Problem SET 4

do question 15

-18 not memorization cuzzzzz

Epigenetics review questions: 8). Describe two ways that methylation of cytosines in DNA can inhibit gene expression (hint: one way involves regional changes in chromatin conformation affecting many genes at a time, and the other can affect one gene at a time).

1. Methylated cytosines promote the formation of heterochromatin: large stretches of DNA that may contain many genes tightly packed in a way where the transcriptional machinery cannot access them (like putting your belongings into a Space Bag). 2. Methylated cytosines in the promoter regions of individual genes in euchromatic regions can block access of transcription factors to the DNA.

c. For any of the mutations you said you would expect to find in tumor cells, would you expect that (exact) same mutation to be found in non-tumor cells of the same individual? Why or why not?

Affected individuals inherited one mutant copy of the gene and are expected to be heterozygous for this mutation in all cells of the body - both tumor and non-tumor cells. In tumor cells, we expect to find two loss of function mutant alleles of Rb - the inherited one will be shared with non-tumor cells but the more recent somatic one will not. As discussed by Julie Law, however, one might not see loss of function mutations in the DNA sequence, since the second copy of Rb might be silenced by methylation.

2). What is the relationship between oncogenes and proto-oncogenes? Provide an example of a genetic change (e.g. one of the examples discussed in class) converting one into the other.

An oncogene arises as a result of a gain of function mutation in a proto-oncogene. Examples discussed in class: 1. Hypermorphic mutations in the EGF receptor ErbB/Her2 proto-oncogene cause cells to proliferate in an EGF-dependent manner (missense allele encoding constitutively active protein) or proliferate too much in response to EGF (local gene amplification >> too much EGF receptor) 3. Philadelphia chromosome: reciprocal translocation in chronic myelogenous leukemia creates the abnormal BCR-ABL fusion protein (ABL is the proto-oncogene, but perhaps BCR could also be considered a proto-oncogene) (skipped 2)

9). What is the enzyme that adds methyl groups to cytosine bases in DNA, and what chemical can be used to block the activity of this enzyme?

DNA methyltransferase (referred to on some slides as Dnmt), blocked by 5-azacytosine

a. What base must be next to a methylated cytosine in order for the methylated state to be propagated?

G

What oncogenes are targeted by the "designer" chemotherapy drugs Herceptin and Gleevec, respectively?

Herceptin: ErbB/Her2 oncogene; Gleevec: BCR-ABL oncogene

i. A missense mutation in Rb encoding a hyperactive form of the protein (G.O.F. or L.O.F? DOMINANT NEG, HYPERMORPH, NEOMORPH ETS??)

Hypermorphic, gain of function mutation - not the kind of mutation in Rb we expect to find in retinoblastoma cells.

iii. An insertion in the regulatory region of the gene that causes >10-fold overexpression

Hypermorphic, gain of function mutation - not the kind of mutation in Rb we expect to find in retinoblastoma cells.

14). Which of the following two events would you expect to see associated with an increase in DNA methylation, and why: loss of heterozygosity for a tumor suppressor gene or conversion of a proto-oncogene to an oncogene?

Loss of heterozygosity for a tumor suppressor gene. Increased DNA methylation is typically associated with a reduction or loss of gene expression. Heterozygosity for tumor suppressor genes is indeed often "lost" via methylation of the functional copy.

13.) If you cross an agouti mouse to a wild type mouse, without folic acid supplementation, do you expect the progeny to be mostly agouti or mostly wild type and why?

Mostly agouti. Loss of methylation of the gene in agouti "mutants" causes overexpression of the gene = gain of function so we expect this epiallele to be dominant to wild type in a heterozygote.

b. Why is the identity of that neighboring base important? Include in your answer the name of the protein that recognizes hemimethylated DNA and how it interacts with the methylase. CG dinucleotides have mirror image symmetry as shown: 5'-CG-3' 3'-GC-5'

NP95 recognizes this dinucleotide in the hemi-methylated state that follows replication of a parent DNA strand containing a methyl-C. NP95 binds to DNA methyltransferase, which adds a methyl group to the C on the new DNA strand opposite the G on the parent strand (pretty clever, eh?)

3). Do proto-oncogenes either promote or inhibit progression of cells along the path toward cancer?

Neither! They carry out normal cellular functions that do not promote or inhibit neoplastic transformation. Promote neoplastic transformation after being converted by mutation into an oncogene.

what is oncogene? fxn?

Oncogene pushes cell toward uncontrolled proliferation by bypassing the processes that would normally restrict cell division. Results from gain of function mutation in a proto-oncogene.

6). Why are oncogenes better targets for chemotherapy drugs than tumor suppressor genes?

Oncogenes are gain of function alleles - they have gained something that could potentially be inhibited by a drug with suitable specificity. Tumor suppressor gene function has already been lost in cancer cells - very hard to use drug therapy to bring something back that has been lost.

10). What is the molecule that donates a methyl group for cytosine methylation by the enzyme you named in 2, above, and what dietary nutrient is it derived from?

S-adenosyl methionine (SAM-CH3), derived from folic acid

iv. A nonsense mutation in Rb.

Simple loss of function mutation - this is the kind of mutation we expect to find in retinoblastoma cells.

ii. A deletion of the Rb gene

Simple loss of function mutation - this is the kind of mutation we expect to find in retinoblastoma cells.

12). In the mouse, what molecular alteration underlies conversion of a wild type coat color allele to an agouti allele, and how can that be that influenced by folic acid in the mouse's diet?

The agouti phenotype results from demethylation of cytosines in the agouti gene, which is heavily methylated in wild type. Demythylation causes the gene to be more highly expressed, so this epigenetic alteration mimics a gain of function mutation. Influenced by folic acid because this nutrient is a precursor of the methyl donor SAM-CH3, as above. If dietary folate is low, agouti gene is more likely to be demethylated to produce the agouti phenotype.

e. What explanation can you offer for the variable expressivity and incomplete penetrance seen here?

To get a retinoblastoma, the second copy of Rb must be lost by somatic mutation (LOH). In some individuals, this never occurs so they don't get retinoblastoma (explains the incomplete penetrance). In individuals where the second copy is lost, it might be lost in only one eye, or it might be lost in both eyes (explains variable expressivity).

What is a tumor suppressor gene? function?

Tumor suppressor protects organism from cancer by repairing DNA damage (mutations) and/or preventing replication or survival of cells with severe genetic damage or an unbalanced genome. Loss of tumor suppressor function pushes cell down the path toward cancer.

Cancer 1). What is the difference between a tumor suppressor gene and an oncogene? In your answer, provide some information about the function of each as it relates to cancer.

Tumor suppressor protects organism from cancer by repairing DNA damage (mutations) and/or preventing replication or survival of cells with severe genetic damage or an unbalanced genome. Loss of tumor suppressor function pushes cell down the path toward cancer. Oncogene pushes cell toward uncontrolled proliferation by bypassing the processes that would normally restrict cell division. Results from gain of function mutation in a proto-oncogene.

b. For any of the mutations you said you would expect to find in tumor cells, would you expect the tumor cells to be homozygous for the mutation (exactly the same mutation on both chromosomes)? Why or why not?

We expect the tumor to have lost the function of both copies of Rb, however since the second mutation must occur somatically, it is extremely unlikely to be exactly the same as the inherited mutation. So no, we don't expect the mutation to be homozygous.

d. Give one example of another mutation you would expect to find in the retinoblastoma tumor cells? That is, what other gene or type of gene would you expect to find a mutation in, and what kind of mutation?

We expect to find loss of function mutations in other tumor suppressor genes, and gain of function mutations in oncogenes (altogether 6-12 "driver" mutations of these types). p53 tumor suppressor is lost in ~50% of all human cancers that this is a good guess for another gene that could be mutated, but for the most part you don't have information here on which to base a prediction of which exact tumor suppressor will be lost or oncogenes activated.

4). What tumor suppressor gene discussed in class functions to induce apoptosis in cells with severe genetic damage or an unbalanced genome?

p53