Gerd P. Pfeifer

Aging and DNA methylation

In this Opinion article, we summarize how changes in DNA methylation occur during aging in mammals and discuss examples of how such events may contribute to the aging process. We explore mechanisms that could facilitate DNA methylation changes in a site-specific manner and highlight a model in which region-specific DNA hypermethylation during aging is facilitated in a competitive manner by destabilization of the Polycomb repressive complex.

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

BackgroundExposure to environmental endocrine-disrupting chemicals during pregnancy reportedly causes transgenerationally inherited reproductive defects. We hypothesized that to affect the grandchild, endocrine-disrupting chemicals must alter the epigenome of the germ cells of the in utero-exposed G1 male fetus. Additionally, to affect the great-grandchild, the aberration must persist in the germ cells of the unexposed G2 grandchild.ResultsHere, we treat gestating female mice with vinclozolin, bisphenol A, or di-(2-ethylhexyl)phthalate during the time when global de novo DNA methylation and imprint establishment occurs in the germ cells of the G1 male fetus. We map genome-wide features in purified G1 and G2 prospermatogonia, in order to detect immediate and persistent epigenetic aberrations, respectively. We detect changes in transcription and methylation in the G1 germline immediately after endocrine-disrupting chemicals exposure, but changes do not persist into the G2 germline. Additional analysis of genomic imprints shows no persistent aberrations in DNA methylation at the differentially methylated regions of imprinted genes between the G1 and G2 prospermatogonia, or in the allele-specific transcription of imprinted genes between the G2 and G3 soma.ConclusionsOur results suggest that endocrine-disrupting chemicals exert direct epigenetic effects in exposed fetal germ cells, which are corrected by reprogramming events in the next generation. Avoiding transgenerational inheritance of environmentally-caused epigenetic aberrations may have played an evolutionary role in the development of dual waves of global epigenome reprogramming in mammals.

Frequent epigenetic inactivation of cystatin M in breast carcinoma.

Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fishers exact test). Treatment of breast cancer cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.

Somatic TP53 Mutations in the Era of Genome Sequencing

Amid the complexity of genetic alterations in human cancer, TP53 mutation appears as an almost invariant component, representing by far the most frequent genetic alteration overall. Compared with previous targeted sequencing studies, recent integrated genomics studies offer a less biased view of TP53 mutation patterns, revealing that >20% of mutations occur outside the DNA-binding domain. Among the 12 mutations representing each at least 1% of all mutations, five occur at residues directly involved in specific DNA binding, four affect the tertiary fold of the DNA-binding domain, and three are nonsense mutations, two of them in the carboxyl terminus. Significant mutations also occur in introns, affecting alternative splicing events or generating rearrangements (e.g., in intron 1 in sporadic osteosarcoma). In aggressive cancers, mutation is so common that it may not have prognostic value (all these cancers have impaired p53 function caused by mutation or by other mechanisms). In several other cancers, however, mutation makes a clear difference for prognostication, as, for example, in HER2-enriched breast cancers and in lung adenocarcinoma with EGFR mutations. Thus, the clinical significance of TP53 mutation is dependent on tumor subtype and context. Understanding the clinical impact of mutation will require integrating mutation-specific information (type, frequency, and predicted impact) with data on haplotypes and on loss of heterozygosity.

RASSF1A interacts with and activates the mitotic kinase Aurora-A

The RAS association domain family 1A (RASSF1A) gene is located at chromosome 3p21.3 within a specific area of common heterozygous and homozygous deletions. RASSF1A frequently undergoes promoter methylation-associated inactivation in human cancers. Rassf1a−/− mice are prone to both spontaneous and carcinogen-induced tumorigenesis, supporting the notion that RASSF1A is a tumor suppressor. However, it is not fully understood how RASSF1A is involved in tumor suppression pathways. Here we show that overexpression of RASSF1A inhibits centrosome separation. RASSF1A interacts with Aurora-A, a mitotic kinase. Surprisingly, knockdown of RASSF1A by siRNA led to reduced activation of Aurora-A, whereas overexpression of RASSF1A resulted in increased activation of Aurora-A, suggesting that RASSF1A is involved in Aurora-A activation. Like other Aurora-A activators, RASSF1A was also a substrate of Aurora-A in vitro. The failure of recombinant RASSF1A to activate recombinant Aurora-A indicates that RASSF1A may not activate Aurora-A directly and suggests that RASSF1A may function as a scaffold to bring together Aurora-A and its activator(s). Inhibition of centrosome separation by RASSF1A overexpression is most likely a consequence of hyperstabilization of microtubules by this protein.

An Intrinsic Epigenetic Barrier for Functional Axon Regeneration

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

Are there specific readers of oxidized 5‐methylcytosine bases?

5‐methylcytosine (5mC) was long thought to be the only enzymatically created modified DNA base in mammalian cells. The discovery of 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxylcytosine as reaction products of the TET family 5mC oxidases has prompted extensive searches for proteins that specifically bind to these oxidized bases. However, only a few of such “reader” proteins have been identified and verified so far. In this review, we discuss potential biological functions of oxidized 5mC as well as the role the presumed reader proteins may play in interpreting the genomic signals of 5mC oxidation products.

Analysis of liver tumor-prone mouse models of the Hippo kinase scaffold proteins Rassf1a and Sav1

The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IκB kinase TBK1 as the most enriched RASSF1A-interacting proteins. The transcriptome of Rassf1a(-/-) livers was more deregulated than that of Sav1(+/-) livers, and the transcriptome of Rassf1a(-/-), Sav1(+/-) livers was similar to that of Rassf1a(-/-) mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several β-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis. Cancer Res; 76(9); 2824-35. ©2016 AACR.

How tobacco smoke changes the (epi)genome

Distinct mutation types are found in diverse cancers associated with smoking Worldwide, more than 1 billion people are tobacco smokers. Cigarette smoking drastically increases the risk of lung cancer. However, many other cancer types also occur more frequently in smokers than in nonsmokers, including cancers of the oral cavity, larynx, pharynx, esophagus, liver, cervix, pancreas, bladder, and kidney. Genome sequencing efforts are beginning to provide more sophisticated clues as to the processes at work that are shaping the mutational landscape of tumors. On page 618 of this issue, Alexandrov et al. (1) focused specifically on smoking-associated cancers and dissected genetic and epigenetic differences in tumors between smokers and lifetime nonsmokers.

High type I error and misrepresentations in search for transgenerational epigenetic inheritance: response to Guerrero-Bosagna.

In a recent paper, we described our efforts in search for evidence supporting epigenetic transgenerational inheritance caused by endocrine disrupter chemicals. One aspect of our study was to compare genome-wide DNA methylation changes in the vinclozolin-exposed fetal male germ cells (nu2009=u20093) to control samples (nu2009=u20093), their counterparts in the next, unexposed, generation (nu2009=u20093u2009+u20093) and also in adult spermatozoa (nu2009=u20092u2009+u20092) in both generations. We reported finding zero common hits in the intersection of these four comparisons. In our interpretation, this result did not support the notion that DNA methylation provides a mechanism for a vinclozolin-induced transgenerational male infertility phenotype. In response to criticism by Guerrero-Bosagna regarding our statistical power in the above study, here we provide power calculations to clarify the statistical power of our study and to show the validity of our conclusions. We also explain here how our data is misinterpreted in the commentary by Guerrero-Bosagna by leaving out important data points from consideration.Please see related Correspondence article: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0982-4 and related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0619-z