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Scottish Chromatin Group |
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9th Scottish Chromatin Group Meeting and 2011 Tenovus-Scotland Medal Lecture
This meeting was held
at the
Charles
Wilson Lecture Theatre,
University of Glasgow on
Wednesday 8th June 2011. It was
organised by Adam West. E-mail
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Programme
9.30
Tea/coffee welcome
9th Scottish Chromatin Group meeting
10.15 - 10.55 Irina Stancheva,
University of Edinburgh
Website
Programming DNA methylation during development
Stable patterns of gene expression during development and differentiation
are achieved by sequence-specific transcription factors as well as
epigenetic mechanisms that facilitate the establishment and propagation of
heritable chromatin states. In the developing mouse embryo, DNA
methylation is extensively reprogrammed and new patterns of DNA
methylation are generated in a lineage-specific manner by de novo DNA
methyltransferases DNMT3a and DNMT3b. The efficiency of this process is
often supported by other proteins. In this talk I will discuss the role of
chromatin remodelling ATPase LSH and G9a/GLP complex of histone methylases
in developmentally programmed DNA methylation.
10.55 - 11.20 Dhaval Varshney (Bob White's group),
University of Glasgow
Website
Methylation of SINE DNA Suppresses Rearrangement but Not Access or
Function of Pol III Transcription Factors
Nearly half the human genome is comprised of repetitive DNA, including
short interspersed nuclear elements (SINEs) such as Alu. SINEs spread by
retrotransposition, which requires their transcripts to be copied into DNA
and then inserted into new chromosomal sites. Gene-rich euchromatic
regions are targeted, which can lead to genetic damage through insertional
mutagenesis and through chromosomal rearrangements between non-allelic
SINEs at distinct loci. SINE DNA is heavily methylated and this is thought
to suppress its transcription and thereby protect against
retrotransposition. However, we found that DNA methylation does not reduce
expression of SINEs or their accessibility to transcription machinery. In
contrast, it does diminish significantly the rate of interchromosomal
translocation between Alu SINEs. This is likely to provide protection
against genetic damage.
11.20 - 12.05 Bas van Steensel,
Netherlands Cancer Institute, Amsterdam
Website
Genome - nuclear lamina interactions
In metazoans, the nuclear lamina is thought to play an important role in
the spatial organization of interphase chromosomes, by providing anchoring
sites for large genomic segments named lamina-associated domains (LADs).
Some of these LADs are cell-type specific, while many others may be
constitutively associated with the lamina. New data will be presented that
provide insight into the nature of these facultative and constitutive LADs.
In addition, we have developed a new approach to track genome-lamina
interactions in living cells.
12.05 - 13.05 Buffet lunch
13.05 - 13.30 Emanuela Sani (Anna Amtmann's group),
University of Glasgow
Website
Changes in whole-genome profiles of histone modifications after salt
priming of Arabidopsis thaliana plants
Priming describes the observations that a short treatment of plants with
non-lethal doses of pathogens or abiotic stress factors (e.g. salt,
temperature, drought) increases the tolerance of plants when these or
their progeny are re-exposed to the pathogen/stress. The fact that a
plant’s ‘memory’ of environmental conditions survives mitosis and meiosis
makes epigenetics and attractive candidate for explaining the effects of
priming. In this project we have confirmed that a short salt pre-treatment
of Arabidopsis seedlings modifies the transcriptional response of mature
plants to salt shock, and we have profiled several histone modifications
immediately after the priming treatment to test the hypothesis that these
could be responsible for altering the transcriptional response. The
obtained ChIPSeq data revealed both expected and novel aspects of
chromatin dynamics in different tissues and in response to environmental
stimuli, which will be presented in this talk.
13.30 - 13.55 Nikolay Pchelintsev (Peter Adams' group),
University of Glasgow
Website
Genome-wide mapping of the histone chaperone HIRA links it with the
regions of highly dynamic chromatin
The HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex preferentially
deposits the histone variant H3.3 into chromatin. Paradoxically, HUCA
contributes to chromatin metabolism associated with both gene activation
and repression. We have performed ChIP-seq analysis of HIRA, UBN1 and
CABIN1. Approximately, 70% of HIRA peaks co-localize with UBN1 and ASF1,
consistent with the notion that these proteins bind as a complex to many
sites throughout the genome. The genome-wide distribution of HUCA will be
described, together with subsequent functional analyses.
13.55 - 14.40 Ali Shilatifard,
Stowers Institute for Medical Research, Kansas City, USA
Website
Supported by Bio-Rad
Lessons learned from yeast about human leukaemia
The mixed lineage leukaemia gene, MLL, is involved in several
translocations common in acute leukaemia. The MLL translocations are most
commonly translocations seen in infant leukaemia, as well as, in acute
leukaemia arising after treatment of a primary malignant disease with
topoisomerase II inhibitors such as etoposide and daunorubicin. To gain a
better insight as to the role of MLL, we have taken advantage of genetics
and biochemical tools in S. cerevisiae to identify the molecular
properties of the yeast MLL homologue, the Set1 protein. We isolated Set1
in a macromolecular complex, which we named COMPASS (COMplex of
Proteins ASsociated with Set1). COMPASS was the first
histone H3 lysine 4 (H3K4) methylase isolated and shown to be associated
with transcribing RNA polymerase II. Using biochemical and genetic studies
in yeast, we have also identified the molecular pathway required for
COMPASS’s function. We have demonstrated that histone H2B
monoubiquitination by Rad6/Bre1 is required for histone H3K4
trimethylation by COMPASS. Moreover, our laboratory and others have
demonstrated that the human MLL complex is also found in a COMPASS-like
complex capable of methylating H3K4 and that the molecular machinery
required for the regulation of this histone crosstalk and H3K4 methylation
is highly conserved from yeast to human. Indeed, there is only one Set1 in
yeast, yet in mammalian cells there are multiple H3K4 methylases including
Set1A/B forming the human COMPASS complexes, and the MLL1-4 forming the
hCOMPASS-like complexes. Work from our laboratory recently demonstrated
that Wdr82, which associates with chromatin in a histone H2B
ubiquitination-dependent manner, is a specific component of the Set1
complexes, but not that of the MLL1-4 complexes. RNAi-mediated knockdown
of Wdr82 results in a reduction in the H3K4 trimethylation levels,
although, these cells still possess active MLL complexes. Comprehensive
in vitro enzymatic studies with the Set1 and the MLL complexes
demonstrated that the Set1 complex is a more robust H3K4 trimethylase in
vitro than the MLL complexes. Given the in vivo and in vitro
observations, it appears that the human Set1 complex plays a more
widespread role in H3K4 trimethylation than the MLL complexes in mammalian
cells. This fundamental observation indicates that MLL1 has specific
functional targets in the mammalian genome and the identification of such
targets could provide us with new avenues for the therapeutic intervention
of MLL translocation-based leukaemia. We have indeed identified many of
these MLL functional targets and plan to describe them in the meeting.
Lee
J.S., et al. (2010) The language of histone crosstalk. Cell. 142: 682-5.
14.40 - 15.20 Tea/coffee
15.20 - 15.30 Introduction to the 2011 Tenovus-Scotland Medal
Lecture
Dr. Sheila Graham, co-chair, Tenvous Medal committee, University of
Glasgow
Prof. Andrew Calder, National Chairman, Tenovus Scotland
15.30 - 16.30 Dr. John Rouse, University of Dundee. Website Forks and molecular knives at the cutting edge of DNA repair The chemical reactivity of DNA contributes to the staggering array of DNA lesions that occur in cellular genomes every day. In addition to their potential mutagenicity, these DNA lesions can block important processes such as DNA replication, which can potentially prevent cell proliferation. It is vital that DNA damage is repaired rapidly to prevent mutations, rearrangements or changes in chromosome number from occurring. Cells have evolved sophisticated pathways that repair DNA damage, and stalled or broken DNA replication forks. Defects in these pathways can have serious consequences that range from cell death and embryonic lethality to a range of debilitating disease syndromes.
Nucleases play important roles DNA repair. In yeast for example, the structure–specific nuclease Rad1-Rad10 is required to trim excess DNA (DNA flaps) at the final stages of termed homologous recombination (HR), an ancient mechanism for repairing DNA damage and stalled or broken replication forks. In 2005 we discovered a regulatory subunit of Rad1-Rad10, termed Slx4, that acts as a scaffold not just for Rad1-Rad10 but for also for Slx1, a structure-specific nuclease of unknown function. In cells lacking Slx4, Rad1-Rad10 cannot cleave DNA flaps during HR-mediated repair of double-strand breaks, and so cells die. Slx4 appears to facilitate Rad1-Rad10 by deforming DNA flaps to render them accessible to attack. In 2009, my lab reported the identification of human SLX1, and SLX4 which binds to and regulates not only SLX1 but two other structure–specific repair nucleases: XPF–ERCC1 (human Rad1-Rad10) and MUS81–EME1 (which is closely related to XPF–ERCC1). We refer to the SLX4 complex is a “molecular toolkit” for DNA repair because the three nucleases can cleave a variety of branched DNA species that resemble HR intermediates. Cells depleted of SLX4 are particularly sensitive to agents that cause inter-strand cross-links (ICLs), toxic lesions that block DNA replication forks. The SLX4 complex processes branched intermediates that occur during the HR step of ICL repair required to regenerate an intact replication fork. In collaboration with Johan de Winter (Erasmus Medical Centre, Rotterdam) we discovered that biallelic mutations in SLX4 cause Fanconi anaemia (see below), further underscoring the importance of the SLX4 complex for human health. Intriguingly, the SLX1 nuclease can cleave Holliday junctions (HJs), DNA structures that arise not only during DNA repair but also during meiosis. Recent data suggests that SLX1 is involved in HJ resolution in vivo in germ cells. Fanconi anaemia (FA) is a rare inherited chromosome instability syndrome accompanied by developmental and skeletal defects, bone marrow failure and predisposition to cancer. There are fifteen FANC proteins, and the central component of the FA pathway is FANCD2, which is mono–ubiquitylated at Lys561 in S–phase and in response to ICLs. This is catalysed by the eight–subunit FA core complex. The mono-ubiquitylation of FANCD2 is essential for the repair of DNA inter-strand crosslinks (ICLs) but despite much work in this area exactly how mono–ubiquitylation of FANCD2 promotes ICL repair at the molecular level was unknown. In 2010, my lab made a major breakthrough by showing that the UBZ domain of the previously uncharacterized FAN1 protein interacts with the mono-ubiquitylated form of FANCD2, and that FAN1 is recruited to sites of DNA damage in a manner that requires FANCD2 mono–ubiquitylation. FAN1 is a structure–specific nuclease that is specific for 5’ DNA flaps. Intriguingly, like the SLX4 complex, FAN1 appears to process DNA repair intermediates during the HR stage of ICL repair to enable regeneration of an intact replication fork. So binding of FAN1 to mono-ubiquitylated FANCD2 at least partly explains how FANCD2 mono–ubiquitylation regulates DNA repair. However, the available evidence indicates that there must be other ligands of mono-ubiquitylated FANCD2 that regulate ICL repair, and we recently identified several such ligands. When replication forks encounter nicks in the DNA backbone, forks can collapse and when this happens there is a high risk of genome instability. Not only must the ends of the broken fork be captured and protected from further degradation, but an intact replication fork must be regenerated and this is achieved by HR. We found that budding yeast Mms22 is required for HR-mediated repair of stalled or broken DNA replication forks, and in 2010 we reported the identification of a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL, of unknown function. Both MMS22L and TONSL bind in the vicinity of distressed replication forks and, by mechanisms that are not yet clear, they promote the loading of the RAD51 recombinase to enable HR–mediated fork repair. My lab is currently using a range of genetic models to understand the molecular modes of action of SLX4, FAN1, MMS22L–TONSL and other new regulators of genome stability we have identified. We are also starting to develop small molecule inhibitors of some of these proteins as next-generation anti-cancer drugs. Key papers:
Stoepker
C. et al. (2011) SLX4, a coordinator of structure–specific endonucleases, is mutated in a new Fanconi anemia subtype. Nat. Genet.
(2011) Jan. 16th Advance online publication.
Questions chaired by Prof. David Gillespie, University of Glasgow
16.30 - 16.40 Medal presentation by Prof. Anna
Dominiczak,
Vice Principal & Head of MVLS College, University of Glasgow
16.40 Wine reception |
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© Adam West 2006-2012
This meeting
is being organised by Adam West and the
