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Genetic imprinting and gene silencing.

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The Three Partners
   There is a well-known statement in the Talmud that: There are three
   partners involved in creating a human being: G-d, the father, and
   the mother. The father sows the seed of "whiteness" whence are
   derived the bones, nerves, fingernails, and toenails, the brain
   inside the head, and the white of the eye. The mother sows the seed
   of "redness" whence are derived the skin, the flesh, the hair, and
   the black of the eye. G-d provides the different aspects of the
   soul, the facial features, the ability of the eye to see, the
   ability of the ear to hear, the ability of the mouth to speak, the
   ability of the legs to walk, understanding, and know-how. (1)


Although the potential for all human traits is inherited from the parental genomes, the genetic information encoded in the DNA of the developing fertilized human egg is never fully expressed equally at all times, nor in all cells and tissues throughout the individual's lifetime. Differentiation is programmed, and the expression of genetic material is under the tight control of an extremely complex signal-transduction system. The sheer number of players and pathways in this system grows larger with every research day. It is truly mind-boggling and miraculous.

DNA can be seen to be the "raw material" or the undifferentiated homer (matter) which arises from G-d's speech, as the Alter Rebbe (Rabbi Shneur Zalman of Liadi) explains. (2) I humbly suggest that the transcription, translation, and post expression modifications could be regarded as the tsurah (form) that flows from G-d's higher will. Every genetically-encoded characteristic inherited from the parents has to be expressed in a manner that G-d has decreed to be appropriate for each of His creations.

Gene Silencing

It is not immediately intuitive that certain traits should be monoparentally inherited, because it is commonly assumed that both chromosomes of each pair are read and contribute to the gene-product pool. We must remember, however, that one of the X-chromosomes in females becomes inactivated early in development. This was first proposed over forty-five years ago; (3) so it has been obvious for a long time that gene silencing does occur. We only began to reexamine it seriously when the molecular basis of genetic developmental diseases in children was uncovered. As it turns out, these defects occur mostly in pathways of development involving almost exactly those faculties enumerated by the Sages of the Talmud !

Today, many assumptions about Mendelian inheritance are being challenged because maternal and paternal inherited alleles are not always identical, and normal function is not always associated with two working copies of a normal gene. There is a lot more to genetic expression than just the sequence of nucleotides in DNA. In fact, the whole notion of "The Central Dogma," i.e., DNA>RNA>Protein, is now being seriously challenged because of the existence of ribozymes, reverse transcription, prions, small interfering RNAs, and so forth.

The availability of complete DNA sequences of many organisms demonstrates that the human genome differs only slightly from that of the mouse, but it is not very difficult to distinguish between the two. Humans have forty-nine pseudogenes (coding sequences for a protein that lack the signals necessary for transcription) for the important oxidative protein cytochrome oxidase, none of which are ever used. One of these human pseudogenes, however, is the exact analog of the functional one in mice.

Not long ago, the prediction was made, based on human cellular DNA content, that humans have at least 100,000 genes. This prediction has proved to be wrong by at least threefold. A cockroach has nearly twice as many chromosomes as a human being. Most of our DNA is looking for a function. This large unused component has been called "selfish" because it insists on replicating itself forever.

We have not yet learned how to decipher all the information encoded in the nucleotide sequence of DNA. In fact, today, it is the three-dimensional structure of proteins that has become the basis for evolutionary and taxonomic studies. The functional shape of a protein can be derived from completely different sequences. Whole databases containing "fuzzy" functional forms (FFF) have been constructed and are available through online searching. I recently described the three-dimensional solution structure of a small protein involved in the morphogenesis of the head structure of a bacterial virus.4 Although its coding sequence is very remote, the derived structure is remarkably similar to proteins involved in human DNA replication. Finally, although we have been accustomed in the past to think of mutations in the genes themselves as the most important factor in alteration of biological expression, we shall have to get used to a whole new way of looking at genetics.

Epigenetics

Recent discoveries related to genetic expression have led to the development of the new scientific field of epigenetics, which involves genetic imprinting and gene silencing. Since this process adds additional information to the DNA sequence, it has been called "epigenetics" (epi=on).

Epigenetics is one way in which the A-lmighty might have superimposed His tsurah (form). Imprinting may be defined as a reversible modification of DNA that can lead to differential expression of maternally or paternally inherited genes. Imprinting does not change the DNA sequence or its homer (matter).

The Mechanism of Gene Imprinting and DNA Methylation

Unlike sex-linked traits, the appearance of which depends on the sex of the offspring, imprinted traits depend on the sex of the contributing parent and may show up in either sex. Normally, imprinting represses gene expression and is imposed during gametogenesis, the process of forming the sperm and the egg. Imprinting must be reimposed following DNA replication and must remain restricted to one chromosome. Moreover, not all chromosomes are imprinted, and where they are imprinted, only specific regions become marked. In humans, more than forty regions have now been pinpointed, distributed among chromosomes 2, 6, 7, 11, 16, 19-22, and, of course, the X chromosome.

If expression of some of these genes is switched from one parental allele to the other during early fetal development, very serious impairments and retardations such as Rett syndrome and fragile-X syndrome can result. Although there is still much research to be done, it appears that at least some of the traits enumerated by the Sages correspond to the parental chromosomes already identified. DNA regions encoding "housekeeping" genes are never imprinted because they must always be expressed, while being strictly controlled by other complex regulatory systems.

What is the mechanism of genetic imprinting? More than forty years ago, I discovered certain enzymatic reactions which lead to the "methylation" of DNA and RNA. (5) One of these reactions involved the addition of a methyl (CH3) group onto the 5-carbon of the DNA base cytosine after the latter had already been incorporated into the polymer. This is an example of "post-replicative modification." I also discovered that the (C[H.sub.3]) donor was the metabolite S-adenosylmethionine. (At this time, there is a monograph in preparation by W.A. Loenen entitled "S-adenosylmethionine: Jack of All Trades and Master of Everything.") It was later found by others that this reaction was sequence specific, and various recognition motifs have been described.

When I first presented this discovery at the annual symposium at Cold Spring Harbor, Long Island, I was pressed to speculate on the significance of my research. I refused to do so because of my youth and strict training that denied teleology. In my written paper, however, I added the following comment: In DNA, at least three possible mechanisms in which methylation could take part may be suggested: replication of DNA, transcription of the DNA into specific messengers in which methylated bases act as points of initiation or cessation of copying, and a recognition system for nuclease activity. (6)

All of these predictions have now been proven to be correct, but there is more to the story. The 5-carbon of the cytosine pyrimidine molecule is located in the major groove of the double helix. All the DNA bases are stacked in the interior of the double helix. An incredible base-flipping mechanism allows the enzyme called DNA methylase to briefly locally unwind or "melt" a small stretch of the DNA and swing the target strand out of the helix and then add a methyl group via a covalent bond.

[FIGURE 1 OMITTED]

A methyl group is hydrophobic and attracts protein binding. It also changes the stability of the double helix, making it more difficult for the DNA to "breathe."

In addition, the methyl group significantly affects the degree to which the DNA bends. DNA can loop, bend, hairpin, and undergo other torsional configurational changes that are important for recombination and transcription. Only one strand of the double helix is ever read at any given time and in only one direction, so the transcription apparatus has to first separate the two strands in order to get at the one that contains the gene to be expressed. This is accomplished by a huge protein complex (much bigger than the gene starting point) made up of a multisubunit enzyme called RNA polymerase, along with a very large number of other proteins. The proteins function to guide the polymerase to exactly the right starting point, to control the rate of transcription, to tell the polymerase to stop at exactly the right terminational signal, and finally to detach the nascent messenger RNA from the complex. These processes are all driven by energy derived from ATP and GTP All of the binding proteins need to "walk" and feel their way or diffuse along the DNA to reach and touch their sequence-specific sites in order to act.

The methylcytosines dramatically change the manner in which these proteins bind, both quantitatively and qualitatively. One of the X chromosomes in the female is inactivated from a specific center by methylation under the guidance of a cis-acting RNA.

Many tumors develop when methylation patterns go awry. Some tumor suppressor genes are actually maternally expressed genes that are mistakenly turned off. This mistake prevents growth-limiting protein from being made. Likewise, many oncogenes (growth promoting genes) are paternally expressed genes for which a single dose of the protein is just right for normal cell proliferation. If the maternal copy of the oncogene loses its methylation and is turned on as well, uncontrolled cell growth can result.

It has become possible to reawaken long-silenced fetal genes in adults by using methylase-inhibiting drugs. Every day there is more evidence implicating DNA-methylation in genetic regulation. There is even an international DNA Methylation Society. The connection between epigenetics and DNA methylation has become the subject of numerous recent papers and reviews. (7-12)

I was led to doubt whether methylation was the key to regulation when it became clear that not all organisms contain 5-methylcytosine or, indeed, any methylated bases in their DNA when they were analyzed with ultrasensitive physiochemical techniques. The organisms that were investigated included the well-studied and completely genetically characterized fruit fly Drosophila, the nematode C. elegans, and certain yeasts. It was known, however, that these organisms exhibited quite rapid mutation rates that were later found to be due to "transposable elements," or short pieces of foreign DNA, which could easily jump around and become stably inserted as new genes into their genomes. As I had speculated, methylation would have protected these genomes from susceptibility to exogenous DNA invasion.

Histones and the Compaction and Packaging of DNA

These and other long-known observations led to the discovery that the basic protein, called histones, which cover the DNA in the chromosomes, were playing a major role in imprinting. The DNA of each chromosome is one very long and flexible molecule, which if stretched out would occupy a space many times larger than its cell of origin. The total DNA of a cell is as much as five feet in length. Therefore, DNA has to be severely compacted and packaged. Because of the resulting enormous repulsion of the negatively charged phosphate backbone, the compact packaging can be accomplished only by coating the DNA with histone proteins containing many positively charged protonated amino-acid lysine side chains. The DNA inside the cell is actually in the form of nucleoprotein chromatin "beads" or nucleosomes, where short stretches of nucleic acid are wrapped around octamers of histone proteins. These "beads" are separated by linker DNA and other histones. It is clear, therefore, that even before the RNA polymerase machine can enter to select a strand to start transcribing, the DNA must be laid bare by "stripping away" the histones. The opposite effect, i.e., stopping transcription of the DNA into a message by RNA polymerase (the enzyme complex which catalyzes DNA>RNA) is shown in figure 2. Here the restoration of the strong positive charge on the histone proteins causes them to bind even more tightly to the DNA.

[FIGURE 2 OMITTED]

Cells have a very interesting mechanism for stripping away the histones. The s-amino groups of certain lysines are modified by acetylation so that the positive charge is removed and the electrostatic attraction between the histone protein and the DNA becomes weakened. This leads to a localized dissolution of the chromatin. This is a reversible reaction because, in addition to several enzymes that can catalyze site-specific acetylation, there are also some that are de-acetylases.

There are some well-known drugs, which have been used for some time for treatment of certain developmental diseases, that have turned out to be inhibitors of these reactions in vivo. There are also several other biochemical reactions taking place that alter the amino groups, such as phosporylation, methylation, and peptidylation, so it is obvious that the cell does not lack resources to modulate chromatin structure.

It is now widely believed that genetic imprinting and gene silencing are mediated through the histones. These reactions, however, may lack the required pinpoint selectivity to be able to account for the high level of precision with which imprinting takes place. Evidence has been accumulating for some time now that the 5-methylcytosine residues act as signals to direct the histone modifications. The methylation state of a given DNA allele is linked inextricably with patterns of histone modification. Methylcytosines near a gene recruit specific DNA-binding proteins, which in turn recruit histone deacetylases resulting in the loss of histone acetylation and silencing of gene expression. In some cases, the histone lysine [epsilon]-amino groups must themselves be methylated in order for the DNA to become methylated. The interactions among the various histone modifications and DNA methylation is so complex that a new thrust in experimental molecular genetics has begun in order to solve the "Histone Code." In those organisms that lack DNA methylation, imprinting proceeds through various histone modifications and specific binding proteins.

Why Are Some Genes Parentally Imprinted?

Why are some genes parentally imprinted? The obvious answer is that they should know whence they came. The Talmud is silent on this matter, but one or two hypotheses have been proposed recently. Historically, the ideas of Lamarck (1744-1829), that evolution occurs when parent organisms pass on to their offspring characteristics they have acquired during their lifetimes began to be ridiculed after they were superseded by Darwinism and Mendelian genetics. However, it was only in the late 1940s that Salvatore Luria finally proclaimed the "fall of the last bastion of Lamarckism" soon after Joshua Lederberg discovered that bacteria practice sexual recombination. Therefore Lamarck was wrong, since any epigenetic changes acquired by adult organisms are unlikely to be passed on. This is because usually germ cells are segregated very early in life and migrate to the embryonic ovaries and testes long before the fetus is born and are thus shielded from these changes.

One generally accepted proposal is that imprinting is a result, or cause, of the "battle of the sexes" or a parental "tug of war." There is a major difference in parental strategies that optimize the number and fitness of their children. Fathers want to have big strong sons, so they contribute genes that enhance growth by extracting as many maternal resources as possible in order to ensure survival. Mothers, on the other hand, are not so particular. Mothers imprint and silence their growth-promoting genes in retaliation because they value daughters as well and try to conserve their resources for future progeny to share equally.

Another hypothesis is based on the principle of "dosage compensation," which has within it the potential to explain why imprinted genes may even affect different social skills in young girls and boys. Imprinting is a tool that can assure that the same parental alleles are present in the same cell since both are essential for survival. Two paternal or two maternal sets of chromosomes lead to lethality, and in fact, even two alleles from the same parent are often deadly and almost always detrimental.

Trisomy of certain chromosomes is the cause of conditions such as Down syndrome. Hemizygosity of certain markers also usually leads to severe developmental diseases. Some manipulated brain cells have only maternal or paternal chromosomes. Maternal cells are prevalent in the neocortex, while paternal cells are found in areas concerned with affective components of behavior like the hypothalamus.

Increased dosages of paternal genes result in hyperkinetic behavior, while increased maternal genes result in hypokinetic behavior. Paternal genes promote stem cell proliferation. On the other hand, maternally imprinted genes promote stem cell differentiation. Imprinting is a logical mechanism for attaining balance. There is also some speculation that imprinting can influence the establishment of distinct species by establishing borders between them and can also provide a barrier to unisexual reproduction and the interbreeding of species.

Summary

What I have presented above is a superficial overview of a part of the process of regulation of gene activity. It must be remembered that imprinting is only a small portion of the regulatory apparatus. Transcription of DNA is controlled by a vast array of inducer, repressor, activator, binding, and other proteins, such as hormone and vitamin receptors. In addition, many small molecules such as ATP, metal ions, nitric oxide, and protons play very important roles. All of these proteins, including the enzymes which synthesize and transport the small molecules, are necessarily also coded by genes, which themselves may or may not be imprinted. The expression of these proteins is also regulated by many other proteins, and this process repeats itself seemingly endlessly.

It is currently beyond our understanding how the whole complex is kept working in complete synchrony and precision. We can only repeat Psalms 104:24, "How manifold are Your works! You made them all in wisdom." This means that for G-d, the wisdom involved in creation is as far removed from His essence as is the lower world of asiyah (the world of work and action). We should always keep in mind what Elijah the Prophet warned Rabbi Shimon Bar Yohai about G-d's wisdom, "[G-d is] wise, but not with wisdom known to us." (13)

Even if G-d's will remains inscrutable, nevertheless, it is certain that many important advances in our understanding of these mechanisms will appear soon from many diverse research laboratories.

In summary, I have tried to show how the addition of a small methyl group to the 5-carbon of certain cytosines in DNA can bring about dramatic changes in the manner in which the information coded in genetic material is expressed without ever changing the sequence in the code. Epigenetics and the solution of the epigenetic and histone codes are now richly funded areas of intensive research. They represent a whole new world of science undreamed of just a few years ago. Long ago, the Sages of the Talmud knew about this because they were imbued with the spirit of truth of the Living G-d. The tsurah (form) becomes superimposed on the homer (matter), and in our lowly world we are led to believe that epigenetics is the mechanism through which this happens.

In a maamar on parashat Wera, (14) the Lubavitcher Rebbe, Rabbi Menahem Mendel Schneerson, discusses the interpretation of the Rebbe Maharash on Exodus 5:3, "I revealed Myself to Abraham, Isaac, and Jacob as G-d A-lmighty (E-l Shad-dai) and did not allow them to know Me by My name YHVH," according to the two explanations of Rashi there. The name E-l Shad-dai reveals miracles bounded in nature, while the name YHVH reveals boundless supernatural miracles. The Rebbe demonstrates, however, that the miracles in nature are more wondrous!

Notes

(1) Talmud Niddah 31a. Similar teachings may be found In Ecclesiates Rabbah 5:13 and She'iltot Yitro 56. See also the Gaon of Vilna on the gemara cited. The Natsiv brings attention to a Tosafot (starting "Kal va homer la'Shekhinah") on Talmud Bava Kama 25:1, wherein Rabbeinu Tam insists that the number of G-d's contributions must be exactly equal to those of the parents and that is why G-d's total is ten in all cases.

(2) Torah Or, Mikets 41:4.

(3) M.F. Lyon, "Gene Action in the X-Chromosome of the Mouse (Mus musculus)," Nature, vol. 190 (1961) pp. 321-322.

(4) K.L. Maxwell, A.A. Yee, V. Booth, C.H. Arrowsmith, M. Gold, and A. Davidson, "Solution Structure of a Small Bacteriophage Protein," Journal of Molecular Biology, vol. 308 (2001) pp. 9-14.

(5) M. Gold, J. Hurwitz, M. Anders, "The Enzymatic Methylation of RNA and DNA I," Biochemical Biophysical Research Communications, vol. 11 (1963) pp. 107-112.

(6) M. Gold, J. Hurwitz, "The Enzymatic Methylation of RNA and DNA," Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963) 156.

(7) Elana de la Casa-Esperon, Carmen Sapienza, "Natural Selection and the Evolution of Genome Imprinting," Annual Review of Genetics, vol. 37 (2003) pp. 349-370.

(8) Yong-hui Jiang, Jan Bresslar, and Arthur L. Beaudet, "Epigenetics and Human Disease," Annual Review of Genomics and Human Genetics, vol. 5 (2004) pp. 479-510.

(9) Peter W. Laird, Rudolf Jaenisch, "The Role of DNA Methylation in Cancer Genetics and Epigenetics," Annual Review of Genetics, vol. 30 (1996) pp. 441-464.

(10) Jay I. Goodman and Rebecca E. Watson, "Altered DNA and Methylation: As Secondary Mechanism Involved in Carcinogenesis," Annual Review of Pharmacology and Toxicology, vol. 42 (2002) pp. 501-525.

(11) K. Si, S. Lindquist, and E. Kandel, "An Epigenetic Hypothesis for Human Brain Laterality, Handedness, and Psychosis Development," Cold Spring Harbor Symposium on Quantitative Biology, vol. 69 (2004) pp. 499-506.

(12) G. Spencer Hamish, "Population Genetics and Evolution of Genetic Imprinting," Annual Review of Genetics, vol. 34 (2000) pp. 457-477.

(13) "Petihat Eliyahu Ha'Navi," found in most Sefardi and some Ashkenazi prayer books.

(14) The Rebbe's explanation of the beginning of parashat Va'era, Shvat 5750. Miracles performed within nature are more awesome than "supernatural" ones. See also Kontres Aharon shel Pesah 5750; and Sefer Ha'Maamarim Melukat, part 4.

Professor Marvin Gold

Presented at the Fifth Miami International Conference on Torah and Science, 16-18 December 2003

Marvin Gold is a professor emeritus of the Department of Medical Genetics and Microbiology, Faculty of Medicine, Graduate Department of Medical and Molecular Genetics, University of Toronto, Ontario, Canada. Born and raised in Toronto, from the age of four he attended the Eitz Chaim Talmud Torah and then Yeshiva Ha'Maharil Grobart.

After studying biochemistry at the University of Toronto, he completed experiments on the effect of X-radiation on DNA replication in mammalian cells, for which he received a doctorate in biophysics. After postdoctoral studies at New York University, he became an assistant professor of molecular biology at the Albert Einstein School of Medicine. In 1967 he returned to the University of Toronto as a member of the Department of Medical Biophysics and later, the Departments of Medical Genetics and Microbiology.

Professor Gold has published over 75 papers, including his discovery of the enzymatic methylation of DNA in bacteria and bacterial viruses. He also made key findings in elucidating the biochemistry of bacteriophage morphogenesis, which were instrumental in the development of in vitro vectors for gene cloning and DNA library formation. At the end of his active career, he devoted his time to solving the mystery of atomic structure of proteins in solution. He was able to use site-directed mutagenesis to engineer enzymes important in metabolism and also for industry. A life-long student of Talmud and halakhah, Professor Gold started his immersion into Habad Hasidism only in 1979, when he spent a year in Boston as a visiting scientist at MIT. Professor Gold and his wife Miriam have five children, eight grandchildren, and five great-grandchildren. He has been active in several synagogues as a Torah reader, teacher, and gabbay. He currently conducts several hevruta study partnerships in Talmud, halakhaah and Hasidism. Professor Gold is enrolled in the rabbinic smikha programs of both the Shema Yisrael Torah Network and Rabbi Dovid Shoichet of Toronto Habad. marvin.gold@utoronto.ca
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Date:Jan 1, 2006
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