Landsberger: Lecture XVI Flashcards
Chromatin Affects Gene Expression - DNA Methylation
Epigenetics is a nonstable code: it needs __, __, & __.
writers, readers and modifiers
Every epigenetic mechanism has to be _____.
transmitted and read by specific proteins
What is the PHO5 promoter?
it is a promoter that has nucleosomes that are positioned precisely so that the TATA box is always assembled into nucleosomes so that TBP (TATA binding protein) cannot bind → promoter is off
PHO5 promoter is generally ___ in promoters.
blocked
Since nucleosomes are repressive, they need to be removed prior to transcription. What does this establish?
chromatin remodeling is a prerequisite and not a consequence of gene transcription
Why is chromatin important?
keeps genes repressed so that they are activated only when it is necessary
Can chromatin participate as an epigenetic signal?
no
Why are nucleosomes important?
they can condense DNA and participate in gene expression
Can chromatin induce gene expression?
yes
What are the 2 reasons Grunstein and Winston decided to use yeast for their experiments?
simple model
only 2 copies of histones (humans have many)
Grunstein and Winston created a yeast strain completely devoid of H4 but carried the gene on a plasmid. What regulated this gene’s expression?
an inducible promoter
What did the yeast strain’s expression rely on?
galactose: activated the gene
glucose: deactivated the gene
H4 expression is shut down →
H4 histone level in strain is low → nucleosome density is reduced → less dense chromatin → linker DNA size increased
Summarize the experiment by Grunstein and Winston:
yeast was modified to have deleted both H4 alleles and supplied by stable extrachromosomal plasmid where the H4 gene was under the control of inducible GAL1 promoter
growth in presence of galactose → H4 is expressed
growth in presence of glucose → H4 not expressed
How do you check after 3 generations what happens to gene expression?
!!
If I don’t have a canidate gene, I have to use a global approach
If I have a canidate gene, I have to use a single approach
What is the best current global approach?
analyzing gene expression using RNA-seq
What could be concluded from the experiments of Grunstein and Winston?
some gene’s nucleosomes are an integral part of regulatory mechanisms
closed chromatin →
gene is not transcribed
open chromatin →
gene is transcribed
How can cells modify the chromatin structure on a gene?
through ATP dependent chromatin remodeling complexes or by epigenetic mechanisms
ATP dependent chromatin remodeling complex
make DNA more accessible
can alter the position or structure of nucleosomes through ATP hydrolysis
Post-translational modifications (PTMs) of histone’s tails (epigenetics)
modifications are induced to recruit proteins that open or closed chromatin
Introduction of histone variants (epigenetics)
resemble canonical histones, with a few different aa and they are characterized by specific structural and functional properties
the tags tell the cycles that a specific region of the chromatin must be highly transcribed or must be repressed
readers read the specific histone variants
What can ATP dependent chromatin remodeling complexes induce?
nucleosome sliding
nucleosome ejection
H2A-H2B dimer ejection
H2A-H2B dimer replacement
Nucleosome Sliding
region which is hidden by a nucleosome becomes accessible to TF
ex: nucleosome can go into linker DNA
Nucleosome Ejection
all nucleosomes are removed and so the DNA becomes accessible
H2A-H2B dimer ejection
nucleosomes are made by DNA wrapped around the octamer
the tetramer made with H3 and H4 is sufficient to have DNA wrapping around but without H2A and H2B, DNA has less contacts so TFs have more access
H2A and H2B removal make the chromatin more transparent for the binding of TFs
H2A-H2B dimer replacement
after the removal of the dimer, it is possible to insert histone variants
What can ATP remodeling complexes do?
work in both directions (open and close chromatin)
What can ATP remodeling complexes be involved in?
every DNA metabolic process and neurological disorder and cancers
What are SWI-SNF (BAF) complexes?
typical ATP dependent chromatin remodelling complexes involved in human tumors
What is a rhabdoid tumor?
a very aggressive pediatric tumor that affects the brain, kidneys and other soft tissues
A deletion in one of the subunits needed for ATP dependent chromatin remodeling complex assembly results in what?
100% of rhabdoid tumors
100% of rhabdoid tumors have a deletion in what?
SMARC5B (SNF2) gene: the lack of protein causes the disassembly of all the related complexes and at least 1 ATP dependent chromatin remodeling complex is always involved
How can ATP dependent chromatin remodeling complexes reach target DNA?
- be recruited by TF, which have access to chromatin thanks to PTMs
- randomly: they slide nucleosomes and if a TF is there, it will have the chance to bind
- readers of PTMs
How can we test which genes are affected by a specific ATP dependent chromatin remodeling complex?
ChIP-Seq can be used to see which complexes are bound, but this will not allows us to known if expression is affected
How can we see if gene expression is affected after doing an analysis with ChIP-Seq?
the best approach is using a knock-down using siRNA or shRNA (short-hairpin RNA)
cells are transfected or infected with siRNA or shRNA expressing vector and we check if the gene is a knock down via RT-PCR or WB (or both) and then RNA-SEq is performed unless we have a candidate gene we want to check
What are the most common modifications on histone tails?
phosphorylation → on Ser, Thr, and Tyr
acetylation → on Lys
methylation → on Lys and Arg
ubiquitination → on Lys
What was the first PTM studied on a histone?
acetylation, which is an epigenetic signal
Histone acetylation correlate with _____.
transcriptional activation
How does acetylation exert its effect on transcription?
acetylation neutralizes the + charge of histones so the tails become less positively charged and the contacts between the nucleosomes are reduced
Are all positive charges neutralized in Lys residues in the presence of acetylation?
no, only 1 or 2 so it cannot be the main model (this artificial effect only occurs in vitro)
What does acetylation do?
creates sites for the recruitment of other proteins
What are some examples of different readers of different PTMs?
bromodomain: reads acetylated Lys (not only in histones…the human proteome contains more than 40 proteins with bromodomain)
chromodomain: reads methylation
What is the histone code hypothesis?
all PTMs on a specific nucleosome must be considered
different combinations of “functional groups” added to the histone tails form specific signals to attract or repel protein complexes directly involved in transcriptional regulation
Why is the combination of PTMs on a specific nucleosome important?
the same modification can mean either condensing or opening…it depends on what is close by
Do prokaryotic cells use DNA methylation?
yes
What are the 2 bases that get modified in prokaryotic cells?
cytosine and adenine
When does DNA-methylation occur?
post-replication
What is the enzyme that methylates DNA called?
DNA methyl transferase
How does DNA methyl transferase work?
uses a donor of a methyl group called SAM (S-adenosyl methionine)
cytosine can be methylated on the 5’ carbon and the adenine gets methylated on the 6’ carbon
What is the role of DNA methylation in prokaryotes?
protect the stability of the genome
How is DNA methylation by prokaryotes used to protect the genome?
avoids the cut from restriction enzymes
distinguish between replicated vs non-replicated DNA (defense mechanism) → cell cycle control to separate the 2 chromosomes into 2 daughter cells
discriminate DNA strands for mismatch repair (defense mechanism) to protect the genome
Is DNA methylation involved in gene expression?
sometimes
How can DNA methylation affect replication?
when replication starts, the origin is hemimethylated → SeqA binds to the origin and can interact with a protein on the membrane → membrane grows → invagination occurs ensuring the 2 genomic DNA will go to 2 different cells
How is DNA methylation involved in DNA repair?
when DNA needs to be repaired, it knows which strand to correct using DNA methylation
Where are DAM methylation sites located?
along the chromosome
What are the nt of DNA methylation sites?
GATC
Describe the figure:
there is a missense mutation and the geometry of the double helix is altered
the mutation is recognized by MutS and it recruits MutL and MutH on the mutated DNA
this complex with endonuclease activity interupts phosphodiesterase bonds in order to degrade DNA
the system moves and loops to the closest parental strand and hydrolyzes it and generates the nick on the opposite strand
the hemimethylated state of DNA allows the repair machinery to recognize which strand carries the mutated nucleotide
*mechanism controls gene integrity
Where is the genome methylated in mammalian cells?
!!
cysteine on the 5’ carbon on the CG dinucleotide
it can also occur on a C followed by another nucleotide ( C A or T, generally A)
CG methylation (most abundant) or CH methylation (C followed by another nt that is not G)
What are the techniques available to detect DNA methylation?
thin layer chromatography (TLC): used to understand if the genome contained methylated nucleotides
high performance liquid chromatography (HPLC): used to understand if adenine is sensitive
restriction enzymes (HpaII and MspI): can recognize palindromic sequences that contain sites for methylation
What is a problem with the TLC technique?
not very sensitive: since it is impossible to lead large amounts of nt on a thin layer, if the methylation is not high in abundance, it won’t be detected
does not give sequence information since the DNA was 1st hydrolysed to single nucleotide level
How can we distinguish if a region is methylated or not and between different types of methylations?
What are the enzymes involved in DNA methylation (writers, readers, and erasers)?
What are the consequences of DNA methylation?
What is the role of DNA methylation in cells?
What happens if DNA methylation does not proceed properly?
What are some disorders associated to DNA methylation?
What do HpaII and MspI recognize?
CCGG but they have different sensitivity for methylation
HpaII is CG methylation sensitive and it cuts CG if it is NOT methylated
MspI is CH methylation sensitive
