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Regulation of Gene Expression > Tonon: Lecture VI > Flashcards

Tonon: Lecture VI Flashcards

Enhancers, Super-Enhancers, and New Technologies

1
Q

What are enhancers?

A

sequences that are usually upstream the promoter (not always though)

located distally from transcription start sites

bound by an activator, TF, and RNA polymerase

cause the loop formation between the enhancer sequence and promoter to begin translation

bind various components, such as acetyl-transferases p300 and CBP

harbour 2 histone modifications, H3Kme1 and H3K27ac

regulate multiple genes

https://www.youtube.com/watch?v=aq8PAM5Sa0s

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2
Q

What kind of specificity do enhancer loops exhibit?

A

tissue-specificity

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3
Q

How do enhancers bind to TF?

A

due to specific motifs located on TF and enhancers

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4
Q

Enhancers hold many nucleotide variations associated with ______________.

A

genetic diseases

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5
Q

Enhancers do not have histones; however there are some regions that have histones: what happens if histones are present?

A

enhancers are shut down, like a promoter

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6
Q

active promoter histone marks

A

H3K4me3
H3K27ac

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7
Q

inactive promoter histone marks

A

K3K27me3

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8
Q

active enhancer histone marks

A

H3K4me1
H3K27ac

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9
Q

inactive enhancer histone marks

A

H3K27me3

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10
Q

What is important to note about me3 (3 methylations)?

A

it is always negative as it represses transcription

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11
Q

What is the histone modification for compact heterochromatin?

A

H3K9me3

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12
Q

What is the histone mark for open or closed enhancers?

A

H3K4me1 (active enhancer)
H3K27me3 (repressed enhancer)

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13
Q

Are RNAs expressed on enhancers?

A

yes, in both directions

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14
Q

What is the name of RNA transcribed from enhancers?

A

eRNA

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15
Q

What is the probable job of eRNAs?

A

increase transcription of corresponding genes

from the slides: promote mRNA synthesis

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16
Q

What are super-enhancers?

A

much bigger enhancers described as “key cell identity genes”

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17
Q

How did they find super-enhancers?

A

by evaluating the Med1 (protein that binds to enhancers) density

Med1 signal was very strong corresponding to super-enhancers

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18
Q

What is the size of an enhancer site and a super-enhancer site?

A

700bp and 9000bp, respectively

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19
Q

What is the mechanism of action of super-enhancers?

A

super-enhancers → many proteins bind →transcription of a downstream gene is upregulated → increased gene expression

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20
Q

What are some genes under the control of super-enhancers?

A

Oct4
Sox2
Nanog
Myc
chromatin-modifying genes

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21
Q

What is Myc (gene controlled by super-enhancers) involved in?

A

tumors like multiple myeloma

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22
Q

What is a protein involved in the machinery that forms super-enhancers?

A

Brd4

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23
Q

Myc is under the control of super-enhancers. In a study, there was the discovery of Brd4’s protein, bromodomain. Why was this discovery important?

A

it was found that dromodomain could disrupt the looping between Brd4 and Myc

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24
Q

What is the Waddington Model?

A

cells can take different trajectories leading to different outcomes and there are components that work together to provide an epigenetic landscape of cells and tissues

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25
What is the single-cell approach?
a method that can be used to detect and analyze specific cells
26
What does every dot on a single-cell transcriptomic graph represent?
a cell
27
How can populations be identified in the clusters of single cell analysis?
using specific markers or a combination of antibodies if trying to find a new subpopulation
28
What has replaced single cell-analysis?
UMAP because it is stronger for a biological point of view
29
What are some assumptions made in RNA seq?
process rates β (in terms of splicing) is the same for all genes and can be set to 1 mRNA degredation rates are gene-specific constants during development, differentiation occurs on a timescale of hours to days, which is comparable to mRNA's half-life
30
What is RNA velocity?
density of a single cell based on the ratio of spliced and unspliced RNA
31
In regards to RNA velocity, what occurs during a dynamic process?
an increase in the transcription rate results in a rapid increase of unspliced mRNA followed by a subsequent increase in spliced mRNA until a new steady state is reached
32
In regards to RNA velocity, what occurs if there is a drop in the rate of transcription?
a rapid drop in unspliced mRNA followed by a reduction in spliced mRNAs
33
In regards to RNA velocity, what happens during the induction of gene expression?
unspliced mRNAs are present in excess based on the equilibrium rate 𝜸
34
In regards to RNA velocity, what happens during the repression of gene expression?
unspliced mRNAs are NOT present in excess based on the equilibrium rate 𝜸
35
What is an indicator of the future state of mature mRNA abundance and thus the fututre state of the cell?
balance of spliced and unspliced mRNA abundance
36
Why are t-SNE and UMAP helpful?
each cell can be described with its own destiny based on the ration between spliced and unspliced
37
Can RNA velocity be used to analyze every process?
no, it can only be used for the phenomena that can be measured in the space of a day or a few days to construct patterns like the one if Figure 16 (which suggests the cell of origin based on arrow direction)
38
Name an application of single-cell RNA velocity:
Single cell analysis was done on 5 patients affected by a deadly brain cancer: glioblastoma paper demonstrated that tumor cell fluctuate between states and do not remain in a single status
39
ATAC-seq
new technique that uses Tn5 transposome
40
What is a Tn5 transposome?
it is a bacterial protein that cuts the DNA where it is open and insets 2 different oligos these fragments are then sequences and information about where the genome is open can be obtained
41
Why is open chromatin important in the genome? Why would you measure it?
it is important to know which parts of the genome are open and expressed
42
Analyze the graph and describe which techniques would use these graphs:
each peak represent short sequences used to read ATAC-seq and GET-seq
43
In this figure of ATAC-seq, what do the peaks represent?
regions that are open
44
What is an advantage of ATAC-seq vs RNA-seq?
ATAC is able to cluster cells better than RNA-seq, so it is much more clear in distinguishing different subtypes and correlating them into groups of cells
45
Describe the ATAC-seq path
put transposes at the single cell level → PCR amplification → profiles are obtained
46
Can ATAC-seq recognize both open and closed chromatin?
no, it can only recognize open chromatin and this is a problem because more than 50% of the genome is occupied by closed chromatin
47
How was the recognition problem of ATAC-seq only recognizing open chromatin solved?
the Tn5 transposome was modified and attached to a domain that could recognize the trimethylation on Lysine 9 of histone 3 (H3K9me3) a chimera was built with Tn5 on one side bound to a domain able to recognize heterochromatin the chimera not only have Yn5, but it also has TnH because TnH has a tail that can recognize heterochromatin through HP1
48
Euchromatin
less condensed at chromosome arms contains unique genes gene-rich replicated through S-phase recombination during meiosis
49
Heterochromain
highly condensed at centromeres and telomeres contains repetition sequences gene-poor replicated in late S-phase no meiotic recombination
50
What is the heterochromatin typical domain?
H3K9me3
51
Name and describe the 3 family members of HP1
HP1a: most specific for heterochromatin and has a chromodomain that recognizes heterochromatin through H3K9me3 HP1b HP1c
52
What can be seen in the following figure?
Tn5 corresponds to H3K4me3 in the genome, which is typical of histones nearby open promoters TnH was very good with H3K9me3, and its profile is very similar to H3K4me3, so it can be concluded that TnH can recognize H3K9me3
53
Which is more reliable: ATAC-seq or GET-seq?
GET-seq
54
Describe this graph:
columns are chromosomes blue depicts losses and red depicts gains
55
What was discovered when analyzing the heterochromatin features on the patient-derived xenograft?
each clone was subdivided into epigenetic subclones, suggesting there is an underlying epigenetic variability that goes beyond genomics and genetics
56
What does each triangle represent on the different genes?
mutations red: missense mutation blue: truncating mutation
57
Of these 4 genes, which ones are the oncogenes and which ones are the tumor suppressors?
IDH1 is an oncogene that hass all of the mutations in one position, which makes it active RB1 and VHL are tumor suppressors
58
Analyze the GET-seq:
the area of iPS that is multicolored shows precursors that are common to iPS, fibroblasts, and NPC
59
What was used when trying to apply the idea of RNA velocity to chromatin?
TnH versus Tn5 was used instead of spliced versus unspliced
60
What was an important finding of the chromatin velocity analysis?
chromatin organization and heterochromatin ratio were analyzed the genes in the iPC area were associated with axon guidance and genesis, neuron projection guidance, and so on this suggests the genes residing in the population were crucial for the evolution of NPC and neuronal products

Regulation of Gene Expression (28 decks)

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