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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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protein. Therefore, our investigation of Mcm10 in
Drosophila
has continued using a collection of three truncation alleles and over 20 missense mutations
generated via a Tilling screen; as well as four deletion alleles resulting from imprecise P-element excisions. This collection of mutants will allow for an in-
depth dissection of this conserved protein’s functions through the analysis of mitotic chromosome phenotypes; EdU incorporation analysis; and evaluation of
chromatin dynamics using PEV analysis, polytene chromosomes, and ovarian tissues. Genetic analyses such as complementation testing, unlinked non-
complementation screens, and yeast two-hybrid analysis will also allow for the evaluation of changes in interactions resulting from these mutations as well.
Throughout this study, we intend to elucidate the domains of the protein responsible for its biological functions, in hopes of better understanding Mcm10’s
roles in these essential biological processes, as well as replication and chromatin biology in general.
357C
HP1a Mediates the DNA Damage Response in Heterochromatin.
Joel Swenson
1,2
, Serafin Colmenares
2
, Irene Chiolo
2
, Cameron Kennedy
2
, Sylvain
Costes
2
, Gary Karpen
2
. 1) Molec & Cell Biol, Univ California-Berkeley, Berkeley, CA; 2) Genome Dynamics, Lawrence Berkeley National Lab, Berkeley,
CA.
Heterochromatin is characterized by the presence of Heterochromatin Protein 1a (HP1a) and is enriched for repeated sequences. Improper repair of DNA
double-strand breaks (DSBs) in repetitive elements leads to expansion or contraction of sequences, translocations, or aneuploidy. Studies from our lab
(Chiolo et al.) have shown that DSBs in heterochromatin are repaired differently from DSBs in euchromatin. We identified several components of this
pathway, but more information about the molecular components of this pathway are required to better understand this process. To address this we performed
a genome-wide RNAi screen to identify components involved in the relocalization of heterochromatic DSBs to euchromatin using pH2Av and HP1a
immuno-fluorescence. Data from our lab and the literature suggest that HP1a plays a key role in regulating the repair of DSBs in heterochromatin via the
homologous recombination (HR) pathway. As a functional secondary analysis we purified HP1a before and after damage and showed a change in associated
proteins. These proteins were identified by mass spectrometry analysis and several of these proteins are shown to be novel heterochromatin components. By
comparing the candidates identified by the biochemical approach and genome-wide RNAi approach we aim to identify components of the HP1a complexes
involved in the heterochromatic DNA damage response as well as a larger subset of proteins involved in this response.
358A
Chromatin remodeling during aging and dietary restriction in Drosophila melanogaster.
Jason G. Wood
1
, Peter V. Kharchenko
2
, Sara Hillenmeyer
1
,
Chengyi Chang
1
, Meyrolin Garcia
1
, Priyan Wickremesinghe
1
, Nan Jiang
1
, Peter J. Park
2
, Nicola Neretti
1
, Stephen L. Helfand
1
. 1) Molecular Bio, Cell Bio,
and Biochemistry, Brown University, Providence, RI; 2) Center for Biomedical Informatics, Harvard Medical School and Children's Hospital, Boston, MA.
Epigenetic changes during development have been widely studied, but relatively little is known about changes in chromatin structure that take place during
aging. We examined the epigenetic state of chromatin and its effect on gene expression during aging in adult Drosophila at both the whole genome and the
cellular level, and found dramatic reorganization of chromosomal regions with age. We observed a robust ChIP-seq enrichment of heterochromatin protein
HP1 as well as the H3K9me3 histone modification at the pericentric heterochromatin, the 4th chromosome, and islands of facultative heterochromatin
throughout the genome in young adult fly heads. In older animals, there is a decrease in HP1 and H3K9me3 signal in heterochromatic regions as compared
to euchromatic regions, resulting in a reduction in enrichment at characteristic heterochromatin loci. However, by Western blot we also observe a significant
increase in overall H3K9me3 signal with age, indicating the decreasing enrichment seen with age likely stems from an increase in H3K9me3 marks in
euchromatin rather than a reduction in heterochromatin. Interestingly, we also observed a similar decrease in enrichment when flies were reared on a dietary
restriction regime, an intervention that extends lifespan, as compared to controls on a richer food source, suggesting that the changes observed in
heterochromatin may be an adaptive rather than a deteriorative effect of aging. In addition, we performed whole transcriptome analysis using mRNA-seq and
observed a number of age and diet-related changes in gene expression. Preliminary experiments suggest that these changes in expression may be coupled to
the observed changes in chromatin structure, especially at heterochromatic loci.
359B
Invadolysin plays a role in the functioning of the SAGA complex.
Michal M. Janiszewski
1
, Shubha Gururaja Rao
2
, Edward Duca
1
, Margarete M.S. Heck
1
.
1) University/BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom; 2) Department of Molecular, Cell and
Developmental Biology, University of California, Los Angeles, California, USA.
In this study, we analyse the role of invadolysin, a novel and essential metalloprotease, in chromosome condensation through its interaction with the
SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex. We have previously shown that invadolysin interacts genetically with
non-stop
(
not
), a de-ubiquitinating
enzyme that has recently been shown to be a component of the SAGA chromatin-remodelling complex. Both
invadolysin
and
non-stop
mutants exhibit
phenotypic similarities in terms of diploid and polyploid chromosome structure abnormalities and accumulation of H2Bub1 and H3K4me3 histone
modifications. We also examined the localization of both H2Bub1 and H3K4me3 in greater detail in wild type polytene chromosomes from salivary glands.
Staining for H2Bub1 and H3K4me3 was noticeably diminished in the polytene chromosome chromocentre, suggesting that these two modifications are not
associated with heterochromatin. In addition, H2Bub1 with H3K4me3 showed a striking lack of co-localization on chromosomes arms, despite their general
‘co-appearance’ in cells. Intriguingly, whole mount immunostaining of
not
1
/IX-14
1
transheterozygous salivary glands revealed that H2Bub1 accumulates in
the cytoplasm, rather than the nucleus. As the SAGA complex exhibits both DUB and HAT activity, it is thus significant that mutants in other SAGA
subunits (
gcn5
,
ada2b
and
sgf11
) suppress an invadolysin-induced rough eye phenotype. Taken together, our data suggest that invadolysin may act to
regulate both the DUB and HAT activities of the SAGA complex.
360C
Investigating the Potential Interaction of SIN3 with Methionine Metabolism.
Mengying Liu, Valerie L. Barnes, Lori A. Pile. Department of Biological
Sciences, Wayne State University, Detroit, MI.
SIN3 is a master transcriptional scaffold and corepressor capable of transcription repression via associated histone deacetylases (HDACs) (Adrienne et al.,
2009). Our laboratory has previously found that a histone H3K4 demethylase named LID also co-immunoprecipitates with SIN3. This indicates that SIN3
may regulate methylation in addition to acetylation. Given that methionine is the major source of the methyl group for methylation (Cellarier et al., 2003),