Insights into interphase large-scale chromatin structure from analysis of engineered chromosome regions.

How chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions that preserve large-scale chromatin structure. Our results confirm the existence of large-scale chromatin domains and fibers formed by the folding of 10-nm and 30-nm chromatin fibers into larger, spatially distinct domains. Transcription at levels within severalfold of the levels measured for endogenous loci occur within these large-scale chromatin structures on a condensed template linearly compacted several hundred fold to 1000-fold relative to B-form DNA. However, transcriptional induction is accompanied by a severalfold decondensation of this large-scale chromatin structure that propagates hundreds of kilobases beyond the induced gene. Examination of engineered chromosome regions in mouse embryonic stem cells (ESCs) and differentiated cells suggests a surprising degree of plasticity in this large-scale chromatin structure, allowing long-range DNA interactions within the context of large-scale chromatin fibers. Recapitulation of gene-specific differences in large-scale chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.

Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia.

The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P =. 006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression. (Blood. 2000;95:738-743)

Comparative analysis of G-banding, chromosome painting, locus-specific fluorescence in situ hybridization, and comparative genomic hybridization in chronic myeloid leukemia blast crisis.

The molecular basis for blast transformation of chronic myeloid leukemia (CML) remains poorly understood. Cytogenetic alterations associated with CML blast crisis have previously been extensively studied by conventional G-banding analysis. However the complexity of some chromosome abnormalities or poor chromosome morphology or both has exceeded the resolution of G-banding analysis in a significant proportion of CML cases, and complex chromosome rearrangements have remained unidentified. In this study, comparative genomic hybridization (CGH) was used to elucidate genome imbalances in chronic phase or blast crisis samples or both from 12 CML patients. CGH and G-banding results were compared, and discrepancies were further clarified by using multipaint chromosome analysis and locus-specific DNA probes. No imbalances were detected in the 4 early disease phase samples studied. Eleven blast crisis samples were analyzed by G-banding and CGH, and the commonest genomic abnormality detected was overrepresentation of the long arm of chromosome 8, which was detected in 5 patients. This overrepresentation was attributable to trisomy 8 in 4 patients, whereas amplification of the entire long arm of chromosome 8 was detected in 1 patient. The formation of isochromosomes of the long arm of chromosome 8 was observed as a mechanism for gene amplification in this patient. Additional material originating from chromosome 8 was also observed intercalated into three marker chromosomes in peripheral blood metaphase spreads from this patient. These markers may further define areas on chromosome 8 that harbor oncogenes implicated in transformation of chronic myeloid leukemia.

Improved sensitivity of BCR-ABL detection: a triple-probe three-color fluorescence in situ hybridization system.

Chronic myeloid leukemia is a clonal stem cell disorder associated with the Philadelphia (Ph) translocation [t(9;22) (q34;q11)]. As a result of the Ph translocation, parts of the ABL and BCR genes become fused. Cytogenetic quantification of Ph+ metaphases can be used to monitor patient response to treatment but is of limited sensitivity and applies only to cycling cells. Fluorescence in situ hybridization (FISH) with probes from the BCR and ABL regions can also identify the Ph translocation in interphase cells. Established systems for the detection of fusion genes by FISH rely on colocalization of two different probes but are associated with a high rate of false-positive results. We have introduced a third probe labeled with a different fluorochrome to create a triple-probe/three-color system that permits identification of both the Ph chromosome and the derivative 9 chromosome in Ph+ cells. This system was used to determine the frequency of interphase cells carrying the BCR-ABL fusion gene in bone marrow and peripheral blood granulocytes from patients showing variable cytogenetic responses to interferon. Our data show that the triple-probe/three-color approach allows highly sensitive detection of residual disease. Moreover, this method is readily applicable to the analysis of other chromosome translocations.