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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
223
337A
Lipid droplets control histone levels and promote mitotic fidelity in syncytial embryos.
Michael A. Welte
1
, Zhihuan Li
1
, Dipak Manna
1
, Katharina
Thiel
3
, Mathias Beller
2,3
. 1) Dept Biol, Univ Rochester, Rochester, NY; 2) Inst Math Modeling Biol Systems, Heinrich-Heine Univ, Düsseldorf, Germany; 3)
Max Planck Inst for Biophys Chem, Göttingen, Germany.
Histones are essential for chromatin packing, yet free histones not incorporated into chromatin are toxic. Early
Drosophila
embryos contain massive
amounts of maternally provided histones: What is the biological function of these histone deposits, and why do they not interfere with development? The
extra-nuclear stores of histones H2A, H2B, and H2Av are bound to lipid droplets, fat storage organelles abundant in embryos. We now report that histones
are anchored to droplets via the novel protein Jabba: Jabba localizes to droplets, co-immunoprecipitates with histones, and is necessary to recruit histones to
droplets.
Jabba
mutant embryos lack the maternal H2A, H2B and H2Av deposits altogether; apparently, these deposits are degraded unless sequestered on
droplets. In yeast, degradation of free histones prevents damage from histone overexpression.
Jabba
embryos develop grossly normally and, by translating
maternal mRNAs, can reach near normal histone levels by cycle 14. Thus, new histone synthesis can be sufficient to sustain life. The maternal protein
deposit nevertheless contributes to chromatin assembly: First, histones can be transferred from droplets to nuclei, as demonstrated by transplantation and
photo-activation experiments. Second, reducing histone mRNA levels in
Jabba
mutants results in lethality during syncytial stages, with widespread mitotic
defects. Histone sequestration on lipid droplets may additionally mitigate the toxicity of supernumerary histones: overexpression of H2Av in
Jabba
embryos
results in synthetic lethality and phenotypes indicative of defects in replication and mitosis, including anaphase bridges and massive nuclear fallout. We
propose that in wild-type embryos sequestration on lipid droplets buffers the histone supply: droplets provide a source of histones for chromatin assembly by
shielding them from premature degradation, yet they limit the pool of free, potentially toxic histones.
338B
Psf2: A Role in Chromosome Condensation.
Jeffrey P. Chmielewski
1
, Laura Henderson
2
, Tim Christensen
1
. 1) Biology, East Carolina University,
Greenville, NC; 2) Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA.
In
D. melanogaster
, the CMG complex is a group of proteins that function as the DNA helicase during replication. The CMG complex is composed of
cdc45, MCM2-7, and the GINS complex. The GINS complex is a heterotetrameric complex composed of the protein subunits Psf1, Psf2, Psf3, and Sld5.
Recent research in human dermal fibroblasts shows GINS is essential for the initiation and elongation stages of chromosomal replication. Working with a
null mutation, I have designed a series of experiments aimed at elucidating the function of Psf2
in vivo
. Using phosphoH3 immunostaining and M-phase
indices, I have shown that heterozygous mutants exhibit a significant M-phase delay. EdU incorporation assays in third in-star larval brains shows no
significant difference in the number of cells in S-phase. However, when compared to wild type, the pattern of EdU incorporation indicates cells take longer
to replicate euchromatin; possibly resulting in the improper packaging of euchromatin as heterochromatin. To corroborate the data seen in the pattern of EdU
incorporation, we designed a novel technique to establish the packing ratio of salivary gland polytene chromosomes. Using this novel technique, we are able
to show that heterozygous mutants exhibit a significant increase in packing ratio compared to WT. Additionally, in the later stages of egg chamber
development, nurse cell nuclei display overly condense polytene chromosomes during the psuedo M-phase at the end of endocycle 5. We have evidence that
indicates these instances of overly condensed chromosomes results in apoptotic egg chambers, seen later in development. We have also taken a genetic
approach to further substantiate our cytological characterizations. Heterozygous Psf2 flies show a significant decrease in viability with complete
homozygous lethality. Position effect variegation analysis show Psf2 enhances variegation an indication that chromosomes are overly condensed. This data,
when combined, suggests Psf2 has a role during replication that ultimately determines how DNA is packaged.
339C
Multiple interactions between Heterochromatin Protein 1 (HP1) and nucleosomes.
Diane E. Cryderman
1
, Abd Elhamid M. Azzaz
2
, Michael W.
Vitalini
1
, Andrew H. Thomas
1
, Adrian H. Elcock
1
, Michael A. Shogren-Knaak
2
, Lori L. Wallrath
1
. 1) Dept Biochemistry, Univ Iowa, Iowa City, IA; 2)
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA.
The Heterochromatin Protein 1 (HP1) family is comprised of conserved nonhistone chromosomal proteins that package chromatin, regulate transcription
and function in DNA repair. HP1 proteins contain an N-terminal chromo domain (CD) and C-terminal chromo shadow domain (CSD), separated by an
unstructured hinge region. The association between HP1 and chromosomes is thought to involve HP1 dimerization and interaction with histone H3 di- and
tri-methylated at lysine 9 (H3K9me2/3). It is not known how HP1 interacts with nucleosomes to form heterochromatin. A multi-disciplinary approach using
in silico
modeling,
in vitro
biochemistry and
in vivo
functional assays was undertaken. Molecular dynamic simulations and atomic resolution modeling
revealed that human HP1
Hsα
is a highly flexible protein possessing the ability to bind both adjacent and non-adjacent methylated nucleosomes within an
array. Models generated
in silico
guided
in vitro
experiments showing that HP1
Hsα
fosters chromatin compaction by interacting with nucleosomes within an
array and enhancing interactions between nucleosome arrays. These
in vitro
findings supported
in vivo
studies in which Drosophila HP1a was found to
promote interactions between distant chromosome sites, akin to inter-array interactions observed
in vitro
. These
in vivo
interactions were reduced upon
expression of mutant forms of HP1a that lacked the hinge or the ability to dimerize. The dimerization mutant failed to support viability of an HP1a null.
Surprisingly, flies expressing only the hinge deletion were viable and possessed nucleosome arrays characteristic of heterochromatin, suggesting the hinge is
dispensable for heterochromatin formation and viability. Taken together, dimerization and the flexibility of HP1 allow for multiple interactions with
nucleosomes necessary for heterochromatin formation.
340A
Understanding the Role of
Topoisomerase 2
in Chromosome Associations.
Amber M. Hohl
1,2,3
, Pamela K. Geyer
2
, Ting Wu
3
. 1) Genetics Program,
University of Iowa, Iowa City, IA; 2) Department of Biochemistry, University of Iowa, Iowa City, IA; 3) Department of Genetics, Harvard Medical School,
Boston, MA.
Homologous chromosomes display associations in many organisms. In Drosophila, chromosomes are paired in all somatic cells throughout development.
For many genes, the degree of homolog association influences gene expression. These effects, collectively referred to as transvection, can promote gene
activation or silencing. The requirements for transvection are poorly understood. Recent studies implicated a requirement for
Topoisomerase 2
(
Top2
) in
chromosome pairing. To expand our understanding of the role of Top2 in chromosome associations, we tested whether mutations in
Top2
disrupted