Maintenance of imprinting and nuclear architecture in cycling cells

Abstract

(14) provided a first case-in-point. They performed FISH experiments in normal, phytohemagglutinin (PHA)-stimulated human lymphocytes with a probe covering a segment of the Angelman syndrome/Prader-Willi syndrome (AS/PWS) locus on HSA 15q11-13 and measured 3D interdistances between the two signals in samples of 50 nuclei at different interphase stages. Approximately 38% of nuclei at G 1 , Ϸ18% at early S phase, Ϸ58% at late S phase, and Ϸ18% at G 2 revealed distances Յ2 m, which were considered as a proximity criterion for a possible functional interaction between the two AS/PWS loci. Notably, the significant increase of associations at late S phase was not found in cells from PWS and AS patients. Furthermore, LaSalle and Lalande (14) mentioned that they obtained similar results for the Beckwith-Wiedemann syndrome (BWS) locus, a second imprinted locus on HSA 11p15.5. Based on these findings, LaSalle and Lalande formulated the kissing hypothesis, which states that a transient spatial association between the oppositely imprinted regions during late S phase is a necessary condition to maintain the imprinted status of cycling cells from one cell cycle to the next. This hypothesis was widely cited and further supported by a 2D FISH study from Riesselmann and Haaf (15), who measured 2D interhomolog distances between oppositely imprinted loci on MMU 7 as a fraction of the nuclear diameter in flattened mouse fibroblast nuclei obtained in cytospin preparations. Somatic pairing was assumed when hybridization signals apparently overlapped or touched each other. By this criterion, significantly higher values were found for BrdU-positive nuclei (13%; 27/208 nuclei) compared with BrdUnegative nuclei (9.2%; 21 of 228 nuclei). No increase of pairing frequencies during S phase was found for control regions not subject to imprinting. In contrast, Nogami et al. (16), who studied the frequency of associations between the two AS loci with probes for the imprinted SNRPN alleles in 3D preserved interphase nuclei of human myeloid leukemia HL60 cells, did not find evidence to further support the hypothesis.Importantly, a large variability of 3D positions was noted for pairs of HSA 11 and HSA 15 chromosome territories (CTs) both within nuclei and between nuclei of different cells. Given this variability, a transient kissing event during late S phase between loci widely separated at other stages of interphase requires specific, long-range, directed chromatin movements. The present study was initiated with the intent to clarify whether such movements lead to the transient juxtaposition of entire homologous CTs or, alternatively, whether homologous CTs stay in place, whereas giant loops harboring genes poised for a kissing event expand from territory surfaces and meet each other in between (1,6,8,17). We visualized the two AS/PWS regions in PHA-stimulated lymphocytes by 3D-FISH with a BAC contig covering most of the AS/PWS locus and measured the 3D distances between the two regions in G 1 , early, mid, and late S phase, G 2 , as well as in...

Chromosome Territories

“…The authors argued that transient "kissing" between the two loci is required for maintaining opposite imprints in cycling cells. This specific case of "kissing," however, could not be confirmed in a later study (Teller et al 2007).…”

Interchromosomal association and gene regulation in trans

“…Homologous associations were also shown to occur at the Beckwith-Wiedemann syndrome locus (BWS) of human chromosome 11p15, which contains which contains imprinted insulin-like growth factor 2 (IGF2) and H19 (a non-coding RNA) genes, suggesting that these associations might represent a general mechanism for regulating imprinting 41 . However, when, Teller et al revisited these findings they found no evidence for an increase of the fraction of nuclei with paired, oppositely imprinted AS/PWS or BWS loci at late S phase 43 . However, in agreement with the findings of LaSalle et al they did observe a significant homologous association between the centromeres of chromosome 11 (approximately 4 megabases away from the AS/PWS locus) during late S phase.…”

“…However, in agreement with the findings of LaSalle et al they did observe a significant homologous association between the centromeres of chromosome 11 (approximately 4 megabases away from the AS/PWS locus) during late S phase. Teller et al predicted that this is mediated by a nucleolar organizer region (NOR) (which are known to undergo paring events) linked to the centromere of chromosome 15 (and by default the AS/PWS region) 43 . Indeed analysis of lymphoblastoid cells from Gorilla gorilla, in which the AS/PWS region is not linked to a NOR they actually observed increased distances between AS/PWS loci during late S phase 43 .…”

Spatial quantitative analysis of fluorescently labeled nuclear structures: Problems, methods, pitfalls

“…An opposite example is the recent study of Angelman syndrome/PraderYWilli syndrome (AS/ PWS) loci by Teller et al (2007). Transient clustering of these loci at the late S-phase has earlier been suggested as a possible mechanism for maintenance of opposite imprinting, while Teller and co-authors argued against kissing of AS/PWS loci.…”

“…For example, this approach helped to demonstrate that NORs (nucleolar organizers) of all NOR-bearing chromosomes tend to cluster (Teller et al 2007). NORs actually cluster around the nucleolus, and the discussed approach compensated for the variation in the position of the nucleolus.…”