Conference Scene: Whistler Epigenetics and Genome Stability Meeting

Abstract

The 55th Annual Canadian Society for Molecular Biosciences Conference on Epigenetics and Genomic Stability in Whistler, Canada, 14–18 March 2012, brought together 31 speakers from different nationalities. The organizing committee, led by Jim Davie (Chair) at the University of Manitoba (Manitoba, Canada), consisted of several established researchers in the fields of chromatin and epigenetics from across Canada. The meeting was centered on the contribution of epigenetics to gene expression, DNA damage and repair, and the role of environmental factors. A few interesting talks on replication added some insightful information on the controversial issue of histone post-translational modifications as genuine epigenetic marks that are inherited through cell division.

The opening plenary lecture was given by Penny Jeggo (Genome Damage and Stability Centre, University of Sussex, UK), an internationally recognized leader in the study of DNA damage responses, and the repair of DNA double stranded breaks (DSBs). She talked about epigenetic marks during DNA replication, and presented her group‘s latest results on how the process of DNA DSB repair is affected by chromatin folding, with special emphasis on the mechanisms that take place in heterochromatic (HC) regions [1]. She showed that the kinetics of DNA repair are much slower in these highly condensed regions of chromatin compared with the transcriptionally active euchromatin. Furthermore, in HC regions, the molecular mechanisms involved require ATM and the DNA damage response mediator proteins (i.e., the MRN complex, H2A.X, MDC1, RNF8, RNF168 and 53BP1), which are dispensable for repair of DSBs in active euchromatin regions. Interestingly, Jeggo described how in the G2 phase HC impacts upon the choice between the homologous recombination (HR) and nonhomologous end joining (NHEJ) repair pathways. The former being the one preferentially used in HC-DSB repair. In addition, she provided novel evidence for the involvement of BRCA1 in HR.

Patrick McGowan (University of Toronto, Ontario, Canada) talked about the fascinating topic of the epigenetic alterations resulting from early-life events that lead to changes in neuroplasticity [2]. He described microarray analysis performed in rat offspring with low and high maternal care over extensive chromosomal regions, showing that epigenetic changes (e.g., DNA methylation and H3K9 acetylation) occur in clusters. They affect genes relevant for development of the stress response (GR and PCDH) in a way that is conserved across different species. Notably, these epigenetic signatures are conserved in humans: the pattern of differences across the syntenic region in abused/nonabused suicide victims mirrors that seen in rat studies.

Alexander Mazo (Thomas Jefferson University, PA, USA) presented revealing recent experimental data that monitor epigenetic marks during DNA replication. This work reveals that most epigenetic histone marks are completely removed before passage of the parental histones through the replication fork. By contrast, PCNA and the trithorax (trx) and polycomb (Pc) proteins are associated with the replicating strands (ssDNA) and may represent and intermediate in transmission of epigenetic markings during replication. Histone epigenetic marks are only re-established 60 min after DNA replication into the G2 phase of the cell cycle in Drosophila embryos. These results significantly challenge the involvement of replication as compared with transcription [3] in the cell inheritance of histone post-translational modifications (PTMs) and question the correct use of the term epigenetics to describe the biological downstream effects of such PTMs [4]. Also addressing the fate of epigentically modified histones during DNA replication, Toshio Tsukiyama (Fred Hutchinson Cancer Research Center, WA, USA) described some new techniques recently developed in his laboratory to measure nucleosome density and occupancy in yeast. During replication, DNA polymerase must transit a nucleosome every 2–3 s. These new methods, which involve comparisons of chromatin structure (micrococcal nuclease access) and histone occupancy (histone chromatin immunoprecipitations), allow analysis of changes in chromatin organization resulting from replication stress at the replication forks in yeast. Interestingly, an increase in nucleosome accessibility was observed under these conditions at the replication fork which was partially dependent on Mec1, an ATR-like kinase in the S-phase checkpoint.

Michael Kobor (University of British Columbia, Vancouver, British Columbia, Canada) described some of their recently published work on the high throughput analysis of different histone PTMs involved in chromatin regulation of genome function (transcription and DNA repair) [5,6]. In particular, the distribution of mono-ubiquitinated H2BK123 (uH2BK123) was described in relation to its role in establishing the trimethylated pattern of H3K4 and H3K79 during transcription and DNA repair. Of note, the genomic distribution of H3K79Me3, which is dependent on the crosstalk with uH2BK123, is nonoverlapping with that of H3K79Me2, even though both methylations are catalyzed by the same enzyme, Dot1.

Robin Allshire (Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK) provided a detailed description of the specificity of CENP-A deposition at centromeres, and how this is prevented from happening fortuitously elsewhere in the genome. He indicated how despite the unique DNA sequences associated with centromeric regions in some organisms, there is no ‘magic‘ underpinning sequence-related feature that is directly responsible for the discrimination between H3- and CENP-A-containing nucleosomes. By the end of his talk, he described how yeast mutants defective in FACT, a histone chaperone involved in the histone dynamics during transcription, exhibit an indiscriminate assembly of CENP-A nucleosomes to different regions of the genome, including actively transcribed genes. This served to highlight how factors acting immediately before, and in the wake of transcribing RNA polymerase II (i.e., Set2 and HATs) are not only important in preventing spurious reinitiation events, but also in abrogating the incorrect deposition of CENP-A. This strongly supports the existence of a crosstalk between chromatin-specific assembly and transcription.

Matthew Lorincz (University of British Columbia) talked about recently published data from his laboratory on ‘writers‘ and ‘readers‘ of H3K9 methylation in mouse embryonic stem cells. His attention focused around mammalian endogenous retroviruses, which comprise 10% of the mouse genome and can be classified into three major types: class I, II and III. While DNA methylation plays an important role in the silencing of somatic endogenous retroviruses, some of the class I and II silencing in mouse embryonic stem cells. is mediated by Setdb1/Eset methyltransferases that methylate H3K9 (H3K9Me2/H3K9Me3) [7]. Interestingly, the silencing histone methylation pathway appears to be independent of all known H3K9Me readers such as HP1 [8], suggesting there is still much to be learned regarding roles for this mark in chromatin regulation.

Benjamin Martin (University of British Columbia) presented some very interesting unpublished results that conclusively show that high levels of histone acetylation at transcriptionally active genes are not the cause, but a consequence of, ongoing transcription. Utilizing RNA polymerase II inhibitors (phenanthroline and thiolutin) in yeast, they observed that within 15 min most of the acetylation is lost from the cell. Furthermore, in mutants lacking HDAC complexes, such as Rpd3, no loss of acetylation was observed, implying that this enzyme is primarily responsible for the removal of transcription-dependent histone acetylation. Thus, global histone acetylation is a mark of ongoing transcription.

Tiffany Quam (University of California San Francisco, CA, USA) described the role of CHD1 in histone H3 turnover. In Drosophila, the protein associates with HIRA to produce the incorporation of histone H3.3 in the paternal pronucleus during the replacement of protamines after fertilization. In salivary glands, the CHD1/HIRA complex, in conjunction with Asf1, participates in the replication-independent incorporation of H3.3 at transcriptionally active regions. Replication-dependent deposition of H3.1 requires Asf1/Caf1. Quam presented data indicating that proper deposition of H3.3 by the HIRA/Asf1 complex requires S87 and G90 of H3.3, whereas H3.1 deposition by Asf1/Caf1complex requires the presence of the N-terminus of H3.1. Furthermore, Quam presented additional unpublished data describing the recruitment of Rpd3 by CHD1 to participate in the histone H3 turnover around RNA polymerase II during transcription.

Continuing with histone H3.3 turnover, Sheila Teves (Fred Hutchinson Cancer Research Center, WA, USA) presented some already published work from Steve Henikoff‘s laboratory on high-resolution mapping of epigenome dynamics centered around this histone H3 variant. In addition, Teves also described a couple of recently developed powerful techniques that permit measurement of nucleosome turnover kinetics and determination of chromatin profiling at a single base pair resolution. Her presentation highlighted the relevance of nucleosome turnover for epigenetic inheritance of gene activity [9].

Gratien Prefontaine (Simon Fraser University, Burnaby, Canada) provided novel evidence for the involvement of the Smc-HD1 protein in the DNA methylation-mediated transcriptional repression of the growth hormone receptor gene.

Different talks by Jennifer Mitchell (University of Toronto) and Josee Dostie (McGill University, Ontario, Canada) discussed most of their recently published observations regarding the 3D nuclear organization of chromosome domains [10]. This constitutes a fascinating topic that adds an extra layer of epigenetic complexity to gene expression. Mitchell focussed on the nuclear organization of RNA polymerase II transcription and interaction clusters. Dostie used the Hox gene clusters to exemplify how loss and gain of contacts among the multiple chromatin loops encompassing this cluster are related to the processes of gene activation.

David Bazett-Jones (Hospital for Sick Children, University of Toronto) presented the results of his recently published work on the global chromatin structure transitions accompanying the reprogramming of fibrobasts into induced pluripotent stem cells [11] using electron spectroscopic imaging. These data provide additional support to the questioning by the same laboratory of the dogmatic existence of a canonical 30-nm folded chromatin fiber in HC regions of somatic cells nuclei [12]. It would appear that for the most part, both euchromatic and HC regions generally consist of polynucleosomal strings with different extent of coalescence. The implication is that the 30 nm chromatin fiber is only observed in vitro with chromatin fibers that have been purified away from their nuclear environment or in the highly compacted organization of chromatin observed within the nucleus of sea urchin sperm.

Michael Hendzel (University of Alberta, Edmonton, Alberta, Canada) presented unpublished data from his laboratory on the role of the PRC1 in DNA DSB signaling cascade through its interaction with DNA repair proteins. He provided evidence that the complex is required for H2A/H2A.X ubiquitination and early recruitment of phosphorylated ATM to DSBs. RNF8, another ubiquitin ligase that has long been involved in the repair process, appears to function downstream of PRC1 di- and poly-ubiquitinating phosphorylated H2A.X.

Michael Skinner (Washington State University, WA, USA) dealt with the interesting data recently published by his laboratory [13] on the impact of environmental contaminants (such as N,N-diethyl-meta-toluamide, bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, among others) on epigenetic transgenerational effects. Both spermatogenic apoptosis and decreases in ovarian primordial follicle pool size in exposed mice were affected by exposure to contaminants. Interestingly, differential DNA methylation regions could be detected in the sperm of the F3 generation, demonstrating that environment-induced epigenetic marks on chromatin can behave like classical imprinted regions. Furthermore, Skinner introduced the field to a new and important concept; that each tissue probably has acquired a different collection of DNA methylation regions with respect to other tissues. Of course, this adds yet another layer of complexity in the interaction of the maternal environment and the epigenome(s) of the developing fetal somatic tissues and germ cells.

Several talks focused on chromosome and genome instability from yeast to plants. Phillip Hieter (University of British Columbia) talked about recently published data on the synthetic lethality of cohesions with PARPs and replication fork mediators and the use of yeast as a model for genes that are mutated and could cause chromosome instability in cancer [14]. Krassimir Yankulov‘s (University of Guelph, Guelph, Ontario, Canada) talk was on interplay of position effect and the passing/-pausing of replication forks [15]: specifically, he discussed the role of CAC1 (a subunit of histone chaperone CAF-1 in yeast) in building nucleosomes from ‘new‘ histones. The recruitment of CAF-1 is mediated by its interaction with PCNA. The presentation provided further insight in the transfer of histone epigenetic marks at stalled replication forks and nicely complemented the earlier talk on this topic by Alexander Mazo. Olga Kovalchuk (University of Lethbridge, Lethbridge, Alberta Canada) focused on the genomic instability (cancer) resulting from diagnostic and therapeutic exposure to radiation with special emphasis on the role played by small RNAs as a result of such exposure [16]. Kovalchuk described some of the transgenerational effects that result in alterations in the levels of DNA methylation and ncRNAs in the paternal germ line. Repeat elements appear to be the major targets of irradiation (long terminal repeats being hypermethylated and SINEs and LINEs being hypomethylated) in response to paternal irradiation. Igor Kovalchuk (University of Lethbridge) also focused on transgenerational changes in genome instability in response to stress in plants [17]. Epigenetic information was gathered from the progenies obtained from seeds of plants exposed to different abiotic and biotic stresses. Changes were observed in the HR frequency, DNA methylation and histone PTMs. For example, seeds of UV-stressed plants were systematically larger and the progeny exhibited a hypermethylated genome at several analyzed genomic regions.

The last session closed on the topic that had been highlighted by the plenary talk on DNA repair and damage at the opening of the meeting. Michael Kruhlak (NIH, MA, USA) provided a detailed microscopic cytological view of the chromatin environment surrounding DNA regions affected by DNA breaks in vivo. An energy-dependent local expansion of chromatin was described that takes place immediately after DNA damage and does neither depend on phosphorylation of H2A.X nor on ATM. Aaron Goodarzi (University of Calgary, Alberta, Canada) referred again to the slow (HC) and fast euchromatic components of DSB DNA repair and focused his attention on the currently ongoing experiments in his laboratory on the involvement of SNF2H-ACF1 on the Artemis-dependent DSB repair in hetrochromatin. Susan Lees-Miller (University of Calgary) described further insights into the NHEJ pathway. In addition, Lees-Miller presented several crystallographic images of different protein complexes involved in this process [18]. She also described the involvement in NHEJ of the infared-induced phosphorylation of human PNKP at S114 and S126 by ATM and DNA-PK [19].

Two talks were included in this section that, although departed from its main subject, brought the meeting to an interesting close. The first, by Alain Verreault (University of Montreal, Montreal, Canada) expanded on their recently published work on histone acetylation and DNA damage during replication. He described how misregulation of H3K56Ac increases sensitivity to genotoxic agents and impairs DNA DSB by homologous recombination [20]. Interestingly, Verreault explained how H3K56Ac is regulated by Hst3 and RTT109 proteins of the sirtuin family in Candida albicans, and how these studies resulted in the design of inhibitors with potential therapeutic use for the treatment of fungi virulence. In closing, Daniel Gottsschling (University of Colorado, CO, USA) provided a comprehensive description about age-associated genome instability and how cellular subsystems break down in budding yeast using their mother enrichment program genetic system [21]. Evidence was provided that aging occurs concurrently with mitochondrial dysfunction and the associated reduced production of iron–sulfur clusters, which ultimately results in a decreased activity of proteins involved in genomic maintenance.

Financial & competing interests disclosure

The authors have no relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.