Week 4

October 20

Epigenetics

What is it?




Papers:
Epigenetics: a historical overview.
hollidayEPI1-2.pdf


A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening | doi:10.1038/ng1841 | VOLUME 38 [ NUMBER 8 [ AUGUST 2006 NATURE GENETICS
ng1841.pdf


Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid
Toxicology Volumes 181-182, 27 December 2002, Pages 433-439
OA_epi.pdf

Links of Interest
Blogs

Wikipedia


Methods to study epigenetics

The concept of Bisulfite sequencing...
external image Wiki_Bisulfite_sequencing_Figure_1_small.png








Video (click on image to play):
epi_videp.png
http://www.pbs.org/wgbh/nova/sciencenow/3411/02-tale-flash.html






GHOST IN YOUR GENES VIDEO




Additional Readings


I thought this was a cool & easy to read overview of epigenetics
ehp_epigenetics.pdf
They mention some good resources in the article too!
- lisa418 lisa418

DIFFERENCES BETWEEN GENETIC AND EPIGENETIC SYSTEMS (from Holliday 2006)
Genetic
Epigenetic
based on cell lineages and clonal inheritance
Single cell à clonal cells
- Epigenetic changes often occur in groups of cells
- Some epigenetic events are clonal,
e.g. X chromosome inactivation
changes are stable and rarely reversed
changes are often reversed, e.g. genomic
imprinting, where the changes imposed on DNA sequences may be lost during development, or if they persist, are erased and re-set during gametogenesis.
No environmental influence
Environmental influence, and heritable; Lamarckian evolution



Creppy et al. (2002)

Key ideas in intro:
- OA is a tumour promoter
- induces DNA adducts
(piece of DNA covalently-bonded to a chemical. This has shown to be the start of carcinogenesis)
- is suspected to promote tumour the human gut

Overall Goal: to study the effects of this marine toxin in several epigenetic mechanisms.
General Method: experiments with human colorectal adenocarcinoma cell line, Caco-2
Other concepts:
Phycotoxin: Marine biotoxins that accumulate in fish and shellfish from their diet (causing shell fish and ciguatera poisoning when the fish are eaten), as distinct from toxins naturally present.
Genotoxicity describes a deleterious action on a cell genetic material affecting its integrity. Genotoxic substances are known to be potentially mutagenic or carcinogenic, specifically those capable of causing genetic mutation and of contributing to the development of tumors. This includes both certain chemical compounds and certain types of radiation.

Concept #1: cytotoxicity is the quality of being toxic to cells.
Question: is OA cytotoxic?
Method: Cytotoxicity assay
- lactate dehydrogenase (LDH) release test kit: is designed to be a simple method to directly quantify cell death in culture. The assay is based on the measurement of LDH released into the growth media when the integrity of the cell membrane is lost. http://www.biocompare.com/Articles/ProductReview/900/ProductReview.html
- MTT assay: is a laboratory test and a standard colorimetric assay for measuring the activity of enzymes that reduce MTT to formazan, giving a blue color. This mostly happens in mitochondria, and as such it is in large a measure of mitochondrial activity. It can also be used to determine cytotoxicity of materials.
Result: release of LDH into culture medium, thus confirming cytotoxicity of OA

Concept #2: Lipid peroxidation refers to the oxidative degradation of lipids. It is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage.
Question: Is there lipid peroxidation of cells during incubation in OA?
Method: Extraction and determination of MDA-TBA adduct
- Quantification of malondihaldehyde (MDA) using the thiobarbiturate (TBA)-MDA-adduct formation
- High-performance, or high pressure, liquid chromatography (HPLC) separation and fluorometric detection
- http://www.chem.vt.edu/chem-ed/sep/lc/hplc.html
- http://www.pharm.uky.edu/ASRG/HPLC/HPLCMYTRY.html
- http://www.forumsci.co.il/HPLC/program.html (short video of chromatography process)

Result: Increased lipid peroxidation
Discussion: peroxidation is thought to inhibit GJIC

Concept #3: Caco-2 cells may react to OA treatment by inducing the synthesis of mRNA encoding oncoproteins, due to modification of the rate of m5dC in the DNA.
Question: How is transcription rate affected?
Method: Cellular [3H]-uridine incorporation
- Transcription quantified by radioactive uridine
Result: OA increases the incorporation of radio-labelled uridine in cells, at a concentration dependent rate (i.e. higher transcription rate with OA).
Discussion: OA induces a concomitant increase in total RNA synthesis as shown by the increase of 3H-uridine incorporation in Caco-2 cells. This was unexpected.

Concept #4: DNA methylation is the addition of a methyl group to DNA. For example, to the number 5 carbon of the cytosine pyrimidine ring, which in this case reduces gene expression. DNA mutagenic or oxidative lesions can interfere with the ability of mammalian cell DNA to be methylated at CpG dinucleotides by DNA-methyltransferases. DNA methylation caused by oxidative lesions can lead to excessive expression of oncogenes and repression of tumour suppressing genes (Wachsman, 1997).

Question: Does a DNA mutagenic lesion increase DNA methylation?

Method: Determination of DNA mutagenic lesion (8-hydroxyguanosine, 8-OH-dG) and of the rate of methylation
- by high-performance, or high pressure, liquid chromatography (HPLC)-electrochemical detection and the rate of m5dC (5-methylcytosine) formation by HPLC-UV detection as previously described by Traore´ et al. (2000)
Results: increase in 8-OH-dG, and increase in m5dC.

Concept #5: Gap junctions are specialised plasma membrane-associated proteins that form intercellular channels that co-ordinate tissue function by allowing cell to cell passage of ions and small molecules.They are composed of connexin. They are involved in cell signaling.
Question: Does OA inhibit connexin-43?
Method: Reverse transcription-polymerase chain reaction (RT-PCR)
- fluoresced and photographed
- http://www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html

Result: Cx-43 mRNA disappearance at very high levels of OA. Thus OA inhibits Cx-43.
Discussion: inhibition through oxidative stress

Question: Does OA inhibit membrane plaques of gap junctions?
Method: Indirect immunofluorescent staining of gap junctions
- Immunostaining
- www.lamondlab.com/f7immunostainprotocol.htm

Result: OA inhibited membrane plaques of gap junction from membranes in a dose-dependent manner.
Another result mentioned in abstract only: OA concentrations also disorganized the cellular cytoskeleton, since both actin and tubulin formations were impaired.

Discussion: Many methods performed to test different levels of epigenetics, but still all related to how OA could induce carcinogenesis.

How do we define epigenetics?


From Holliday:
1. The study of changes in gene expression which occur in organisms with differentiated cells, and the mitotic inheritance of given patterns of gene expression
2. Nuclear inheritance which is not based on changes in DNA sequence

Both definitions deal with inheritance – a definition that is biased towards the interest of the discipline of evolution. If you are interested in non-heritable traits (like carcinogenesis in Creppy et al.) epigenetics should also include non-heritable mechanisms.

Some ways gene expression can be modified outside of nucleotide sequence change
-methylation
-post-transcriptional modification of mRNA (alternative splicing)
-alternate protein folding
-chromatin structure (modification of histones)
-RNA molecules that affect 3D distribution of proteins
-environmental influences (e.g. oxidative stress, chemicals that block receptors)
-epistasis (multiple genes interact to affect a phenotype)

Manning et al. 2006

Overview
1. Authors identified ORF 7 as a candidate for the Cnr gene
2. Cnr mutants do not ripen and underexpress ORF7 at the time of ripening
3. Cnr mutants had no sequence differences from wild type, but do show differences in methylation, supporting methylation as a mechanism for difference in gene expression and the ripening phenotype
4. Cnr mutants from both genetic lines have a much higher rate of methylation supporting their hypothesis that methylation prevents ripening
5. When ORF7 is silenced in wild type fruits they are unable to ripen.

Definitions:
Pericarp: the ripened and variously modified walls of a plant ovary

Breaker stage: first sign of lycopene accumulation on the blossom end of the fruit (blossom end turns pink)

Figure 1: High resolution genetic mapping to identify a candidate gene that inhibits the ability of Cnr mutants to ripen.
The authors crossed mutated and non-mutated plants in different schemes to identify which region of the genome was associated with the non-ripening phenotype. They were able to narrow down to a 95kb region and by sequencing they identified 14 possible open reading frames (ORFs). Crossovers (recombination events) are used to further narrow down the region where they think the gene is and in this region the authors found 3 possible promoter regions – ORFs 7, 8, 9 &10.
• BACs: Bacterial artificial chromosome – bacterial clones that can hold large pieces of DNA and are used for genomic DNA libraries
• Comsid: bacterial clones that can hold large pieces of DNA and are used for genomic DNA libraries
• Gene walking: sequencing the ends of BACs and aligning sequences to reconstruct a part of the genome
• Alleles: variations in a gene (a diploid organism will have 2 alleles – one inherited from the father & one inherited from the mother)
• Isogenic lines: breeding organisms so you produce offspring that have the same genetic background (the same alleles)

Figure 2: Expression studies that show Cnr mutants that do not ripen do not express ORF7 as much at the time of ripening, supporting the idea that ORF7 expression is part of the ripening pathway.
Expression of ORF7 at different stages of development using quantitative real time PCR. Liberto line shows differences in expression of ORF7 between individuals that do ripen (wild-type) and don’t ripen (Cnr mutants). Alisa Craig represented a line of individuals who are almost completely homozygous (almost all alleles for a given gene are the same) – this accounts for any other genes that may modify the phenotype that lie outside the candidate region the authors identified.

Figure 3: Establishes methylation as a potential mechanism for differences in gene expression of ORF7 and the ripening phenotype.
The authors had already established that the Cnr mutants had no nucleotide sequence differences, and this figure shows a difference in methylation pattern between the mutants and wild type.

Figure 4: Cnr mutants (tomatoes that don’t ripen) from both genetic lines have a much higher rate of methylation supporting their hypothesis that the methylation prevents ripening. It also shows that the Liberto control shows a reduction in methylation during ripening.
The authors predicted that in order to suppress expression of ORF7, the sequence upstream of ORF7 would have to be methylated to prevent transcription proteins from accessing the gene.
a. Liberto – wild type control
b. Alicia Craig – wild type control, but a different line and therefore different genetic background
c. Liberto segregant – Cnr mutant, genetically identical to Liberto wild type except at ORF7
d. NIL Alicia Craig – Cnr mutant, nearly isogenic background from Alicia Craig line

Bisulphate sequencing: The upstream region was sequenced, then the same genomic DNA was treated with sodium bisulfate which converted unmethylated cytosines to uracils. The treated DNA was sequenced and they compared the sequences pre and post treatment. If a cytosine was unmethylated, then the pre-treatment DNA would show up as a C and post-treatment would show up at a T.
See http://www.zymoresearch.com/products/dna/ez_dna_methylation_kit.asp

Figure 5: Further supports their hypothesis that methylation prevents expression of ORF7, plants with methylated ORF7 alleles are unable to ripen.
The authors used a viral construct to silence the expression of ORF7 in Alicia Craig control fruits and found that when this gene is silenced the fruits are unable to ripen

As advertised...
The Healthing Power of Methylation