Tonon: Lecture VI Flashcards
Enhancers, Super-Enhancers, and New Technologies
What are enhancers?
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
What kind of specificity do enhancer loops exhibit?
tissue-specificity
How do enhancers bind to TF?
due to specific motifs located on TF and enhancers
Enhancers hold many nucleotide variations associated with ______________.
genetic diseases
Enhancers do not have histones; however there are some regions that have histones: what happens if histones are present?
enhancers are shut down, like a promoter
active promoter histone marks
H3K4me3
H3K27ac
inactive promoter histone marks
K3K27me3
active enhancer histone marks
H3K4me1
H3K27ac
inactive enhancer histone marks
H3K27me3
What is important to note about me3 (3 methylations)?
it is always negative as it represses transcription
What is the histone modification for compact heterochromatin?
H3K9me3
What is the histone mark for open or closed enhancers?
H3K4me1 (active enhancer)
H3K27me3 (repressed enhancer)
Are RNAs expressed on enhancers?
yes, in both directions
What is the name of RNA transcribed from enhancers?
eRNA
What is the probable job of eRNAs?
increase transcription of corresponding genes
from the slides: promote mRNA synthesis
What are super-enhancers?
much bigger enhancers described as “key cell identity genes”
How did they find super-enhancers?
by evaluating the Med1 (protein that binds to enhancers) density
Med1 signal was very strong corresponding to super-enhancers
What is the size of an enhancer site and a super-enhancer site?
700bp and 9000bp, respectively
What is the mechanism of action of super-enhancers?
super-enhancers → many proteins bind →transcription of a downstream gene is upregulated → increased gene expression
What are some genes under the control of super-enhancers?
Oct4
Sox2
Nanog
Myc
chromatin-modifying genes
What is Myc (gene controlled by super-enhancers) involved in?
tumors like multiple myeloma
What is a protein involved in the machinery that forms super-enhancers?
Brd4
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?
it was found that dromodomain could disrupt the looping between Brd4 and Myc
What is the Waddington Model?
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
What is the single-cell approach?
a method that can be used to detect and analyze specific cells
What does every dot on a single-cell transcriptomic graph represent?
a cell
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
What has replaced single cell-analysis?
UMAP because it is stronger for a biological point of view
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
What is RNA velocity?
density of a single cell based on the ratio of spliced and unspliced RNA
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
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
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
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
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
Why are t-SNE and UMAP helpful?
each cell can be described with its own destiny based on the ration between spliced and unspliced
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)
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
ATAC-seq
new technique that uses Tn5 transposome
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
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
Analyze the graph and describe which techniques would use these graphs:
each peak represent short sequences
used to read ATAC-seq and GET-seq
In this figure of ATAC-seq, what do the peaks represent?
regions that are open
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
Describe the ATAC-seq path
put transposes at the single cell level → PCR amplification → profiles are obtained
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
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
Euchromatin
less condensed
at chromosome arms
contains unique genes
gene-rich
replicated through S-phase
recombination during meiosis
Heterochromain
highly condensed
at centromeres and telomeres
contains repetition sequences
gene-poor
replicated in late S-phase
no meiotic recombination
What is the heterochromatin typical domain?
H3K9me3
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
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
Which is more reliable: ATAC-seq or GET-seq?
GET-seq
Describe this graph:
columns are chromosomes
blue depicts losses and red depicts gains
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
What does each triangle represent on the different genes?
mutations
red: missense mutation
blue: truncating mutation
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
Analyze the GET-seq:
the area of iPS that is multicolored shows precursors that are common to iPS, fibroblasts, and NPC
What was used when trying to apply the idea of RNA velocity to chromatin?
TnH versus Tn5 was used instead of spliced versus unspliced
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
