MECHANISMS GENERATING TEMPORAL EMBRYONIC GENE EXPRESSION

Project: Research project

Project Details

Description

The long range goals of the experiments outlined in this proposal are to
understand at the molecular and biochemical level what determines temporal
patterns of gene expression during early embryogenesis. It has been made
clear from numerous studies that cells committed to different lineages
differ in the pattern of genes they express. A fundamental question
concerning developmental biologists is to dissect just how these different
patterns are generated in the daughters of a single cell, the fertilized
egg. Our approach to this problem has been to dissect the cis-acting
regulatory sequences and the trans-acting regulatory proteins of families
of genes encoding histone H1 proteins that are differentially regulated
during early embryogenesis and in adult tissues of the sea urchin. The
expression of the early or embryonic histone genes, which are encoded by
300-500 tandem arrays, is confined to a period up to the blastula stage of
development about 12 hrs. following fertilization. The late histone gene
family consists of 2 single copy genes whose transcripts are expressed
from a basal promoter until the blastula stage when their transcription
rate increases and about 1 million additional late H1 mRNAs per embryo
accumulate during the next 8 hrs. Our experimental approach to the
questions outlined above has been to identify the DNA sequences required
for the accurate stage-specific initiation of transcription and to purify
and characterize the proteins that bind to these sequences. We will test
the biological activity of these factors, produce antisera, isolate cDNAs
and determine when and where these proteins are present in oocytes, eggs,
embryos and adult tissues. This reverse genetic approach has led to the
identification of both promoter specific elements and an enhancer element
that act as molecular timing switches for stage specific embryonic
transcription. For example, the late H1 gene is activated at the mid-
blastula stage by an enhancer element that consists of 3 binding sites for
a single protein, Stage Specific Activator Protein (SSAP). We have
purified SSAP and obtained cDNA clones encoding this protein. SSAP is a
novel transcription factor because it can bind to single stranded DNA and
it has a transcription activation domain that is 6-10 fold more potent
than VP16 in mammalian cells. We hope to understand how this potent
transcription activation domain functions as a molecular timing switch by
mutagenesis, identification of interacting proteins, and post-
translational modification. Since this is such a potent transcription
activator, this suggests that it could interact with unique components of
the transcription machinery that we hope to identify and isolate. SSAP is
related to two human proteins, EWS and TLS, of unknown function except
they are involved in chromosomal translocations in Ewings Sarcoma and
Liposarcoma respectively. We have identified and have candidate clones for
a human homologue of SSAP and we will ask if this homologue is a candidate
for involvement in human disease. These studies pertain directly to
understanding the precise and detailed mechanisms underlying differential
gene expression during embryogenesis and differentiation.
StatusFinished
Effective start/end date12/31/896/30/00

ASJC

  • Genetics
  • Molecular Biology

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