Chapter 3: Gene expression (lecture 2) Flashcards
Thus far we discussed 1) Transcription 2) Chromatin structure And we’re going to look into these topics now: 3) DNAmethylation 4) microRNAs (miRNAs) 5) Telomeres and telomerase 6) Therapeutic options
okay good to know
DNA methylation is an epigenetic modification to the DNA. Which enzyme is involved in this proces?
DNA methyltransferases (DNMT’s)
There are two types of DNA methyltransferases. What are they and where are they used?
- De novo methyltransferases recognize something in the DNA that allows them to newly methylate cytosines. These are expressed mainly in early embryo development and they set up the pattern of methylation.
- Maintenance methyltransferases add methylation to DNA when one strand is already methylated. These work throughout the life of the organism to maintain the methylation pattern that had been established by the de novo methyltransferases.
On what sequence does methylation occur?
On Cs preceding a G (CpG)
Are CpG’s under- or overrepresented in DNA?
Underrepresented
Why are CpG’s underrepresented in the DNA?
This underrepresentation is a consequence of the high mutation rate of methylated CpG sites: the spontaneously occurring deamination of a methylated cytosine results in a thymine, and the resulting G:T mismatched bases are often improperly resolved to A:T; whereas the deamination of thymine results in a uracil, which as a foreign base is quickly replaced by a cytosine by the base excision repair mechanism. The C to T transition rate at methylated CpG sites is ~10 fold higher than at unmethylated sites.
Where are clusters of CpGs (CpG islands) often found?
In gene promoters (about 50% of genes)
Fill in: DNA methylation … transcription
Inhibits
How does DNA methylation inhibit transcription?
- Binding of proteins that recognize methylated CpG DNA
- prevent binding of TFs
- recruit HDACs (eg. MeCP1,2)
- Remember that HDACs enforce compression/packaging of the DNA
- Direct interference with binding of TFs
- Compression of the DNA (packaging)
The cooperation in epigenetics is also depicted in the following figure
Note how the recruitment of HDAC (instead of HAT) results in the packaging of the DNA. This is all under influence of the methylation (red small circles)
Tumors make use of the effects of DNA methylation. Do they do this by hypermethylation or hypomethylation? Is this done for onco- or suppressor genes?
hypermethylation (on promoters) that silences tumor suppressor genes
Interesting enough, tumors globally hypomethylate. What does this lead to?
Re-expression of silenced genes
Why is the hypermethylation of suppressor genes and the hypomethylation of silenced genes beneficial for a tumor cell?
So it can grow fasters
Sodium bisulfite can be used to detect DNA methylation. How is this seen?
Sodium bisulfite specifically deaminates CpG’s that are not methylated. The unmethylated CpG’s will be mutated from C -> U, whereas methylated DNA is protected from this
What is the easiest method to see the effects of sodium bisulfite (aka see which sequences are methylated)?
Read-out by sequencing, a T will be formed instead of a C, as can be seen by the arrows in the figure
Besides read-out sequencing, read-out by PCR can also be used to detect methylated sites. How does this work?
Primers are used that specifically detect either the methylated or unmethylated strand of DNA (the difference of course is the C/T). An example of this is shown in the figure, where can be seen that the cancer cells have highly regulated the mir-3663 promoter.
What are micro RNAs (miRNAs)?
Very small (~22 nucleotides) non-coding RNA molecules
True/false: Transcription of miRNA genes is regulated by the same (epi)genetic mechanisms as protein-coding genes
True
What is the function of miRNA? How?
post-transcriptional regulation of gene expression
1) Repression of translation (imperfect match)
2) mRNA destabilisation or cleavage (perfect match)
There can be deregulated expression of miRNA in cancer. Where are miRNAs often downregulated, and where are miRNAs often upregulated?
Downregulated miRNA: oncogene (resulting in more protein)
Upregulated miRNA: suppressor gene (resulting in less protein)
How are miRNAs processed right after they’re made by the DNA polymerase?
They are taken up and cleaved by the DGCRB/DROSHA (don’t remember this name) to form a hair-pin like structure. They are then transported outside the nucleus where a DICER will cleave the ‘head’ off, creating a mature miRNA. This can then be taken up by a RISC protein to fulfill its function.
Which side of the miRNA specifically reacts with which side of an RNA?
The 5’ end of the miRNA often reacts with the 3’ (UTR) of the RNA.
Earlier we saw that there can be an increased or decreased expression of miRNA in cervical cancer. What conclusion can be drawn, when looking at this figure? Slide 41
That especially the ratio between the two is a good indicator for the prediction of malignancy in a cell
An example that was given for a long non-coding RNA was the Xist. This is quite an important molecule. Why?
It is a X-inactive specific transcript (Xist) on the X chromosome that plays a role in the X-inactivation process
Illustrate how the telemore extension looks per cell type over a period of time (e.g. germ line, stem cells, normal cells)
Don’t learn this by heart but understand the concept
senescence = cell rest, will not divide anymore
