Lesson 22 Flashcards
what is it called when DNA is injected directly into the nucleus to be transcribed?
blind method → you cannot see the nucleus
when are histones synthesized?
during S phase
what is the direct model proposed by Adrian Bird?
proposed that if we have a promotor, that will have some CG - if the binding site of the transcription factors is not methylated, the transcriptional factor can bind and therefore the gene is expressed
if the consensus sequence of your tf is methylated, the methyl group overruns into the major groove and then directly interferes with the bind of the factor that therefore will not bind = we will have repression
what is the direct model based off of?
the fact that the methyl group is overhanging into the major groove of DNA
if you are given a tf that has a Cg consensus sequence and you want to test if it binds to its recognition sequence when it undergo methylation in vitro, what do you use?
electrophoretic mobility shift assay → use probes, a piece of the promotor that contain a piece of the binding site for your factor, you put it in a tube and do different samples: not methylated with no factor, methylated with no factor, that will migrate just the same; but for sure one non- methylated with the factor and one methylated with the factor, then observe. What you are expecting is that if the factor can bind to the promotor or to the sequence, the DNA will be delayed because it will be heavier in your electrophoresis.
if we have sick cells and the promotor in the sick cells is methylated, how can you check if the protein (reader) is binding or not to the non-methylated or to the methylated promotor?
do a chromatin immunoprecicitation assay followed by PCR to see if the sequence of interest is the one that has been bound, or follow through real time PCR
what are methyl binding proteins?
bind to methylated DNA and by binding they inhibit the binding of the transcriptional factor and therefore gene expression
what is the indirect model?
its not the methylation that directly blocks the binding of tfs, but methylation attracts readers, methyl binding proteins, and and the binding of these blocks the binding of transcription factors → same outcome (blocks transcription) but different method
what types of things would you get in a nuclear extract?
a huge amount of proteins which are going to bind to DNA (because they like DNA) - transcription factors, nucleosomes, DNA polymerase, RNA polymerase, etc
what does MeCP1 & 2 stand for?
methyl CpG binding protein 1 and 2 depending on the shift
what happens if we have more DNA than readers?
we will lose capacity to cover all the methylated DNA
when does chromatin assembly usually occur?
after DNA replication
describe the speed of chromatin assembly based on the structure of DNA:
if we have double stranded DNA this happens more slowly, if the DNA is single stranded it happens immediately
after experimentation was performed it was found that we have inhibited transcription only when chromatin is assembled onto our plasmid, what does this indicate?
methylation inhibits transcription through readers of methylation, but also using in part chromatin structure and nucleosome assembly
what is GAL4?
a transcription factor - this protein was already shown to be able to bind to its own sequence even if it was methylated (does not care about he nucleosome or methylation) → when its own sequence is assembled into chromatin it binds and activates
what is the reporter gene of thymidine kinase (TK) promotor?
CAT → if TK is methylated CAT is repressed, if TK is not CAT is expressed
what happens if you have a gene with only one single CpG methylation?
the gene is most likely expressed, maybe a little less than having nothing
what specific region was discovered when AB analyzed MeCP2, and what was its function?
MBD (methyl binding domain) → it is a region that is necessary and sufficient to bind to the DNA that contains at least one symmetrically methylated CpG dinucleotide
what is the function of MeCP2?
it is a reader of methylation because it contains a methyl binding domain but in addition its a transcriptional repressor because it contains a transcriptional repressor domain (TRD)
what are some general characteristics of MeCP2?
nuclear protein, highly abundant, ubiquitously expressed → through MBD it binds a single CpG dinucleotide symmetrically methylated
what genetic disorder is cause by MeCP2?
Rett Syndrome
where is MeCP2 most abundant?
in the CNS
describe MeCP2 localization:
it goes into the nucleus because it has a nuclear localization signal, but then it gets properly localized because DNA is methylated (if DNA is not methylated you lose your localization)
what is the best assay to use if you want to prove that a protein is interacting with another protein?
co-immunoprecipitation → you can immunoprecipitate your protein and check by Western blot how they react
what happens if you increase the amount of inhibitor of histone deacetylases?
you can start to relieve the repression of the inhibition → on that piece of DNA that is methylated, readers are bound and lead to a deacetylated chromatin structure
how does Landsberger’s beliefs differ from AB concerning MeCP2?
she agrees that MeCP2 is abundant and probably organizes a very compacted chromatin structure, but there is no proof that it is more condensed
how is MeCP2 a transcription factor?
it has been found that its involved in alternative splicing because it might interact with factors that are involved in splicing
what is another hypothesized function of MeCP2 besides its potential as a tf?
it is involved in protein synthesis, either directed or undirected
what did AB think the most important feature of MeCP2 was (also L)?
its capacity to recruit histone deacetylases
what binding proteins are similar to MeCP2?
MBD family (MBD1-6) - all share methyl binding domains and are characterized from a portion of their secondary and tertiary structure that is quite similar to MeCP2
what is another family made of 2 proteins that are similar to MeCP2?
Uhrf1 (aka Np95) → proteins that brings the DNA methyltransferase 1 to hemimethylated DNA, so the protein that binds either to methylated or hemimethylated DNA (pair?)
describe the structure of MBD1:
has a methyl binding domain, zinc finger, a long sequence, and the transcriptional repression domain
what are the functions MBD1?
- able to bind to methylated DNA so it is a reader of methylation and goes through its methyl binding domain, also able to bind to non methylated DNA due to its zinc finger domain
- contains transcription repressor domains that can repress transcription both from methylated and non methylated DNA (depending where the protein is found)
- interacts with proteins that are able to modify histones
how is MBD1 able to repress transcription from methylated DNA?
it recruits a histone methyl transferase
what biological process is MBD1 involved in?
neurodevelopment
clinically, what is a mutation in MBD1 associated with?
autism
what is the function of MBD2 and 3?
bind to methylated DNA and help recruit proteins that modify chromatin structure
what are MBD2 and MBD3 mutually exclusive in binding?
NuRD (nucleosome remodeling deacetylase) complex
what happens if animals are synthesized to be null for MBD3?
they die very early in conception (~8 days)
what is MBD3 important for?
not only establishing the right signature of DNA methylation but also reading it in the proper way
what does MBD4 contain that is different from the others?
does not contain a TRD but contains a glycosylase domain which is a domain important for DNA repair
what is the function of MBD4?
not a repressor of transcription but is generally considered one of the proteins that protects our genome from the damage that occurs during the deamination of methylated DNA
how does MBD4 work?
when DNA is methylated and unmethylated, MBD4 will go there are start to repair with the glycosylase activity, removing the base and then DNA would be fixed
what could happen with a lack of MBD4?
tumor progression
describe MBD5 and 6:
very little characterized or known → neurological issues if they are missing
what is the overall mechanism of how DNA methylation inhibits gene expression?
it recruits methyl binding protein and the methyl binding protein recruits protein that condense chromatin structure, so this structure that you have on methylated DNA is more close. Therefore, methyl binding protein and chromatin structure are 2 independent epigenetic mechanism that talk to each other
overall, how does methylation relate to chromatin?
no methylation leads to a chromatin that is more open; although more methylation leads to the recruitments of methyl binding protein and to a close
chromatin structure
what can a mutation in MBD1 cause?
neurological alteration or cancer
what can a mutation in MBD2 cause?
cancer or issues with hematopoesis
what can a mutation in MBD3 cause?
neurologic issues and cancer
what is the effect of more compact DNA?
if the DNA is more compact it will recombine less, it will break less, it will be less accessible to nuclease, it doesn’t permit to activate transposon, provirus sequences and so on and so forth
what is a gene body?
where the RNA polymerase transcribes in exons → DNA methylation is more present in gene bodies when the genes are much more active
what is the effect of transcription occurring at a regulatory region?
it is fine → the mechanism is that methyl binding proteins bind and recruit condensed chromatin structure, meaning transcription factors cannot bind and inhibiting transcription
