Ch. 7: Tumor Suppressor Genes
How do Epigenetic Changes Work Together to Shut Down Transcription?
-Methylated DNA at promoters may physically impede binding of Tx factors -methylated DNA also attracts histone deacetylase (HDAC), which stabilizes chromatin structure -when acetyl groups are removed from histones, a positive charge is restored to lysine residue of the histone tails. A+ charge increases the tail's ability to interact with DNA, thus creating a more compact form
RFLP experiment
-a single base-pair substitution creates an EcoRI site in the maternal chromosome and no restriction site in the paternal chromosome -probe for RFLP in normal tissue and tumor: single band indicates LOH
histone acetylation
-acetylation of histones: may attract or repules chromatin-modifying factors 1. histone acetyltransferases (HATs add acetyl groups to lysine tail of histones and relax chromatin) 2. Histone Deacetylases (HDACs, remove acetyl groups and stabilizes chromatin)
two types of DNA methyltransferases (DNMTs)
-de novo methylation: the methylation of previously unmethylated DNA via DNMT3a and DNMT3b -maintenance methylaton: CpG methylation to continue methylation pattern already in place via DNMT1
retinoblastoma tumors
-early childhood tumor of retina cells -incidence: 1 in 20,000 children 2 forms 1. sporadic: no family history, unilateral; radiation or surgery results in complete remissio with no increased risk of cancers 2. familial: children with one affected parent, bilateral multiple foci; radiation or surgery cures original tumor, but risk for osteosarcomas and other tumors increase 500-fold
role of methylation in carcinogenesis
-global hypomethylation: during tumor progression, tumor cells become less methylated -inappropriate methylationL DNA regions with high density of CpG islands (at promoters) are methylated
histones & histone tails
-histones: proteins that form a "spool" for DNA to wind around, allows for chromatin compaction -histone tailes: both the NH2-terminal lysine rich tail and COOH-terminal tails may be post-translationally modified via acetylation, methylation or phosphorylation
methylation of RASSF1A promoter
-inappropriate promoter methylation silences the RASSF1A tumor suppressor gene -promoter sequence of normal, control tissue: unmethylated -promoter sequence of tumor DNA: highly methylated
chromosomal localization of Rb locus
-interstitial deletions found in chromsome 13 of a few retinoblastoma patients Rb gene was then further localized between bands 13q12 and q14
viral oncogenes display a dominant phenotype in normal cell
-led to the thinking thta cancer was a dominanct phenotype; however, since viruses are not the only cause of human tumors, the possibiity that cancer cell phenotype was dominant or recessive is equally likely
methylation state of promoters of 12 genes in a variety of human tumor types
-methylation of specific gene varies upon tumor type -both TSGs and "caretaker" genes have methylated promoters -promoter methylation may shut down multiple genes in a given tumor
inactivating TSGs: DNA Methylation at Promoters
-methylation state: DNA can be methylated a cytosine residues that lie 5' to guanine=CpG -CpG islands found in promoter regions of 50% of all genes -promoter methylation: can cause Tx repression of associated gene, such as a TSG
some TSGs are never inactivated by somatic mutation
-runx3 gene found methylated, but not mutated, in 45-60% of stomach cancers scenario -1st copy: loss of function through promoter methylation 2nd copy: frequently lost through LOH, not independent methylation event
LOH loss of heterozygosity
-since tumor suppressor genes are recessive, they still produce a wildtype phenotype in the heterozygous state -LOH refers to the inactivation of the second homologous allele, reducing the locus to a nonfunctional state of homozygosity
tumor suppressor genes
-unlike oncogenes, tumor suppressor genes are involved in tumor formation when inactivated or lost -cell loses its ability to suppress growth
TSGs regulate cell proliferation through many biochemical mechanisms-look at diverse functions of proteins
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epigenetic regulation of tumor suppressor genes
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histone modification also alters gene expression
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in unilateral cases, a second "hit" must occur for onset of disease. the probability of incurring 2 mutations follows a quatratic-like curve since it is the square of incurring single mutation (linear relationship)
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modifying histones can change the compation or relacation of chromatin structure, allowing it to have a regulatory role in transcription
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promoters of many tumor suppressor genes have been found in methylated state, and may be equally as important as mutation in shutting down TSGs
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process of gene conversion
1. DNA polymerase begins replication on template strand of red chromosome 2. DNA polymerase jumps to template strand of homologous, green chromosome 3. after copying segment of green template strand, DNA polymerase jumps back to template strand of red chromosome and continues copying
nucleosome
147 bps DNA wrapped 1.7 times around an octamer core of histone proteins (2 copies of H2A, H2B, H3 and H4)
modern TSG mapping
PCR used to amplify polymorphisms rapidly once polymorphism with LOH detected, continue PCR mapping to hone in on exact location of a tumor suppressor gene first primer anneals, elongation proceeds if first primer fails to bind completely, no elongation & no product
retinoblastoma formation
Rb gene found on human chromosome 13 in familial form, a single somatic mutation suffices to eliminate RB function, hence multiple cells in both eyes are affected. Tumors originate from many focal points
tumor suppressor genes (TSGs)
TSGs: inactivating mutations easy to incur...many vulnerable sites -easier to inactivate a gene than to hyperactivate one via a specific mutation (ex. Ras pt muts) problem: if TSGs act recessively, both alleles of a diploid organism need to incur mutations for phenotype to express
maintenance methylases
attach methyl groups to newly synthesized DNA strands hemi-methylated DNA is recognized by maintenance methylases methylation state of CpGs is a heritable, non-genetic mechanism for controlling gene expression
Rb cloned and DNA probe created--Southern Blotting revealed homozygous mutant RB alleles
deletion of Rb cause mutational inactivation
LOH and the Rb locus
during these recombination events, Rb+/- may become Rb+/+ or Rb-/- remember: it is not only the RB locus that has undergone recombination; flanking regions are also exchanged
epigenetic regulation of transcription
epigenetic: changes in cell or chromatin that does not involve a change in nucleotide sequence of DNA 1. DNA methylation 2. Histone modification both mechanisms can affect the structure and conformation of chromatin...and thus influence transcriptional activitiy
if the probability of a cell incurring 2 successive mutation events is 10^-12, how could both wildtype copies of Rb gene be eliminated in sporadic Retinoblastoma?
first hit: mutational inactivation of 10^-6 frequency, heterozygosity at Rb second hit: independent mutational event with higher frequency?, homozygosity=loss of heterozygosity
colon cancer RFLP analysis
for each tumor, probe for LOH at PFLP sites on each arm of 22 chromosomes. Measure LOH indicated on y-axis. High percentage of LOH indicates there might be a TSG in the region 8p, 17p and 18q
How was recessive phenotype reconciled with cancer cell's genetics?
hypothesis -normal cell: proliferation suppressing gene -tumor cell: these genes are lost, tumor grows in cell fusion experiment, the wildtype version of this gene can rescue the mutant allele and act in a dominant manner to stop tumor growth
testing LOH at the Rb locus
if the 2nd hit was an LOH even, then the genetically linked Esterase D gene (13q14) should also undergo LOH -patients heterozygous at the esterase D locus made 2 forms of the enzyme, each with a different mass -evidence that the growth suppressing gene was removed before cells grow uncontrollably
knudson's hypothesis
kinetic studies on rate of tumor appearance familial retinoblastoma: single random even one mutant "Rb" allele inherited via germline mutation, only one to inactivate via somatic mutation sporadic retinoblastoma: two random events, neither allele is inherited defective and 2 inactivating somatic mutations must knock out the function of both alleles
unifying theme of TSGs
loss of any one of them increases the change of neoplastic transformation
gene conversion
non-reciprocal transfer of information from a donor allele to a recipient allele
finding tumor suppressor genes
oncogenes--comparably easy to fine-retrovirus genome, transfection-focus assay, gene amplification TSGs-need to find a gene whose phenotype is expressed when it's missing
clinical appearance
opaque mass obscures retina
RFLP analysis: Restriction Fragemtn Length Polymorphism
polymorphisms: functionally silent differences in DNA sequences-are abundant throughout our genome some of these polymorphisms create a cleavage site for a restriction enzyme in one individual, but resist cleavage in another
cell fusion experiments
purpose: to test dominance and recessiveness of tumorigenic phenotype result: fusion cell was non-tumorigenic, as long as the parental cancer cell was not from a virus-induced cancer conclusion: cancer cell phenotype was recessive to normal phenotype
cross section of retina
retinoblastoma originates in the precursors or stem cells fo any of the multiple retinal cell types
general homologous recombination
several models exist: common steps- 1. initiation (strand nick or break) 2. invasion (DNA 3' end invades homolog) 3. resolution (strand cleavage)