Decondensation of a heterochromatin
amplified chromosome arm is observed after targeting the acidic activation
domain of the VP16 viral transcription factor. DNA is stained with DAPI
(blue) and the lac repressor_VP16 fusion protein is in red. A cell containing
an amplified chromosome region containing lac operator repeats was transiently
transfected with a lac repressor- VP16 AAD expression vector (see text above).
Decondensation appears to occur through the uncoiling of a ~80 nm chromonema
Tumbar T, Sudlow G, Belmont AS. Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J Cell Biol. 1999 Jun 28; 145 (7) :1341-54. PubMed PMID:10385516; PubMed Central PMCID: PMC2133171.
The effects of transcriptional
activators on local chromatin structure is now well documented. Many transcriptional
activators have been shown to have histone modifying activities or to recruit
other protein complexes which have histone modifying activities. What is
not clear is the effect transcriptional activators might have on higher
levels of chromatin organization.
Previously, we described
the folding of 30 nm chromatin fibers into still larger scale fibers, in
the range of 60-130 nm in diameter, which we have called chromonema fibers
(see Large-scale chromatin structure and chromonema
fibers:) Given the large molecular weights of many macromolecular protein
complexes involved in transcriptional regulation, we wondered what effect
this large-scale chromatin organization might have on transcriptional activation.
One possibility was
that during the process of transcriptional activation, an activity which
decondenses large-scale chromatin organization might be recruited by some
To test this possibility,
we designed an experiment to observe the effect on large-scale chromatin
organizaton of targeting a large amount of a transcriptional activator to
a specific chromosome region. We exploited a system we had developed previously
to create gene amplified chromosome regions containing lac operator direct
repeats which we could visualize using lac repressor binding (see In
vivo tagging of chromosome sites and regions). We chose a cell line
that contained an ~ 90 Mbp amplified chromosome region which shared some
properties of heterochromatin. Specifically it remains highly condensed
through most of interphase and replicates in middle to late S phase.
We targeted the acidic
activation domain (AAD) of the strong viral transcription factor, VP16,
using a fusion protein of lac repressor, or GFP-lac repressor, with the
VP16 AAD. This targeting was accomplished either by microinjecting purified
GFP-lac repressor- VP16 AAD protein into cells or by transient transfection.
A dramatic decondensation
of the heterochromatic amplified chromosome region was observed. Interestingly,
this decondensation occurred through an extension and straightening of chromonema
fibers such that the condensed chromosome arm unfolded into an ~ 25 um long
chromonema fiber. This unfolding was accompanied by a high level of transcriptional
activation. Decondensation was still observed after inhibition of transcription,
implying that decondensation was not simply the result of high transcriptional
activity. Histone acetylation and recruitment of histone acetyltransferases,
including GCN5, CBP/p300, and PCAF was observed.
Our results demonstrate
that targeting large-amounts of a transcriptional factor can lead to large-scale
chromatin decondensation. Although our system is artificial, we propose
that it may represent an exaggerated response which occurs on a smaller
scale adjacent to natural gene loci. In this model, changes in large-scale
chromatin organization might be induced by elements of the transcriptional
machinery over distances of tens to hundreds of kb. Experiments are in progress
to test this model. Key questions include whether this decondensation occurs
through action of the same type of chromatin modifying activities know to
operator on local nucleosome structure, or whether novel chromatin modifying
activities are involved. Interestingly, high levels of transcription occur
although the chromatin remains packaged in large-scale chromatin fibers.
This may be characteristic for transcription from many pol 2 genes within
diploid cell nuclei, as suggested by in situ localization of transcription
adjacent to condensed chromatin domains.