Epigenetic Regulation: From Mechanism to Intervention 2012
MRC Clinical Sciences Centre Symposium
The CSC Symposium on Epigenetic Regulation was held in London and Yale’s Lauren Blair was on the scene to cover the whole thing. (She also attended this Abcam Chromatin Meeting). Check out her report below to get the full conference experience:
CSC Symposium Epigenetic Regulation: From Mechanism to Intervention Summary
The goal of this meeting was to examine the link between the basic science of epigenetic mechanisms and the therapeutic value of drugs targeting these processes. The venue was a charming building right on the River Thames in London. Lunches were served in various rooms where conference attendees mingled and visited sponsor booths. Coffee and tea breaks were welcome as food and drink were not allowed in the auditorium. The poster session provided an excellent opportunity for intimate mingling and for a broadening of topics not covered in the talks. The program was heavy on identifying and interpreting enhancer regions and in polycomb complex studies. Below are summarized a few of the more intriguing presentations.
Epigeneome Status Controlled by Gentics or Epigenetics?
Emma Whitelaw, Queensland Institute of Medical Research
Dr. Whitelaw’s lab is interested in addressing the question of whether or not cell type specific epigenome status is controlled by genetics or epigenetics. To address this issue, her lab has used an inbred mouse model where coat color corresponds to promoter methylation state. They have performed random mutagenesis to find genes responsible for epigenetic changes. A human blood gene promoter was inserted upstream of a gene coding for a fluorescent protein. This protein presents as green in mouse blood cells. They then followed this protein using FACs sorting. An altered FACs profile indicated a heritable change. They then sequenced the gemones of the mice that showed a heritable change and pinpointed the location of these mutations. They have termed their model Modifiers Of Murine Metastable Epialleles (MOMMES). Dr. Whitelaw specifically highlighted the identification of Rif1, which is implicated in breast cancer, as an example of a gene that undergoes a heritable change.
Epigenetic Enhancers in Neural Crest Cells
Joanna Wysocka, Stanford University
Dr. Wysocka studies neural crest cells. Neural crest cells form early and migrate throughout the body. The main mechanism for determining what each cell will become is epigenetics. These cells can differentiate into over 100 different cell types. Dr. Wysocka addressed the neural crest cells that develop into facial features, specifically. The Wysocka lab derived neural crest cells from hESC cells and used the model to find putative enhancers. To define enhancers, they used noted enhancer marks p300, H3K4me1 and H3K27ac. After identifying putative enhancers, they used zebrafish as a model system to determine if the enhancers were legitimate.
They found that many of the enhancers were real but that they were not active in all cell types. They then searched these enhancers for DNA sequence motifs. The motif they found to be most highly enriched was one bound by the transcription factor TFAP2A. ChIP-seq of TFAP2A revealed that it is widely bound to enhancers and 90% of these sites lack an active chromatin signature. When they compared TFAP2A binding sites that had or did not have the enhancer signature, they found that the sites that are not enhancers had significantly less H3K27ac. The sites that did overlap enhancers were correlated with gene expression. The lab also found NR2F1 and NR2F2 binding sites enriched as motifs. They determined that these proteins bind similar chromatin to TFAP2A. They hypothesized that allelic variants in these enhancer regions (SNPs) could affect transcription of genes involved in craniofacial development.
Polycomb Recruitment to Target Genes
Kristian Helin, University of Copenhagen
Dr. Helin touched a bit upon TET proteins, which are thought to be crucial to the removal of methyl groups from DNA. TET proteins work by hydroxylating the methyl group resulting in hydroxymethyl cytosine which is then processed to unmethylated DNA. Dr. Helin noted that a loss of TET proteins causes a local, but not global, decrease in gene expression but that it doesn’t seem to affect mouse embryonic stem cell proliferation or differentiation. He suggests that the primary purpose of TET proteins is to erase methyl groups from where they should not exist.
Dr. Helin spent more time talking about polycomb recruitment to target genes. In stem cells, polycomb proteins are poised on genes, keeping them off. The removal of PCG proteins then activates these genes. How PCG proteins are initially recruited to genes, however, is not well understood. Removal of the PRC2 complex causes a loss of H3K27me3 but not H2AK119ub. Previously this ubiquitin mark had been thought to recruit the PRC1 complex. The Helin lab asked if the Ring 1B ubiquitin ligase could be recruited independently of ubiquitin. One non-canonical PRC1 complex contains KDM2B (FBXL10/JHDM1B) which is an H3K36 demethylase.
The Helin lab hypothesized that KDM2B could be recruiting PCGs because it binds to non-methylated regions of DNA. They found that KDM2B is a component of a non-canonical PRC1 complex with Ring1B. They found that the c-terminus of Ring1B is required for binding KDM2B and that KDM2B recruits Ring1b to target genes. They also showed that if you remove KDM2B, ubiquitin levels decrease in a manner similar to what is seen in Ring1b deletion strains. KDM2B binds GC rich regions of DNA and its binding correlates with H3K4me3. It also colocalizes with Ring1B on bivalent chromatin. If you downregulate KDM2B you lose Ring1B and NSPc1 binding to targets and you see a decrease in H2A ubiquitinylation. The cxxc domain in KDM2B and NSPc1 is specifically required for ubiquitinylation. They conclude that KDM2B is required for differentiation but not proliferation of stem cells.
RNA polymerase II Promoter Occupancy
Asifa Akhtar, Max Planck Institute
Dr. Akhtar discussed dosage compensation in flies. This is a phenomenon in which the male chromosome has to be twice as active as the female chromosome. The method by which this occurs is not well understood. The MSL complex regulates this dosage compensation. MSL2 specifically is not expressed in female flies so its role in the process is particularly interesting. Dr. Akhtar wondered if enhanced RNA polymerase II occupancy of promoters was related to this phenomenon. She found that RNApolII is increased at promoters of x-linked genes in males but otherwise the distribution is not changed. This occupation results in an increase in short 5’ transcripts. She has also discovered that MSL1 is only structured when it is bound to MOF or MSL3 (other components of the MSL complex). MOF also interacts with the non-specific lethal complex (NSL) which made the Akhtar lab hypothesize that this interaction was at the same site as the MOF/MSL1 interface. They found that, indeed, this interaction does occur at the same site as the NSL interaction. They also noted that MSL1 can form homodimers or heterodimers with MSL2 but MSL2 cannot form heterodimers.
lncRNAs in Cardiomyocyte Regeneration
Laurie Boyer, MIT
As we age, are cardiac cells are lost and not replaced. Therefore, it is important to study cardiomyocyte regeneration. The Boyer lab uses embryonic stem cells to make cardiomyocytes in different stages: embryonic, mesoderm, cardiac progenitor, and cardiomyocyte. They performed RNA-seq and ChIP-seq on all of these cell types and studied the enhancers. They found many tissue specific enhancers. RNA polymerase II positive enhancers were enriched for non-coding transcripts and neighboring genes had higher gene expression patterns. Enhancers tend to show stage specific enrichment of highly conserved transcription factor binding motifs. These enrichments in active enhancers identify target gene networks. The combination of transcription factors bound to these enhancers defines stage specific expression. In other words, if all the transcription factors are there, the enhancer is “on.”
Dr. Boyer also talked about the possibility that long non-coding RNAs could be regulators of lineage commitment. They looked for lncRNAs that were highly expressed in cardiac stem cells but not in differentiated tissues. They found one called AK143260 which they re-named “Braveheart.” Braveheart is enriched in the nucleus and is not required for embryonic stem cell self-renewal. When you remove Braveheart, however, cardiomyocytes stop beating. Deletion of Braveheart leads to changes in gene expression, mostly correlating to downregulation of genes involved in heart morphogenesis. Finally they note that Braveheart appears to function upstream of MesP1, which drives cardiovascular differentiation.
The Histone Modification and Transcription Factor Relationship
Rick Young, Whitehead Institute
Dr. Young set out to address the issue of collaboration between transcriptional and epigenetic regulators. Transcription and histone modification are coupled process. Traditionally, we think of histone modifications being responsible for transcription factor variation but Dr. Young suggests that it could be the other way around. Maybe transcription factors are responsible for histone modifications. He uses c-myc as an example. C-myc regulates many different cell process and these processes differ between cell types. Dr. Young’s lab used a system where they could turn c-myc off and then turn it back on. When they turned it back on, they counted the number of c-myc molecules they could see.
They saw high levels of c-myc after 24 hours. They noticed that c-myc saturates core promoter E-box sequences and then binds lower affinity sites nearby. They followed this binding from high to low levels and noticed that the set of actively transcribed genes changed little with changes in c-myc binding. They did, however, see changes in the elongation factor pTefb which led to an increased number of transcripts. A threefold increase in transcripts led to a threefold increase in cell size. They saw no changes in silent genes. They noted that c-myc binding occurs only at actively transcribing genes, specifically in promoters and enhancers. When they studied c-myc binding in different cancers, they found that it binds to most actively transcribing genes, not to specific genes like people tend to think. He suggests that c-myc is more transcriptional amplifier than transcriptional activator and that looking for myc target genes is a flawed system because most people use microarray which is based on a median and is therefore not altogether reliable.
Dr. Young then used the relationship between c-myc and the chromatin associated protein BRD4 to illustrate the important relationship between transcription factors and epigenetic regulators in treating cancer. The Young lab used the IGH/MYC fusion to test JQ1, a drug targeting the bromodomain of BRD4. The IGH enhancer is very large and is hyperoccupied by BRD4. Cells containing the IGH/MYC fusion were highly sensitive to loss of BRD4 in the form of JQ1 treatment. Transcriptional elongation across the myc gene is reduced upon treatment with JQ1. Here Dr. Young presents us with an example of targeting chromatin regulators therapeutically in an effort to control a transcription factor disregulated in cancer.
**EpiGenie would like to thank Lauren Blair, a Postdoc Associate in the Yan lab at the Yale School of Medicine, for providing coverage of this conference.