Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: DNA Habitats and Their RNA Inhabitants
Epigenetic heredity: RNA-mediated modes of phenotypic variation Minoo Rassoulzadegan and Franc¸ois Cuzin Universite´ de Nice Sophia Antipolis, Nice, France Address for correspondence: Minoo Rassoulzadegan, Universite´ de Nice Sophia Antipolis, Inserm U1091 – CNRS U7277, 06034 Nice, France. [email protected]
In addition to the Mendelian mutations, several instances of heritable phenotypic variation have been reported. We have observed, in mice, a role for sperm RNAs in the induction of such stable phenotypic variation. When experimentally transferred by RNA microinjection into fertilized mouse eggs, the noncoding RNAs homologous in sequence to the target locus are efficient inducers of variation at the transcriptional level. Transmission of the phenotypic variation to progeny is highly efficient and independent of gender. Here, we have summarized these finding and how they relate to other reports of epigenetic variation. Keywords: embryos; mouse; oocytes; RNA; spermatozoa; inheritance; fertilized egg; non-Mendelian
For the biologist, and currently also for the layman, “hereditary” means “determined by the nucleotide sequences of chromosomal DNA.” Such heredity is the basis of Mendelian analysis and one of the key processes in living organisms. However, 21stcentury genetics still cannot explain several welldocumented instances of inheritance. A distinct type of heredity was discovered, initially in plants, but more recently in other species ranging from nematodes to mice and, interestingly, humans. This peculiar mode of heredity may generate subtle variations, but it may also create visible markers, which can be followed by pedigree analysis, similarly to the mutations in classical genetic studies. A unique feature of these non-Mendelian variations is that a number of them occur as a response to an environmental change, such as modified diet availability1 or insufficient maternal care2 (see below). These variations are often qualified as epigenetic, but the term does not, by itself, provide an effective explanation of the heredity process. Two decades ago, embryologists fascinated by the deployment of developmental programs felt the need to reinvent the word “epigenetic” first proposed by Conrad Hal Wadington in 1942. At that time, DNA had not yet been identified as the constituent of the gene, and virtually nothing was known of the regulation
of gene expression. All of that unknown territory composed the realm of epigenetics. Currently, “epigenetics” has different meanings, and these differences generate confusion rather than clarification. For instance, “epigenetic” is sometimes used to refer to any unknown regulatory mechanisms. More frequently, “epigenetics” refers to the specific features of eukaryotic chromatin, modifications of histones, and methylation patterns in DNA (the so-called epigenome). Our group favors a more restrictive definition. We define epigenetic variations as longterm phenotypic changes that depend on neither the maintenance of temporary controls of transcription on the basis of ligand–receptor interactions nor on sequence variations in genomic DNA. The extreme long term is hereditary maintenance across generations. We and others have created experimental models, but the notion of epigenetic heredity was first established by epidemiological studies of human cohorts that showed inheritance of pathologies generated by food shortage and hyperlipidic food.1,3 A central goal is to identify the transgenerational vector of information responsible for the abnormal phenotype, especially in the case of paternal transmission. Our proposal was that noncoding RNAs (ncRNAs) in sperm act as transgenerational determinants.4 This notion is currently not accepted doi: 10.1111/nyas.12694
172
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
Rassoulzadegan & Cuzin
by some of our colleagues; only a few years ago, it was not accepted at all. The two most frequent alternatives offered are the transfer of stable chromatin structures (i.e., patterns of chemical modifications in histones) to the embryo and the transgenerational maintenance of cytosine-methylation profiles in DNA. Differences in chromatin structure between native and epigenetically modified genes, which correspond to distinct modes of expression, are clearly expected. Whereas the chromatin structures of the somatic genome are extensively erased during spermatogenesis, with histones replaced by protamines, a minor portion of the genome remains in the form of DNA–histone complexes.5 The hypothesis that local structural variations may be maintained in the sperm and play a role in the control of postfertilization expression may therefore not be excluded on a theoretical basis, but it has thus far failed to garner experimental support.6 The possible role of DNA-methylation patterns is more challenging to address. Several examples of clear transgenerational maintenance of methylcytosine distribution have been reported in plants and mice.7 It is not yet understood how de novo methylation is initiated, with the exception of a preference for repetitive sequences, transposon elements, and transgenes. A trend toward new and often nonreversible methylation through successive generations is often observed for newly transposed or transgene elements. It is not clear why some of these modifications escape the waves of demethylation that occur during gametogenesis and embryonic development, but the fact is that they are quite stable. The pioneering case of the paramutation of the maize B locus involves the induction of methylation in a 5 -upstream repetitive element. This methylation is stable over successive generations, with very low frequencies of spontaneous demethylation. Although the detailed mechanisms are not completely clear, de novo methylation of the locus, which results in silencing of the gene, requires elements of the RNA machinery.8 The metastable epialleles agouti viable yellow (Avy ) and axin fused (AxinFu ) are often referred to as examples of epigenetic heredity, although the main observation is not the transgenerational transfer of the variation per se, but rather an reprogramming effect in the progeny that modulates DNA methylation and gene expression from a transposed element.9 Recent reports compared DNAmethylation and chromatin-modification profiles of
RNA vector of epigenetic heredity
the soma and of sperm epigenomes at the genomic level to evaluate the possibility of their maintenance as transgenerational signals.6,7 A comprehensive understanding of transgenerational epigenetic variation on the basis of either one of these mechanisms has not yet been established. We have reported three independent cases of RNA-mediated heredity in mice.10–12 These instances were referred to as paramutations, which now appears somewhat misleading, as their mechanisms differ in several aspects from the phenomena originally documented in plants, with no change in DNA methylation detected in the loci of interest.13 The key experimental results were generated by exposure of naive fertilized mouse eggs to small RNAs by the classical microinjection procedure. This experimental method differs in several aspects from the transfer of sperm RNA, as it uses pure unmethylated RNA rather than sperm RNA–protein complexes. In fact, we observed a rapid degradation of most of the injected material within the first hour after injection. Nevertheless, efficient generation of the epigenetic phenotypes and strict sequence specificity provided reliable assays for the induction capacity of a given RNA. The three phenotypes analyzed, namely, fur color variation, cardiac hypertrophy, and gemelity and gigantism, were induced by short ncRNAs with sequence homology to the locus, such as the cognate microRNAs. In contrast to the silencing in the study of the maize paramutation, transcription of the three affected genes, namely, Kit, Cdk9, and Sox9, was upregulated in differentiated tissues by an unknown mechanism initiated by exposure of the genome to the homologous small RNAs at an early developmental stage. A similar result was recently reported by Gapp and colleagues in a study of the inheritance of stress behaviors.14 Our current work analyzes the inheritance of the pathologies generated by an unhealthy diet, obesity, and type 2 diabetes mellitus. Such heredity was first shown in humans in epidemiological studies1,3 that reported paternal transmission of the disease in the progeny of ancestors who experienced severe food restriction. These observations were transposed into experimental protocols in which mice or rats fed a hyperlipidic diet developed a metabolic syndrome, which was efficiently transmitted by the males to their progeny. Our contribution was to test this inheritance by RNA microinjection into fertilized oocytes from healthy parents. When RNA
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
173
RNA vector of epigenetic heredity
Rassoulzadegan & Cuzin
Figure 1. Summary of the hereditary modes of variations: different modes of variation with different degrees of sensitivity to environmental pressure.
was prepared from the sperm of obese and diabetic males, all of the offspring generated by reimplantation in healthy foster mothers and raised on a normal diet developed the complete syndrome. Control offspring generated by microinjection of sperm RNA from healthy males had normal health (V. Grandjean & M. Rassoulzadegan, manuscript in preparation). As outlined in Figure 1, it therefore appears that a variety of phenotypes can be generated from the stock of RNA accumulated in the spermatid and maintained in the sperm. Hereditary transmission is effected by RNA molecules devoid of coding capacity, short fragments of gene transcripts, or microRNAs. The notion of a regulatory function of ncRNA molecules is in full agreement with the current interest in the role of the major part of our genome that encodes ncRNAs.15 One important question is whether RNA accumulation in sperm is a selective process or, alternatively, whether sperm RNA just reflects the profile of somatic cells. The accumulation of defined species of ncRNAs not detected in somatic cells, such as a novel RNA product of a Piwi-interacting RNA (piRNA) locus16 and a class of tRNA-derived small RNAs,17 would argue for a choice among the RNAs in the immediate precursor of sperm, the round and elongated spermatids. Of possible relevance is the presence at these stages of a unique organelle, the chromatoid body, which contains the elements of the RNA-processing machinery.18 The mechanism underlying the regulatory effect following targeting of RNA to the locus by its homology to the DNA sequence remains unclear. A possible clue is provided by the observation that 174
RNA-mediated hereditary transmission did not occur in a mouse mutant lacking the RNA methyltransferase Dnmt2.13 This is a novel notion in the epigenetics field; the large number of RNA modifications identified so far have been in highly expressed RNAs, such as ribosomal RNA or tRNA, from bacteria to humans. Dnmt2 is an evolutionarily well-conserved protein, so far known to efficiently methylate several tRNAs, in vitro and in vivo. First experiments were performed on the paramutation induced at the Kit locus, which was not inherited from a mutant lacking Dnmt2. While no change was observed in the DNA-methylation profiles of the Kit locus between the wild-type and paramutant mice, RNA bisulfite sequencing indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Other RNA-mediated epigenetic variations were subsequently found to depend on Dnmt2 expression. Not exclusive of other modifications, cytosine methylation of RNA is crucial for induction of the epigenetic variation. Concluding remarks Modes of epigenetic heredity vary from worms to mice and humans. Control of inheritance protects the genome to preserve the species. Organisms have adapted responses to the environment to protect the integrity of the genome, with important differences between groups, such as the absence of DNA methylation in worms and flies and of RNA amplification in mice. The extent of the possible epigenetic changes remains a matter of speculation. Beyond the long and bright history of DNA-dependent genetic analysis, one may expect that an increase in
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
Rassoulzadegan & Cuzin
RNA-mediated experimentation will uncover new variant phenotypes. As a mutation in the DNA sequence of a gene may essentially affect one protein but may create multiple phenotypes, one may expect ncRNAs to affect several loci, thereby creating complex sets of phenotypic modifications. The initial suggestion of a role for sperm RNA in the fertilized embryo19 was met with a degree of skepticism. Currently, sperm RNA is a focus of research, including molecular determination by microarray and deep sequencing, analysis of RNA alterations in infertile men, and studies in mouse experimental models. A variety of ncRNAs are present in the sperm, with some differences in expression relative to that in somatic cells. How these RNA variants transmit information to the next generation remains to be established, and a precise experimental design will be required to further our understanding of the transgenerational signals. Sperm-RNA sequencing combined with RNA microinjection into fertilized eggs will be important methods to uncover the function of these RNA molecules in the transgenerational transmission of information. Acknowledgment Our laboratory is part of the French program «LAB EX SIGNALIFE» (ANR-11-LABX-0028-01). Conflicts of interest The authors declare no conflicts of interest. References 1. Pembrey, M.E. et al. 2006. Sex-specific, male-line transgenerational responses in humans. Eur. J. Hum. Genet. 14: 159– 166. 2. Weiss, I.C. et al. 2011. Inheritable effect of unpredictable maternal separation on behavioral responses in mice. Front. Behav. Neurosci. 5: 3. 3. Veenendaal, M.V. et al. 2013. Transgenerational effects of prenatal exposure to the 1944–45 Dutch famine. BJOG 120: 548–553.
RNA vector of epigenetic heredity
4. Cuzin, F. & M. Rassoulzadegan. 2010. Non-Mendelian epigenetic heredity: gametic RNAs as epigenetic regulators and transgenerational signals. Essays Biochem. 48: 101–106. 5. Hammoud, S.S. et al. 2009. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460: 473–478. 6. Carone, B.R. et al. 2014. High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm. Dev. Cell 30: 11–22. 7. Radford, E.J. et al. 2014. In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science 345: 1255903. 8. Alleman, M. et al. 2006. An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 442: 295– 298. 9. Whitelaw, E. & D.I. Martin. 2001. Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27: 361–365. 10. Grandjean, V. et al. 2009. The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth. Development 136: 3647–3655. 11. Rassoulzadegan, M. et al. 2006. RNA-mediated non-Mendelian inheritance of an epigenetic change in the mouse. Nature 441: 469–474. 12. Wagner, K.D. et al. 2008. RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 14: 962–969. 13. Kiani, J. et al. 2013. RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2. PLoS Genet. 9: e1003498. 14. Gapp, K. et al. 2014. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nat. Neurosci. 17: 667–669. 15. Mattick, J.S., R.J. Taft & G.J. Faulkner. 2010. A global view of genomic information—moving beyond the gene and the master regulator. Trends Genet. 26: 21–28. 16. Kawano, M. et al. 2012. Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos. PLoS One 7: e44542. 17. Peng, H. et al. 2012. A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm. Cell Res. 22: 1609–1612. 18. Kotaja, N. & P. Sassone-Corsi. 2007. The chromatoid body: a germ-cell-specific RNA-processing centre. Nat. Rev. Mol. Cell Biol. 8: 85–90. 19. Krawetz, S.A. 2005. Paternal contribution: new insights and future challenges. Nat. Rev. Genet. 6: 633–642.
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
175
Copyright of Annals of the New York Academy of Sciences is the property of WileyBlackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: DNA Habitats and Their RNA Inhabitants
Epigenetic heredity: RNA-mediated modes of phenotypic variation Minoo Rassoulzadegan and Franc¸ois Cuzin Universite´ de Nice Sophia Antipolis, Nice, France Address for correspondence: Minoo Rassoulzadegan, Universite´ de Nice Sophia Antipolis, Inserm U1091 – CNRS U7277, 06034 Nice, France. [email protected]
In addition to the Mendelian mutations, several instances of heritable phenotypic variation have been reported. We have observed, in mice, a role for sperm RNAs in the induction of such stable phenotypic variation. When experimentally transferred by RNA microinjection into fertilized mouse eggs, the noncoding RNAs homologous in sequence to the target locus are efficient inducers of variation at the transcriptional level. Transmission of the phenotypic variation to progeny is highly efficient and independent of gender. Here, we have summarized these finding and how they relate to other reports of epigenetic variation. Keywords: embryos; mouse; oocytes; RNA; spermatozoa; inheritance; fertilized egg; non-Mendelian
For the biologist, and currently also for the layman, “hereditary” means “determined by the nucleotide sequences of chromosomal DNA.” Such heredity is the basis of Mendelian analysis and one of the key processes in living organisms. However, 21stcentury genetics still cannot explain several welldocumented instances of inheritance. A distinct type of heredity was discovered, initially in plants, but more recently in other species ranging from nematodes to mice and, interestingly, humans. This peculiar mode of heredity may generate subtle variations, but it may also create visible markers, which can be followed by pedigree analysis, similarly to the mutations in classical genetic studies. A unique feature of these non-Mendelian variations is that a number of them occur as a response to an environmental change, such as modified diet availability1 or insufficient maternal care2 (see below). These variations are often qualified as epigenetic, but the term does not, by itself, provide an effective explanation of the heredity process. Two decades ago, embryologists fascinated by the deployment of developmental programs felt the need to reinvent the word “epigenetic” first proposed by Conrad Hal Wadington in 1942. At that time, DNA had not yet been identified as the constituent of the gene, and virtually nothing was known of the regulation
of gene expression. All of that unknown territory composed the realm of epigenetics. Currently, “epigenetics” has different meanings, and these differences generate confusion rather than clarification. For instance, “epigenetic” is sometimes used to refer to any unknown regulatory mechanisms. More frequently, “epigenetics” refers to the specific features of eukaryotic chromatin, modifications of histones, and methylation patterns in DNA (the so-called epigenome). Our group favors a more restrictive definition. We define epigenetic variations as longterm phenotypic changes that depend on neither the maintenance of temporary controls of transcription on the basis of ligand–receptor interactions nor on sequence variations in genomic DNA. The extreme long term is hereditary maintenance across generations. We and others have created experimental models, but the notion of epigenetic heredity was first established by epidemiological studies of human cohorts that showed inheritance of pathologies generated by food shortage and hyperlipidic food.1,3 A central goal is to identify the transgenerational vector of information responsible for the abnormal phenotype, especially in the case of paternal transmission. Our proposal was that noncoding RNAs (ncRNAs) in sperm act as transgenerational determinants.4 This notion is currently not accepted doi: 10.1111/nyas.12694
172
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
Rassoulzadegan & Cuzin
by some of our colleagues; only a few years ago, it was not accepted at all. The two most frequent alternatives offered are the transfer of stable chromatin structures (i.e., patterns of chemical modifications in histones) to the embryo and the transgenerational maintenance of cytosine-methylation profiles in DNA. Differences in chromatin structure between native and epigenetically modified genes, which correspond to distinct modes of expression, are clearly expected. Whereas the chromatin structures of the somatic genome are extensively erased during spermatogenesis, with histones replaced by protamines, a minor portion of the genome remains in the form of DNA–histone complexes.5 The hypothesis that local structural variations may be maintained in the sperm and play a role in the control of postfertilization expression may therefore not be excluded on a theoretical basis, but it has thus far failed to garner experimental support.6 The possible role of DNA-methylation patterns is more challenging to address. Several examples of clear transgenerational maintenance of methylcytosine distribution have been reported in plants and mice.7 It is not yet understood how de novo methylation is initiated, with the exception of a preference for repetitive sequences, transposon elements, and transgenes. A trend toward new and often nonreversible methylation through successive generations is often observed for newly transposed or transgene elements. It is not clear why some of these modifications escape the waves of demethylation that occur during gametogenesis and embryonic development, but the fact is that they are quite stable. The pioneering case of the paramutation of the maize B locus involves the induction of methylation in a 5 -upstream repetitive element. This methylation is stable over successive generations, with very low frequencies of spontaneous demethylation. Although the detailed mechanisms are not completely clear, de novo methylation of the locus, which results in silencing of the gene, requires elements of the RNA machinery.8 The metastable epialleles agouti viable yellow (Avy ) and axin fused (AxinFu ) are often referred to as examples of epigenetic heredity, although the main observation is not the transgenerational transfer of the variation per se, but rather an reprogramming effect in the progeny that modulates DNA methylation and gene expression from a transposed element.9 Recent reports compared DNAmethylation and chromatin-modification profiles of
RNA vector of epigenetic heredity
the soma and of sperm epigenomes at the genomic level to evaluate the possibility of their maintenance as transgenerational signals.6,7 A comprehensive understanding of transgenerational epigenetic variation on the basis of either one of these mechanisms has not yet been established. We have reported three independent cases of RNA-mediated heredity in mice.10–12 These instances were referred to as paramutations, which now appears somewhat misleading, as their mechanisms differ in several aspects from the phenomena originally documented in plants, with no change in DNA methylation detected in the loci of interest.13 The key experimental results were generated by exposure of naive fertilized mouse eggs to small RNAs by the classical microinjection procedure. This experimental method differs in several aspects from the transfer of sperm RNA, as it uses pure unmethylated RNA rather than sperm RNA–protein complexes. In fact, we observed a rapid degradation of most of the injected material within the first hour after injection. Nevertheless, efficient generation of the epigenetic phenotypes and strict sequence specificity provided reliable assays for the induction capacity of a given RNA. The three phenotypes analyzed, namely, fur color variation, cardiac hypertrophy, and gemelity and gigantism, were induced by short ncRNAs with sequence homology to the locus, such as the cognate microRNAs. In contrast to the silencing in the study of the maize paramutation, transcription of the three affected genes, namely, Kit, Cdk9, and Sox9, was upregulated in differentiated tissues by an unknown mechanism initiated by exposure of the genome to the homologous small RNAs at an early developmental stage. A similar result was recently reported by Gapp and colleagues in a study of the inheritance of stress behaviors.14 Our current work analyzes the inheritance of the pathologies generated by an unhealthy diet, obesity, and type 2 diabetes mellitus. Such heredity was first shown in humans in epidemiological studies1,3 that reported paternal transmission of the disease in the progeny of ancestors who experienced severe food restriction. These observations were transposed into experimental protocols in which mice or rats fed a hyperlipidic diet developed a metabolic syndrome, which was efficiently transmitted by the males to their progeny. Our contribution was to test this inheritance by RNA microinjection into fertilized oocytes from healthy parents. When RNA
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
173
RNA vector of epigenetic heredity
Rassoulzadegan & Cuzin
Figure 1. Summary of the hereditary modes of variations: different modes of variation with different degrees of sensitivity to environmental pressure.
was prepared from the sperm of obese and diabetic males, all of the offspring generated by reimplantation in healthy foster mothers and raised on a normal diet developed the complete syndrome. Control offspring generated by microinjection of sperm RNA from healthy males had normal health (V. Grandjean & M. Rassoulzadegan, manuscript in preparation). As outlined in Figure 1, it therefore appears that a variety of phenotypes can be generated from the stock of RNA accumulated in the spermatid and maintained in the sperm. Hereditary transmission is effected by RNA molecules devoid of coding capacity, short fragments of gene transcripts, or microRNAs. The notion of a regulatory function of ncRNA molecules is in full agreement with the current interest in the role of the major part of our genome that encodes ncRNAs.15 One important question is whether RNA accumulation in sperm is a selective process or, alternatively, whether sperm RNA just reflects the profile of somatic cells. The accumulation of defined species of ncRNAs not detected in somatic cells, such as a novel RNA product of a Piwi-interacting RNA (piRNA) locus16 and a class of tRNA-derived small RNAs,17 would argue for a choice among the RNAs in the immediate precursor of sperm, the round and elongated spermatids. Of possible relevance is the presence at these stages of a unique organelle, the chromatoid body, which contains the elements of the RNA-processing machinery.18 The mechanism underlying the regulatory effect following targeting of RNA to the locus by its homology to the DNA sequence remains unclear. A possible clue is provided by the observation that 174
RNA-mediated hereditary transmission did not occur in a mouse mutant lacking the RNA methyltransferase Dnmt2.13 This is a novel notion in the epigenetics field; the large number of RNA modifications identified so far have been in highly expressed RNAs, such as ribosomal RNA or tRNA, from bacteria to humans. Dnmt2 is an evolutionarily well-conserved protein, so far known to efficiently methylate several tRNAs, in vitro and in vivo. First experiments were performed on the paramutation induced at the Kit locus, which was not inherited from a mutant lacking Dnmt2. While no change was observed in the DNA-methylation profiles of the Kit locus between the wild-type and paramutant mice, RNA bisulfite sequencing indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Other RNA-mediated epigenetic variations were subsequently found to depend on Dnmt2 expression. Not exclusive of other modifications, cytosine methylation of RNA is crucial for induction of the epigenetic variation. Concluding remarks Modes of epigenetic heredity vary from worms to mice and humans. Control of inheritance protects the genome to preserve the species. Organisms have adapted responses to the environment to protect the integrity of the genome, with important differences between groups, such as the absence of DNA methylation in worms and flies and of RNA amplification in mice. The extent of the possible epigenetic changes remains a matter of speculation. Beyond the long and bright history of DNA-dependent genetic analysis, one may expect that an increase in
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
Rassoulzadegan & Cuzin
RNA-mediated experimentation will uncover new variant phenotypes. As a mutation in the DNA sequence of a gene may essentially affect one protein but may create multiple phenotypes, one may expect ncRNAs to affect several loci, thereby creating complex sets of phenotypic modifications. The initial suggestion of a role for sperm RNA in the fertilized embryo19 was met with a degree of skepticism. Currently, sperm RNA is a focus of research, including molecular determination by microarray and deep sequencing, analysis of RNA alterations in infertile men, and studies in mouse experimental models. A variety of ncRNAs are present in the sperm, with some differences in expression relative to that in somatic cells. How these RNA variants transmit information to the next generation remains to be established, and a precise experimental design will be required to further our understanding of the transgenerational signals. Sperm-RNA sequencing combined with RNA microinjection into fertilized eggs will be important methods to uncover the function of these RNA molecules in the transgenerational transmission of information. Acknowledgment Our laboratory is part of the French program «LAB EX SIGNALIFE» (ANR-11-LABX-0028-01). Conflicts of interest The authors declare no conflicts of interest. References 1. Pembrey, M.E. et al. 2006. Sex-specific, male-line transgenerational responses in humans. Eur. J. Hum. Genet. 14: 159– 166. 2. Weiss, I.C. et al. 2011. Inheritable effect of unpredictable maternal separation on behavioral responses in mice. Front. Behav. Neurosci. 5: 3. 3. Veenendaal, M.V. et al. 2013. Transgenerational effects of prenatal exposure to the 1944–45 Dutch famine. BJOG 120: 548–553.
RNA vector of epigenetic heredity
4. Cuzin, F. & M. Rassoulzadegan. 2010. Non-Mendelian epigenetic heredity: gametic RNAs as epigenetic regulators and transgenerational signals. Essays Biochem. 48: 101–106. 5. Hammoud, S.S. et al. 2009. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460: 473–478. 6. Carone, B.R. et al. 2014. High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm. Dev. Cell 30: 11–22. 7. Radford, E.J. et al. 2014. In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science 345: 1255903. 8. Alleman, M. et al. 2006. An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 442: 295– 298. 9. Whitelaw, E. & D.I. Martin. 2001. Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27: 361–365. 10. Grandjean, V. et al. 2009. The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth. Development 136: 3647–3655. 11. Rassoulzadegan, M. et al. 2006. RNA-mediated non-Mendelian inheritance of an epigenetic change in the mouse. Nature 441: 469–474. 12. Wagner, K.D. et al. 2008. RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 14: 962–969. 13. Kiani, J. et al. 2013. RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2. PLoS Genet. 9: e1003498. 14. Gapp, K. et al. 2014. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nat. Neurosci. 17: 667–669. 15. Mattick, J.S., R.J. Taft & G.J. Faulkner. 2010. A global view of genomic information—moving beyond the gene and the master regulator. Trends Genet. 26: 21–28. 16. Kawano, M. et al. 2012. Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos. PLoS One 7: e44542. 17. Peng, H. et al. 2012. A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm. Cell Res. 22: 1609–1612. 18. Kotaja, N. & P. Sassone-Corsi. 2007. The chromatoid body: a germ-cell-specific RNA-processing centre. Nat. Rev. Mol. Cell Biol. 8: 85–90. 19. Krawetz, S.A. 2005. Paternal contribution: new insights and future challenges. Nat. Rev. Genet. 6: 633–642.
C 2015 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1341 (2015) 172–175
175
Copyright of Annals of the New York Academy of Sciences is the property of WileyBlackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
