We were lucky to have Dr. Fabio Spada ready to go in Munich to cover this year’s Epigenetics Europe Conference, which ran on September 8th , 2011. Read on to see his full report on the event’s highlights: The Epigenetics Europe Conference ran in parallel to the RNAi and miRNA Europe Conferences in downtown Munich.
Despite a relatively small number of participants the conference exhibited an excellent scientific program with talks by top European researchers working on diverse themes of Epigenetics ranging from DNA methylation, imprinting, histone modification and dynamics, long term epigenetic effects on metabolism, transcriptional control by long range chromatin interactions and mathematical modeling of epigenetic regulation. In addition, three talks were given by scientists from biotech companies promoting products relevant to epigenetics research. Scientific discussions were lively and stimulating and the limited number of attendees facilitated contact with the speakers during the breaks.
Effects of Foreign DNA on the Epigenome
Walter Doerfler, University of Erlangen-Nürnberg
In the keynote presentation Dr. Doerfler reported on his long standing interest in the consequences that insertion of foreign DNA has on the host epigenome as well as work on methylation of the FMR1 gene in the Fragile X syndrome. Using both adenovirus infected and transgenic cell lines his group has shown that insertion of foreign DNA has striking effects on expression profiles of the host cells, which are reflected by long range alterations of DNA methylation both in cis and trans (that is, on the same as well as other chromosomes). These alterations may be subject to selection, are stable even after excision of the foreign sequences and their extent seems to depend on the insertion site. This is relevant not only to the consequences of viral infection (such as potential oncogenic transformation), but also to all experimental procedures involving insertion of transgenes, including induction of pluripotent stem cells using integrating vectors.
In addition, as mobilization of endogenous transposable elements was shown to occur in the germ line, early development and neural progenitors, it may be a source not only of genomic, but also epigenetic mosaicism in animal progeny as well as within somatic tissues of the same individual.
DNA Methylation in Honey Bees
Frank Lyko, German Cancer Research Centre
Dr. Lyko presented recently published work on changes in DNA methylation patterns upon ageing, sun exposure, stem/progenitor cell differentiation and honey bee castes (Lyko et al., PloS Biology, 8:e1000506; Bocker et al., Blood, 117:e182; Grönniger et al., PloS Genetics, 6:e1000971). Their genome-scale analyses of methylation patterns in human epidermis, hematopoietic progenitors and differentiated myeloid progeny showed high similarity among individuals of the same age in a given tissue/cell type, but substantial change among different tissues, supporting the idea that methylation patterns reflect tissue specific expression programs. However, stratification of data from epidermis by age revealed hypermethylation of a small fraction of analyzed sequences, with changes resembling those seen in tumors (indeed, age is a major risk factor for cancer). Instead, stratification by sun exposure showed a trend towards hypomethylation. Interestingly, comparison of methylation patterns between young and old hematopoietic progenitors revealed a bimodal pattern, with demethylation of genes involved in differentiation and sparse acquisition of methylation elsewhere.
These data support the idea that epigenetic changes underlie the loss of phenotypic plasticity associated with stem cell ageing. Another example of epigenetic plasticity translating into phenotypic plasticity was revealed by genome-wide survey of methylation patterns in distinct honey bee castes. 550 genes were found to be differentially methylated in the brains of queen (egg-lying) and worker bees, which develop from an isogenic population as a consequence of a different diet.
DNA Demethylation Dynamics in Mouse Development
Jörn Walter, University of Saarland
Dr. Walter reported on a recent breakthrough by his group after a decade of investigation on the elusive dynamics of DNA demethylation during mouse preimplantation development (Wossidlo et al., Nature Commun, 2:241). Several groups have shown that genomic methylcytosine (mC) can be converted into hydroxymethylcytosine (hmC) by the three members family of Tet dioxygenases.
The Walter lab could show that the rapid disappearance of mC immunostaining from the male pronucleus is paralleled by a rapid appearance of hmC staining. Their knock down experiments showed that Tet3, the largely preponderant Tet family member in oocytes and one cell stage embryos, is responsible for mC hydroxylation in the early zygote, which has very recently been confirmed by others using a conditional knockout strategy (Gu et al., Nature, epub ahead of print). The Walter lab could also show that, although some mC hydroxylation occurs also in the female pronucleus, the maternal genome seems to be largely protected against it by PGC7/Stella/Dppa3, a factor also expressed in naïve pluripotent stem cells and primordial germ cells. Important questions remain to be answered as to the mechanism(s) that spares selected sequences from mC hydroxylation (including most of the maternal genome), the fate of the abundant hmC in the paternal genome and how this leads to the establishment of pluripotency in the early epiblast.
In addition, Walter presented unpublished data on non‑CpG methylation and the contribution of different factors to the maintenance of DNA methylation patterns in mouse embryonic stem cells using knockout cell lines. DNA methyltransferases Dnmt3a and 3b were found to be required for methylation of distinct CpA sites in major satellite repeats. Maintenance of methylation in repeated elements was investigated using hairpin bisulfate sequencing to determine the methylation state of CpG sites on both strands of the same DNA molecule. This revealed specific contributions by Dnmt3a and 3b and unanticipated differences in the roles of Dnmt1 and Uhrf1.
Paul Cloos, University of Copenhagen
The seminal discovery of mC hydroxylation by Tet proteins has raised many fundamental questions, including the function of genomic hmC invarious cellular contexts and how Tet proteins are targeted to specific methylated sites. There is actually growing evidence for a role of hmC as DNA demethylation intermediate. Dr. Cloos showed recently published data on the function of Tet1 in embryonic stem cells (ESCs; Williams et al., Nature 473:343) as well as unpublished results on targeting of Tet2. In ESCs both hmC and Tet1 are enriched around the transcription start sites of CpG‑rich promoters and hmC is also found in gene bodies.
Knockdown of Tet1 results in partial reduction of hmC and both up- and down‑regulation of a fraction of Tet1‑bound genes. While increased expression is consistent with a role of hmC as intermediate in the erasure of repressive cytosine methylation, down-regulation seems to be independent from Tet1 catalytic activity and could be explained by either indirect effects or a role of Tet1 in gene silencing. Indeed, Cloos and collaborators detected an interaction of Tet1 with the Sin3a corepressor complex and found substantial overlap of target genes between Tet1 and Sin3a as well as Polycomb repressive complex 2. Most of these data have been confirmed by other groups, supporting a double life of Tet1 as a transcriptional activator and corepressor. Several writers and readers of histone and DNA modification contain CXXC-type zinc finger domains with DNA binding activity.
A Tet1 fragment containing such a CXXC domain was recently shown to recruit Tet1 to genomic target sites. Interestingly Tet2 and 3 do not harbor a CXXC domain, but the proteins CXXC4 and 5 contain CXXC domains highly homologous to that present in Tet1. Interestingly, CXXC5 has been involved in normal and malignant myelopoiesis and Tet2 mutations have been found in several myeloid malignancies. Cloos and collaborators detected an interaction between Tet2 and CXXC5 and found a large overlap of their target genes as well as a very similar sequence signature at their binding sites. Importantly, knockdown of CXXC5 reduced binding of Tet2 at genomic targets, supporting a role for CXXC5 in targeting Tet2. In addition, the chromosomal region spanning the CXXC gene is often deleted in myelodisplastic syndromes and acute myeloid leukemias, the same malignancies associated with Tet2 mutations.
Lymphoid Specific Helicase
Irina Stancheva, University of Edinburgh
Dr. Stancheva showed recently published as well as unpublished work from her lab on lymphoid specific helicase (Lsh, also called Hells). Ironically, Lsh is neither lymphoid specific nor a helicase and, although it is closely related to SNF2 family chromatin remodeling factors, so far no evidence has been shown for its chromatin remodeling activity. Previous work had shown that Lsh deletion is postnatal lethal and affects the establishment of DNA methylation patterns early in development, with drastic hypomethylation of repetitive elements (satellites and endogenous retrotransposons).
Accordingly, both the Muegge and Stancheva labs had shown that Lsh interacts with de novo DNA methyltransferases Dnmt3a and 3b and indirectly also with Dnmt1. Recent genome-scale DNA methylation and expression analyses by the Stanceva lab revealed that a fraction of promoters are either hypo- or hypermethylated in Lsh null fibroblasts (Myant et al., Genome Res 21:83), resulting in a substantial number of misexpressed genes (recently confirmed by the Muegge lab; Tao et al., PNAS 108:5626). The hypermethylated genes include pluripotency, trophoectoderm-specific, and reproduction (Rhox) genes as well as genes involved in differentiation.
Primed by a number of observations pointing to a potential role for Lsh in DNA repair (defects in meiotic chromosome synapse in the germ line of Lsh null females, premature ageing of Lsh hypomorph mice and Lsh homologues in yeast and plants associated with DNA repair), Stancheva and collaborators found that Lsh depleted fibroblasts do not efficiently repair DNA double strand breaks (DSBs) induced by irradiation. This occurs independently of DNA methylation defects, that is, in both fibroblasts derived from Lsh null mice (hypomethylated) and fibroblasts subjected to Lsh knockdown (normal methylation). Further investigation revealed that in Lsh depleted fibroblasts phosphorylation of histone H2AX (a marker of DSBs) and accumulation of MDC1 and p53BP1 at DNA damage foci is impaired. These defects ultimately reflected impaired activation of Chk2, a key mediator of the DNA damage checkpoint that responds to DSBs, establishing a role for Lsh in the DSB response pathway. Interestingly, this phenotype is similar to that of cells lacking Brg1, a bona fide chromatin remodeler, and could be rescued by exogenous expression of an Lsh mutant that lacks ATPase activity, suggesting that the elusive ATP‑dependant chromatin remodeling activity of Lsh may be required for activation of the DSB damage response.
Histone Modification and Dynamics
Fred van Leeuwen, Netherlands Cancer Institute
For chromatin states to be inherited through cell generations, patterns of histone posttranslational modification (PTM) must be maintained through replication. Although it is known that parental histones segregate roughly randomly to the daughter genomes, the underlying mechanism is unknown, including how and where they are repositioned after replication and how their PTMs are “copied” onto newly incorporated histones. In addition, although histones are less dynamic than most other proteins, they are known to be exchanged independently of DNA replication through chromatin remodeling and during transcription. However, detailed in vivo analysis of these dynamics poses major technical challenges.
Dr. Van Leeuwen presented very recently published work that addresses dynamics of histone H3 and its PTMs using RITE (recombination induced tag exchange), a very elegant Cre/lox based approach established by his lab to inducibly switch genetic tags on proteins, thus allowing biochemical tracking of protein molecules synthesized before and after the switch (Verzijlbergen et al., PNAS 107:64). This was possible in the budding yeast as there are only two genes encoding histone H3, of which one was deleted and the other targeted with the RITE cassette. Using cell cycle synchronization they showed that replication independent histone exchange occurs at surprisingly high rates, which are however different in distinct cell cycle phases (G0, G1 and G2/M) and are proportional to transcription rates, suggesting that transcription-coupled histone exchange provides a means to replace compromised histones or to frequently reset histone PTMs. Global tracking of “old” H3 histones over several cell generations showed that these accumulate near the 5’ end of ORFs, especially in long and poorly transcribed genes (Radman-Livaja et al., PLoS Biol 9:e1001075). Mathematical modeling supported a combination of replication-independent histone replacement, transcription-coupled 3’ to 5’ movement and spreading during replication, resulting in an average displacement of old histones by 400 bp per cell cycle with respect to their original location before replication.
Analysis of mutants showed no effect of several candidates among histone modifiers, while it identified a role for topoisomerase I and the N‑terminal tail of histone H4 in transcription-coupled retrograde (3’>5’) histone movement (with more pronounced effects of the latter on TFIID- than SAGA‑dominated genes), supporting a role for acetylation of the H4 tail in this process. Thus, old histones would make their way upstream until they would reach the histone depleted region around the promoter where they would be finally evicted. Interestingly, CAF-1 was found to impact replication independent histone turnover. The Van Leeuwen group used RITE also to analyze the methylation dynamics of lysine 79 in histone H3 (H3K79; De Vos et al., EMBO Rep 12:956). Interestingly, no demethylase has been identified for H3K79, which is exclusively methylated by Dot1 in a distributive (non-processive) manner. The methylation state of H3K79 was found to increase progressively (mono>di>tri-methylation) after histone H3 deposition, which is not compatible with a mechanism for copying H3K79 methylation patterns with single nucleosome precision after replication and provides a mechanism for H3K79 methylation to time and possibly signal cell cycle length at the chromatin level.
Protein Recognition of PARP1 Via Macrodomains
Andreas Ladurner, Ludwig Maximilans University
Dr. Ladurner showed recent work of his group on poly‑ADP‑ribose (PAR) polymerase 1 (PARP1) and proteins that recognize PAR through their macrodomains. PARP1 accumulates very rapidly at sites of DNA damage where it PARylates itself as well as several other chromatin bound factors using NAD as ADP‑ribose donor. The binding kinetics of PARP1 at damage sites is faster than the typical life time of DNA double strand breaks (DSBs), suggesting that PARP1 is not directly involved in the repair mechanism.
Although it is not known how PARP is activated, they could show that two N-terminal zinc finger domains (ZnF1 and 2) on distinct molecules of a PARP1 homodimer are required for recruitment at DNA damage sites and mediate binding to the free 3’ end of DNA double strand breaks. The evolutionary conserved macrodomain is present in a large group of proteins with diverse cellular functions, including PARP family members and several variants of histone H2A (although only macroH2A1.1 binds PAR). As binders of NAD metabolites macrodomain proteins may connect cellular metabolism and chromatin states. To exemplify the point that PARP1 controls the activity of macrodomain proteins Ladurner reported findings on Alc1 (Gottschalk et al., PNAS 106:13770), a chromatin remodeling factor of the SNF2 ATPase superfamily that is activated in the presence of PARP1 and NAD and requires an intact macrodomain for its activity. Thus, PARylated PARP1 (or other PARylated chromatin components) likely targets Alc1 activity to specific genomic sites.
**EpiGenie would like to thank Dr. Fabio Spada, who is a Professor in the Dept. of Biology II at Ludwig-Maximilians-University (LMU) in Munich for his detailed coverage of this conference.