Lecture 32

Question 1

X rays as mutagens

Answer 1

They damage DNA a lot, can lead to breaking of chromosomes and otherwise loss of genes
In the case of drosophila they were looking for mutations in the eyes, and they got a phenotype of mostly white (recessive, loss of genes function) with some red patches (so wildtype gene maintained)

Question 2

Why did the drosophila start expressing the recessive eye color post x ray mutagen

Answer 2

Turns out the x ray mutagen damaged DNA, caused breaks, but as the cell was repairing it caused an inversion, and this inversion but the wild type gene in a place that is usually silenced, heterochromatin, so the gene is intact but not expressed in part of the eye
Note that the inversion put the eye color gene right next to the Centromeric chromatin, which as we know is always tightly bundled

Question 3

In drosophila with the inversion involving the color eye gene, what can lead to less wildtype expression in subsequent generations

Answer 3

Less wildtype expression means less cells are able to actually unwind their DNA enough to expose the wildtype gene
Mutations in HATs, the ones that add acetyls that lead to euchromatin and active DNA transcription
Less opening of chromatin leads to less transcription leads to less red eyed cells

Question 4

In drosophila with the inversion involving the color eye gene, what can lead to more wildtype expression in subsequent generations

Answer 4

More wildtype expression means we get more opening of the chromatin, or less closure, so mutated HMTs
Less HMTs means less methylation means less heterochromatin means more of the gene is expressed
Also mutations in HP1, heterochromatin protein 1, because it propagates heterochromatin and its formation once HMTs get that ball rolling

Question 5

What are epigenetics

Answer 5

The study of heritable traits that cannot by explained by changes in DNA sequence
So its not about having or not having an allele but rather actually expressing it
Usually not expressing it is due to it being in heterochromatin, and the markers that make it that way (like methylation), those are heritable

Question 6

Barr body

Answer 6

Inactivated X chromosome in mammals, ensures that males and females have the same gene dosage of X genes
Note that the decision of which X chromosome to inactivate occurs at the 8 to 32 cell stage of embryogenesis in females, every subsequent cell will have the same inactivated chromosome
So statistically the split will be like half half

Question 7

Are hétérozygote females for an x linked recessive disease automatically not showing the phenotype

Answer 7

Not necessarily, it depends the split, its possible that is was not exactly a 50 50 split in X chromosomes being inactivated, and in the case that more of the good allele was repressed, you’ll have less healthy cells to compensate for a given mutation
However you do still have like 40% function, so you aren’t totally recessive either, so most women will experience partial phenotype, variances in penetrance and expressivity

Question 8

DNA methylation

Answer 8

Catalyzed by DNA methyltransferase, DNMT, these add methyls mostly to CpG dinucleotides, note this is on DNA directly, not histones
Note that this modification does not affect the affinity of the base pair, still binds to G

Question 9

CpG dinucleotide

Answer 9

CG sequences such that the complement is GC, like specifically the doubles, the pair of them, the Cs in these pairs are methylated
60-80% of these CpGs are methylated, tho the distribution is not random

Question 10

Which CpG regions tend to be methylated

Answer 10

Mainly intergenic regions, so CpGs found in intergenic regions, theres a high correlation with these methylations (liek at these kinds of locations) and the repressor chromatin state (heterochromatin)
Also occurs at CpG islands, CpG rich clusters near promoters, 95% if not all are unmethylated and therefore transcriptionally active

Question 11

Why does having methylated CpGs lead to less transcription

Answer 11

Affects the binding of transcription factors, if they cant bind they cant initiate transcription, or at least not as often/efficiently
May also affect HDAC recruitment, like increase, and HDAC removes acetyl and increases DNA interactions with itself, as well as HMTs that have the same effect when methylating histones

Question 12

During replication, how are DNA methylations maintained, like to daughter cells

Answer 12

DNMTs, the enzymes that catalyze the addition of methyl groups to CpGs, have a very high affinity for hemimethylated sites, as in CpGs (the doubles) that only have 1/2 Cs methylated, which would be the case during replication because one of those strands would be new
So the DNMT uses the parental strand as a guide in this way, and thats how these methylations are heritable and how the expression of certain genes is heritable even if sequence is the same

Question 13

Monoallelic inheritance

Answer 13

A form of inheritance where you have 2 alleles, one from each parent, but only one will be expressed as if you only had one
There’s about 200 genes in which either the maternal or paternal (but not both) gene is expressed in this monoallelic way

Question 14

Imprinted alleles

Answer 14

Non-expressed, could be due to the monoallelic inheritance, if thats the case then the non expressed allele is the imprinted one
Note: this term is associated with phenotype

Question 15

Why is the paternal allele H19 imprinted every time

Answer 15

Happens before the offspring is even there, is happens in the germline of the father, theres methylation of the ICR (imprinting control region) and of the promoter for the H19 gene, methylation is associated with heterochromatin and just overall lower levels of transcription, so that gene is repressed from day one
Only the mothers H19 allele, which does not have these methylations, will be expressed, and because theres no methylation in the way the TF CTCF will be able to binds enhancer and actually elicit transcription but only on the mothers allele
Note: CTCF needs the ICR as a binding site, CTCF activates the enhancer, so blocked binding site by methylation= dead enhancer=dead transcription

Question 16

Are there imprints in early gematogenesis

Answer 16

Not initially, the germ cells are completed stripped of all methylations, even X chromosome inactivation is undone, its all basically fair game
It’s further into the gematogenesis process (making of gametes) that certain genes get imprinted, and at that only in certain genders, so like in males gene X is methylated but in females its gene y, net result is one active and one inactive copy from each so monoallelic inheritance (phenotypically anyway)

Question 17

How can imprinting affect disease inheritance

Answer 17

In some cases, like recessive diseases (but applies to dom too), where Gg would normally be considered phenotypically normal, if the good compensatory gene is imprinted (say cuz it came from mom) it will be silenced and wont be able to do its job, so all thats left is the mutant gene and an individual that should’ve been normal is now diseased, but on the genomic level is heterozygous
Basically it’ll change how offspring are affected (phenotypically), genotypes are sufficient to predict who is affected
Refer to slide 21

Question 18

Silver Russel syndrome

Answer 18

Dwarfism caused 50% of the time by mutation in Igf2 gene rendering non-functional, but 50% of the time due to hypomethylation of H19 gene in paternal copies
Maternal is supposed to make H19 (no methylations) and paternal Igf2 (methylation required), but since theres a failure in methylation, paternal also produces H19, so we have extra H19 and no Igf2, its repressed, leads to dwarfism

Question 19

Epimutations

Answer 19

Mutations that result in disease or changes as if a gene was rendered non functional, but its actually fine it just cant be expressed because of an epigenetic mutation making it accidentally repressed of active
It’s a change in phenotype due to a change in epigenetic marks, in our case methylation