Exam 5

6. We discussed several kinds of epigenetic phenomena that have been observed in different organisms. List five different epigenetic phenomena.

1 imprinting 2 Position Effect Variegation 3 mammalian x-chromosome inactivation 4 centromere function/heterochromatin formation 5 transposon silencing/heterochromatin formation

25. During the semester, we discussed enhancers and promoters and how they contribute to gene expression. List TWO ways in which the DNA sequences in enhancers and promoters can "communicate" (or, be brought together).

A) Promoter and enhancer sequences might be bridged via protein/protein interactions involving Mediator B) Promoter and enhancer sequences might be bridged via noncoding RNAs transcribed from enhancer regions, and associated RNA binding proteins and other bridging proteins

19. What are CpG shores?

CpG shores are G+C-rich regions of DNA within 2000bp, but not inside, a CpG island. Methylation of CpG island shores is also associated with inhibition of gene transcription in cancer cells.

4. We discussed Ron Jirtles' experiments on Agouti mice that were fed a diet that included large amounts of folic acid, choline, betaine, etc. You force Drosophila strains that display Position Effect Variegation to ingest this diet as well. What effects would you predict the diet would have on their progeny? (Circle one) No effect Suppression of PEV (redder eyes) Enhancement of PEV (whiter eyes) WHY did you select the answer above????

Enhancement of PEV (whiter eyes) This is a high methyl diet that would be predicted to lead to increased heterochromatin marks.

20. List TWO examples of histone variants and their association with specific biological activities/functions.

H2A.Z is a variant of Histone 2A that is incorporated into nucleosomes that typically flank promoter regions. The presence of H2A.Z plays a key role in gene expression, DNA repair and centromere function and its presence is inversely correlated with methylated DNA. CENP-A is a variant of the Histone H3 protein. This histone localizes to centromeres. In yeast, RNAi mechanisms that act on RNA transcribed from centromeric DNA help to recruit CENP-A to this region

24. Please list TWO non-cannonical Histones, or Histone variants, and the nature of the specific DNA sequences/chromosomes/chromatin regions with which they are associated:

H2A.Z is a variant of Histone 2A that is incorporated into nucleosomes that typically flank promoter regions. The presence of H2A.Z plays a key role in gene expression, DNA repair and centromere function and its presence is inversely correlated with methylated DNA. CENP-A is a variant of the Histone H3 protein. This histone localizes to centromeres. In yeast, RNAi mechanisms that act on RNA transcribed from centromeric DNA help to recruit CENP-A to this region

1. You have a strain of Drosophila that displays Position Effect Variegation. To study this effect in greater detail, you dissect the eyes and separate the cells into two groups based on the amount of pigment (red versus white). Now you would like to compare the methylation status of the white gene in the red cells versus the white cells. What kind of experiment or technique would you use to investigate this? --Describe the reagents you will need. --Describe how specific the reagents need to be. --Describe your detection method. --Describe what results you expect.

anti-H3K9 Me or other antibody that recognizes a specific histone modification associated with heterochromatin. Use ChIP to bring down the chromatin with the modified histones. PCR the sequence to see if the white region is there. some folks described strategies to bring down methylated DNA. if the strategy made sense, credit was given. But there is not much DNA methylation in Drosophila.

17. Which of the following mutations is seldom associated with cancer? a) hypermethylation of CpG islands b) defective histone methyltransferases c) defective histone acetyltransferases d) defective histone deacetylases e) defective histone demethylases

d) defective histone deacetylases

16. Which of the following is NOT a post-translational modification that has been associated with histones/nucleosome? (at least, not yet) a) phosphorylation b) ubiquitination c) citrullination d) pepperonylation (addition of pepperoni) e) methylation

d) pepperonylation (addition of pepperoni)

5. We discussed several well known chromatin-associated functions that are affected by temperature. What are these chromatin-associated functions?

1.recombination 2.segregation/disjunction in meiosis and mitosis 3. transposon silencing

27. In the context of gene expression, what is 5-hydroxyMethylCytosine? How is it formed? What is its functional relationship to 5-methylCytosine?

5-HydroxymethylCytosine is enzymatically formed from 5- MethylCytosine by the action of TET enzymes. 5hmC is thought to be part of an oxidative demethylation pathway that results in unmodified bases in DNA. Alternatively 5hmC could b a new epigenetic "mark" that is capable of recruiting 5hmC-binding proteins, for example, although evidence for this is lacking. (5hmC can be observed in most cells. It is abundant in the CNS, in primordial germ cells, and the levels increase with age. 5hmC is associated with regions of unstable nucleosomes—those that are repositioned or replaced with histone variants.)

10. Chromatin in sperm is different from that in other cells, with most of the genome packaged by protamines not nucleosomes. In human sperm, about 4% of the genome is bound up by nucleosomes, affording sperm the ability to potentially transmit paternal epigenetic information. What regions of the genome in sperm are bound by nucleosomes, and not protamines? Recent studies indicate that nucleosomes are retained in regions of the genome that are highly GC-rich. In particular, these regions include transcription start sites and genes that regulate development, as well as most housekeeping genes. Sperm nucleosomes also help establish DNA methylation-free regions in the early embryo. Taken together, this suggests that paternal nucleosome transmission may facilitate robust gene regulation in the early embryo. Would you expect a gene that is paternally imprinted to be bound by protamines or nucleosomes in sperm? Why? Would you expect a gene that is maternally imprinted to be bound by protamines or nucleosomes in sperm? Why?

Because sperm nucleosomes are associated with GC-rich and methylation-free regions, one might assume that these regions would NOT be silenced by sperm, as methylation is associated with silencing. So such an allele, bound by nucleosomes and inherited via sperm, would be expected to EXPRESS in the developing embryo. If the gene is imprinted, it would be imprinted maternally. Genes bound by protamines are not necessarily methylation free. Methylation is associated with silencing, so DNA bound by protamines might be paternally imprinted.

2. Picture: We discussed a model for imprinting at the Igf2 locus (an "enhancer competition" model). Important insights into the regulation of Igf2 (which is maternally imprinted) came from the observation that the nearby H19 locus is oppositely imprinted and that the two genes share a common enhancer. Equally important was the realization that DNA methylation in the Differentially Methylated Domain (DMD) region contributes to enhancer activity. A) What would you expect for the expression of these two genes in a DNA methyltransferase mutant?(Assume that mouse DMT strains are viable and fertile. (In other words, what would happen to the expression of Igf2 from the maternal chromosome, what would happen to the expression of Igf2 from the paternal chromosome? Similarly, what would happen to H19 expression?) B) WHY??

Biallelic expression of H19, no/little expression of Igf2 (see drawing above--this is the condition you might expect in a DMT mutant) Since the DMD region is not methylated on EITHER of the chromosomes(due to the DMT mutation), the CTCF repressor protein can bind to this region of the DNA and act as a boundary element, preventing the enhancer from acting on the Igf2 gene. The enhancer will act on the H19 gene on both chromosomes.

26. In class discussions and in the reading material, the nature of methylated DNA was discussed in the context of gene expression, especially in cancer cells versus normal cells. Describe the results of the experiment in which the oncogene c-myc was overexpressed in "normal" cells (with respect to DNA methylation and a histone variant).

From observations of DNA methylation and the presence of H2A.z in cells, methylation and H2A.z deposition seem to be mutually antagonistic. Yet, cells with H2A.z deletions do not display drastic losses in DNA methylation. H2A.z deletion strains do display misregulation of many genes, including mRNAs with aberrant transcriptional start sites. In cells overexpressing c-myc (and displaying an overgrowth phenotype), the H2A.Z histone variant was found to re-distribute from promoters to gene bodies. Also the amount of DNA methylation in gene bodies was found to be reduced in cells overexpressing c-myc. These results support the hypothesis that DNA methylation in gene bodies precludes deposition of H2A.z, and these mechanisms are important to safeguard against cellular overproliferation.

15. List THREE important factors/enzymes/proteins that contribute to chromatin-based regulation of gene expression in eukaryotes.

Histone modifications (Histone methyltransferases/acetyltransferases) DNA methylation (DNA methytransferases) Non-coding RNAs ATP-dependent Chromatin remodeling factors such as SWI/SNF 3D rearrangements of genes/chromatin within the nucleus (location) replacement of canonical histones by histone variants

18. What are CpG islands?

Regions of the genome, mostly in promoters, that are composed of 50% G+C content in a stretch of 200 bases. CpG islands are unmethylated in normal cells, some genes acquire methylation in CpG islands during development, and these regions are commonly hypermethylated in cancer—especially in regulatory regions of tumor suppressor genes.

11.Picture: the Prader-Willi/Angelman region is an example of nearby genes on the same chromosome that are imprinted oppositely. The imprinted status of the genes is maintained in the brain such that the Prader-Willi and Angelman genes are expressed in a mono-allelic manner. There are a number of mechanisms that help to maintain the imprinted status of these genes in the brain. List two of those mechanisms.

Regulation by proteins that can bind methylated DNA Regulation by anti-sense RNAs The Imprinting Control Region directs the allele-specific expression of imprinted genes. One gene (the UBE3a) gene is regulated by MeCP2 (methyl-CpG-binding protein 2). When MeCP2 is absent, this leads to the production of an anti-sense UBE3a transcript from both alleles.

8. Rainbow, the calico cat, was cloned. The nucleus from a somatic cell from Rainbow was transplanted into an enucleated oocyte. The coat color pattern in the clone did not resemble Rainbow. Why not?

Remember, Rainbow was originally a single nucleus from a female adult cat. The nucleus was transplanted into an Enucleated donor oocyte. (remember that imprints are re-set in the germline) The oocyte had sufficient machinery to re-set the X chromosome inactivation marks. Later in development, when Rainbow was a few hundred cells, the random decisions to inactivate one X chromosome per cell were made. Thus Rainbow had a different pattern from Rainbow's clonal mom. (If a somatic cell were taken from a mammalian female and grown in culture, the X chromosome inactivation marks would not be reset. It is the cycling through a germline cell that re-sets the inactivation marks.)s

13. Genomic imprinting was discovered in 1984 by two groups who were studying parthenogenesis (development from an egg with no paternal contribution). Surani and colleagues collected nuclei from oocytes and from sperm. When sperm nuclei were injected into oocytes, viable mouse offspring could be produced. However, when oocyte nuclei were injected into oocytes, no viable offspring were produced. Using your modern knowledge of epigenetic imprinting, explain why both maternal and paternal contributions are required for normal development.

Some genes are imprinted maternally. These genes are normally expressed from the paternal chromosome, and the genes are epigenetically silenced in the maternal ovary, where the gene and associated nucleosomes become methylated (histone marks for silenced genes). Thus, in order for the gene to be expressed, one chromosome has to come from the male. Genes that are imprinted include genes that are important for metabolism, growth, development, etc.; thus, if the embryo inherits two sets of chromosomes from the female parent, none of the maternally-imprinted genes will express and the embryo will die.

23. Fabry Disease is lipid storage disorder characterized by an accumulation of globotriaosylceramide with blood vessls and other organs. Fabry symptoms are noticed in early childhood and include full body pain or pain in extremities, GI pain, kidney and cardiac complications, high blood pressure, appearance of angiokeratomas, clouding of the cornea, and other manifistations. Fabry disease is associated with a genetic mutation in the gene coding for the enzyme alpha galactosidase A, a gene that is located on the X- chromosome. Most Fabry disease patients are males, and females who are heterozygous for the mutation suffer a range of symptoms—from severe to mild. What is the molecular explanation behind WHY some heterozygous female might display a milder form of the disorder, while other heterozygous females might display a severe form of the disease?

The Fabry disease gene is located on the X-chromosome. One X-chromosome is randomly inactivated in females. In a heterozygous female, if the gene for wild-type alpha galactosidase A is located on the Inactivated chromosome, then no AGA will be synthesized in that cell (because the 2nd allele is mutant). If most of the cells in the female have this pattern of X-inactivation, then the female will display a more severe form of Fabry Disease.

21. Hemophilia is a blood clotting disorder characterized by excessive bleeding as a result of injury. Hemophilia patients do not form clots as easily as non-hemophilia patients. Some forms of hemophelia are caused by a genetic mutation in the Factor IX gene, a gene that is located on the X- chromosome that encodes a protein that is secreted into the blood stream. Most hemopheliacs are males, and most females are heterozygous. A few rare heterozygous females have been observed to display the hemophilia trait. What is the molecular explanation behind WHY a heterozygous female might display the blood clotting disorder?

The Factor IX gene is located on the X-chromosome. One X-chromosome is randomly inactivated in females. In a heterozygous female, if the gene for wild-type Factor IX is located on the Inactivated chromosome, then no Factor IX will be synthesized in that cell (because the 2nd allele is mutant). If most of the cells that secrete Factor IX have this pattern of X-inactivation, then the female will display hemophelia. Fortunatly, for females, the Factor IX protein is synthesized in the liver, which is a relatively large organ and enough cells have the opposite X-inactivation pattern to allow for a sufficient amount of blood clotting.

22. A girl displays a mutant phenotype because she inherited a mutant allele for a gene that is normally maternally imprinted. From which parent did she inherit the mutant allele? WHY? 22b. The girl (above) grows up, marries, and plans to have children. Will any of her children be affected by the disorder?

The gene is maternally imprinted. Since the girl has the phenotype, she inherited the imprinted version of the gene from her mother, and the mutant gene from her father. Neither allele will allow for expression of a functional gene product. Since the gene is maternally imprinted, and since the "girl" is now planning to be a mother, the gene will be imprinted in ALL those chromosomes that derive from her. They will NOT be imprinted in the chromosomes that are inherited from her husband. Their children will not display the phenotype. (But 50% of them will be heterozygous for the mutation.)

12a. A boy displays a mutant phenotype because he inherited a mutant allele for a gene that is normally paternally imprinted. From which parent did he inherit the mutant allele? WHY? 12b. The boy (above) grows up, marries, and plans to have children.

The gene is paternally imprinted. Since the boy has the phenotype, he inherited the imprinted version of the gene from his father, and the mutant gene from his mother. Neither allele will allow for expression of a functional gene product. Since the gene is paternally imprinted, and since the "boy" is now planning to be a father, the gene will be imprinted in ALL those chromosomes that derive from him. They will NOT be imprinted in the chromosomes that are inherited from his wife. Their children will not display the phenotype. (But 50% of them will be heterozygous for the mutation.)

3. There are two types of DNA methylation patterns that are associated with cancers. TYPE A patterns appear in the borders/shores of CpG islands and are age-related (commonly observed in p16 and hMLH1 genes). Type A (aging-specific) methylation patterns form as a result of age in normal cells and are believed to result from inflammation, viral infections and other environmental stressors. Type A patterns predominately affect genes that regulate growth or differentiation of these cells and may contribute to the hyperproliferative state that is thought to precede tumor formation. TYPE C patterns (cancer-specific) are specific to some forms of cancer and are found in the CpG islands, which are densely methylated in the promoters of cancer-associated genes, leading to a reduction in gene expression. In this situation, concordant methylation of multiple genes is observed, and may be due to a genetic defect in the cancer cells, for example, a mutation in a DNA methyltransferase. Chronic use of nonsteroidal anti-inflammatory drugs have been demonstrated to lower the risk of colon cancer, while consumption of excess folic acid by colon cancer patients can be harmful (see question 4). Provide a molecular explanation that would explain how these environmental components affect cancer. (What process do these dietary components affect? What molecules do they affect?)

The hypothesis is that nonsteroidal anti-inflammatories might prevent the progression of Type A methylation patterns to Type C patterns, and would therefore slow cancer progression in susceptible patients. For patients with Type C patterns in their cancer cells, the high-methyl diet would enhance the aberrant methylation patterns. There are several epigenetic drugs on the market and in trials that are designed to remove the Type C methylation patterns. These methylations prevent expression of important genes. Re-activation of these genes can decrease the growth of the cancer cells. Folic acid (high-methyl diet) are predicted to lead to increased methylation (as in Ron Jirtle's mice). For patients who already have cancer, with Type C patterns, additional methylation, or maintaining the methylation patterns, in the CpG islands would be detrimental, as such modifications are often observed in tumor suppressor genes. (New cancer therapies that target epigenetic mechanisms can re-activate CpG island hypermethylation, re-activating tumor suppressor genes and decreasing cancer growth.)

14. Genetics studies of the IGFR2 locus in mice performed in the 1990s identified the first paternally imprinted genes. Soon afterwards, a few more imprinted genes were discovered, but progress was slow, as their identification required the use of specific mutations. Nowadays, it is possible to identify thousands of genes with imprinting patterns in one experiment. Describe how such an experiment was performed to identify genes that are imprinted (display mono-allelic expression patterns) in the brain.

Two different mouse strains were used in the study. Complete genome sequences were available for these two mouse strains and SNPs were identified such that the scientists would know which parent donated the allele in question. Brains were taken from F1 cross progeny of reciprocal crosses and the brain mRNAs were sequenced. A particular mRNA with SNPs ONLY from one parent (father) is likely imprinted, and this could be confirmed by looking at the data in progeny derived from the opposite reciprocal cross—the SNP would correspond to the other parent (mother).

28. In the context of gene expression, list TWO different histone post-translational modifications (be specific) and the gene expression status for the DNA sequences with which they are assocated. a)_______________is a modification that is associated with genes that are____________ b)______________is a modification that is associated with genes that are ______ expressed___

a) H3K9Methyl; silenced/not expressed b) H3K9Acetyl; expressed

7. Which of the following histones undergo post-translational modifications that can affect gene expression? (circle all that apply) a) Histone H1 b) Histone H2a c) Histone H2b d) Histone H3 e) Histone H4

a) Histone H1 b) Histone H2a c) Histone H2b d) Histone H3 e) Histone H4