Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

Abstract

Abstract.A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos y Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop S whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Breakpoint cluster regions of the AML-1 and ETO genes contain MAR elements and are preferentially associated with the nuclear matrix in proliferating HEL cells

“…Analysis of spatial positions of BCRs present in AML-1 and ETO genes by in situ hybridization of BCR probes with nuclear halos Visualization of specific DNA sequences on nuclear halos by hybridization opens the possibility of studying their partitioning between matrix-bound and looped-out DNA fractions (Balajee et al, 1996;Gerdes et al, 1994;Ratsch et al, 2002). In our recent experiments, this method was successfully used to study the partitioning of the human dystrophin gene into loop domains (Iarovaia et al, 2004).…”

Chromatin loops are selectively anchored using scaffold/matrix-attachment regions

“…Nuclear halos were prepared essentially as described (Gerdes et al, 1994;Cai and Kohwi-Shigematsu, 1999). In brief, cultured mouse spleen cells and transfected mouse cells were harvested; then immersed in 4°C CSK buffer containing 0.5% Triton X-100 for 10 minutes.…”

“…Following histone extraction, these loops can be visualized as a DNA halo anchored to the densely stained nuclear matrix or chromosomal scaffold (Vogelstein et al, 1980;Gerdes et al, 1994;Bickmore and Oghene, 1996). As a basic unit, the loop is essential for DNA replication, transcription regulation and chromosomal packaging (Gasser and Laemmli, 1987;Berezney et al, 1995;Bode et al, 1995;Nickerson et al, 1995;Razin et al, 1995;Jackson, 1997;Stein et al, 1999;Sumer et al, 2003).…”

Chromatin supraorganization and extensibility in mouse hepatocytes with development and aging

“…Because young mice have transcriptional rates higher than those of adult mice (32), this chromatin may mostly be attached to the nuclear matrix, thus explaining absence of ECF formation in these nuclei, despite relatively low condensation of this chromatin in relation to that of the adult mice. Considering the hypothesis of Gerdes et al (52), the hepatocyte nuclei from old mice, which showed higher ECF formation compared to that of adults, could have a reduced number of DNA-nuclear matrix interactions, reflecting reduced gene activity and low metabolism.…”

“…In the present study, the absence of ECF formation in fixed hepatocyte nuclei of young mice could not be associated with chromatin condensation. Gerdes et al (52) stated that DNA sequences attached to the nuclear matrix (MARs), which are resistant to saline extraction, are located in regions where the DNA is being transcribed or replicated. Because young mice have transcriptional rates higher than those of adult mice (32), this chromatin may mostly be attached to the nuclear matrix, thus explaining absence of ECF formation in these nuclei, despite relatively low condensation of this chromatin in relation to that of the adult mice.…”