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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
Q

What is the single-cell approach?

A

a method that can be used to detect and analyze specific cells

26
Q

What does every dot on a single-cell transcriptomic graph represent?

A

a cell

27
Q

How can populations be identified in the clusters of single cell analysis?

A

using specific markers or a combination of antibodies if trying to find a new subpopulation

28
Q

What has replaced single cell-analysis?

A

UMAP because it is stronger for a biological point of view

29
Q

What are some assumptions made in RNA seq?

A

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
Q

What is RNA velocity?

A

density of a single cell based on the ratio of spliced and unspliced RNA

31
Q

In regards to RNA velocity, what occurs during a dynamic process?

A

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
Q

In regards to RNA velocity, what occurs if there is a drop in the rate of transcription?

A

a rapid drop in unspliced mRNA followed by a reduction in spliced mRNAs

33
Q

In regards to RNA velocity, what happens during the induction of gene expression?

A

unspliced mRNAs are present in excess based on the equilibrium rate 𝜸

34
Q

In regards to RNA velocity, what happens during the repression of gene expression?

A

unspliced mRNAs are NOT present in excess based on the equilibrium rate 𝜸

35
Q

What is an indicator of the future state of mature mRNA abundance and thus the fututre state of the cell?

A

balance of spliced and unspliced mRNA abundance

36
Q

Why are t-SNE and UMAP helpful?

A

each cell can be described with its own destiny based on the ration between spliced and unspliced

37
Q

Can RNA velocity be used to analyze every process?

A

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
Q

Name an application of single-cell RNA velocity:

A

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
Q

ATAC-seq

A

new technique that uses Tn5 transposome

40
Q

What is a Tn5 transposome?

A

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
Q

Why is open chromatin important in the genome? Why would you measure it?

A

it is important to know which parts of the genome are open and expressed

42
Q

Analyze the graph and describe which techniques would use these graphs:

A

each peak represent short sequences

used to read ATAC-seq and GET-seq

43
Q

In this figure of ATAC-seq, what do the peaks represent?

A

regions that are open

44
Q

What is an advantage of ATAC-seq vs RNA-seq?

A

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
Q

Describe the ATAC-seq path

A

put transposes at the single cell level → PCR amplification → profiles are obtained

46
Q

Can ATAC-seq recognize both open and closed chromatin?

A

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
Q

How was the recognition problem of ATAC-seq only recognizing open chromatin solved?

A

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
Q

Euchromatin

A

less condensed
at chromosome arms
contains unique genes
gene-rich
replicated through S-phase
recombination during meiosis

49
Q

Heterochromain

A

highly condensed
at centromeres and telomeres
contains repetition sequences
gene-poor
replicated in late S-phase
no meiotic recombination

50
Q

What is the heterochromatin typical domain?

A

H3K9me3

51
Q

Name and describe the 3 family members of HP1

A

HP1a: most specific for heterochromatin and has a chromodomain that recognizes heterochromatin through H3K9me3

HP1b
HP1c

52
Q

What can be seen in the following figure?

A

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
Q

Which is more reliable: ATAC-seq or GET-seq?

A

GET-seq

54
Q

Describe this graph:

A

columns are chromosomes

blue depicts losses and red depicts gains

55
Q

What was discovered when analyzing the heterochromatin features on the patient-derived xenograft?

A

each clone was subdivided into epigenetic subclones, suggesting there is an underlying epigenetic variability that goes beyond genomics and genetics

56
Q

What does each triangle represent on the different genes?

A

mutations

red: missense mutation
blue: truncating mutation

57
Q

Of these 4 genes, which ones are the oncogenes and which ones are the tumor suppressors?

A

IDH1 is an oncogene that hass all of the mutations in one position, which makes it active

RB1 and VHL are tumor suppressors

58
Q

Analyze the GET-seq:

A

the area of iPS that is multicolored shows precursors that are common to iPS, fibroblasts, and NPC

59
Q

What was used when trying to apply the idea of RNA velocity to chromatin?

A

TnH versus Tn5 was used instead of spliced versus unspliced

60
Q

What was an important finding of the chromatin velocity analysis?

A

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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