“DNA methylation is like a storyteller. We believe that it keeps a memory of the cell of origin during development and it is informative about activities that take place during tumor development,” says Jose Ignacio Martin-Subero, Ph.D., principal investigator and leader of the epigenomics group at the University of Barcelona.
As part of a collaborative endeavor established by two major initiatives, the EU-funded Blueprint Project and the chronic lymphocytic leukemia (CLL) genome project, Dr. Martin-Subero and colleagues examined DNA methylation changes in 139 patients with CLL. The availability of the methylome, exon sequencing, and gene expression profiling data for this patient group, and access to their clinical reports, provided an unprecedented opportunity to examine methylation, gene expression, and clinical parameters in parallel.
One of the features that made this study unique is that, instead of using normal B cells as controls, it compared the full methylomes between pure populations of naïve and memory B cells, two subtypes that were isolated from the peripheral blood of a single donor.
“We found approximately 1.7 million CpG sequences that were significantly and clearly differentially methylated, indicating that a massive modulation of the DNA methylome occurs during B-cell differentiation, a finding that was quite unexpected,” notes Dr. Martin-Subero.
Patients with CLL belonging to one of two subtypes, either with very favorable prognosis, related to memory B-cell origin, or with a slightly worse prognosis, related to naïve B-cell origin, exhibited very distinct DNA methylome signatures, despite expressing very similar gene sets, and DNA methylation did not correlate well with gene expression.
A comparison revealed that approximately 90% of the changes were hypomethylation, which was enriched in gene bodies and intergenic regions, while the less frequently occurring hypermethylation was enriched at transcriptional start sites.
By using the ENCODE data to examine the genomic distribution of hypomethylation, the investigators found an enrichment of this modification at enhancer regions during both B-cell differentiation and CLL pathogenesis. In addition, a consensus cluster analysis that used 10,000 permutations of the slightly over 1,600 CpG sites found that approximately 15% of the patients did not belong to any of the two groups, but showed an intermediate DNA methylation profile, and had an intermediate prognosis.
“We believe that DNA methylation helps us better classify this disease, and the cell of origin seems to be associated with prognosis, because the more undifferentiated it is, the worse the prognosis seems to be,” explains Dr. Martin-Subero.
While alternative splicing was estimated to occur in approximately 95% of human genes, and has increasingly been implicated in gene regulation during development and disease, understanding the propagation and maintenance of alternative splicing patterns during cell division and differentiation remains elusive.
“Alternative splicing is the last of the major steps in gene expression that we still do not understand, and the idea that these patterns could be determined by epigenetic marks is very attractive,” says Tom Misteli, Ph.D., head of the ccll biology and genomes group at the National Cancer Institute.
A model that emerged several years ago examined splicing patterns for the same alternatively spliced reporter gene that was expressed from promoters of variable strength, and proposed that splicing is shaped by the RNA polymerase elongation rate. More recently, several groups found, during genome-wide histone modification analyses, that specific histone modifications accumulate over exons, marking their boundaries.
“This was intriguing as it drew a lot of attention to the idea that chromatin and epigenetic marks could affect splicing and brought the two areas together,” says Dr. Misteli.
Recent work in his lab made important contributions toward unveiling the interface between splicing and epigenetic modifications. In a survey of histone modifications on the alternatively spliced FGFR2 gene, Reini Luco, Ph.D., at the time a post-doctoral fellow in the Misteli lab, found that enrichment in a particular histone modification, H3K36 trimethylation, recruits, through a protein adaptor protein, a polypyrimidine tract binding protein that suppresses the inclusion of an exon, leading to mesenchymal stem cell splicing patterns.
The alternative outcome, in which recruitment of another chromatin factor causes the exclusion of a different exon, specifies epithelial stem cell lineage commitment.
“Additional layers of regulation most likely exist, because we know, for example, that if certain splicing factors are phosphorylated, their propensity for being recruited to RNA molecules changes,” explains Dr. Misteli.
As a field that unveils new concepts and complements genetic and genomic approaches, epigenetics promises a novel framework to dissect cellular and molecular networks shaping development, differentiation, homeostasis, and pathogenesis.
Integrating multiple sources of information assumes a key role during these endeavors. Findings that uncover the involvement of epigenetic modifications in many biological processes, the complex regulatory pathways, and the crosstalk between them, are paving the way for one of the most exciting times in biology and medicine.