Lesson 16 Flashcards
describer the structure of the PHO5 promotor:
there are precisely positioned nucleosomes so that the TATA box is always assembled into nucleosomes and TBP cannot bind → promotor is off
describe one of the two binding sites for Pho4:
one site is free of nucleosomes so it is accessible for transcription factors and the other is occluded
what is chromatin important for?
keeping genes repressed so that they are activated only when it is necessary → participates in gene expression but not in epigenetic terms
describe how genes are effected by chromatin in regards to expression:
some genes are only repressed by chromatin, whereas other genes are affected more meaning that chromatin participates in the regulation
what are two reasons why yeast is a good genetic model:
it is very simple and it has only a few copies of histone genes
when they wanted to know if the depletion of nucleosomes affects general transcription or specific expression in yeast, what did they set as the control and experimental variables?
control: yeast cells grown in the presence of galactose (H4 is present)
experimental: yeast cells in the presence of glucose (down regulation of H4 gene)
when both H4 alleles were deleted in yeast and supplied by stable extrachromosomal plasmids, what did galactose stimulate?
growth in the presence of galactose stimulated H4 expression
when both H4 alleles were deleted in yeast and supplied by stable extrachromosomal plasmids, what did glucose stimulate?
growth in the presence of glucose caused H4 not to be expressed
if you have a candidate gene, how can you check gene expression multiple generations later?
use a single cell approach
if you don’t have a candidate gene, how can you check gene expression multiple generations later?
use a global approach
what is the best global approach to analyze gene expression?
RNA-Seq
what are some nucleotides integral for?
an integral part of the regulatory mechanism
what did histone H4 depletion cause in yeast cells?
- reduced H4 levels
- reduced number of nucleosomes (ab 50%)
- increased accessibility of chromatin to nucleases
what were the three effects on gene expression in yeast cells?
- expression of endogenous genes were de-repressed
- the affected genes were all inducible genes
- constitutive genes were not affected
how is chromatin related to transcription?
in most cases the chromatin is closed when the gene is not transcribed and it is open when the gene is transcribed
what are ATP-dependent chromatin remodeling complexes?
make DNA more accessible → alter the position and structure of nucleosomes through ATP hydrolysis
what do the post-transcriptional modification of histone tails cause?
induced to recruit proteins that open or close chromatin
what does the induction of histone variants do?
tags that tell the cell the a specific region of the chromatin must be highly transcribed or repressed
why are the induction of histone variants epigenetics?
there are readers that read specific histone variants
name four ATP-dependent chromatin remodeling complexes:
- nucleosome sliding
- nucleosome ejection
- H2A-H2B dimer ejection
- H2A-H2B dimer replacement
what is nucleosome sliding?
a region which is hidden by a nucleosome becomes now accessible to transcription factors; the nucleosome for example can go into the linker DNA
what is nucleosome ejection?
all nucleosomes are removed
and so the DNA becomes accessible
what is H2A-H2B dimer ejection?
the tetramer made with H3 and H4 is sufficient to have DNA wrapping around, but without H2A and H2B DNA has less contacts with what remains of the nucleosome and so transcriptional factors can have more access → H2A and H2B removal make the chromatin more transparent for the binding of transcriptional factors
what is H2A-H2B dimer replacement?
after the removal of the dimer it is possible to insert histone variants
How do ATP-dependent chromatin remodeling complexes function?
work in both directions → they can either open or close chromatin
what are typical ATP-dependent chromatin remodeling complexes that are involved in a huge amount of human tumors, and also have the highest rate of mutations?
SWI-SNF (BAF) complexes
describe rhabdoid tumors:
100% of these tumors have a deletion in the SMARC5B (SNF2) gene: the lack of the protein causes the disassembly of all the related complexes and at least one ATP dependent chromatin remodeling complex is always involved
how can ATP-dependent chromatin remodeling complexes reach the target DNA?
- They can be recruited by transcriptional factors, which in turn can have access to chromatin thanks to PTMs
- It can happen randomly: they slide nucleosomes and if a transcriptional factor is there, it will have the opportunity to bind
- They can be readers of PTMs
what is the best way to see which genes are affected by specific ATP-dependent chromatin remodeling complexes?
The best experiment to perform is knock down, using siRNA or shRNA (short hairpin RNA): cells are transfected or infected with siRNA or shRNA expressing vector; we check if the gene is knocked down by RT-PCR or WB, then we perform RNA-Seq experiments
what part of the histone is most easily accessible to histone modifications?
the tails → they are exposed on the surface of nucleosomes
what PMTs are most common for histone tails?
- Phosphorilation→ on Ser, Thr and Tyr
- Acetylation→ on Lys
- Methylation→ on Lys and Arg
- Ubiquitination→ on Lys
what do the different histone PMTs depend on?
depend on the positions and histone type
what type of signal is histone acetylation?
an epigenetic signal
what does histone acetylation correspond with?
transcriptional activation
how does acetylation exert its effect on transcription?
it neutralizes positive charges of histones, meaning that the tails become less positively charged and reduces the contact between the nucleosomes
what does acetylation allow?
allows readers to get in, read the modified sequence, and bring other post-translational modifiers or ATP-dependent chromatin reading complexes → creates sites for the recruitment of other proteins
what are the two different readers for different PTMs?
bromodomains and chromodomains
what is the function of bromodomains?
reads acetylated Lys (not only on histones)
what is the function of a chromodomain?
reads methylation
what is the histone code hypothesis?
different combinations of “functional groups” added to the histone tails to form specific signals to attract or repel protein complexes directly involved in transcriptional regulation
what does phosphorylation of histones involve?
a covalent modification of histones that involves mainly serine 10 of histone H3 leading to completely opposing effects
how can you find out where enhancers are located?
using omic approaches (RNA, ChIP, or ATAC-Seq)
what enzyme methylates DNA?
DNA methyl transferase
what does DNA methyl transferase use to methylate DNA?
a donor of methyl groups called Sam (S-adenosyl methionine)
in prokaryotic organisms, what is the purpose of methylation?
protect the stability of their genome
in what ways do prokaryotic organism use methylation?
- to avoid the cut from restriction enzymes, (defends mechanism for the bacterial genome)
- to distinguish between replicated versus not replicated DNA (also important to separate chromosomes into daughter cells)
- to discriminated DNA strands for mismatch repair
the OriC (origin of replication in E. coli) is made of a sequence containing GATC repeated 11 times, what is this called?
a DNA methyl transferase - DAM methylase
in the original formation of E. coli DNA, what state is it in and what does this mean?
both strands are methylated - DNA can replicate
what happens when the E. coli DNA replicates?
the origin becomes hemimethylated because one strand is newly synthesized and does not contain any methylation → cannot replicate
at the molecular level, what happens when the origin becomes hemimethylated?
become the binding site of a protein called SeqA that can somehow read DNA hemimethylated → SeqA and DAM compete for binding
how do DAM and SeqA compete?
when SeqA is bound, the DAM can’t methylate the origin ; when it goes away, it can resume function
when do eukaryotic cells go through mitosis?
eukaryotic cells don’t go through mitosis up to the moment that all DNA is replicated (only once), and they don’t proceed to the end of mitosis if there is not a control that the two mitotic fibers pull with the same strength the chromosomes, because if one pulls more the result will be aneuploid cells; so eukaryotes control a lot that each daughter cells receive equally amount of DNA
besides transcription, what else is DNA methylation involved in?
DNA repair - change in the regulatory of the double helix is a signal for the mismatch repair system
how does the difference in DNA repair change the outcome?
if the parallel strand that is degraded the mutation is fixed forever ; if its the newly synthesized strand that is fixed, the mutation is corrected
in E. coli, what happens if there is a missense mutation in the geometry of the double helix?
this is recognized by a protein, MutS, that recruits on the mutated DNA MutL and MutH, and then this complex with its endonuclease activity interrupt phosphodiester bonds in order to degrade the DNA
how do MutS, MutH, and MutL work?
system moves and loops to the closest methylation site that will be on the parental strand, and it hydrolyses and generates the nick on the opposite strand
when dealing with MutS, MutH, and MutL in E. coli, what does hemimethyaltion allow for?
allows the repair machinery to recognize which strand carries the mutated nucleotide
where does most methylation occur in prokaryotes?
on the cystine on the 5’ carbon
what type of modification is DNA methylation in eukaryotes?
post-replicative
where does the vast majotity of methylation occur (think base)?
occurs in a C that is followed by a G → CG dinucleotide
what other type of methylation is common besides CG, that happens especially in neurons?
CH methylation → (which is any methylation that occurs on a C followed by a nucleotide different from G
how is our genome methylated?
globally methylated
give an example of global methylation:
all hepatocytes have probably a similar pattern of CG methylation, that will be different from the one of glutamatergic neurons, which will be different form the one of GABAergic neurons, etc
why are there much less methylated cysteines, and therefore much less CG dinucleotides than expected?
Cs can be deaminated becoming a uricil, but U can’t be present in our DNA so the repair system fixes it - a methylated cysteine can also become a thymine which is a natural component of DNA
what happens if they repair system dos not repair the mutation before replication?
there will be a cell with a WT and the other will have the mutation fixed - many genetic disorders have this mutation
what occurs in Rhabdoid syndrome (first epigenetic disorder discovered)?
most of the time C to T transitions occur in germinal cells (generally in sperm) - sporadic genetic disorder: does not originate from parental DNA
what are CpG islands?
regions of our genome in which there is an abundance of CG dinuicleotides (more than expected) - often associated with promoters so they are a signature of a presence of a gene
describe the natural state of a CpG island:
most of the time they are not methylated → these regions are bound by a transcription factor or other proteins that protect them from methylation
what is a very old way to detect methylation?
thin layer chromatography - way to separate the molecules depends on features such as charge, size, etc
why do C and G produce a similar spot on TLC like A and T do?
the C doesn’t lead to only one spot, but to two spots → the second spot represented the methylated cytosines
what two issues are associated with TLC?
the technique is not super sensitive and it does not give any sequencing information
what technique is used now instead of TLC?
high performance liquid chromatograph (HPLC) → still requires the hyrdolysation of DNA to obtain nucleotides, but is much more sensitive
what is another method to detect methylation besides HPLC?
the use of restriction enzymes → they can recognize palindromic sequences that contain sites for methylation
what are the most popular restriction enzymes used to detect methylation?
HpaII and MspI → They are istochizomer, which means that they recognize and cut exactly the same sequence, but they come from two different organisms. They both recognize CCGG but they have a different sensitivity for methylation
How does HpaII work?
reads CCGG and cuts it if it is not methylated and it does not cut it if it’s methylated on the C followed by G: so this enzyme is CG methylation sensitive
how does MspI work?
is its control, since it can cut not only when the C followed by G is methylated but also if it is methylated - MspI is CH methylation sensitive
describe the cuts made by HpaII and MspI if the DNA is unmethylated?
HpaII and MspI make the same 3 fragments (300+500+50 bp)
describe the cuts made by HpaII and MspI if the DNA is methylated?
if the DNA is methylated the digestion with MspI still results in three fragments, whereas the cut with HpaII results in a full length fragment (850bp), because the C is methylated and so this enzyme can’t cut it
