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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
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nuclei without their chromatin modification.
353B
Functional Characteristics of HP1a.
Deanna L Mendez
1
, Sepideh Khorasanizadeh
2
, Sarah CR Elgin
1
. 1) Department of Biology, Washington University,
CB-1137, St. Louis, MO 63130; 2) Sanford-Burnham Medical Research Institute, 6400 Sanger Road, Orlando, FL 32827.
Eukaryotic genomes are roughly partitioned into heterochromatin, gene-poor domains that remain highly packaged throughout the cell cycle, and
euchromatin, gene-rich domains that are more accessible for transcription. Heterochromatic silencing is thought to be essential to maintain the integrity of
the genome, minimizing transposition of endogenous DNA transposons and retroviruses found in eukaryotic genomes. Heterochromatin Protein 1a (HP1a)
plays a key role in establishing and maintaining heterochromatin structure. HP1a binds to a wide array of protein partners among which are HP2, a non-
histone chromosomal protein and PIWI, an RNA binding protein. We have found that HP2 binds 66-fold more tightly to HP1a compared to HP2 in a
fluorescence polarization assay, consistent with their predicted roles. This differential affinity corresponds to a more extensive binding surface for HP2
compared to PIWI, as shown by mapping the chemical shift perturbations of the HP1a residues in a complex with HP2 or PIWI respectively. In addition,
deleting the HP1a C-terminal extension differentially impacts HP2 and PIWI binding. We are continuing to investigate the binding surface of HP1a by
systematically mutating hydrophobic patches with the goal of identifying the patch responsible for the preferential binding of HP2. Our results are consistent
with a model where heterochromatin is initiated through a transient interaction between HP1a and PIWI, but once initiated, heterochromatin is maintained
through the more stable interaction between HP1a and HP2. Thus HP1a has a number of built in mechanisms to discriminate between its binding partners as
a means to coordinate its various functions. This research is supported by NIH GM068388 to SCRE and GM 070558 SK.
354C
Identifying Proteins that Interact with Drosophila melanogaster Heterochromatin Protein 2 (HP2) and Characterizing Their Contribution to
Heterochromatin Formation.
Patrick C. Ng
1
, Elizabeth E. Slawson-Tempel
1
, Hien P. Nguyen
2
, Chris D. Shaffer
1
, Sarah C. Elgin
1
. 1) Biology, Washington
Univ, St. Louis, MO; 2) Biochemistry and Molecular Biology, Saint Louis Univ, St. Louis, MO.
Heterochromatin Protein 2 (HP2) interacts and co-localizes with Heterochromatin Protein 1 (HP1a) within D. melanogaster chromatin, and is itself
involved in heterochromatin formation. Several mutations in the HP2 gene cause suppression of position effect variegation (PEV), a loss of reporter gene
silencing. Of the 17 HP2 mutations recovered, three missense mutations, 288, P2763L, and 230, have been identified, one each in exons 6, 8, and 9 of HP2;
the latter two have been selected for further study. We postulate that interactions between HP2 and its binding partners at the sites of these mutations impact
heterochromatin formation. A Yeast-2-Hybrid (Y2H) mating screen was utilized to find proteins potentially interacting with HP2 exon 9, and identified 37
protein-coding clones from the D. melanogaster library. None of these clones displayed a loss of protein interaction with the mutant form of HP2 exon 9
relative to its wild type counterpart. However, a Y2H screen did identify interacting proteins that distinguish between the wild type and missense mutation in
HP2 exon 8. We examined one of these proteins,
cheerio
, for possible Su[var] effects by comparing expression of a
lacZ
reporter in D. melanogaster stocks
with wild type or mutant
cheerio
. In these experiments the
lacZ
gene has been juxtaposed with a region of heterochromatin to produce PEV. Differences in
the β-galactosidase activity are observed qualitatively in tissue staining and quantitatively in whole fly assays. Differences in wild type and mutant
suppression of PEV for
white
activity are observed in another exon 8 interacting protein,
sinuous
. The results indicate that
cheerio
and
sinuous
are associated
with gene silencing due to heterochromatin formation. Supported by grant GM068388 to SCRE and a WU/HHMI SURF to PCN.
355A
Studying the functions of the hybrid lethality proteins- LHR and HMR.
Satyaki P. Rajavasireddy, Nathan L Clark, Tawny Cuykendall, Shuqing Ji,
Hojoong Kwak, Daniel Barbash. Molecular Biology and Genetics, Cornell University, Ithaca, NY.
The Dobzhansky-Muller (DM) model suggests that hybrid incompatibilities are caused by negative epistasis between divergent genes among sister species.
However, this model does not suggest a cause for the interspecific divergence between these hybrid incompatibility (HI) genes. Further, it makes no
prediction for the molecular mechanism underlying HI. Our lab is utilizing hybrids of
D. melanogaster
mothers and
D. simulans
fathers to address these
questions. In this system, hybrid male progeny die as larvae. This lethality is caused in part, by two rapidly evolving genes-
Lethal hybrid rescue
(
Lhr
) and
Hybrid male rescue
(
Hmr
). We seek to understand the functions and interactions of
Lhr
and
Hmr
in
D.melanogaster
in order to answer the questions of what
is causing them to rapidly evolve and how they cause lethality in hybrids.
Hmr
encodes a putative nuclear protein necessary for normal levels of female
fertility in
D. melanogaster
. LHR is an HP1 interacting protein with extensive localization to pericentric heterochromatin and the telomeres. We have
previously shown that both
Lhr
and
Hmr
are required for normal levels of female fertility. We report here that
Lhr
and
Hmr
orthologs show highly correlated
rates of evolution along different
Drosophila
lineages suggesting that they may be coevolving. These signatures of co-evolution suggest that LHR and HMR
may have similar functions. We make multiple observations that support this model. First, mutations in both genes affect female fertility. Second, we find
that LHR and HMR can be co-immunoprecipitated showing that they are members of the same complex. Third, we find that both HMR and LHR, localize to
pericentric heterochromatin and the telomeres. However, we find that HMR additionally localizes to the nucleolus. We are now carrying out experiments to
test if LHR and HMR affect either structure or function at pericentric chromatin, at the telomeres or the nucleolus.
356B
A dissection of Mcm10’s functions in
D. melanogaster
.
Michael C. Reubens, Casi Strickland, Tim W. Christensen. Biology Dept, East Carolina
University, Greenville, NC.
Highly efficient DNA replication is essential for the accurate transmission of genetic material from cells to their progeny; likewise, the maintenance of
epigenetic chromatin states is essential for the faithful reproduction of the transcriptional state of the cell. Improper regulation, and coordination, of these
essential processes can result in genomic instability, which can manifest in disease or potentially the death of the organism. It is becoming more apparent
that these two processes are linked through interactions between DNA replication proteins and chromatin associated proteins. Recently our lab demonstrated
that Mcm10 not only plays a role in DNA replication, but also has a role in heterochromatic silencing and chromosome condensation; thus the
D.
melanogaster
homolog Mcm10 provides an excellent subject to study the connections of these two essential processes. Interaction studies in yeast, as well as
phenotypic and genetic analyses in Drosophila, imply that the conserved C-terminus is important for the many interactions carried out by this promiscuous