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Full Abstracts – CHROMATIN AND EPIGENETICS
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79
Mapping of chromosomal proteins of the bithorax complex in single parasegments.
Welcome Bender
1
, Heber Domingues
1
, Sarah Bowman
2
, Robert
Kingston
2
. 1) BCMP Dept, Harvard Medical Sch, Boston, MA; 2) Dept. of Molecular Biology, Massachusetts General Hospital, Boton, MA 02114.
The Drosophila bithorax complex (BX-C) is thought to have a different chromosome structure in each of the parasegments that it regulates. Thus,
chromatin immunoprecipitation experiments done on whole embryos or cultured cells are of limited value for the study of the homeotic complexes. We have
created embryos that produce GFP in PS4, PS5, PS6, or PS7. These are the parasegments most important for the expression of
Ultrabithorax
and
abdominal-
A
. Enhancer trap P elements were swapped into the BX-C and the Antennapedia complex; these produce GAL4 or GAL80 with anterior expression limits at
specific parasegmental borders. P elements with embryonic enhancers derived from the BX-C have also been constructed, which give similarly restricted
expression patterns. Appropriate combinations of GAL4 and GAL80 producers restrict GAL4 activity to single parasegments. We have used FACS to isolat
nuclei making GFP under GAL4 control, and ChIP/Seq experiments are in progress to map histone modifications and chromosomal proteins.
80
Spliceosomal Dynamics is Required for Nurse-Cell Chromatin Dispersal In the
Drosophila
Germ Line.
Stephen M. Klusza, Shirelle Figueroa, Amanda
Novak, Billy Palmer, Wu-Min Deng. Dept Biological Sci, Florida State Univ, Tallahassee, FL.
In
Drosophila
oogenesis, nurse-cell nuclei display a significant progressive change in their chromatin morphology, from a highly-compact polytenic
configuration to a diffuse, dispersed state largely devoid of any visible structures. Mutations in splicing proteins such as Hrb27C, Squid, and PUf68 have
been found to affect nurse-cell chromatin dispersal (NCCD) through persistence of the intermediate ‘5-blob’ phenotype in later stages of oogenesis,
suggesting that splicing is an integral part of chromatin dynamics in polytene-polyploid nurse cells. In an effort to identify cellular processes that may be
significantly affected by NCCD failure, we identified mutations in
peanuts
(
pea
), the
Drosophila
homolog of the yeast Prp22p DEAH-box RNA helicase, as
an enhancer of the condensed '5-blob' morphology phenotype of
ovarian tumor
(
otu
) heterozygotes. Prp22p has been shown to be involved in RNA exon
ligation and recycling of spliceosomal components through unwinding of the spliceosome from spliced RNA. FLP-FRT-induced whole germline clones of a
null allele of
pea
invariably arrest at stages 2-3 of oogenesis, while half-germline clones display NCCD failure in
pea
-null nurse-cell nuclei at stages 6-7 of
oogenesis. By using a Prp38 antibody that recognizes activated spliceosomes, we also discover that enrichment of spliceosomes in the inter-chromatin
spaces, as well as a subset of chromatin, occur during the transiently-condensed phase of NCCD; in
pea
-null nurse-cell nuclei, the spliceosomes remain
enriched in these areas, suggesting a defect in the re-distribution and/or recycling of spliceosomal fractions as a result of NCCD failure. Previous research in
yeast spliceosomal mutants have identified splicing aberrations in many ribosomal proteins, and cursory examination of ribosomal-protein loss-of function
(LOF) in
Drosophila
ovaries also retain similar NCCD defects. Therefore, we are currently examining ribosomal function through spliceosomal studies as a
potential general mechanism for NCCD that may implicate chromatin dynamics as a regulator of translation in the cell.
81
Phylogenomic analysis of the Heterochromatin Protein 1 gene family defines new germline-restricted functions.
Mia Levine
1
, Connor McCoy
1
,
Danielle Vermaak
1
, Mary Alice Hiatt
1
, Frederick Matsen
1
, Harmit Malik
1,2
. 1) Fred Hutchinson Cancer Research Center, Seattle, WA; 2) Howard Hughes
Medical Institute.
Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes, from chromosome
segregation to genome defense. Extensive tracts of repetitive DNA in heterochromatin, however, challenge traditional methods of sequence assembly and
experimental manipulation. Fortunately, the functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence mirrors
the diverse functions supported by this enigmatic genome compartment. To identify new such surrogates for dissecting heterochromatin function and
evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family over 40 million years of Drosophila
evolution. Our study expands this gene family from a modest 5 genes to at least 27 genes, including several uncharacterized HP1 genes in
D. melanogaster
.
The 22 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family.
We find that dynamic evolution occurs via prolific gene gains and losses while HP1 gene number per lineage remains remarkably constant, consistent with a
‘revolving door’ model. The high resolution dating of one such loss event—that of HP1E in the obscura group, introduces the notion that heterochromatin
content and/or distribution across Drosophila evolution may drive at least some of these gain/loss dynamics. We have begun to test this hypothesis by
functionally and cytologically characterizing the testis-restricted HP1E, which is dispensable over evolutionary time scales but essential for male fertility, at
least in
D. melanogaster
. Our preliminary analyses are consistent with HP1E acting to epigenetically transfer information via the male germline to the egg.
These data support the utility of this expanded compendium of ovary and testis-restricted HP1 genes guiding functional analyses of germline chromatin
dynamics.