Chromatin loop structure of the human X chromosome: relevance to X inactivation and CpG clusters.

Abstract

Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and a-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.Sex dosage compensation in mammalian females is achieved by random inactivation of one X chromosome (reviewed in reference 26). Active and inactive X chromosomes differ in a number of characteristics, including degree of condensation (3), timing of replication (18), and transcriptional activity (19). In addition, genes that are constitutively expressed are methylated differently on the two chromosomes (24,46,49,50). The functional differences in DNA methylation reside in the clusters of CpG dinucleotides, the so-called CpG islands, that are commonly found at the 5' ends of many housekeeping genes (4). These clusters are methylated only in genes on the inactive X chromosome, and methylation helps maintain the inactive state (23). Furthermore, since these clusters are nuclease hypersensitive in active but not in inactive genes (51), the chromatin structures of the two chromosomes must differ. Finally, methylation and gene activity are coordinately regulated at three CpG clusters near the glucose 6-phosphate dehydrogenase (G6PD) locus (46), which suggests that CpG islands may help define functional domains along the chromosome.Although the differences in structure of the homologous X chromosomes during interphase are striking, little is known about the organization of X chromatin at the molecular level. The 300-A (30-nm) chromatin fiber is thought to be organized into 30-to 100-kilobase-pair (kbp) loops that are anchored to a protein structure variously termed the nuclear matrix (7,8,37,(41)(42)(43)48), scaffold (9, 15-17, 21, 22, 25, 33, 34). or cage...

DNA loop domains in a 1.4-Mb region around the human hprt gene mapped by cleavage mediated by nuclear matrix-associated topoisomerase II

“…2 and 4). This is consistent with the reported absence of SARs near the hprt gene (Beggs and Migeon 1989); the ®nding that restriction fragments from the gene itself show MAR activity (Sykes et al 1988;Chong et al 1995) can be understood if genomic DNA contains a repertoire of MARs from which only certain loop domain attachments are selected according to the cell's status, as suggested by the change of loop domain length during development in Xenopus (Micheli et al 1993). Since K562 cells are derived from a female leukemia, the partial topoisomerase II cleavage in some fragments (Figs.…”

Forming facultative heterochromatin: silencing of an X chromosome in mammalian females

“…Finally, the inactive X chromosome is less sensitive to DNase I than the active X chromosome [175,176], consistent with active chromatin being preferentially sensitive to this enzyme [177]. In addition, general DNase sensitivity and the presence of DNase hypersensitive sites at individual genes corresponds to their activity (e. g. [178 -180]); however, there does not appear to be a difference between the active and inactive X chromosomes in their scaffold-associated regions [181].…”

A major positive regulatory region located far upstream of the human alpha-globin gene locus.

“…In contrast, the oL genes are less typical in that they have an unusually high GC content (>60%), are associated with unmethylated CpG-rich islands (Bird et al 1987;Fischel-Ghodsian et al 1987a, b), and replicate early in all cell types (Holmquist 1987). Similarly, the higher order structures of the two gene clusters are quite different; whereas nuclear matrix-associated regions can be demonstrated within and around the 13-globin complex, no such attachment sites have been identified within 180 kb around the a complex (Jarman and Higgs 1988), in keeping with other GC-rich regions of the genome (Beggs and Migeon 1989).…”