Dr Esteban Ballestar leads the Chromatin and Disease Group at Bellvitge Biomedical Research Institute (IDIBELL) in Barcelona, and is a member of the Cancer Epigenetics and Biology Programme (PEBC). His research focuses on epigenetic deregulation of immune cells in both autoimmune and autoinflammatory diseases as well as primary immunodeficiencies. Dr Ballestar has authored around one hundred papers and is also a member of numerous international scientific societies. His research has identified the role of methyl-CpG binding domain (MBD) proteins in the epigenetic deregulation in cancer. He has also contributed to seminal publications regarding specific alterations in histone modifications and DNA methylation in cancer and the age-dependent accumulation of epigenetic changes.
We spoke to Esteban to find out about his latest research and to get his expert insight into the field and future of epigenetics.
What are your current research interests?
One of my main research interests in molecular biology focuses on understanding how certain epigenetic modifications are targeted to specific genomic regions to achieve transcriptional control and, ultimately, define cell fate. By targeting, not only do I mean how different transcription factors recruit and target epigenetic enzymes to a given genomic sequence but also how extracellular factors (through their receptors) and downstream signalling pathways participate in such targeting of epigenetic changes. In other words, I am interested in understanding how extracellular factors, cell signalling pathways and nuclear factors engage cells in a specific transcriptional programme (and, ultimately, a given phenotype) through epigenetic modifications. These questions are particularly relevant in the context of the immune system, especially myeloid cells, which are highly plastic and respond to a wide variety of extracellular signals in the bone marrow, blood, and tissues. How these processes are disrupted in immune diseases is another major topic in my lab.
Can you tell us more about how epigenetics is involved in immune response
Various immune cell types undergo further terminal differentiation or activation in response to external influences or insults. Some examples include the activation of monocytes or macrophages in the presence of bacteria or other pathogenic agents, the perversion of the immunogenic responses of macrophages and dendritic cells in the tumoral microenvironment and the activation of naïve B cells to become memory B cells. Epigenetics is essential in specifying the fine gene regulation that it is needed for the acquisition of the final phenotype. The epigenomes of these cells are profoundly reprogrammed and different factors and pathways specify such remodelling.
What first ignited your interest in epigenetics?
My interest in epigenetics started during my early years in research, as the concept of a histone code was developed. The dual role of histones as both assembling unit blocks of chromatin and active players in the interphase between genetic information and transcriptional regulation was very attractive to me. In fact, my master thesis was on histone modifications. I continued working on histone modifications during my PhD under the supervision of Luis Franco in Valencia, Spain. During my postdoc, in Alan Wolffe’s lab at NIH, I eventually became attracted to DNA methylation and nuclear factors that mediate the interplay between DNA methylation and histone modifications, highlighting the interconnection between different epigenetic marks.
At Cambridge Epigenetix we’ve a long standing interest in DNA modifications. Is understanding DNA modifications important in your current research?
DNA modifications are central to our studies. For years, DNA methylation has been seen as a relatively stable epigenetic mark in comparison with histone modifications. However, cells of innate immunity, like monocytes and macrophages undergo vast changes in DNA methylation in terminal differentiation processes. In these cells, DNA methylation-related enzymes such as DNMT3A and TET2 are highly expressed and these enzymes play a central role in specifying the function of these cells. The relevance of DNA methylation events in this lineage is also highlighted by the frequent occurrence of mutations of DNMT3A and TET2 in myeloid leukaemia.
What do you think are the main outstanding challenges associated with studying DNA modifications and epigenetics, and how are you approaching them?
Epigenetic modifications are not only cell type- but also ‘individual cell’- specific. It is essential to ensure that the epigenetic profiles that we generate correspond to the actual cell type or cell that we are interested in understanding. In immune cells, for instance, this can be approached by using flow cytometry and cell sorting, using precise sets of antibodies to purify the cell type of study. However, this can be challenging in many cases.
What excites you most about the future of epigenetics?
Currently, we are witnessing the generation of a huge amount of information as the epigenomes of different cell types and tissues are determined. By epigenome I mean the whole map of all DNA and histone modifications along the entire genomic sequence. There are so many epigenomes! Every cell has a different epigenome at a specific moment and, therefore, the task of obtaining full maps of all epigenetic modifications is virtually endless. In addition, each modification is set by a variable number of enzymes that are targeted to specific genomic regions though the association with different sequence-specific nuclear factors. Understanding the functional contribution of different combinations of epigenetic marks at the sequence and three-dimensional level can still take many years. Grasping the relevance of individual epigenomes (at the single cell level) and the functional consequences of the epigenetic heterogeneity of cell populations is another challenge. We still have a huge amount of work in generating thousands of datasets and the challenge ahead is, first, interpreting in an integrated manner all that amount of information, and, second, being able to selectively manipulate parts of the epigenome of a given cell or group of cells to modify its behaviour.
Given epigenetics covers a whole range of mechanisms, what techniques do you typically use in your research?
We use a wide range of standard techniques to study epigenetics. Regarding DNA modifications, we mainly use bisulfite- and oxidative bisulfite-based methods to interrogate 5mC and 5hmC. In many cases, we combine the aforementioned methods with bead arrays, particularly when we have a high number of samples, especially in the case of patient cohort samples. For selected samples, we use whole genome bisulfite sequencing, or in some cases we generate libraries of enriched sequences and then perform deep sequencing.
We consider BS/oxBS the gold standard for DNA methylation analysis. We have also used MeDIP and Methyl-CAP (the last one based on capture of methylated DNA using the methyl-CpG-binding domain of MeCP2) in combination with arrays or quantitative PCR, but we prefer using BS or oxBS, as they provide single nucleotide resolution.
To map histone modifications or to test the binding of epigenetic enzymes or transcription factors, we use chromatin immunoprecipitation, which we have also combined in some cases with deep sequencing. We have also performed some experiments at the single cell level.
You mentioned studying 5hmC in your research. How important do you think it is to study this DNA modification in addition to 5mC?
Nowadays, it is important to take both 5mC and 5hmC into account when investigating DNA modifications. 5hmC is transient in some processes, but it has also demonstrated to be stable in some genomic regions and cell types and represents a separate epigenetic mark in its own merit. For years, standard bisulfite modification was used as the method to determine DNA methylation without considering that it measures the sum of 5mC+5hmC. In certain cases, the levels of 5hmC are negligible and therefore using bisulfite modification is sufficient). Bisulfite-based analysis can provide a good approximation when testing certain processes like the hypermethylation of CpG islands in cancer cells. However, researchers need to decide for their specific process whether bisulfite modification is sufficient or not.
I am interested by the potential use of 5hmC as a biomarker for diagnosis, prognosis and monitoring of cancer and various immune-related diseases. This epigenetic modification has a high potential in those diseases for which mutations or defects in TET proteins has been found, such as myeloid malignancies.
How do you handle the analysis of 5mC and 5hmC data?
Currently, data analysis is one of the main challenges in the field. There is a high need for bioinformaticians in epigenomics laboratories. Challenges not only include the capability to analyse datasets but also to integrate different data types. We do carry out some bioinformatics analysis in our lab but in many cases we need the support of dedicated bioinformatics teams and we have on-going collaborations with specialists in data analysis, including statisticians and bioinformaticians.
What breakthroughs in methodologies do you think are needed to move the field of epigenetics forward?
The availability of methods for the analysis of very small amounts of material or even single cells have been a major breakthrough. Currently, the main challenge is still the possibility of simplifying bioinformatics methods and the development of more tools in the same ways that kits helped to simplify the work in wet labs. Another challenge is the possibility of generating compounds that specifically inhibit different a wider range of epigenetic enzymes
Can you recommend any recent papers to researchers interested in this field?
There are so many papers that I could recommend that I think it may be best to provide a few keywords. I would recommend researchers to look for papers on methyladenine, an old DNA modification but very new in relation to its functional roles in eukaryotes. The field has exploded since 2015 and there many good papers to read, which also provide an excellent perspective on the development of the field of DNA modifications. A similar level of excitement happened around 5 years ago with the discovery of the oxidised forms of cytosine (5hmC and also 5fC and 5caC). Also, the many papers published by the Blueprint consortium illustrate very well the development of the field. These papers can be found on the Blueprint website.
What advice would you give any young researchers entering the field of epigenetics today?
My first piece of advice for young researchers is the need to read a lot, focusing on epigenetics papers in good journals which would help them to identify outstanding unanswered questions. There is a lot of literature with confusing concepts around epigenetics that need to be filtered out. Read, read, and read! And I would recommend them to attend dedicated basic epigenetic conferences besides attending those more specific to their field of research.
Where can we hear more about your research?
I will be presenting my work this year at a number of basic and clinical immunology conferences and you can follow my presence at these meetings on twitter at @eballestar.
Speaking of conferences, which ones do you recommend for epigenetics?
I would recommend the Gordon Research Conferences and Keystone Symposia on Epigenetics, which are among the best. In Europe, I recommend the EMBO Conference on Chromatin and Epigenetics at the EMBL.
Find out how CEGX can support your epigenetics biomarker development and research projects.