Clinical Epigenetics Workshop & Symposium
The DIA-ChiP workshop and symposium offered a unique combination of wet lab training, and cutting edge epigenetic seminars that we just couldn’t resist. Elisabetta Fratta was on hand in Swansea, Wales to give us a first hand account of how the multi-faceted event played out. Check out her Report:
DIA Chip Overview
The DIA ChIP meeting, which was EC funded through a Framework Programme 7 project entitled ‘ChIP and MIRA for clinical diagnosis’ (DIA-ChIP), took place on June 11th-13th, 2012 in Swansea, Wales. The aim of the DIA-ChiP workshop was to create a venue to stimulate participants to discuss developments and gain practical experience in new high throughput molecular screening techniques, as well as to promote innovative research in Clinical Epigenetics. The workshop was very successful, attracting approximately 100 scientists and clinical researchers, was well organized and kept a very high standard for oral and poster presentations. This workshop gave also a chance for industry representatives from well known companies like Active Motif, Porvair Filtration Group, Merck Millipore and Biorad to share their own research with academic researchers.
The workshop included a lab session which was hosted by the Reproductive Biology Group (RBG) lead by Dr. Steve Conlan at the Institute of Life Science and Centre for NanoHealth. The overall atmosphere during the lab session was familiar and people working in the Conlan lab were very nice and well prepared. The lab session gave the opportunity to get lab hands-on, wet lab experience through the key stages of chromatin immunoprecipitation (ChIP), a powerful and widely applied technique for studying interactions of individual proteins with specific genomic regions within the cell. The session focused on chromatin extraction techniques from cell lines to clinical samples (fresh frozen tissue and FFPE material) and on chromatin preparation strategies, including fragmentation of chromatin. A more theoretical section was also included that provided an overview on the choice of adequate controls for ChIP experiments and for PCR technologies that can be used to specifically analyze the gene or chromosomal region of interest, plus common methods used to normalize ChIP-qPCR data.
At the end of the lab session all participants moved to the Sketty Hall, a Georgian mansion set within superb ornamental gardens for the rest of the talks.
Understanding the Impact of Epimutations in Cancer
Adele Murrell, Cambridge Research Institute
The DIA-ChIP symposium was opened by the excellent keynote lecture from Dr. Murrel who underlined the importance of determining baseline normal epigenetic profiles in normal cell-biology conditions in order to identify epigenetic markers inherently stable in most normal cells and use them to monitor the overall epigenetic health of cells. Murrell closed off by saying there are new technologies that must be used to interrogate different mechanisms involved in cell epigenetic reprogram with the aim to understand the impact of epimutations in cancer.
Epigenetic Regulation of ESR1
Steve Conlan, Swansea University
In the initial phase Dr. Conlan briefly described DIA-ChiP, a project that includes collaborative partnership between academic, SME and public health partners. The aim of DIA-ChiP is to develop molecular techniques for the routine high-throughput analysis of clinical samples for advanced molecular diagnosis and biomedical research. Subsequently, Conlan spoke about epigenetic regulation of ESR1, the gene encoding the estrogen receptor ( ERa) protein variants ERa-66 and ERa-36.The role of DNA methylation in the expression of ERa isoforms was investigated in endometrial cancer cell lines treated with 5-aza-2’deoxycytidine (5-AZA-CdR). Bisulfite sequence analysis revealed that hypomethylation of the ERa promoter induced by 5-AZA-CdR treatment associated with an increased expression of ERa-66 and ERa-36 depending on the endometrial cancer cell line analyzed.
Brg1 and Chromatin Remodeling in Cancer
Aliaksei Holik, Cardiff University
Dr. Holik focused his talk on Brg1, an ATPase subunit of the SWI/SNF class of chromatin remodelling complexes, which may contribute to cancer development due to its role in the trans-activation of Wnt-pathway target genes. In order to investigate Brg1’s potential role in vivo, Brg1 was deleted in the murine small intestinal epithelium under normal physiological conditions and in the context of a Wnt-activated background. Results obtained suggested that in the small intestinal epithelium i) Brg1 deﬁciency impedes Wnt-driven tumourigenesis by impairing Wnt signalling ii) Brg1 is a crucial factor for stem cell maintenance. Moreover, simultaneous loss of Brg1 and negative Wnt pathway regulator Apc resulted in reduced tumourigenesis and increased animal survival compared to Apc-deficient animals.
Breast Cancer Treatment Using PARP Inhibitors
Jurgen Veeck, Aachen University
Dr. Veeck discussed poly(adenosine diphosphate)-ribose polymerase (PARP) inhibitors, a novel drug class most promising for the treatment of hereditary BRCA-associated breast cancer. Data reported demonstrated that inactivation of the BRCA1 gene by CpG island hypermethylation could be a potential biomarker of sensitivity to these compounds in breast cancer cells (BCL), being BRCA1 mutated and BRCA1 methylated BCL most sensitive against PARP inhibitors. Also, epigenomic profiling identified several other potential candidate genes next to BRCA1 that might confer PARP inhibitor sensitivity in BCL.
Telomerase: A Potential Cancer Drug Target
Evelyne Segal-Bendirdjian, INSERM
Dr. Segal-Bendirdjian spoke about human telomerase, an enzyme that is highly expressed and active in most malignant tumors (85%), thus indicating that its regulation may represent a promising target for anticancer drug discovery. Telomerase consists of two components, a RNA component (hTR), which is used as a template for the elongation of telomeres and a protein reverse transcriptase (hTERT), which represents a key regulator of this enzyme. Regulation of telomerase operates at several biological levels: transcriptional, post-transcriptional and post-translational. Acute promyelocytic leukemia (APL) was found to be particularly sensitive to therapy using ATRA (all-trans retinoic acid), which induces remission of APL patients by stimulating differentiation of leukemic cells. Segal-Bendirdjian’s laboratory found out that ATRA not only transcriptionally repressed hTERT in differentiation-associated pathways, but also independently of differentiation. These findings are of importance for therapy of APL because they suggest that a telomerase-dependent mechanism can be targeted by retinoids in maturation-resistant cells. Based on these data maturation-resistance cells were used to study telomerase activity.
Two distinct functional domains were identified in hTERT promoter: a distal region where hTERT repressor signaling likely converges and a proximal domain that encodes binding sites essentially for activators of hTERT expression. However, DNA methylation might have a key role in hTERT expression, but in an opposite manner than has been previously proposed. In fact, the analysis of the hTERT promoter demonstrated that when the proximal domain is hypomethylated, WT1 or ATRA-inducible hTERT repressors could bind the promoter thus preventing gene expression, even in presence of appropriate transcription factors acting downstream on the promoter. On the other hand, methylation of CpG islands within the distal domain could prevent the binding of WT1 or ATRA-inducible hTERT repressors, allowing the promoter to remain activated by regulatory factors whose binding would be methylation dependent. In addition to DNA methylation, histone modifications have a critical role in the modulation of hTERT expression, through affecting the accessibility of the regulatory sequences to the transcriptional factors. Recent studies have demonstrated that also mammalian telomeres and subtelomeric regions are enriched in epigenetic marks of repressed heterochromatin, thus indicating the importance of the epigenetic status of the telomeric chromatin in mammaliam telomere length regulation.
Differential miRNA Expression Patterns in Trachoma Samples
Tamsyn Derrick, London School of Hygiene and Tropical Medicine
Dr. Derrick investigated conjunctival miRNA expression in scarring trachoma (TS) and inflammatory trachoma (TSI) utilizing conjunctival swabs selected from a large archive of samples collected from individuals resident in trachoma endemic communities, like The Gambia. In total, five miRNAs were found differentially expressed between TS and control groups, while 27 mRNAs resulted differentially expressed between TSI and control groups.
The ENCODE Project
Ian Dunham, EMBL-European Bioinformatics Institute
Dr. Dunham concluded the session by speaking about the public consortium named ENCODE, the Encyclopedia of DNA Elements, which is carrying out a project aimed to identify all functional elements in the human genome sequence. You can find more details at the ENCODE website. (www.encodeproject.org).
Optimizing Analysis of Chromatin Alterations in Clinical Samples
Saverio Minucci, European Institute of Oncology and University of Milan
Dr. Minucci presented compelling data indicating that even epigenetic drugs (histone deacetylase inhibitors and DNA demethylating agents) are currently widely propagandized as an example of successful targeted approaches, they have been effective only in a few tumor types. The lack of consistent clinical results could be due to their employment in a poorly targeted approach, since little is known about targets of epigenetic alterations during tumor development. Therefore, both basic and applied research should focus on the identification of epigenetic alterations in tumor cells that are critical for cell survival. This aim can be pursued by optimization and development of technological approaches enabling the analysis of chromatin alterations in patient samples.
Pathology Tissue ChIP Technique: PAT-ChIP
Mirco Fanelli, University of Urbino
Dr. Fanelli presented a ChiP protocol, called PAT-ChiP (Pathology Tissue – Chromatin Immunoprecipitation) that can be applied to epigenetic studies using formalin-fixed paraffin-embedded (FFPE) sections of pathological tissues. This protocol includes different steps of sonication in association with the controlled enzymatic cleavage of DNA by micrococcal nuclease activity in order to efficiently isolate chromatin fragments with an average size compatible with the final step of DNA analysis. The quality of the antibody influences the feasibility of the entire PAT-ChiP procedure, so the best antibody for subsequent PAT-ChiP steps must be found by testing them against the target of interest in a screening procedure (e.g., immunofluorescence) using experimental conditions as close as possible to those applied to perform the PAT-ChIP assay. Non- specific binding is avoided by progressive increase of the washing step stringency. The immunoprecipitated DNA is then subjected to de-crosslinking and phenol/chloroform extraction and finally analyzed through different procedures, such as qPCR.
EZH2 Alters Tumor Resistance to Chemotherapy
Robert Brown, Imperial College London
In his talk, Dr. Brown discussed the importance of defining epigenetic profiles of individual cancer subtypes for improving diagnosis and epigenetic therapies. Along this line, Brown’s group recently demonstrated that DNA methylation at promoter CpG islands (CGIs) of Wnt pathway genes is strictly associated with progression-free survival (PFS) of ovarian cancer patients. DNA methylation profiling at promoter GPIs of other key pathways/families also revealed methylation at NKD1 and DVL1 as independent predictors of PFS. Furthermore, the histone methyltransferase EZH2, known to be involved in epigenetic regulation, was found over-expressed in ovarian tumour sustaining cells. Inhibition of EZH2 expression by siRNA knock-down reduced tumour growth, whereas overexpression of EZH2 associated with acquired cisplatin resistance. Together these data support a key role for EZH2 in the maintenance, during chemotherapy, of a drug-resistant tumour-sustaining subpopulation of cells in ovarian tumours that can potentially influence the subsequent acquisition of drug resistance and tumor progression.
Epigenetic Consequences of Hypoxia
Amanda McCann, University College Dublin
Dr. McCann explained that increasing levels of tissue hypoxia have been reported as a key tumor microenvironment associated with chemio-resistence. In a recent study McCann’s group investigated phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. Results indicated a significantly altered cellular phenotype in response to chronic hypoxia, characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL6 and IL8. These phenotypic changes associated with increases in global levels of DNA methylation and H3K9 histone acetylation, concomitant with the increased expression of DNA methyltransferase DMNT3b. Moreover, expression of the tumor suppressor gene MAD2 was reduced in hypoxia, suggesting that MAD2 down-regulation might be a crucial mechanism by which hypoxic cells become chemorefractory to Paclitaxel treatment. In this regard, treatment with epigenetic drugs, such as 5-AZA-CdR and TSA, was able to restore MAD2 expression.
**EpiGenie thanks Elisabetta Fratta for kindly providing this conference coverage. Elisabetta is a PhD Medical Biotechnologist in the Department of Medical Oncology at the Centro di Riferimento Oncologico in Italy.