| Summary: | Essentially all higher organisms are made up of two or more types of
tissues. The specific identity of those tissues is dependent on the genes that are
expressed within the cells of the particular tissue type. The correct set of genes
must be expressed and genes, that are not part of the set specific to that tissue,
must be kept silenced. In addition, in most cells, the decision whether a gene
will be active or not is made early in development and therefore must be passed
on to daughter cells. The focus of this thesis is an investigation into the
mechanism or mechanisms employed by eukaryotes to silence genes and to
maintain that silenced state throughout development. The model system our
laboratory has been using to investigate silencing is position effect variegation
(PEV) in D. melanogaster. In PEV a gene is silenced in a certain proportion of the
cells of a tissue in which it is normally expressed due to its proximity to an
heterochromatic breakpoint. The decision whether a gene will be active or
inactive is made early in development and that decision is passed on to
daughter cells with reasonable fidelity. Thus PEV mimics normal development
in many ways. This has led our lab, and several others, to try to dissect the
mechanisms underlying PEV with the hope they will shed some light on the
more general silencing mechanisms that occur during normal development.
In Chapter 2 of this thesis I describe the cloning and characterization of a
gene identified in a screen for dominant suppressors of the variegation
associated with PEV [Su(var)s]. The gene encodes HDAC1 , an histone
deacetylase homologous to HDAC1 from mammals and Rpd3 from S. cerevisiae. Specific mis-sense mutations in HDAC1 cause strong dominant suppression of
PEV while null or hypomorphic mutations have no effect on the variegating
phenotype. I present a model proposing that the mis-sense mutations are
acting as anti-morphic mutations that "poison" the deacetylase complex.
The level of variegation of a gene subject to PEV is very sensitive to a
wide variety of factors, some, which may be acting directly and some, which
may be acting indirectly. HDAC1 localizes to a large number of sites on the
polytene chromosomes of D. melanogaster (Pile and Wasserman, 2000) and
therefore appears to regulate a large number of genes. Thus it is a possibility
that the Su(var) mutations in HDAC1 are affecting PEV indirectly. In Chapter 3
I present data from chromatin immuno-precipitation experiments (X-ChIP) that
provides compelling evidence that HDAC1 is acting directly on the
euchromatic region subject to silencing in PEV. I propose a model linking the
histone deacetylase activity of HDAC1 to the function of other proteins known
to be involved in the silencing associated with PEV.
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