Course Title: Epigenetics

Course Title: Epigenetics Principle Lecturer: Professor Bao Liu （刘宝） E-mail: baoliu@nenu.edu.cn Homepage: http://www.nenu.edu.cn/professor/pro/show.php?id=138 Other Lecturers: Dr. Ningning Wang（王宁宁） Dr. Jinsong Pang （庞劲松） Dr. Yu Zhang (张瑜） Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, 130024, China

Course Title:Epigenetics Lecture Titles: Lecture 1: General Overview and History of Epigenetics Lecture 2: DNA methylation Lecture 3: Alteration in DNA methylation and its transgenerational inheritance Lecture 4: DNA methylation and genome stability Lecture 5: Histone modifications Lecture 6: Noncoding small RNAs Lecture 7: Epigenetic control of gene expression Lecture 8: Epigenetic variation in genome evolution and crop improvement Lecture 9: Epigenetics and human health Lecture 10: Summary

Father of Genetics, discovered the basic laws of heredity. Gregor Mendel (1822-1884)

Thomas Hunt Morgan (1966-1945) Discovered the 3rd basic genetic law, together with Mendel’s two laws, they form the basis of what is now known as classical genetics.

James Watson & Francis Crick Elucidation of the “double helix structure” of DNA molecule is one of the most important scientific discovery in the 20th century, which symbols the birth of Molecular Genetics.

Mendelian Genetics Change in nucleotide sequence Alteration in gene expression and/or function Novel phenotype

Epigenetics ---- non-Mendelian genetics Lamarckian phenomenon

Charles Darwin Jean-Baptiste Lamarck (1809-1882) (1744-1829)

Before 1800, Lamarck was an essentialist who believed species were unchanging; however, after working on the molluscs of the Paris Basin, he grew convinced that transmutation or change in the nature of a species occurred over time. He set out to develop an explanation, which he outlined in his 1809 work, Philosophie Zoologique. Lamarck developed two laws to explain evolution: The law of use and disuse: In every animal which has not passed the limit of its development, a more frequent and continuous use of any organ gradually strengthens, develops and enlarges that organ, and gives it a power proportional to the length of time it has been so used; while the permanent disuse of any organ imperceptibly weakens and deteriorates it, and progressively diminishes its functional capacity, until it finally disappears. The law of inheritance of acquired characteristics: All the acquisitions or losses wrought by nature on individuals, through the influence of the environment in which their race has long been placed, and hence through the influence of the predominant use or permanent disuse of any organ; all these are preserved by reproduction to the new individuals which arise, provided that the acquired modifications are common to both sexes, or at least to the individuals which produce the young. The idea of passing on to offspring characteristics that were acquired during an organism's lifetime is called Lamarckian.

Traditional examples considered as Lamarckian inheritance Giraffes stretching their necks to reach leaves high in trees (especially Acacias), strengthen and gradually lengthen their necks. These giraffes have offspring with slightly longer necks (also known as "soft inheritance").

Traditional examples considered as Lamarckian inheritance Ablacksmith, through his work, strengthens the muscles in his arms. His sons will have similar muscular development when they mature.

Like many of his generation, Kammerer undertook numerous experiments, largely involving interfering with the breeding and development of amphibians. He interested himself in the Lamarckian doctrine of acquired characteristics and eventually reported that a Midwife toad was exhibiting a black pad on its foot - an acquired characteristic brought about by adaptation to environment. Claims arose that the result of the experiment had been falsified. The most notable of these claims was made by Dr. G. K. Noble, Curator of Reptiles at the American Museum of Natural History, in the scientific journal Nature.[1] He reported that the black pad actually had a far more mundane explanation: it had simply been injected there with Indian ink. Six weeks later, Kammerer committed suicide. Paul Kammerer (1880-1926) 1971. , ISBN 0-394-71823-2. An account of Paul Kammerer's research on Lamarckian evolution and what he called "serial coincidences".

“Some authors use the term “variation” in a technical sense, as implying a modification directly due to the physical conditions of life; and “variations” in this sense are supposed not to be inherited; but who can say that the dwarfed condition of shells in the brackish waters of the Baltic, or dwarfed plants on Alpine summits, or the thicker fur of an animal from far northwards, would not in some cases be inherited for at least a few generations?” (Darwin, 1859)

Darwin wrote in 1861: Lamarck was the first man whose conclusions on the subject excited much attention. This justly celebrated naturalist first published his views in 1801. . . he first did the eminent service of arousing attention to the probability of all changes in the organic, as well as in the inorganic world, being the result of law, and not of miraculous interposition.

Science7 April 2000:Vol. 288. no. 5463, p. 38 Was Lamarck Just a Little Bit Right? Michael Balter Although Jean-Baptiste Lamarck is remembered mostly for the discredited theory that acquired traits can be passed down to offspring, new findings in the field of epigenetics, the study of changes in genetic expression that are not linked to alterations in DNA sequences, are returning his name to the scientific literature. Although these new findings do not support Lamarck's overall concept, they raise the possibility that "epimutations," as they are called, could play a role in evolution. Lamarck was a true pioneer of evolutionary theory!

Lecture I: General Overview and History of Epigenetics Various aspects of the modern understanding of epigenetic inheritance are reminiscent of Lamarck's ideas about evolution.

The current concept of epigenetics: Changes in phenotype that are inheritable but do not involve DNA mutation. Lecture I: General Overview and History of Epigenetics The Historic and modern definitions of “Epigenetics” The term 'epigenetics' was introduced by Conrad H. Waddington in 1942 to describe “the interactions of genes with their environment that bring the phenotype into being”. Conrad H. Waddington (1905-1975)

Lecture I: General Overview and History of Epigenetics The chromatin structure plays an important role in regulation of gene expression, while the tail modifications in the histones play an important role in the chromatin structure.

Lecture I: General Overview and History of Epigenetics Epigenetic inheritance Epigenetic inheritance is the transmission of information from a cell or multicellular organism to its descendants without that information being encoded in the nucleotide sequence of the genes. Epigenetic inheritance occurs in the development of multicellular organisms: dividing fibroblasts for instance give rise to new fibroblasts even though their genome is identical to that of all other cells. Epigenetic transmission of traits also occurs from one generation to the next in some organisms, though it is comparatively rare. It has first been observed in maize.

Champion of Chromatin and Pioneer of Epigenetics: Alan Wolffe (1959-2001)

Changes in gene expression and/or function and new phenotypes Genetics Epigenetics alterations mutations

Non-coding RNAs (samll RNAs) How are epigenetic variations accomplished？ Epigenetic effects can be accomplished by several self-reinforcing and inter-related covalent modifications on DNA and/or chromosomal proteins, such as DNA methylation and histone modifications, and by chromatin remodeling, such as repositioning of nucleosomes. These heritable modifications are collectively termed “epigenetic codes” (reviewed in Richards and Elgin, 2002).

Three types of Epigenetic variations:

Four classical epigenetic phenomena: • Position-Effect Variegation (PEV) (H.J. Muller, 1930) • Paramutation (R.A. Brink, 1958) • X-chromosome Inactivation (M.F. Lyon, 1961) • Genomic Imprinting

B' plants (light pigmented plant, colorless kernels) were crossed with B-Peru plants (nearly green plant, purple kernels). The resulting seeds (B'/B-Peru; purple kernels) gave rise to B' colored F1 plants. The F1 plants were crossed to B-I plants (dark pigmented plant, colorless kernels), giving rise to an ear segregating colorless (B'/B-I) and purple (B-Peru/B-I) kernels. When the colorless seeds were planted, the vast majority of the resulting plants showed a B' plant phenotype (B'/B-I' plants; the paramutation of B-I in these plants is indicated asB'*). Two dark individuals were isolated in which the B-I allele was not paramutated. The B' allele in these individuals is neutral for paramutation (B'-n). ~9000 colorless seeds (B’/B-I) planted Stam et al. (2002) Genetics ~6500 B’/B’* plants 2 B’-n/B-I plants (100% paramutation) (No paramutation) Paramutation (R.A. Brink, 1958) Brink described his stunning observations of “paramutation” at the R locus in maize in 1958. Several similar loci were later again discovered in maize.

The mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Mop1 is an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. It was proposed the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation. Alleman et al. (2006) Nature The mop1(mediator of paramutation1) mutation (A) B’ Mop1/mop1 (B) B’ mop1/mop1 (C) B-I Mop1/Mop1 (D) B’ mop1/mop1 with B’-like sectors (F) Pl’ mop1/mop1 (E) Pl’ Mop1/mop1

Molecular mechanism of X chromosome inactivation Chow et al. (2005) Annu. Rev. Genomics Hum. Genet. 6: 69-92.

Human genes escaping from X inactivation 624 genes were tested in nine Xi hybrids. Each gene is linearly displayed. Blue denotes significant Xi gene expression, yellow shows silenced genes, pseudoautosomal genes are purple, and untested hybrids remain white. Positions of the centromere (cen) and XIST are indicated. Carrel and Willard, 2005, Nature 434: 400-4.

Genomic imprinting Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. Imprinted genes are either expressed only from the allele inherited from the mother (eg. H19 or CDKN1C), or in other instances from the allele inherited from the father (eg. IGF2). Forms of genomic imprinting have been demonstrated in insects, mammals and flowering plants.

Body color (hypothetical) Genomic imprinting can be defined as the gamete-of-origin dependent modification of phenotype. Paternal imprinting means that an allele inherited from the father is not expressed in offspring. Maternal imprinting means that an allele inherited from the mother is not expressed in offspring.

"parent-of-origin effects" discovered ~3000 years ago by mule breeders in Asia.

Imprinted genes in plants Decades after imprinting was demonstrated in the mouse, a similar phenomena was observed in flowering plants (angiosperms). During fertilisation of the embryo in flowers, a second separate fertilisation event gives rise to the endosperm, an extraembryonic structure that nourishes the seed similar to the mammalian placenta. Unlike the embryo, the endosperm often contains two copies of the maternal genome and fusion with a male gamete results in a triploid genome. This uneven ratio of maternal to paternal genomes appears to be critical for seed development. Some genes are found to be expressed from both maternal genomes while others are expressed exclusively from the lone paternal copy.[30]

What do we learn from the last four classical epigenetic cases? Controlled by non-coding RNA and DNA/histone modification Significant variability/stability (PEV, ina-X) Reversible and/or transmittable through germ cells

Imprinting mechanisms Imprinting is a dynamic process. It must be possible to erase and re-establish the imprint through each generation. The nature of the imprint must therefore be epigenetic (modifications to the structure of the DNA rather than the sequence). In germline cells the imprint is erased, and then re-established according to the sex of the individual; i.e. in the developing sperm, a paternal imprint is established, whereas in developing oocytes, a maternal imprint is established. This process of erasure and reprogramming is necessary such that the current imprinting status is relevant to the sex of the individual. In both plants and mammals there are two major mechanisms that are involved in establishing the imprint; these are DNA methylation and histone modifications.

Some other important epigenetic phenomena

Bookmarking In genetics and epigenetics, bookmarking is a biological phenomenon believed to function as an epigenetic mechanism for transmitting cellular memory of the pattern of gene expression in a cell, throughout mitosis, to its daughter cells. This is vital for maintaining the phenotype in a lineage of cells so that, for example, liver cells divide into liver cells and not some other cell type.

Soft inheritance Soft inheritance is the term coined by Ernst Mayr to include such ideas as Lamarkism. It contrasts with modern ideas of inheritance, which Mayr called hard inheritance. Since Mendel, modern genetics has held that the hereditary material is impervious to environmental influences (except, of course, mutagenic effects).[1] In soft inheritance "the genetic basis of characters could be modified either by direct induction by the environment, or by use and disuse, or by an intrinsic failure of constancy, and that this modified genotype was then transmitted to the next generation."[2] Concepts of soft inheritance are usually associated with the ideas of Lamarck and Geoffroy. Recent work in plants and mammals on the role of the environment on epigenetic modifications of DNA have led to the argument that inherited epigenetic variation is a kind of soft inheritance.[1]

Most recently reported important epigenetic phenomena

Too big! Apparently as a result of abnormal imprinting, the cloned lamb at left is bigger than the normal lamb at right. Cloned animals often have other health problems as well.

Epigenetic variation among monozygous twins Monozygous twins are considered genetically identical, but significant phenotypic discordance between them exist, which is particularly noticeable for psychiatric diseases. Although MZ twins are epigenetically indistinguishable during early years of life, older MZ twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene expression portrait. Fraga et al. 2005, PNAS 102: 10604-9

Trans-generational inheritance of epigenetic variation • Consider an epigenetic mark (e.g. DNA methylation) that exists at a hypothetical locus in the primordial germ cells of the parent. Most aberrant epigenetic marks will be erased during the genome-wide epigenetic reprogramming during gametogenesis, and the mature gametes will not carry this mark. Occasionally, epigenetic marks escape reprogramming and are maintained in the mature gametes. These marks are transmitted to the offspring. There is a second wave of genome-wide epigenetic reprogramming around the time of blastocyst formation and some marks transmitted by the parent are erased at this stage. Marks that survive this reprogramming are then inherited by the offspring and have the potential to influence phenotypic outcomes.

The agouti locus in mouse Epigenetic regulation of the agouti gene in Avy/a mice. Phaeomelanin (the product of the agouti gene) is not produced from the ‘a’ allele because the agouti gene is mutated. Two potential epigenetic states of the ‘Avy’ allele can occur within cells of Avy/a mice. The IAP that lies upstream of the agouti gene can remain unmethylated, allowing ectopic expression of the gene from the IAP and resulting in a yellow coat colour (top). Alternatively, the IAP can be methylated, so that the gene is expressed under its normal developmental controls, leading to a brown coat colour. If the IAP methylation event occurs later in development and does not affect all embryonic cells, the offspring will have a mottled appearance (illustrated on the right). Right: Genetically identical week 15 Avy/a mouse littermates are shown, representing five coat-colour phenotypes. Mice that are predominately yellow are also clearly more obese than the brown mice Jirtle and Skinner 2007 Nat. Reviews Genetics 8: 253-262

Epigenetics and hybrid speciation O'Neill RJ, O'Neill MJ, Graves JA. 1998. Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid.Nature 1998 393: 68-72

S L PL How widely exist about such environmentally induced variation in the nature? To what extent such variation contributed to evolution?

Epigenetic Variation – significance and implications 1. Phenotypic variation is traditionally parsed into components that are directed by genetic and environmental variation. Now the line between these two components is blurred by inherited epigenetic variation. 2. How widely exist about the inheritable epigenetic variation in the nature? Could inheritance of epigenetic variants be an important means of adaptive evolution in the face of environmental change, without a permanent alteration in the DNA? What’s the difference between inheritable epigenetic variation and neo-Lamarckian? 3. There is an increasing belief that epigenetic variants and inheritance could provide the missing piece of the puzzle for understanding the basis of many complex phenotypes. 4. Our understanding of epigenetic variation and inheritance is still in its infancy, and it is unclear what proportion of heritable phenotypic variability can be ascribed to epigenetic factors.

Course Title: Epigenetics