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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
232
retrotransposons (HTT array). This array contains three elements,
HeT-A
,
TART
, and
TAHRE
, of which
HeT-A
is the most abundant. Transposition and gene
conversion are the two major mechanisms of telomere elongation. A dominant mutation
Telomere elongation, Tel
, causes cytologically visible elongation of
telomeres.
Tel
was mapped to 69 on the genetic map, corresponds roughly to 92 on the cytological map. It was further localized to a 318 kb region between
92A2 to 92A11, using
P
element mediated male recombination. To further map the
Tel
locus, we selected 3
P
elements and 2
Minos
elements that subdivide
this region. Recombination was induced in males that carried each of these elements individually, and recombinants were put into stocks and DNA collected
at intervals for 12 generations. Quantitative PCR was performed to estimate
HeT-A
copy number in these recombinants. The results show that
Tel
maps
between the transposons
P{PZ}Dl
05151
and
P{XP}Ino80
d10097
, a 77 kb region that contains
Ino80
, a ncRNA, a pseudo gene, a miRNA, 8 unannotated genes,
and a 40 kb well-conserved intergenic region. Next generation sequencing of
Tel
and a
y w
control was done to identify signature differences in the sequence
by comparing them with FlyBase reference sequence and with DGRP sequences. DGRP is a resource panel of 192 wild-caught, inbred lines, whose
sequences were available in a public database.
HeT-A
copy number analysis of DGRP lines identified 5 lines that showed elongated telomeres, on par with
Tel
, and 3 others with moderately increased copy number. We expect that mapping of the genetic factor(s) responsible for the increased
HeT-A
copy number
in these DGRP lines, coupled with subdivision of the 77 kb region using
Minos
element insertions that have recently become available will identify the
Tel
mutation.
373A
An RNA-seq screen for allele-specific parent-of-origin effects in
Drosophila melanogaster
.
Kevin H.C. Wei
1
, Julien F. Ayroles
1,2
, Daniel A. Barbash
1
,
Andrew G. Clark
1
. 1) Molecular Biology and Genetics, Cornell, Ithaca, NY; 2) Harvard Society of Fellows, Harvard, Cambridge, MA.
Genomic imprinting is a non-Mendelian mode of inheritance where the expression of each allele depends on the parent-of-origin. In classical cases of
imprinting, characterized mostly in mammals, epigenetic modifications are deposited in the parental germline causing parent-specific silencing of one allele
in the offspring. Evidence for imprinting in
Drosophila melanogaster
comes from complex genetic crosses, where mutations that induce position effect
variegation produce different variegating phenotypes depending on transmission from the mother or father. However, to date, no naturally occurring variants
have been found to display genomic imprinting. Moreover, no genes have been identified to show parent-specific silencing. We generated transcriptome
sequences by RNA-seq for offspring of reciprocal crosses and used read counts to identify distortion of allele proportions characteristic of imprinted
expression. Chi-square tests of these counts show that in the reciprocal crosses between lines Beijing11 (B11) and Netherland4 (N04), 507 and 665 genes
have significant distortions in daughters and sons respectively. As the chi-square test is expected to yield many false positives due to the compound binomial
sampling in library preparation, cluster generation and sequencing, we focused on the 147 genes significantly distorted in both sexes of offspring. In this set,
140 (95%) share the same direction of distortion, and they cluster broadly along chromosome arms. Many of these display parent-specific silencing of only
B11 but not N09 alleles, some of which we confirmed with allele-specific restriction enzyme digestion and pyrosequencing. However, in a separate
reciprocal cross between Beijing14 (B14) and Tasmania9 (T09), the same tests fail to detect significant distortions of allele proportions. Unlike classical
cases in mammals with species-wide imprinted loci, these results suggest that in flies “imprinting” is a strain-, and perhaps cross-specific phenomenon.
374B
De novo establishment of Polycomb-mediated repression.
Jumana AlHaj Abed, Siddhi Desai, Judith Benes, Richard Jones. Biology, Southern Methodist
University, Dallas, TX.
Polycomb group proteins (PcG) are epigenetic regulators that control target genes by taking over repression from gene-specific transcription factors. Once
PcG-mediated repression is established, it is maintained through many cell divisions. Although maintenance of repression by PcG proteins is well
characterized, the mechanism by which PcG proteins initially recognize repressed state of a gene remains ambiguous. The challenge to understanding PcG
silencing mechanisms in vivo is the difficulty of acquiring a homogeneous population of cells in which all cells are exhibiting PcG-mediated repression of a
particular gene. We will report on our progress towards characterizing the molecular and biochemical events that lead to the initiation of PcG-mediated
repression of
giant
(
gt
) in a genetic background with a homogenous population of cells with respect to PcG-mediated repression. Embryos will be collected
over a time course where initially
gt
is repressed by maternally expressed Hb, and in which the PcG is required to maintain its ubiquitous repression at a later
stage. Chromatin immunoprecipitiation (ChIP) will allow analysis of distribution of PcG proteins in addition to
gt
activators and repressors during the
developmental window at which repression transitions from Hb to repression by PcG proteins. Whole-mount embryo staining of transgenic reporter lines
will allow the functional identification of Polycomb Response Elements (PREs) within the upstream regulatory region of
gt
, and will aid in the interpretation
of the ChIP data.
375C
Stuxnet destabilizes Polycomb-associated PRC1 complex to facilitate Notch receptor gene transcription.
Juan Du
1
, Junzheng Zhang
1
, Feng Tie
2
, Ying
Su
1
, Min Liu
1
, Peter Harte
2
, Alan Zhu
1
. 1) Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; 2) Department of
Genetics, Case Western Reserve University, Cleveland, OH, USA.
The activities of developmental signaling are controlled by a large array of post-translational modification events. By contrast, very little is known about
the mechanisms that regulate the expression of their core pathway components. Using an in vivo RNAi screen, we identified a new gene, stuxnet, that
functions as a key component of Notch signaling, a process at the core of cell fate decisions in development, adult tissue homeostasis and cancer. We
generated a lethal null allele of stuxnet through an FLP/FRT-based technique and demonstrated that stuxnet is required for transcription of the Notch
receptor gene in the wing imaginal disc. We found that this stuxnet lethal mutation can be rescued by reducing the activity of Polycomb (Pc), an essential
component of the Polycomb Repressive Group complex 1 (PRC1) that is known to epigenetically silence target genes critical for animal development.
Consistently, we found that Stuxnet protein physically interacts with and subsequently destabilizes Pc protein and its associated PRC1 complex in vivo.
Thus, Stuxnet protein facilitates Notch signaling by destabilizing the PRC1 complex, thereby reducing the repressive chromatin modification marker
imposed on the Notch receptor locus. We named this novel gene stuxnet after the powerful virus that destroys PC computers. Our work identified a novel
mechanism for the control of the activity and stability of the PRC1 transcriptional silencing machinery in development.
376A