCF to the unmethylated maternal ICR creates a physical boundary, blocking the interaction of downstream enhancers with the remote Igf2 promoters and silencing the maternal allele (4,13,15). When this ICR is deleted (35) or mutated (32, 34), the maternal Igf2 allele is expressed, leading to biallelic expression. In addition, CTCF has recently been shown to act as a tethering protein, serving as a molecular glue to secure long-range intrachromosomal (17) and interchromosomal (18) interactions.By chromosome configuration capture (3C) methodology, it has been shown that CTCF participates in the formation of a long-range chromosomal loop to the upstream Igf2 DMRs when it is bound to the maternal ICR (17,42,21). This model suggests that CTCF may not only function as a physical insulator but also actively participate in the regulation of the imprinted Igf2 allele. We were interested in learning how CTCF mediates the suppressi...

Alternate interactions between the H19 imprinting control region (ICR) and one of the two Igf2 differentially methylated regions has been proposed as a model regulating the reciprocal imprinting of Igf2 and H19. To study the conformation of this imprint switch, we performed a systematic structural analysis across the 140 kb of the mouse Igf2-H19 region, which includes enhancers located both between the two genes as well as downstream of H19, by using a scanning chromosome conformation capture (3C) technique. Our results suggest that on the active paternal Igf2 allele, the various enhancers have direct access to the Igf2 promoters, whereas the imprinted silent maternal Igf2 allele assumes a complex three-dimensional knotted loop that keeps the enhancers away from the Igf2 promoters and allows them to interact with the H19 promoter. This complex DNA looping of the maternal allele is formed by interactions involving differentially methylated region 1, the ICR, and enhancers. Binding of CTC-binding factor to the maternal, unmethylated ICR in conjunction with the presence of multicomplex components including interchromosomal interactions, create a barrier blocking the access of all enhancers to Igf2, thereby silencing the maternal Igf2. This silencing configuration exists in newborn liver, mouse embryonic fibroblast, and embryonic stem cells and persists during mitosis, conferring a mechanism for epigenetic memory.

Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.