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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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Heterochromatin Protein 1a (HP1a). Though traditionally associated with gene silencing or position-effect variegation, heterochromatin also contains many
genes that are transcriptionally active despite high levels of HP1a. KDM4A, a member of the jumonji family of protein demethylases exhibits specificity for
the tri- and di-methylated forms of H3K36 and binds HP1a. In this study, we show that KDM4A is enriched in heterochromatin, particularly over active
genes, and functions as a suppressor of variegation of reporter genes. We further show that KDM4A exhibits differential effects in H3K36 methylation levels
between euchromatic and heterochromatic genes, and that loss of KDM4A alters organization of the heterochromatin landscape. Surprisingly, we find that
KDM4A also plays a role in the clearing of DNA repair foci from heterochromatin, which is thought to proceed through movement of damaged
heterochromatic DNA into euchromatin in a HP1a-dependent manner. We propose that KDM4A functions to regulate heterochromatin organization and
dynamics of HP1a complexes.
349A
Functions of the RNAi system and heterochromatin components in heterochromatin formation.
Tingting Gu, Sarah Elgin. Biology, Washington
University, St Louis, MO.
The RNAi system is believed to be involved in post-transcriptional silencing of transposable elements (TEs) both in germline and somatic cells, but
whether it also operates through a chromatin-based transcriptional silencing mechanism in Drosophila is not clear. To explore whether and when the RNAi
system and other heterochromatin components are essential for silencing by heterochromatin formation, we use the GAL4-UAS system to knock down genes
specifically in early embryos, when heterochromatin is first observed, or at later stages, to study their function in initiation and maintenance of
heterochromatin during development. We focus on PIWI (piRNA system), HP1a (heterochromatin component), and EGG (histone methyltransferase). Using
the germline specific nos-GAL4 driver and the shmiR hairpins produced by the Perrimon lab, we are able to knock down the mRNA levels of these proteins
in 1.5-3 hr early embryos (the critical time for heterochromatin initiation); in all three cases this leads to elevated expression of hsp70-lacZ PEV reporters
inserted in the Y chromosome or at the pericentric region of 3L in both 3rd instar larvae and adult flies, suggesting that early depletion of PIWI, HP1a or
EGG has a long-lasting effect in later stage animals. ChIP-qPCR assays show that the increased expression of hsp70-lacZ is coupled with HP1a depletion on
the hsp70 promoter region, arguing that this long-lasting effect is chromatin-based. Knock down of two of these protein products in the developing eye
(using the eye lineage-specific ey-GAL4 driver) results in loss of silencing of the wm4 reporter, suggesting that HP1a and EGG, but not PIWI, are also
crucial in the maintenance of heterochromatin structure in developing animals. On-going experiments are examining the distribution of HP1a and H3K9me2
in piwi mutant animals, to look in detail at the impact of PIWI on heterochromatin. Our efforts to dissect the roles of RNAi and heterochromatin components
in different developmental stages will shed light on how heterochromatin is established and maintained.
350B
dSet1 acts as the main global H3K4 di- and tri-methyltransferase throughout Drosophila development.
Graham Hallson, Robert E. Hollebakken,
Taosui Li, Monika Syrzycka, Inho Kim, Shawn Cotsworth, Kathleen A. Fitzpatrick, Donald A. R. Sinclair, Barry M. Honda. Dept MBB, Simon Fraser Univ,
Burnaby, BC.
In eukaryotes, the post-translational methylation of H3 lysine 4 (H3K4) is catalysed by the Set1 and MLL classes of histone methyltransferases (HMTs),
and correlates with active transcription and open chromatin. The MLL classes of HMTs Trithorax (Trx) and Trithorax-related (Trr), were thought to be
responsible for this modification in Drosophila, along with possible contributions from the trithorax group protein Ash1. In our efforts to functionally
annotate essential genes within the centric heterochromatin of the Drosophila third chromosome, we have linked a genetically defined heterochromatic locus
(
lethal 5
/
l(3L)h5
) to the Drosophila ortholog of
set1
(
dSet1
). Surprisingly, we observe that dSet1 acts as the main H3K4 di- and trimethylase throughout
development: levels of di- and trimethyl H3K4 are significantly reduced in
dSet1
mutants but not in animals carrying mutations in or expressing dsRNAs
targeting
trr
,
trx
, or
ash1
. We also provide biochemical evidence that dSet1 interacts within an evolutionary conserved protein complex (COMPASS), and
demonstrate the functional requirement of other members of this complex for H3K4 methylation. Our results establish a model system for studying the
functional roles of and mechanisms underlying H3K4 methylation in animal development.
351C
Loss of heterochromatic repression with age in Drosophila.
Nan Jiang, Guyu Du, Ethan Tobias, Stephen Helfand. Molecular Biology, Cell Biology and
Biochemistry, Brown University, Providence, RI.
In the nucleus, chromatin structure is an essential player in regulation of gene expression and maintenance of genome stability. It has recently been
observed that certain chromatin marks undergo changes in distribution in aged organisms, raising a hypothesized link between the chromatin changes and the
age-related dysregulation of genes. To test the possible effect of these chromatin changes on gene regulation in flies, we used an inducible reporter gene
residing near heterochromatin to measure its expression in individual cells from different tissues during aging. With age there is a strong increase in reporter
gene expression in diverse tissues, suggesting a loss of heterochromatic repression in those genomic regions. Furthermore, this age-related increase in loss of
gene silencing is delayed by calorie restriction, an intervention that is shown to extend the life span. These results provide evidence for a change in gene
regulation with age resulting directly from the changes of chromatin, highlighting the role of chromatin in the aging process.
352A
Separation of stem cell maintenance and transposon silencing functions of Piwi protein.
Mikhail S. Klenov, Olesya A. Sokolova, Evgeny Y. Yakushev,
Sergey A. Lavrov, Vladimir A. Gvozdev. Dept Molecular Gen of Cell, Inst Molecular Genetics, Moscow, Russia.
piRNAs and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germlines. The founder of the Piwi
subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germline stem cells (GSCs). It remained unknown whether
the failure of GSC maintenance is related to transposon derepression or GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein.
We have revealed a new mutation piwiNt removing the nuclear localization signal of the Piwi protein. piwiNt females retain the ability of GSC self-renewal
and a near to normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their
transcripts in the germline. piwiNt mutants are sterile most likely due to the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin
modifications in the piwiNt ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the