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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
230
function together in regulating cell cycle and developmental pathways.
364A
Studies into functional aspects of two isoforms of Drosophila SIN3.
Nirmalya Saha, Lori Pile. Wayne State University, Detroit, MI.
SIN3 along with the histone deacetylase (HDAC) RPD3 form a co-repressor complex in which SIN3 functions as a scaffold protein. Previous studies
identified two isoforms of SIN3, SIN3 220 and SIN3 187, which differ only in the presence of extra C-terminal domain in SIN3 220. The two isoforms are
found in distinct complexes with some unique proteins (though many proteins are common in both SIN3 isoforms). Additionally the complexes showed
differences in deacetylation potential. Given the differences in structure and protein binding capabilities, we hypothesize that SIN3 220 and SIN3 187 will
exhibit significant differences in function in the context of repression of transcription. Assuming the functional differences, we are studying the binding sites
of SIN3 isoforms using the ChIP-qPCR technique. Previous studies also suggested that acetylation and deacetylation affect the nucleosomal density along
the gene. We are performing MNase mapping after over expressing the isoforms and knocking down SIN3 in Drosophila S2 cells to study the changes in
nucleosomal density along various regions of a gene. Results from these experiments are anticipated to elucidate functional differences between the isoforms
and provide insight into the mechanism of action by which the isoforms repress transcription.
365B
Elucidating the contribution of distinct Su(Hw) zinc fingers in DNA association and female fertility.
Ryan M. Baxley
1
, Alexey A. Soshnev
1
, Michael
W. Klein
2
, Ashley B. Gaeth
2
, Joel A. Morales-Rosado
2
, Bing He
3
, Kai Tan
3
, Pamela K. Geyer
1,2
. 1) Molecular and Cellular Biology Program, University of
Iowa, Iowa City, IA; 2) Biochemistry Department, University of Iowa, Iowa City, IA; 3) Internal Medicine Department, University of Iowa, Iowa City, IA.
Suppressor of Hairy-wing [Su(Hw)] is a twelve zinc finger (ZnF) DNA binding protein that localizes to ~3,000 broadly distributed sites genome wide.
Su(Hw) has two known functions. First, Su(Hw) establishes an insulator within the
gypsy
retrotransposon. Second, loss of Su(Hw) results in female sterility,
characterized by apoptosis during mid-oogenesis. To better understand the role of Su(Hw) in oogenesis, an EMS mutagenesis screen was performed. This
screen differed from previous studies because
su(Hw)
mutations were identified based on female fertility and insulator functions. From ~8,000 mutagenized
chromosomes screened, four new
su(Hw)
alleles were identified. These include two new alleles that genetically separate Su(Hw) functions. One allele falls
into a novel class that retains insulator function but is female sterile. Molecular characterization demonstrated that these novel alleles carried mutations in
ZnF4 and ZnF8, respectfully. Our current studies focus on defining how changes in individual ZnFs alter Su(Hw) function. To this end we are analyzing
DNA binding of Su(Hw) mutants in vitro and in vivo. We find that loss of a single ZnF decreases in vitro binding and alters the distribution of retained sites
in the genome. In general, retained sites are enriched for Su(Hw) interaction partners Mod67.2 and CP190. Together, our data suggest that genome wide
occupancy of Su(Hw) binding sites have distinct ZnF requirements.
366C
In vivo
function of Homie, the
eve
insulator: Is it a PRE blocker?
Miki Fujioka, James B Jaynes. Dept Biochem & Molec Biol, Thomas Jefferson Univ,
Philadelphia, PA.
Recently, we described the properties of an
even skipped (eve)
insulator, named Homie (Homing insulator at eve), located at the 3’ end of the
eve
locus,
between an
eve
PRE and the adjacent gene. Homie has both enhancer blocking activity and P-element transgene homing activity. The third feature of Homie
is that it is capable of causing long-range enhancer-promoter (E-P) communication between genes located several megabases apart. Our studies indicate that
a transgenic copy of Homie interacts physically with the endogenous Homie, which causes a transgenic promoter to communicate with the endogenous
eve
enhancers. Two transgenic copies of Homie can also interact to facilitate long-range communication. How do these properties of Homie relate to its
in vivo
function? In order to test this, we created a “native context” transgene that contains the entire
eve
locus and the 5’ end of the adjacent gene
TER94
, including
its 1st 2 introns, which contain enhancers that drive the near-ubiquitous
TER94
embryonic expression. In this transgene, the coding regions are replaced with
reporter genes, so that promoter activity can be easily monitored. Using this construct, we see that Homie has the ability to block the activity of the
eve
PRE.
Monitoring
TER94
promoter activity, when Homie is removed, the initially strong, ubiquitous expression that mimics
TER94
expression changes to mimic
the
eve
pattern. When the PRE is also deleted, the pattern reverts to that of
TER94
. This suggests that one function of Homie is to shield the
TER94
promoter
from repressive effects of the
eve
PRE. Furthermore, in keeping with its ability to facilitate long-range communication, results with these same transgenes
suggest that in addition to blocking PRE-dependent repression, Homie facilitates interaction between the
eve
promoter and the set of enhancers located 3’ of
the
eve
coding region. Our working model is that Homie interacts with the
eve
upstream promoter region, looping out the intervening DNA.
367A
A Role for dCAP-D3/Condensin II in Preventing Natural Transposon Mobilization.
Michelle Longworth, Andrew Schuster. Department of Molecular
Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH.
Condensin complexes are conserved from bacteria to humans and are well known for their role in promoting chromatin condensation at the beginning of
mitosis. Recently we discovered a non-mitotic role for the
Drosophila
Condensin II complex: the Condensin II component, dCAP-D3, regulates transcription
of clusters of genes, partly in combination with the
Drosophila
retinoblastoma protein homolog, RBF1. Surprisingly, approximately 1/3 of all dCAP-D3
targets are positioned within 5 kb of a natural transposon. We analyzed a number of loci containing dCAP-D3 target gene clusters in close proximity to
natural transposons and found that the transposon DNA in the area had mobilized in dCAP-D3 mutants. Transposon mobilization events increase with
decreasing expression levels of dCAP-D3 protein. In addition, mutants heterozygous for two other Condensin II subunits also demonstrate similar
transposon mobilization events. Finally, we present data that acute knockdown of dCAP-D3 by RNAi results in partial loss of Transposon DNA sequence
and generation of possible intermediates of a Homologous Recombination event. Our lab and others have previously demonstrated that
Drosophila
Condensin II subunits can influence the spread of chromatin marks in Position Effect Variegation assays and we are currently analyzing whether decreased
dCAP-D3 expression levels result in changes in histone modifications following transposon mobilization. We propose a working model whereby dCAP-
D3/Condensin II prevents the homologous recombination of transposons by acting to maintain a repressive chromatin state.
368B