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Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
233
Epigenetically-confused, an unusual Trithorax Group SET domain-containing protein, functions like a Polycomb Group gene.
Hector Rincon-Arano,
Jessica Halow, Jeff Delrow, Jorja Henikoff, Steven Henikoff, Susan Parkhurst, Mark Groudine. Basic Sciences Division, Fred Hutchinson Cancer Research
Center, Seattle, WA.
Polycomb (PcG) and trithorax (TrxG) group of proteins are the basic machinery of the transcriptional memory. PcG genes maintain gene silencing by
modulating chromatin structure via repressive marks like H3K27m3, whereas TrxG genes support gene expression by establishing activating histone marks
including H3K4m3. Here we describe the functions of the protein we named Epigentically-confused (Epic), which possesses a SET domain related to the
H3K4 methyltransferase Trx. DamID chromatin profiling of Epic shows preferential recruitment to transcriptionally active genes overlapping with RNA pol
II and H3K4m3, a typical feature of TrxG proteins. Surprisingly, in genetic interactions Epic behaves as a PcG protein. Accordingly, lack of Epic does not
affect H3K4 methylation regardless its similarity to the Trx SET domain. By co-immunoprecipitation, we find that Epic interacts with the histone
deacetylase RPD3 and Sin3. Chromatin immunoprecipitation experiments indicate that Epic is required for controlling acetylation levels of active promoters
and their gene units, as its knock down increases H3K9Ac and H4K16Ac levels over promoter and neighbors regions. Paused genes are also targeted by Epic
including the heat shock genes. Although the activation of heat shock genes is properly set up in Epic knock down cells, their turn off is impaired.
Additionally,
epic
mutant flies are female sterile and exhibit an up-regulation of the Notch pathway, which affects cell lineage specification. Thus,
continuous presence of Epic at active genes could be a fast way of modulating chromatin architecture during transcriptional shut down. Altogether, our data
suggests that Epic belongs to new class of PcG gene that use their Trx features to bring the HDAC machinery to transcriptionally active regions in order to
modulate the chromatin opening.
377B
The histone demethylase UTX and the chromatin remodeler BRM bind to Drosophila CBP and modulate the acetylation of histone H3 lysine 27.
Feng Tie, Rakhee Banerjee, Patty Conrad, Peter Scacheri, Peter Harte. Dept Genetics, Case Western Reserve Univ, Cleveland, OH.
Trithorax-group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. We previously showed
that the histone acetyltransferase (HAT) CREB-binding protein (CBP) acetylates histone H3 lysine 27 (H3K27), thereby directly blocking its trimethylation
(H3K27me3) by the Polycomb Repressive Complex 2 (PRC2) in Polycomb target genes. Here we show that H3K27ac levels also depend on other TrxG
proteins, including the histone H3K27-specific demethylase UTX and the chromatin-remodeling ATPase Brahma (BRM). We show that endogenous CBP is
physically associated with UTX and BRM in vivo and that UTX, BRM and CBP exhibit genome-wide co-localization on Polycomb Response Elements
(PREs) and on many Polycomb target genes marked by H3K27ac. UTX and BRM bind directly to the conserved zinc fingers of CBP, suggesting that their
individual activities are functionally coupled in vivo. BRM and histone H3 bind cooperatively to the PHD finger (C4HC3-type) of CBP, an integral part of
the CBP HAT domain. BRM enhances in vitro acetylation of H3K27 by recombinant CBP. Drosophila brm mutations and RNAi knockdown of UTX and
BRM reduce H3K27ac levels and increase H3K27me3 levels. Direct binding of UTX and BRM to CBP in vitro, and their physical association in vivo
suggest that demethylation of H3K27me3 by UTX, acetylation of H3K27 by CBP and remodeling activity by BRM complex are coordinated for a rapid and
efficient reversal of Polycomb silencing. We propose that UTX and BRM play an important role in antagonizing Polycomb silencing by positively
modulating acetylation of H3K27 by CBP.
378C
Drosophila
Myb interacts with NURF to repress cell cycle genes and transposons in non-mitotic tissues.
Juan Santana
1
, Stephen Butcher
1
, Scott
McDermott
1
, Mrutyunjaya Parida
1
, Kristen Jogerst
1
, J Robert Manak
1,2
. 1) Dept of Biology, Univ of Iowa, Iowa City, IA; 2) Dept of Pediatrics, Univ of
Iowa, Iowa City, IA.
c-
Myb
is encoded by a proto-oncogene associated with leukemias and lymphomas in birds and mammals. Vertebrates have three representatives of the
Myb gene family consisting of A-, B- and c-
Myb
, all of which encode DNA-binding factors that are important for the proper expression of large numbers of
genes including those that regulate cell cycle progression and cell differentiation.
Drosophila melanogaster
contains a single Myb gene (Dm-
Myb
), mutants
of which die before reaching adulthood. siRNA knockdown of Dm-
Myb
was shown to reduce expression of genes with prominent roles in coordinating cell
division in an embryonic
Drosophila
cell line. Along the same lines, Dm-
Myb
mutant flies display cell cycle defects such as aneuploidy and polyploidy, both
hallmarks of cancer. Additionally, the Dm-Myb protein was identified in a complex containing a large number of proteins including the nucleosome
remodeling factor NURF. Through yeast two-hybrid and genetic screens, as well as co-immunoprecipitations, we have now established that Dm-Myb
specifically interacts with the major subunit of NURF (Nurf301). In light of these results, we performed gene expression analyses in Dm-
Myb
and
Nurf301
mutant animals under the assumption that a significant number of genes are co-regulated by both proteins. As expected but nonetheless striking, there is an
almost 50% overlap of the genes regulated by these two proteins and, in contrast to the dogma in the field, we have observed a prominent transcriptional
repression function for Myb and Nurf301 in non-mitotic tissues. These data suggest that, in addition to activation of cell cycle genes in dividing cells, Myb
and NURF work to repress such genes in non-dividing cells. Even more surprising, tiling microarray and RNA-seq data indicate that Dm-Myb and Nurf301
are working in concert to silence transposable elements.
379A
Brahma (SWI/SNF) complex regulation of transcript elongation and pre-mRNA splicing is mediated by the SNR1 regulatory subunit.
Claudia B.
Zraly. Oncology Institute and Department of Pathology, Stritch School of Medicine, Loyola University of Chicago, Maywood, IL.
Drosophila Brahma (SWI/SNF) complex dependent chromatin remodeling is essential for the precise regulation of in vivo target genes during Drosophila
development. Transcriptome studies in flies and mammals identified cell cycle and hormone responsive genes as important targets, while loss of SWI/SNF
function has been linked to developmental abnormalities and aggressive cancers. The Brahma complex assists in reprogramming and coordinating gene
expression in response to hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and
conditional mutant alleles to identify unique and important functions of two conserved Brahma (Brm) complex core subunits, SNR1/SNF5 and BRM/SNF2-
SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase
elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-
dependent nucleosome remodeling activities downstream of the promoter region. Our results provide evidence for critically important roles of the