Visualization of individual DNA loops and a map of loop domains in the human dystrophin gene

The organization of the human dystrophin gene into
loop domains has been studied using two different
experimental approaches: excision of DNA loops
mediated by nuclear matrix-bound topoisomerase II
and in situ hybridization of different probes with histone-
depleted nuclei (nuclear halos). Our objective
was to examine if the DNA loops mapped by this
biochemical approach coincide with loops visualized
by microscopy. The results obtained using
both approaches were in good agreement. Eight
loops separated by attachment regions of different
length were mapped in the upstream part (up to
exon 54) of the gene by topoisomerase II-mediated
excision. One of these loops was then directly
visualized by in situ hybridization of the corresponding
bacmid clone with nuclear halos. This is
the ®rst direct demonstration that a DNA domain
mapped as a loop using a biochemical approach
corresponds to a loop visible on cytological preparations.
The validity of this result and of the whole
map of loop domains was con®rmed by in situ
hybridization using probes derived from other
attachment regions or loops mapped by topoisomerase
II-mediated cleavage; these probes hybridized
on the core or halo region, respectively, of nuclear
halos. Our results demonstrate that a single transcription
unit may be organized into several loops
and that DNA loop attachment regions may be fairly
long. Three out of four replication origins mapped in
this gene co-localize with loop attachment regions,
and the major deletion hot spot is harbored in an
attachment region. These results strongly suggest
that partitioning of genomic DNA into speci®c loops
attached to a skeletal structure is a characteristic
feature of eukaryotic chromosome organization in
interphase.