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Full Abstracts – CHROMATIN AND EPIGENETICS
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Dynamic and Tissue Specific Regulation of Gene Activation and Silencing by Noncoding PRE/TRE Transcription.
Leonie Ringrose
1
, Adelheid
Lempradl
1,2
, Frank Ruge
1
, Helena Okulski
1
, Christina Altmutter
1
, Gerald Schmaus
1
, Karin Aumayr
1,3
, Hasene Basak Senergin
1
, Andrew Dimond
1,4
. 1) IMBA
(Institute of Molecular Biotechnology), Vienna, Austria; 2) MPI (Max Planck Institute of Immunology and Epigenetics), Freiburg, Germany; 3) IMP
(Institute of Molecular Pathology), Vienna, Austria; 4) Nuclear Dynamics ISP, Babraham Institute, Cambridge, UK.
Many Drosophila Polycomb/Trithorax Response Elements (PRE/TREs) give rise to noncoding transcripts, but whether these transcripts are involved in
gene activation or silencing is currently unclear. Here we show that the Drosophila vestigial PRE/TRE undergoes a developmental switch, in which
embryonic transcription of the PRE/TRE reverse strand is linked to vestigial gene activation, whereas larval transcription of the PRE/TRE forward strand is
involved in vestigial gene repression. Transgenic reporter constructs identify a 100bp region of the PRE/TRE as a key switching point for this developmental
transition. High ectopic transcription of a transgenic PRE/TRE leads to tissue specific misregulation of the vestigial gene, demonstrating a transcription
dependent and strand specific interaction between the transgenic and the endogenous loci. By showing that PRE/TRE transcription is involved in both
activation and silencing, and that this is dependent on developmental context, these findings have broad implications for understanding the dynamic dual
nature of PRE/TRE elements.
76
An RNA Memory Mechanism to Inherit Epigenetic Marks.
Maria Cristina Onorati, Walter Arancio, Davide F.V. Corona. STEMBIO, Telethon Dulbecco
Institute c/o University of Palermo, Italy.
A central question in epigenetics is to understand how, terminally differentiated daughter cells can inherit complex patterns of chromatin modifications
from their mother cell. Even if several mechanisms have been hypothized to explain the establishment and maintenance of cell identity, it is still unclear how
during mitosis covalent and ATP-dependent chromatin modifications are transmitted after DNA replication. Indeed, a simple way for daughter cells to
restore the transcriptional profile of mother cells is to directly ‘sense’ the transcriptome of their mother cells. In order to unveil the molecular nature of
somatic cell epigenetic memory, we used classic Position Effect Variegation assays to check if non functional alleles of the white gene could modify the eye
color variegation caused by an heterochromatin inversion of the white gene called white-mottled 4 (wm4h). Our data show that several white alleles suppress
the variegation of the wm4h line. Unexpectedly, the presence of white alleles causes an increase in the white gene transcript as well as an opening in the
chromatin structure at the wm4h locus. Remarkably, this effect is inheritable, a phenomenon highly reminiscent of RNA mediated paramutation. The
changes in the levels of expression of the wmh4 gene, induced in trans by several white alleles, indicate that the presence of a non functional gene that does
not produce a coding transcript but potentially only ncRNA, could influence in trans the expression of a functional copy of the same gene silenced by
heterochromatin. Our data indirectly indicates that cells can 'sense' the presence of non coding RNA’s inherited from their mother cells and can use them to
epigenetically reset their transcriptional program after DNA replication.
77
Insulators bring active genes into transcription factories in Drosophila.
Hua-Bing Li, Vincenzo Pirrotta. Molecular Biology & Biochemistry, Rutgers
University, Piscataway, NJ.
Transcription factories are nuclear sites where active genes undergo transcription but it is still not clear how genes are targeted to a given transcription
factory. Insulators might play a key role in the formation of this kind of nuclear body. We previously have used transgenes containing the Mcp or Fab-7
(insulator+PRE) elements to show that two remote transgene copies interact and co-localize in around 7% of the nuclei, as shown by live-imaging and by 3C
assays. This interaction is not caused by the binding of Polycomb complexes but depends on the presence in both copies of the insulator element. We
observed a much higher frequency of co-localization in eye imaginal discs (in 50-90% of the nuclei) but not in wing imaginal discs when both transgenes
contained the eye enhancer, indicating that interaction is much stronger when the transgenes are transcriptionally active. This high-level interaction requires
also the insulator and the binding of Trithorax but not Polycomb to the PRE. The high-level interactions require CTCF but are modulated by CP190 and by
RNAi genes, particularly by Ago2. The two transgenes do not co-localize with each other when one copy is active and the other is silenced. The transgenes
also interact with endogenous homeotic genes, co-localizing with them when both transgene and endogenous gene are active but not when one is active and
the other repressed. From a combination of genetics, live imaging, 3C, ChIP, and transcriptional data, we conclude that the insulators mediate the contacts
but target the genes to different nuclear structures, depending on the state of activity. When both genes are repressed, they co-localize in Polycomb bodies.
When both genes are active, they are targeted to transcription factories in a Trithorax-dependent fashion that is not directly related to strength of
transcriptional activity.
78
A genetic screen for recessive Polycomb group mutants.
James A. Kennison, Mark A. Mortin, Monica T. Cooper. Program in Genomics of
Differentiation, NIH, Bethesda, MD.
The Polycomb group genes were first identified because mutations in them fail to maintain HOX gene silencing in Drosophila. In an F1 screen for new
Polycomb group genes, we are generating clones of homozygous mutant cells in the eye using the yeast FLP/FRT system. The mutagenesis is done in a
genetic background in which Polycomb group Response elements (PREs) in transgenes silence the mini-white reporter gene by pairing-sensitive silencing.
New silencing mutations are identified by derepression of the mini-white reporter in the homozygous mutant clones. On the second chromosome, we have
recovered 32 mutations in seven previously-identified Polycomb group genes and 39 mutations in at least 16 new sileincing genes. The transcription units
for three of the new silencing genes have been identified.