Research Topics
Department of Cell & Structural Biology
M.D., Ph.D. (1983) Temple University
Research Topics
Research Interest
The highest levels of chromatin organization, consisting of the arrangement of
30 nm chromatin fibers within mitotic and interphase chromosomes, are unknown.
Our laboratory is trying to understand both the structure and the
biological implications of these highest levels of chromatin organization, which
we have termed large-scale chromatin structure.
This is being accomplished through a combination of molecular, genetic,
biochemical, and structural experimental approaches.
Our long-term goals are not only to understand the basic biology
underlying this level of chromosome organization but also to apply this
knowledge towards improved methods for transgene expression and gene therapy.
In particular we are focused on the following questions: What are the folding motifs underlying mitotic and interphase chromosome structure? What is the large-scale chromatin structure of specific gene loci and replication units? What are the functional implications for this large-scale chromatin structure- to what degree does this level of chromatin organization impact regulation of transcription, replication, and recombination? What are the cis and trans determinants of this large-scale chromatin organization?
In previous work, our laboratory described the folding of 10 and 30 nm
chromatin fibers into larger, ~100 nm "chromonema" fibers.
To dissect the folding of these fibers within interphase and mitotic
chromosomes we have developed a novel approach which involves engineering
chromosomes to contain labeled regions with characteristic and homogenous
folding patterns which can be directly visualized within living cells.
These labeled regions were created by gene amplification using a DHFR
construct containing 256 copies of the lac operator.
The lac operator repeats can be visualized through in vitro or in vivo
lac repressor binding.
A green fluorescent protein-lac repressor-nuclear localization signal
fusion protein allows direct in vivo
imaging.
We have isolated a number of lines containing labeled chromosome regions
of varying sizes and levels of interphase condensation.
Light microscopy analysis of these lines has revealed a precise
choreography of chromosome structural changes through the interphase cell cycle.
These structural changes occur synchronously throughout the chromosome
arm and involve transitions between the different folding intermediates
identified earlier from our studies of cell cycle changes of bulk chromatin.
Moreover, we have shown that specific decondensation events immediately
precede initiation of DNA replication and movement of the amplified chromosome
arm within the nucleus.
A detailed structural analysis of these changes using EM tomography
is now beginning.
In a separate project we are beginning to manipulate effects on
chromosome structure of specific DNA sequences.
We have begun to investigate changes in large-scale chromatin
organization associated with transcriptional activation.
A lac repressor-VP16 fusion protein was constructed and directed to the
lac op repeats contained within amplified chromosome regions.
A striking decondensation of the amplified chromosome regions into
extended large-scale fibers was observed as well as a change in position within
the nucleus. Similar decondensation
was obtained using a lac repressor- estrogen receptor fusion protein. Experiments
are now in progress to dissect which parts of the transcriptional machinery are
required for these changes in large-scale chromatin decondensation and movements
within the nucleus.
At the same time we are beginning to apply this technology to visualize the ultrastructure of specific endogenous gene loci, in both Drosophila and mammalian cells in order to examine structural changes associated with induction of transcription.
Key Words
Recombinant DNA, Introduction of DNA into Animal Cells, Gene
Expression Systems, Genome Organization/RFLP Mapping, Supramolecular Systems, Insect Tissue Culture,
Insect Development and Physiology, Structural
Biology, Hormones, Animal Cell and/or Tissue Culture, Image Analysis,
Transgenic Animals
Current Research Funding NIH