Lecuture 10 - Regulation of Gene expression Flashcards by 1a agostin

What are the 4 levels of regulation in which gene expression occurs?

Methylation of DNA
histone modification
post transcriptional regulation via micaro RNAs
post translational modification

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a) DNA methylation is generally associated with transcriptional _______. (activation/repression)
b) DNA methylation occurs on ___’s followed by a ___. (A/C/G/T)
c) In CpG islands, the lowercase p represents the __________ bond. so 5’-C-p-G-3’
d) Methylated DNA recruits Histone ________ (acetylases/deacetylases (HDAC) and Histone ___________ (HMT) which functions to compact the DNA, __________ (enhancing/supressing) transcription
e) DNA methylation patterns can be inherited. What does this mean?
f) DNA Methylation works in conjunction with Histone
modifications to __________

a) repression
b) C’s, G
c) phosphodiester
d) deacetylase, methyl transferase, suppressing
e) this means that gene expression patterns can be inherited
f) regulate gene expression

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what 2 enzymes are recruited by methylated DNA to compact DNA (suppress it)

histone deacetylase
histone methyl transferase

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HDAC?

histone deacetylase

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HMT?

histone methyl transferase

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What does the p in CpG island represent

the phosphodiester bond btw the C and G bases

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What is going on here?

The C within the CpG islands is being methylated, resulting in that associated gene being silenced

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Over evolutionary time, methylated __s tend to turn into ___s (A/T/C/G) because of spontaneous ________

C, T, deamination

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The result of spontaneous deamination is that CpGs are relatively ______ (common/rare) unless there is selective pressure present to ________ (prevent/encourage) methylation

rare, prevent

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In the mammalian genome, what percentage of the cytosine residues are methylated?

2-7%

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Due to spontaneous deamination over time, the genome will become more _____ (AT/CG) rich unless _________ is present

AT, selective pressure

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nt?

nucleotides

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What is a CpG count?

The number of CG dinucleotides in the island

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What are the 2 conditions for the presence of a CpG island

Within the region of 200nt, there has to be greater than 50% GC content
The ratio greater than 0.60 of the observed number of CpG dinucleotides to the expected number based on GC content.

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a) Over ____% of human genes have a CpG island
b) almost all ________ genes have a CpG islan

a) 60
b) housekeeping

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What are housekeeping genes?

Genes are contantly expressed in almost all cells due to them being essential for basic cellular function

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T or F - All genes have a CpG island

F - not all genes

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What does the 54 represent here?

The CpG count

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T or F - almost all housekeeping genes have a CpG island

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What is the equation to calculate the number of expected CpG islands for a given gene?

((# of C + # of G)/2) ^2/ length of sequence

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Based on this, what is the expected CpG count?

= ((# of C + # of G)/2)^ 2/ length of sequence
= (410/2)^2/624
= 67.3

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Based on this, what is the observed CpG count?

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The ratio of observed to expected needs to be greater than 0.60 to be considered a CpG island. Therefore, based on this information, is a CpG island?

The observed: expected ratio is 54:67.3, which is 0.8 (54/67.3) ;therefore yes
expected ratio –> ((# of C + # of G)/2)^ 2/ length of sequence

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T or F - Unmethylated cytosines are converted to uracil by bisulfite

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T or F - Unmethylated cytosines are resistant to bisulfite conversion

F - methylated C

T or F - Methylated cytosines are resistant to bisulfite conversion

T or F - Methylated cytosines are converted to uracil by bisulfite

F - unmethylated C

Is this Cytosine methylated?

yes

What are the 4 regions that methylation can occur in the genome

1. Transcription start site (TSS) 2. CpG island shores 3. in the gene body 4. in repetitive sequence

Match the following wrt regions of methylation a) unmethylated transcription start site b) Unmethylated CpG island shore c) methylated TSS d) methylated CpG island shore

a) blue b) yellow c) green d) pink

What benefit is there to having methylation in the gene body?

to prevent alternative TSSs

What is the benefit of methylating repetitive sequences?

to silence them, preventing them from being involved in recombination (prevents genomic instability)

How do DNA methylation and cancer relate a) TSG b) repeats

a) hypermethylation of CpG islands associated with TSG can lead to its suppression, reducing resistance to cancer b) hypomethylation of repeats leads to mitotic recombination which results in genetic instability (can lead to cancer as a result)

TSG?

tumor suppressing gene

DNA can be modified by the methyl groups specifically on the ____ (A/G/C/T)

T or F - a methylated cytosine will be converted to a U

F - nonmethylated

T or F - a methylated cytosine is protected and now cannot be converted into a U and instead remain as a C

Methylation protects _____ from becoming a ____. a) U, C b) C, U c) T, G d) G, T

what level of methylation matches the following red boxes a) 10% unmethylated and 90% methylated b) 60% unmethylated and 40% methylated

a) right - C >> T b) left - C < T

______ C's are protected from conversion to ____'s (base). _____ (base) are converted to _____'s during the sequencing process.

methylated, U, Uricil, T

T or F - all methylated cytosine are a part of CpG islands

T or F - you can have a methylated C that is not a part of a CpG island

methylated cytosines typically occur in which region of DNA

promoter region

DNMT

DNA methyl-transferase

T or F - both the hypermethylation and the hypomethylation of certain genes can lead to cancer

based on this image answer the following a) where does it indicate methylation is occurring? b) where does it indicate no methylation is occurring c) what does methylation do/stop d) why is methylation high in that certain region but low in the other region

a) gene body as indicated by the orange b) transcription start site (beginning left) as indicated by the green c) it represses transcription d) it is low at the start site as transcription is supposed to occur here while it is high in the gene to prevent false transcription initiation.

Based on the methylation pattern, I would predict that ______ is actively transcribed in the _____ (majority/minority) of the cell line lines. Further, I would predict the absence of an alternative _____ since the gene body is ____ (methylated/unmethylated).

NEU1 (the gene), majority, TSS, methylated

a) what are the arrows pointing at b) what could prevent this from occurring?

a) false transcription initiations b) methylating cytosines in those areas

what are alternative txn start sites

different positions a within a gene where transcription can begin, leading to the production of different mRNA isoforms (one gene generates multiple transcript variants)

a) AIDA Prediction: actively transcribed in cell lines shown. A low %GC throughout the gene suggests that alternative txn start sites (if any) _______ (would/wouldn't) be regulated by CpG methylation. b) MIA3 Prediction: expression ________ (is/isn't) regulated by CpG methylation Legend - green/blue = unmethylated - orange = methylated - purple = partially methylated

a) wouldn't, isn't

TBX2 Prediction based on CpG methylation alone; (high/low) a) ______ expression in embryonic stem cells (hESC) b) ______ expression in leukemia cells (K562) c) ______ expression in HepG2 legend - green = unmethylated - orange/red = methylated

a) high b) low c) high

what is this?

CpG island shore

Which of the following takes priority when deciding expression level based on methylation? a) upstream CpG shore b) gene body c) transcription start site d) CpG island e) all of them have similar priorities you just need to go based on the majority

what is the box indicating?

the transcription start site

So we know that TBX2 is not expressed in any of the cell line. But it appears the image is not reflecting this properly. Explain which cell line(s) are not accurately predicting the expression levels and which one(s) are - green = unmethylated - red/orange = methylated

as shown the hESC 1 and the Hep G2 cell lines are portraying this accurately as they are showing unmethylation at the start sight indicating high expression levels. While the K562 is showing low expression levels (methylated TSS)

With the exception of TBX2 in MCF7 cells, there is a good correlation between ______ (region) methylation, levels of ________ and transcription factor binding.

promoter, expression

a) not expressed b) newborn (NB) or adult c) stem cells (ES) d) lowly e) highly

a) blue b) green c) yellow d) orange e) pink

a) Genes that are highly expressed in the ES cells are ________ (hypo/hypermethylated) at the Transcriptional Start Site (TSS) and Gene body, compared to the same genes in NB or adult cardiomyocytes. b) why?

a) hypomethylated b) low methylation leads to higher expression levels and the stem cells require a high transcriptional activity. vs the adult genes which are now developed and need a lower activity

The ________ region is susceptible to digestion by an ________

linker, endonuclease

a) blue b) green c) yellow d) orange e) pink

a) 2nM b) 11nm c) 146 d) 1.75 e) 8 to 114

The packaging of DNA into nucleosomes is the _____ level of condensation a) first b) second c) third d) fourth e) only

DNA is wrapped around a ________ of 8 __________ and anchored by a 9th histone called Histone H1 to form a ________.

nucleosome core, histone proteins, complete nucleosome

This image shows a a) histone protein b) nucleosome core c) complete nucleosome d) wrapped nucleosome e) closed DNA

These Histones can be post-translationally modified to alter What? (3 things)

1. DNA tertiary structure 2. Protein binding 3. DNA modifications

What are the 4 histone protein types that make up the nucleosome core?

1. H2a 2. H2b 3. H3 4. H4

Which of the following histone proteins are NOT part of the nucleosome core a) H1 b) H2a c) H2b d) H3 e) H4

Which of the following histone proteins is a part of the complete nucleosome but not a part of the core nucleosome? a) H1 b) H2a c) H2b d) H3 e) H4

Which of the following histone proteins are we the most interested wrt modifications a) H1 b) H2a c) H2b d) H3 e) H4

T or F - The core histone tails are extensively modified

T or F - The core histone tails are rarely modified

F - they are extensively modifed

What are the 4 modifications that can occur on DNA?

1. phosphorylation 2. acetylation 3. methylation 4. ubiquitylation

Which histone protein contains the most modifications

T or F - histone modification can only occur in the promoter

F - they can also occur in genes or other regions

Locate the following histone modifications a) H3K4 b) H3K9 c) H3K27 d) H3K36

Which of the following modified histone proteins are involved in expressing genes and which for repressing genes a) H3K4 b) H3K9 c) H3K27

expressing: a +c repressing: b + c

How is it possible that H3K27 is used to both express and suppress genes?

When you acetylate K27 it expresses genes but when you methylate it is represses

What does the following stand for a) 5-hmC b) 5-mC

a) hypomethylated cytosine b) methylated cytosine

So we know that the modifications will eventually lead to the DNA expression being changed, but what exactly does the histone protein modification do?

They allow for recruitment of certain proteins and depending on which protein recognizes that modification it will lead to either the enhancement or the suppression of that part of the DNA being expressed

Match the following: a) HP1 b) TAF3 c) H3K4 d) H3K9 e) SETD1A f) KDM3A/B

a) orange b) yellow c) green d) pink e) blue f) purple

indicate if it is part of activation or repression a) HP1 b) TAF3 c) H3K4 d) H3K9 e) SETD1A f) KDM3A/B

activation: b+c+e represssion: a+d+f

For the modified histone protein H3K9me3 answer the following a) What does H3 stand for b) what does K9 stand for c) what does me3 stand for d) does this modification lead to activation or suppression e) which histone protein modification will lead to the opposite effect

a) modification is done on the tail of the histone 3 protein b) the 9th lysine on the histone tail c) modification involved trimethylation (3 methyl groups on that K) d) suppression e) H3K4me3 (activation)

What state is this in? a) activation b) repression c) transition from activation to repression d) transition from repression to activation

What state is this in? a) Activation b) Repression c) Transition from activation to repression d) Transition from repression to activation

If you performed a histone modification ChIP-seq using an H3K4me3 antibody, what type of DNA would be isolated?

activated genes within the promote --> it will let you know what genes are being activated

If you performed a histone modification ChIP-seq using an H3K9me3 antibody, what type of DNA would be isolated?

repressed genes in the promoter --> it will let you know what genes are being repressed

a) Where are nucleosome-free enhancer regions located? b) What happens in these regions

a) the regions of DNA that are not associated with nucleosomes b) activation/repression depending on which protein binds to it

What are DNase I hypersensitive sites?

They are regions of chromatin that are highly accessible to DNase I digestion, indicating open chromatin that is typically associated with regulatory elements like promoters, enhancers, insulators, and other transcription factor binding sites.

T or F - nucleosome-free regions are linker DNA

F - linker DNA is a type of nucelosome-free region but any part of the DNA can be nucelosom-free. While linker DNA is specifically the region of DNA between two nucelosomes

which of the following could provide you with DNase I hypersensitive sites a) DNA-binding protein ChIP-sq b) Histone modification ChIP-seq c) DNase-seq d) FAIRE-seq

If you performed a DNase-seq what type of DNA will be isolated

Protein bound DNA

Open or Euchromatin is ________, while closed or Heterochromatin is _________ a) hypersensitive b) hyposensitive

a then b

a) which histone modification leads to activation b) which histone modification leads to repression c) Which gene is being activated d) which gene is being suppressed

a) H3K36 b) H3K27 c) FBX07 d) SYN3

a) Which ChIP has the lowest resolution? How do you know b) which one has the highest resolution?

a) ChIP-chip as it is unable to distinguish the difference in activity of the TSS for the NPC1 gene and the Pros35 gene b) ChIP-seq

Which of the following only influences the gene body? a) H3K4me3 b) H3K36me3 c) H3K9me d) H3K27me

match the following a) H3K4me3 b) H3K36me3 c) H3K9me d) H3K27me 1. activation at TSS/promoter 2. activation at gene body 3. suppression at TSS/promoter 4. suppression at gene body

a) 1 b) 2 c) 3 d) 3

which of the following influences the TSS and the promoter a) H3K4me3 b) H3K36me3 c) H3K9me d) H3K27me

Take Home: Lots of Histone Modifications affecting ______ and chromatin structure.

gene transcription

Which of the following modifications influence the TSS. Indicate which activates vs repression a) H3K4me3 b) H3K36me3 c) H3K9me d) H3K27me

a) activates c + d represses

what does the following stand for a) DM b) DNMT c) HAT d) HDAC e) HDM f) HMT

a) DNA demethylase b) DNA methyl transfer c) histone acetyltransferase d) histone deacetylase e) histone demethylase f) histone methyltransferase

A(n) _______ (increase/decrease) of H3K27Me3 modification in the region of TBX2 is consistent with its ________ (increased/decreased) level of trancription in MCF-7 cells

decrease, increased

A(n) _______ (increase/decrease) of H3K27Me3 modification in the region of TBX2 is consistent with its ________ (increased/decreased) level of trancription in H1-hESC cells

increase, decreased

T or F - H3K27me3 modifications lead to gene repression while H3K27ac lead to gene activation

T or F - H3K27me3 modifications lead to gene activation while H3K27ac leads to gene repression

F - the opposite it true

indicate whether the following represses or activates gene expression a) H3K27me3 b) H3K27ac c) H3K36me3 d) H3K9me3 e) H3K4me3

a) represses b) activates c) activates d) represses e) activates

Based on this image a) is the TBX2 gene being activated or repressed in the MCF-7 cell line? b) Which histone modification would you associate with TBX2 transcription in the MCF-7 cell line?

a) mainly activation as there appears to be the most activity with the H3K27ac modification at the TSS and activity for the H3K36me3 modification in the gene body b) H3K27ac

T of F - the absence of a repressor histone modification marker is enough to prove that a certain gene in a given cell is being trancribed?

F - There needs to be an activator histone modification marker present for this to be proven

Where is the H3K36me3 modification marker located within a given gene? a) TSS b) the promoter c) the gene body d) Upstream of the TSS e) a and b

For transcription of a gene to occur within a cell, you need a(n) _______ (absence/presence) of repressors and a(n) _______ (absence/presence) of activators

absence, presence

CBSs expression levels are being _________ (activated/suppressed). Positive histone markers for transcription (___________ and _________) correlate well with transcription levels. Negative histone markers of transcription (___________ and ________) are negative.

activated, H3K27Ac and H3K36me3, H3K9me3 and H3K27me3

DNase-FLASH and MNase-seq are both described as non-specific what does this mean?

it means that they are just going to isolate all open DNA (only DNA that is free from bound proteins)

Match the following terms to the definitions below a) DNase-Seq b) DNas-FLASH c) FAIR-seq d) Mnase-seq

a) green b) blue c) orange d) pink

txn factors are?

transcription factors

What are the 4 factors that influence transcription

1. trancription factor binding 2. DNA methylation 3. histone modification 4. chromatin state (open/closed)

a) define post-transcriptional regulation b) define post-translational modifications

a) Regulators that affect mRNA stability (after transcription occurs) b) factors that affect protein function (after protein is translated)