Faculty Bibliography

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

'Aging epigenetics' is an emerging field that promises exciting revelations in the near future. Here we focus on the functional and biological significance of the epigenetic alterations that accumulate during aging and are important in tumorigenesis. Paradigmatic examples are provided by the global loss of DNA methylation in aging and cancer and by the promoter hypermethylation of genes with a dual role in tumor suppression and progeria, such as the Werner syndrome (WRN) and lamin A/C genes. Another twist is provided by sirtuins, a family of NAD-dependent deacetylases that act on Lys16 of histone H4, which are emerging as a link between cellular transformation and lifespan.

Human cancers exhibit genomic instability and an increased mutation rate due to underlying defects in DNA repair genes. Hypermethylation of CpG islands in gene promoter regions is an important mechanism of gene inactivation in cancer. Many cellular pathways, including DNA repair, are inactivated by this type of epigenetic lesion, resulting in mutator pathways. In this review, we discuss the adverse consequences suffered by a cell when DNA repair genes such as the DNA mismatch repair gene hMLH1, the DNA alkyl-repair gene O(6)-methylguanine-DNA methyltransferase, the familial breast cancer gene BRCA1 and the Werner syndrome gene WRN become epigenetically silenced in human cancer.

In the last few years, microRNAs have started a revolution in molecular biology and emerged as key players in the cancer process. For these reasons, it is extremely important to understand the physiological and disease-associated mechanisms underlying the regulation of these small, single-stranded RNAs. Thus, it was merely a matter of time before microRNAs and epigenetics coincided. In cancer, aberrant DNA hypermethylation of tumor suppressor genes, global genomic DNA hypomethylation, and disruption of the histone modification patterns are the main epigenetic alterations, and have consequently been widely studied. Some microRNAs are downregulated in cancer and act as bona fide tumor suppressor genes, and this knowledge led to the proposal of the hypothesis that miRNAs could be silenced by epigenetic mechanisms. It has recently been shown that miR-127 and miR-124a, two putative tumor suppressor miRNAs, are methylated in tumor cells. Epigenomic tools can be effectively used in the search for new methylated tumor suppressor microRNAs. Furthermore, this aberrant methylation can be reversed by epigenetic drugs, such as DNA demethylating agents and histone deacetylase inhibitors, restoring microRNA expression levels and reverting the tumoral phenotype. In the coming years we will come to realize more fully the relevance of this expected encounter between two forces-epigenetics and microRNAs-that are currently at the forefront of biology.

Glioblastoma multiforme (GBM) is an incurable malignancy with inherent tendency to recur. In this study, we have comparatively analyzed the epigenetic profile of 32 paired tumor samples of relapsed GBM and their corresponding primary neoplasms with special attention to genes involved in the mitochondria-independent apoptotic pathway. The CpG island promoter hypermethylation status was assessed by methylation-specific polymerase chain reaction and selected samples were double checked by bisulfite genomic sequencing. Thirteen genes were analyzed for DNA methylation: the pro-apoptotic CASP8, CASP3, CASP9, DcR1, DR4, DR5 and TMS1; the cell adherence CDH1 and CDH13; the candidate tumor suppressor RASSF1A and BLU; the cell cycle regulator CHFR and the DNA repair MGMT. The CpG island promoter hypermethylation profile of relapsed GBM in comparison with their corresponding primary tumors was identical in 37.5% of the cases, whereas in 62.5% of patients, differences in the DNA methylation patterns of the 13 genes were observed. The most prominent distinction was the presence of previously undetected CASP8 hypermethylation in the GBM relapses (P = 0.031). This finding was also linked to the observation that an unmethylated CASP8 CpG island together with methylated BLU promoter in the primary GBM was associated with prolonged time to tumor progression (P = 0.0035). Our data strongly suggest that hypermethylation of the pro-apoptotic CASP8 is a differential feature of GBM relapses. These remarkable findings may foster the development of therapeutic approaches using DNA demethylating drugs and activators of the extrinsic apoptotic pathway to improve the dismal prognosis of GBM.

The balance of histone acetylation and deacetylation is an epigenetic layer with a critical role in the regulation of gene expression. Histone acetylation induced by histone acetyl transferases (HATs) is associated with gene transcription, while histone hypoacetylation induced by histone deacetylase (HDAC) activity is associated with gene silencing. Altered expression and mutations of genes that encode HDACs have been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis. Thus, HDACs are among the most promising therapeutic targets for cancer treatment, and they have inspired researchers to study and develop HDAC inhibitors.

We have performed a methylation-specific PCR approach to comparatively analyze the MGMT promoter methylation status in 186 glioblastomas (GBM) from patients with classic survival and nine from patients with long-term survival (LTS GBM). The methylation rate in LTS GBM was significantly higher (77.8% vs. 39.2%, P = 0.033) which suggests that MGMT hypermethylation is a frequent hallmark of LTS GBM and contributes to characterize this intriguing GBM subtype.

Monozygotic twins share the same genotype because they are derived from the same zygote. However, monozygotic twin siblings frequently present many phenotypic differences, such as their susceptibility to disease and a wide range of anthropomorphic features. Recent studies suggest that phenotypic discordance between monozygotic twins is at least to some extent due to epigenetic factors that change over the lifetime of a multicellular organism. It has been proposed that epigenetic drift during development can be stochastic or determined by environmental factors. In reality, a combination of the two causes prevails in most cases. Acute environmental factors are directly associated with epigenetic-dependent disease phenotypes, as demonstrated by the increased CpG-island promoter hypermethylation of tumor suppressor genes in the normal oral mucosa of smokers. Since monozygotic twins are genetically identical they are considered ideal experimental models for studying the role of environmental factors as determinants of complex diseases and phenotypes.

Epigenetic gene inactivation in transformed cells involves many 'belts of silencing'. One of the best-known lesions of the malignant cell is the transcriptional repression of tumor-suppressor genes by promoter CpG island hypermethylation. We are in the process of completing the molecular dissection of the entire epigenetic machinery involved in methylation-associated silencing, such as DNA methyltransferases, methyl-CpG binding domain proteins, histone deacetylases, histone methyltransferases, histone demethylases and Polycomb proteins. The first indications are also starting to emerge about how the combination of cellular selection and targeted pathways leads to abnormal DNA methylation. One thing is certain already, promoter CpG island hypermethylation of tumor-suppressor genes is a common hallmark of all human cancers. It affects all cellular pathways with a tumor-type specific profile, and in addition to classical tumor-suppressor and DNA repair genes, it includes genes involved in premature aging and microRNAs with growth inhibitory functions. The importance of hypermethylation events is already in evidence at the bedside of cancer patients in the form of cancer detection markers and chemotherapy predictors, and in the approval of epigenetic drugs for the treatment of hematological malignancies. In the very near future, the synergy of candidate gene approaches and large-scale epigenomic technologies, such as methyl-DIP, will yield the complete DNA hypermethylome of cancer cells.