RESEARCH SUMMARY:
Research in my lab concerns several aspects
of transcriptional regulation. We use the fruit fly, Drosophila
melanogaster, as a model to study mechanisms of gene
activation and gene silencing.
Histone biotinylation and gene expression. The DNA in eukaryotic
cells is complexed with proteins that facilitate DNA folding
as well as access to specific regions for transcription. The
primary level of DNA packaging involves the wrapping of DNA
around a complex of small basic proteins called histones.
The histones are covalently modified at a variety of sites
to facilitate transcriptional regulation, DNA repair or compaction
for mitosis. The known histone modifications include acetylation,
methylation, phosphorylation, ubiquitination, ADP-ribosylation
and biotinylation. Biotinylation of histones is unique among
histone modifications in that a metabolic co-factor also functions
as a chromatin mark. Histone biotinylation could act as a
metabolic sensor, mediating changes in transcription in response
to changes in nutritional status. We are collaborating with
the Janos Zempleni lab (University of Nebraska--Lincoln) to
determine the genetic role of histone biotinylation. Histone
biotinylation is mediated by holocarboxylase synthetase (HCS)
and by biotinidase (BTD). Both enzymes are found in the nucleus.
We found that HCS binds to specific sites on Drosophila chromosomes,
consistent with a role for targeted HCS in histone biotinylation.
We showed that knockdown of HCS in transgenic flies results
in shorter lifespan, reduction in certain biotinylated histones
and altered gene expression, including 18S rRNA. We are currently
investigating the distribution of specific biotinylated histone
isoforms, to determine the chromosomal regions that are targets
of biotinylation. We are using specifically engineered transgenic
flies to test the roles of HCS and BTD in gene expression.
We are also purifying and characterizing nuclear complexes
containing HCS, to identify candidates for targeting proteins.
Transcription activation and chromatin remodeling.
The initiation of transcription by RNA Polymerase II in eukaryotes
requires a number of factors that make the DNA available for
Polymerase binding. One such factor is the mammalian SRCAP
protein, which is believed to use the energy of ATP hydrolysis
to modify the chromatin of certain genes to promote gene expression.
In collaboration with Dr. John Chrivia (Department of Pharmacology
and Physiological Sciences), we showed that human SRCAP can
complement genetic defects (recessive semi-lethality; female
sterility) associated with mutations in the Drosophila gene
domino. We also showed that this activity requires a functional
ATPase domain. These studies uncovered a role for the Domino/SRCAP
family proteins in signaling by the Notch pathway, a developmental
regulator of cell differentiation. Our current studies are
aimed at identifying other factors that cooperate with Domino/SRCAP
family proteins in transcription activation.
RNA Polymerase elongation factors and gene regulation.
RNA Polymerase II binds a variety of protein factors while
it transcribes DNA into RNA. Several of these factors work
to enhance the rate of RNA chain biosynthesis, and are thus
called "elongation factors." In collaboration with
Dr. Ali Shilatifard in the department, we have characterized
the elonagation factor ELL, which was first identified as
part of a chimeric protein expressed in certain leukemias.
We showed that ELL is associated with RNA Polymerase II on
chromosomes at sites of active transcription. We showed that
ELL is encoded by a previously characterized locus, Suppressor
of Triplo-lethal, an essential gene that is required for proper
gene expression throughout development. We showed that ELL
is required for both the Notch and ras signaling pathways,
which are both required for normal development and are mutated
in many human cancers. Current studies are aimed at identifying
other factors that cooperated with ELL in transcriptional
regulation.
Heterochromatin and gene regulation. In
the nuclei of all higher eukaryotes, certain regions fail
to decondense after telophase in the cell cycle. These regions--termed
"heterochromatin"--are associated with gene silencing.
We are currently characterizing a heterochromatin-associated
protein, "heterochromatin protein 1", or HP1. HP1
is the founding member of a highly conserved family of chromosomal
proteins. In collaboration with Drs. Howard Worman (Columbia
University) and Jean-Claude Courvalin (Institut Jacques Monod),
we showed that human HP1 functions in Drosophila, arguing
that studies of the Drosophila protein will be relevant to
human gene regulation. We were the first to report that HP1
binds DNA and nucleosomes in vitro, and subsequent work by
others using mammalian HP1-family proteins suggests that HP1
binds methylated histone H3. One area of research in my lab,
in collaboration with Dr. Tomasz Heyduk in the department,
focuses on biochemical characterization of HP1-nucleosome
interactions. We were also the first to identify HP1-regulated
genes. Surprisingly, HP1 has different effects on different
genes. In collaboration with Dr. Arthur Hilliker (York University),
we showed that the heterochromatin genes light and rolled
require HP1 for their normal activation. In collaboration
with Dr. Worman, we identified four euchromatic genes that
are normally repressed by HP1. We are now in the process of
identifying the DNA sequences responsible for HP1 targeting
to specific genes, using microarray expression profiling and
chromatin immunoprecipitation analysis.
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