Lesson 6 Flashcards
where are enhancers most often located?
upstream to the promotor, but they can be also found downstream or in the first intron as well
where are enhancers located in regards to the transcription start sites (TSS)?
distally
what are enhancers bound to an activator by?
a transcription factor and an RNA polymerase
what do enhancers cause?
the formation of a loop between the enhancer sequence and the promotor to initiate trancription
what are some examples of components bound by enhancers?
acetyl-transferases (p300 and CBP)
which two important histone modifications are harbored by enhancers?
H3K4me1 and H3K27ac
describe the amount of genes regulated by enhancers:
regulate multiple genes at once, usually regulate in cis (on the same sequence of DNA) but also in trans (rarely - different chromosomes)
where are histones located on enhancers when activated?
only at the boundaries of the enhancers
what is it called when an enhancer loops in the 3’ direction in respect to a certain gene?
sense
what is it called when an enhancer loops in the 5’ direction in respect to a certain gene?
antisense
describe the typical spatial orientation of enhancers:
typically relatively close, but they could also be intronic or very far away (on rare occasion even on different chromosomes)
name two histone markers that activate promotors:
H3K4me3 and H3K27ac
name a histone marker that inactivates promotors:
H3K27me3
name two histone markers that activate enhancers:
H3K4me1 and H3K27ac
Name one histone marker that inactivates enhancers:
H3K27me3
what is the significance of me3 in a histone?
always a negative signal which stands for the repression of transcription
what is different about a poised or closed enhancer?
H3K4me1 is an activated enhancer and H3K27me3 is a repressed enhancer
what has been discovered about RNAs and enhancers?
there are RNAs expressed on enhancers in both directions → eRNA
what does the level of eRNA expression at neuronal enhancers positively correlate with? And what does this suggest?
correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis
what are super enhancers described as?
key cell identity genes
what protein is specifically bound to enhancers and aided in the discovery of super enhancers?
Med1
describe the composition of super enhancers and their effect on the downstream gene:
many proteins bind to the site and upregulate the transcription of a downstream gene → you have a much longer sequence with more proteins bound
what does the binding of a super enhancer cause to the gene?
a high increase in gene expression
what are enhancers almost comparable to in terms of effect on expression?
housekeeping genes
where do most super enhancers reside in the genome?
in regions of the genome that we used to call “junk”, and instead are extremely important for some of the most common genetic diseases
what diseases do super enhancers play an important role in?
cancers (Myc)
what is the function of the bromodomain of Brd4?
it can block the activity of Brd4 and disrupt the looping which is formed by the connection between Brd4 and Myc
what is the hypothesis for treating the bromodomain of Brd4?
the looping could be completely disrupted so that the super enhancers would no longer be able to enhance gene expression → shutting down Myc expression and increase survival rate
what does the Waddington model state?
the cell can take different trajectories leading to different outcomes or cell fates, to a much more complicated one in which there are more components that work together to provide an epigenetic landscape of cells and tissues
describe the single cell approach:
cell gets sliced → RNA binds to different RNA linked to each bead → creates a series of many beads each of which defining a single cell (each bead has a unique barcode corresponding to a cell)
what do they use in immunology to find new subpopulations?
a combination of different antibodies
nowadays, what do we uses instead of single cell analysis?
UMAP
why is UMAP better than single cell analysis?
more consistent across experiment and is stronger from a biological point of view
what three assumptions can be made due to the fact that when sequencing is performed, spliced sequences can be seen inside the gene as well?
- processing rates B (in terms of splicing) are the same for all genes (and therefore can be set to 1)
- mRNA degradation rates are gene-specific constants
- during development, differentiation occurs on a timescale of hours to days, which is comparable to the typical half-time of mRNA
what does RNA velocity show?
the destiny of a single cell based on how the ratio between spliced and unspliced RNA is
describe the process of splicing in terms of its components:
at the beginning, there is more unspliced than spliced because when arriving at a steady state, an equilibrium of spliced and unspliced is reached - if you stop transcription, there will be less unspliced than spliced
in general, which lasts longer, spliced or unspliced?
spliced
during a dynamic process, what does an increase in transcription rate result in?
results in a rapid increase in unspliced mRNA, followed by a subsequent increase in spliced mRNA until a new steady state is reached
what does a drop in the rate of transcription lead to in terms of splicing?
leads to a rapid drop in unspliced mRNA, followed by a reduction in spliced mRNAs
during the induction of gene expression, how are unspliced mRNA present?
present in excess of the expectation based on the equilibrium rate γ, whereas the opposite is true during the repression
what is the balanced between spliced and unspliced mRNA an indicator of?
an indicator of the future state of mature mRNA abundance, and thus the future state of the cell
what can you estimate based on the ratio between spliced and unspliced RNA?
where a cell is going be in terms of rhythm or maturation
what is the limitation of using RNA velocity when determining cell progression?
RNA velocity can be used only for phenomena that can be measured in the space of a day or few days
what was discovered about the states of tumor cells in regards to transcriptomes?
cells do not stay in one single status, but they fluctuate among different states
describe the cell states of tumors:
contain multiple cell states with distinct transcriptional programs and provides inferential evidence for dynamic transitions.
describe the ATAC-Seq technique:
based on the use of a Tn5 transposeome → a bacterial protein that cuts the DNA were it is open and inserts two different oligos → identifies the open regions of the genome
why do we want to look at open chromatin?
shows which parts of the genome are expressed
in terms of clustering cells, which common program is ATAC-Seq better than, and why?
RNA-seq → much more clear and more capable of distinguishing different subtypes
what path does single cell ATAC-Seq follow?
put transposases, in this case at the single cell level, and you do amplification by PCR, then you get the profiles
what percentage of the genome is occupied by closed chromatin?
over 50%
list some characteristics of euchromatin:
- Less condensed
- At chromosome arms
- Contains unique sequences
- Gene-rich
- Replicated throughout S phase
- Recombination during meiosis
list some characteristics of heterochromatin:
- Highly condensed
- At centromers and telomers
- Contains repetitions sequences
- Gene-poor
- Replicated in late S phase
- No meiotic recombination
which protein recognizes H3K9me3?
HP1
which HP1 protein is the most specific for heterochromatin and has a chromodomain that recognizes heterochromatin through H3K9me3?
HP1a
what can TnH recognize?
H3K4me3
what is even more reliable than ATAC-Seq?
GET-Seq
what is a patient-derived xenograft? (PDX)
a model in which a patients tumor is transplanted into a mouse, where it can keep growing and be studied
when studying PDX models, what was discovered?
when the mice reproduced, each of these clones was subdivided into epigenetic subclones, suggesting that there is an underlying epigenetic variability that goes beyond genomics and genetics.
what was discovered when scientists tried to apply the idea of RNA velocity to chromatin?
they discovered lots of genes associated with axon guidance and genesis and neuron projection guidance, suggesting that the genes residing in the population are crucial for the evolution of NPC and in general for neuronal products
how is GET-Seq different that ATAC-Seq?
they built a chimera that doesn’t only have Tn5 but also TnH because the tail can recognize heterochromatin through HP1 → can recognize both open and closed chromatin
they took fibroblasts of two donors and reprogrammed them to induce pluripotent stem cells to differentiate towards neuronal progenitor, then looked at the cells with both GET-Seq and ATAC-Seq - what was the difference?
both techniques divided the cells into three groups very clearly, but with GET-Seq they got a small area of mixed cells which gave more sensitive information (suggested that we have common precursors in IPS that are common to IPS, fibroblasts, and NPCs)
