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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
231
Variegated transvection by the enhancer
GMR
.
Jack R. Bateman, Justine E. Johnson, Melissa N. Locke. Dept Biol, Bowdoin College, Brunswick, ME.
In Drosophila, maternal and paternal homologs are intimately paired in virtually all somatic cells. Pairing of homologs can permit
trans
-interactions
between an enhancer on one homolog and a promoter on another, a phenomenon known as transvection. We recently established a transgenic system that
uses fluorescent reporters to assess the capacity of diverse enhancers to act in
trans
on a paired homolog. Using this system, we have shown that the eye
enhancer
GMR
is capable of activating expression of
GFP
via the
hsp70
promoter in
trans
. However, when we compare fluorescent intensities of individual
cells in the eye disc, we find that there is great variability in the strength of
GFP
activation from cell to cell. Such variation is not observed when
GMR
acts
in
cis
at this genomic position, implying that the variability results from mechanisms of transvection and not from local chromatin effects. Furthermore,
using quantitative RT-PCR, we show that activation in trans is decreased when a competing promoter is placed in
cis
to
GMR
, implying that the capacity of
an enhancer to act in
trans
is influenced by its local genetic environment.
369C
Histone Recognition and Nuclear Receptor Coactivator Functions of Drosophila Cara Mitad, a Homolog of the N-terminal Portion of Mammalian
MLL2/3.
Andrew K. Dingwall, Chhavi Chauhan, Megan Parilla, Manuel O. Diaz, Claudia B. Zraly. Stritch School of Medicine, Oncology Inst & Dept
Pathology, Loyola Univ Med Ctr, Maywood, IL.
MLL2/3 histone lysine methyltransferases are conserved components of COMPASS-like nuclear hormone receptor coactivator complexes. In vertebrates,
the paralogous ALR/MLL2 and HALR/MLL3 contain multiple domains required for proper epigenetic reading and writing of the histone code involved in
hormone-stimulated gene programming, including receptor binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and
MLL3 arose from a common ancestor. Phylogenetic analysis of MLL family proteins suggests that the ancestral gene underwent a fission event in some
Brachycera dipterans including Drosophila spp., creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila the C-
terminal SET domain is encoded by trithorax-related (trr), required for hormone dependent gene activation. We identified the cara mitad (cmi) gene that
encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor binding motifs. The cmi gene is essential and its
functions are dosage sensitive. CMI associates with TRR, as well as the EcR/USP receptor heterodimer and is required for hormone dependent transcription.
Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone stimulated transcription requires chromatin binding by
CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of ALR/MLL2 into two distinct genes in
Drosophila allows for important insight into the distinct epigenetic functions of the conserved readers and writers of the histone code.
370A
The piRNA is sufficient to guide Piwi to specific genomic sites to induce epigenetic changes.
Xiao Huang, Haifan Lin. Yale Stem Cell Center, New
Haven, CT.
Although the function of many epigenetic factors has been intensively studied, little is known about mechanisms that guide them to specific sites in the
genome. Previously, we showed that a Piwi-piRNA complex in Drosophila specifically binds to a genomic site complementary to that particular piRNA1
and that Piwi recruits Heterochromatin Protein 1a (HP1a) to chromatin2. This led us to propose that the piRNA guides PIWI and its associated epigenetic
factors to target genome1-3. To test this hypothesis, here we demonstrate that inserting piRNA-complementary DNA to an ectopic site either on the same
chromosome or on a different chromosome leads to the recruitment of Piwi to these sites; whereas inserting a non-complementary sequence to the same
ectopic site does not lead to Piwi recruitment. Piwi recruitment is abolished by RNase treatment, supporting the role of piRNAs in recruiting Piwi to
chromatin3. The ectopic site without the piRNA-sequence insertions shows no Piwi binding; therefore the Piwi-piRNA complex is recruited to, but not
originated from, the ectopic site. These observations together demonstrate that piRNAs are both necessary and sufficient to guide Piwi to specific genomic
sequences by sequence complementarity. Furthermore, ectopic Piwi recruitment is accompanied by the enrichment of HP1a, Su(var)3-9, and repressive
chromatin marks H3K9me2, H3K9me3 as well as reduction in Pol II transcription activity. These data implicate that Piwi-piRNA complexes guide
epigenetic factors to specific genomic sties.
371B
PNUTS-PP1 associates with transcriptionally active sites on interphase chromosomes and is required for cell survival.
Louise Rebecca Rawling
1
,
Anita Lucaci
1
, Andrey Rudenko
2
, Peter Glenday
1
, Luke Alphey
2
, Daimark Bennett
1
. 1) Inst Integrative Biology, Univ Liverpool, Liverpool; 2) Dept
Zoology, Oxford University, Oxford.
Tight regulation of gene expression is critical for cells to respond normally to physiological and environmental cues and to allow cell specialization.
Reversible phosphorylation of key structural and regulatory proteins, from histones to the transcriptional machinary, is acknowledged to be an important
mechanism of regulating spatial and temporal patterns of gene expression. Protein Phosphatase 1 (PP1), a major class of serine/threonine protein
phosphatase, is found at many sites on
Drosophila
polytene chromosomes where it has many important roles in controlling gene expression and chromatin
structure. PP1 is targeted to different chromosomal loci through interaction with a variety of different regulatory subunits, which are thought to modify PP1’s
activity towards specific substrates. Here we describe the
in vivo
role of PNUTS (PP1 Nuclear Targeting Subunit), one of the most abundant PP1-binding
proteins in the mammalian nucleus.
Drosophila
PNUTS is an essential gene. In proliferating tissues mutant cells become basally localised, express activated
caspase and undergo cell death. Binding to PP1 is essential for PNUTS function. PNUTS protein is found in the nucleus and, during interphase, is
chromosomally localised and associates with PP1 at active sites of transcription in an RNA-dependent manner, suggesting a role in transcriptional
regulation. We will present here our results from genetic and cell biological experiments to identify potential substrates of chromosome-associated PNUTS-
PP1, which help to explain the requirement for this holoenzyme in cell survival.
372C
Mapping the
Telomere elongation
mutation in Drosophila.
Hemakumar M. Reddy, James M. Mason. Laboratory of Molecular Genetics, NIH/NIEHS,
Research Triangle Park, NC.
Telomeres are necessary to prevent activation of the DNA damage response and to maintain chromosome length. Drosophila telomeres differ from those in
mammals in the mechanism of telomere maintenance, as Drosophila lacks telomerase. Drosophila telomeres contain a terminal array of non-LTR