Zeybel M, Hardy T, Wong YK, Mathers JC, Fox CR, Gackowska A, et al. Multigenerational epigenetic adaptation of the hepatic wound-healing response. Nat Med 2012;18:1369-1377. (Reprinted with permission.)
We investigated whether ancestral liver damage leads to heritable reprogramming of hepatic wound healing in male rats. We found that a history of liver damage corresponds with transmission of an epigenetic suppressive adaptation of the fibrogenic component of wound healing to the male F1 and F2 generations. Underlying this adaptation was less generation of liver myofibroblasts, higher hepatic expression of the antifibrogenic factor peroxisome proliferator-activated receptor g (PPAR-g) and lower expression of the profibrogenic factor transforming growth factor b1 (TGF-b1) compared to rats without this adaptation. Remodeling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for the histone variant H2A.Z and trimethylation of histone H3 at Lys27 (H3K27me3) at PPAR-g chromatin. These modifications to the sperm chromatin were transmittable by adaptive serum transfer from fibrotic rats to naive rats and similar modifications were induced in mesenchymal stem cells exposed to conditioned media from cultured rat or human myofibroblasts. Thus, it is probable that a myofibroblast-secreted soluble factor stimulates heritable epigenetic signatures in sperm so that the resulting offspring better adapt to future fibrogenic hepatic insults. Adding possible relevance to humans, we found that people with mild liver fibrosis have hypomethylation of the PPARG promoter compared to others with severe fibrosis.
Jean Baptiste de Lamarck proposed that characteristics acquired due to environmental effects could be inherited beyond the given generation; this is now known as Lamarckian inheritance.1 Charles Darwin supported Lamarckian inheritance and proposed the pangenesis theory, which was based on the inheritance of speculative tiny particles, which he called gemmules, which could be transmitted from parents to their progeny.2 However, the concept of Lamarckian inheritance was subsequently rejected. August Weismann, who proposed the theory that characteristics are transmitted only through the germ cells, but not the somatic cells, the so-called germ plasm theory, rebutted Lamarckian inheritance.3 He claimed that germ cells are strictly segregated from somatic cells, a separation that came to be referred to as the “Weismann Barrier.” In his theory, the characteristics that somatic cells learned and acquired during their lifetime could not be transmitted into germ cells, which is inconsistent with Lamarckian inheritance. However, recent progress in epigenetics provided evidence that environmental factors change epigenetic information, such as DNA methylation and histone tail modifications, and the alteration of epigenetic information is indeed inherited beyond the given generation, so-called transgenerational effects.4, 5
Zeybel et al.6 have uncovered that hepatic fibrosis leads to epigenetic changes in the sperm, which exert a suppressive function against fibrosis in subsequent male offspring. Surprisingly, this epigenetic information was transmittable through serum, derived from fibrotic rats, to normal rats, which implies that memorized epigenetic information can be transmitted across the Weismann barrier, mediated by serum. This report supports Lamarckian inheritance in which the traits acquired from environmental cues can be transmitted to the next generation.
The authors focused on the difference in susceptibility in the progression of liver cirrhosis among patients. They hypothesized that ancestral liver damage confers adaptive traits that are transmitted between generations through heritable epigenetic, rather than genetic, changes. They treated F0 and F1 male rats with the hepatotoxin carbon tetrachloride (CCl4) to induce hepatic injury, and then compared the degree of liver injury and fibrosis in F2 male rats after the administration of CCl4. There were no obvious differences in liver injury, but there was a difference in wound healing among F2 male rats. Male rats whose father and grandfather both had liver injury showed the least fibrosis, male rats whose father and grandfather both did not have liver injury showed the most severe fibrotic phenotype. These results indicate that ancestral liver injury provides heritable characteristics for the suppression of wound healing that occurs after liver injury. Liver fibrosis is caused by the overproduction of collagen derived from myofibroblasts that arise from hepatic stellate cells. Transdifferentiation from hepatic stellate cells to myofibroblasts is repressed by nuclear receptors PPAR-γ (Pparg), and the expression of Pparg was upregulated at peak fibrosis in livers of F2 rats whose ancestors received liver injury, compared to those of F2 rats whose ancestor had no liver injury.
The authors focused on the difference in epigenetic states within a regulatory region of Pparg in livers derived from normal and injured offspring. Consistent with adaptive changes in the expression of Pparg, they detected hypomethylation in the regulatory region of Pparg in livers at peak fibrosis from injured offspring. According to germ plasm theory as proposed by Weisman, transmission of a father's epigenetic information to offspring is mediated only by the sperm. Therefore, the authors verified the possibility that liver fibrosis induces changes in DNA methylation of Pparg in sperm derived from injured male rats. However, they could not detect DNA methylation of Pparg in the sperm. Therefore, they focused on other epigenetic marks, histone variants H2A.Z and H3K27me3, which are reported to be mutually exclusive with DNA methylation. Both the distribution of H2A.Z and the modification of H3K27me3 on Pparg were enriched in sperm derived from both CCl4-treated and bile duct-ligated male rats compared to controls. They propose that the enrichment of H2A.Z and H3K27me3 on Pparg in sperm is a form of epigenetic memory for the inheritance of adaptive information against a liver wound-healing response to offspring. Epigenetic information in germline cells is extensively reprogrammed at several stages, spermatogenesis, early embryo, and primordial germ cells (PGCs), the source of both oocytes and spermatozoa. Although canonical histones are largely exchanged for protamine, a small basic protein that is involved in the packaging of sperm DNA during spermatogenesis, about 4% of the haploid genome retains nucleosomes consisting of H2A.Z and H3K27me3.7 Furthermore, it has been shown that genome-wide epigenetic modifications, including DNA methylation and H3K9me2, are erased in early embryo and/or PGCs, while the genome-wide H3K27me3 erasure is not observed in germ cell development.8 These findings led me to consider that epigenetic information consisting of H3K27me3 is relatively more transmittable to the next generation, compared to DNA methylation and H3K9me2. Consistent with my consideration, another study about intergenerational transmission of epigenetic information has also suggested that changes in H3K27me3 modification in sperm by the intake of a low-protein diet affect offspring behavior.9
Recently, several reports have shown that changes in epigenetic information induced by environmental cues are inherited through the germline.9, 10 However, the precise route of transmission of epigenetic information from father to offspring has been largely unknown. The latest finding, demonstrated in this study, is that serum acts as a carrier of the characteristics, acquired due to environmental effects, to the sperm. The transfer of serum derived from injured male rats to uninjured male rats gave rise to enrichment of H2A.Z and H3K27me3 modification in Pparg in sperm from uninjured male rats. Unfortunately, the authors did not investigate whether recipient rats receiving the serum from injured rats acquired adaptive characteristics of the fibrogenic component of wound healing. They next showed that conditioned media, derived from activated human hepatic stellate cells, have the ability to enhance H2A.Z distribution and H3K27me modifications in Pparg in human mesenchymal stem cells (Fig. 1). This study provides new evidence that acquired characteristics can be transferred from somatic cells to germ cells through the serum, passing through the Weismann barrier, which strongly supports Lamarckian inheritance.
This study raises many questions and further investigation is required. The authors claim that the enrichment of H2A.Z and H3K27me3 at the Pparg locus in sperm from injured male rats were the epigenetic source for adaptation of the hepatic wound-healing response in offspring. If this consideration is true, this epigenetic information ought to spread across all cells that make up individuals in the next generation. The authors monitored the localization of H2A.Z and H3K27me3 at the Pparg locus only in sperm from injured rats and livers (at peak fibrosis) from their injured offspring. To test their hypothesis, epigenetic modifications at the Pparg locus should also be traced from sperm derived from injured rats to early embryos and various cell types in the offspring.
The authors focused on the effect of injured liver only on the adaptation to hepatic disease. However, it is possible that the serum from injured rats may alter other types of epigenetic information, nonadaptive effects to hepatic disease in their sperm. To test this possibility, genome-wide approaches, such as chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq), should be used to map the liver injury-induced global changes in epigenetic modification in sperm. Such studies may more comprehensively elucidate epigenetic and nonadaptive effects induced by liver injury.
I consider that there are two important issues in the study of epigenetic transgenerational effects. One is the identification of the “primary epigenetic marks” that are written or erased by environmental cues in germ cells; the other is the extraction of “inheritable epigenetic marks” among primary epigenetic marks beyond a given generation. Epigenetic information is established by the complex crosstalk of transcription factors, epigenetic modifiers, and signal transduction. To identify primary epigenetic marks, it is necessary to exclude secondary effects. Cultured germline stem (GS) cells, which can yield offspring, are a suitable resource with which to identify the primary epigenetic marks established by environmental conditions in germ cells. In the case of the study discussed here, culturing of GS cells with conditioned media derived from activated hepatic stellate cells may lead to the identification of both primary epigenetic marks and signal transduction from the extracellular environment to such primary epigenetic marks.