Lecture 9 - ENCODE Flashcards by 1a agostin

What is this - Highly organized, consists of subdomains involved in specific functions (Nucleoli, Chromosomal domains and Interchromosomal channels).

a Nucleus

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_________ are positioned
in the nucleus.

chromosomes

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T or F - DNA is randomly thrown in the nucleus

F - certain regions are positioned next to each other as a means of the chromosomes regulating the activity of those regions relative to each other

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ENCODE

encyclopedia of DNA elements

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To identify all regions of transcription, ______ factor association, ________ structure, and _______ modification in the human genome sequence

transcription, chromatin, histone

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What are the genetic mechanisms of transcriptional regulation?

Cis regulatory elements
transcription factor avaliability

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Define epigenetic mechanisms

mechanisms that alter the conformation fo teh DNA and/or the ability of transcription factors to bind

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What are the 3 epigenetic mechanisms of transcriptional regulation?

methylation of DNA
Chromatin Accessibility
Histone modification

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in humans, ______ represent the largest family of proteins, accounting for around ______% of genes

transcription factors, 8-10%

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what does TF stand for

transcription factors

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What are the two types of TFs

general
sequence-specific

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Define general TFs - give an example

TF that cooperate with RNA pol II and are ubiquitously involved in the transcription of a large fraction of genes - TBP

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define sequence-specific TF - give an example

TF that bind specific subsets of target genes, leading to distinct spatiotemporal patterns of gene expression - Myc

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Seq-specific TFs lead to distinct spatiotemporal patterns of gene expression. What does this mean

This refers to how genes are activated/suppressed in specific locations (spatial) at particular times (temporal) within an organism

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Although _________________ has been available for a decade from microarray experiments, only recently has the genome-wide identification of __________ become possible, owing to the development of ___________ followed by microarray (ChIP-chip) and sequencing (ChIP-seq) technologies.

systematic gene expression quantification, TF-binding sites, chromatin immunoprecipitation

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T or F - sequence-specific TF are specific to the expression of given genes. This means that it dictates whether the gene is turned off/on and by how much

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T or F - General TF are specific to the expression of given genes. This means that it dictates whether the gene is turned off/on and by how much

F - sequence-specific TF do this not general TFs

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a) _______ bind DNA in a sequence specific and non-sequence specific manner
b) Myc/Max are ________ TFs (general/sequence-specific) but can also bind DNA generically

a) TFs
b) sequence-specific

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GOI?

gene of interest

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Match the following terms: transcritpiaon activation, Max, Non-productive for transcripion, Myc
a) blue
b) pink
c) yellow
d) green

a) myc
b) max
c) non-productive for transcription
d) transcriptional activation

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“DNA altered under different conditions” (open/closed) <– What type of modification is this referred to?

histone modification

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ChIP_Seq?

Chromatin ImmunoPrecipitaiton_Sequence

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What is the purpose of ChIP-Seq?

to help identify promoter regions of a gene by analyzing protein-DNA interactions

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Steps of ChIP_seq

1. crosslink/fix protein of interest (already bound to DNA 2. Fragment DNA 3. add protein specific antibody 4. purify sample to only contain DNA bound to protein of interest 5. reverse cross link and remove protein (and antibody) 6. amplify and sequence DNA fragment

During the Chip-seq process, what is formaldehyde used for?

for fixing the protein of intest to the DNA (cross linking)

in chip-seq, how does purification work

a magnet can draw in any of the proteins of interest that are attached to an antibody because the antibody contains a bead that is magnetic

T or F - in Chip-seq, we are analyzing a protein of interst

F - sequencing the DNA that that protein is bound to

T or F - in Chip-seq, we are analyzing the DNA that a specific protein of interest is bound to

T or F - chip-seq can be used to identify histone modification as well

As we know, chip-seq can be used to identify the DNA bound to a certain protein of interest, but we can also seq DNA that is bound to certain modified histones. How can this be?

Just treat the modified histone as a protein of interest, making the antibody specific to the modification of that histone (the methylated part,) and continue from there

T or F - chip seq is used to sequence what gene is bound to the protein of interest

F - It's used to identify the promoter region of that gene, not the gene itself

Match the following with the colour a) DNase-seq b) histone modification Chip-seq c) DNA-binding protein chip-seq d) FAIRE-seq

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

Define the following a) DNA-binding portein chip-seq b) histone modification chip-seq c) DNase-seq d) FAIRE-seq

a) DNA sequence that binds to a specific TF b) DNA sequence associated with specific histone modification c) DNA that is bound to all protein in general d) DNA that is not bound to a protein

DNase-seq is a chip-seq tech used to find all the DNA that is bound to protein (in general). How is this possible?

they use a DNase enzyme to digest all free DNA to isolate any DNA that is not free (bound to a protein) and then they just remove the proteins from the sample to seq the DNA

As we know FAIRE-seq is a chip-seq tech where you are trying to analyze all the DNA not bound to a protein. How is this possible

theyuse phenol-chloroform extraction of DNA to segragate the DNA bound to proteins (sinks) and the lone standing DNA (floats) then they remove the protein bound DNA so the lone DNA can be seqenced

In chip-seq, what is phenol-chloroform used for

Extraction of DNA by segregating protein bound DNA and stand-alone DNA

a) What are the 4 chip-seq tests b) which ones are considered complementary tests

a) DNA-binding protein chip-seq, histone modification chip-seq, DNase-seq, and FAIRE-seq b) DNase-seq and FAIRE-seq

Match the following to the image a) Prepare sequence library b) immunoprecipitated c) DNA + bound protein d) fragment DNA e) Sequence f) Release DNA g) Map sequence stages to genome and identify peaks

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

Chip-seq overview: Put the following steps in order a) Prepare sequence library b) immunoprecipitated c) DNA + bound protein d) fragment DNA e) Sequence f) Release DNA g) Map sequence stages to genome and identify peaks

c->d->b->f->a->e->g

chip-seq a) What does each red dot represent here? b) What does the black line represent? c) What does the peak mean?

a) DNA fragments b) DNA c) shows the intensity, which shows where on the DNA that protein of interest is bound do

what does POI stand for

protein of interest

What is the difference between the following a) chip-chip b) chip-seq

a) ChIP-Chip relies on hybridizing DNA to a microarray, meaning it can only detect sequences that are on the chip. b) ChIP-Seq uses next-gen sequencing, offering higher resolution, genome-wide coverage, and better sensitivity.

T or F - Chip-Chip is better than chip-seq

F - chip-seq has a higher resolution and better genome wide coverage

T or F - Chip-seqis better than chip-chip

T or F - chip-chip and chip-seq tend to give similar results

T - chip-chip just tends to show more noise

Define the following parts of H3K27me3 a) H3 b)K27 c) me3

a) histone 3 b) lysine 27 (27th lysine on histone tail) c) trimethylation (3 methyl groups added to histone tail)

Related to the computational analysis of chip-seq, what does motif analysis do?

They help to identify putative transcription factors

a) What are putative TFs b) What part of Chip-seq's computational analysis involves a)?

a) TF that are suspected to function as a TF but have not been fully confirmed yet b) motif analysis

Looking at this chromatin state annotations which histones would you expect to be involved in the genes activation vs repression?

activation: H3K4me3 repression: H3K4me1 or H3K9me3

T or F - long non-codoing RNA are important for the regulation of genes

How are biotinylated antisense oligos used in chip-seq?

they are the adaptors that attached themselves to a long non-coding target RNA bound to the DNA to allow for purification to occur

What do the folloiwng stand for a) Chip-seq b) chip-exo c) chirp

a) chromatin immunoprecipitation-seq b) chromatin immunoprecipitaiton-exonuclease c) chromatin isolation by RNA purification

define and differentiate the following a) Chip-seq b) chip-exo c) chirp

a) ChIP-Seq: Maps where proteins (TFs, histones) bind to DNA. b) ChIP-Exo: A higher-resolution version of ChIP-Seq that trims excess DNA to define precise binding sites. c) ChIRP: Captures lncRNA (long non-coding RNA) interactions with chromatin rather than protein-DNA interactions.

lncRNA?

long non-coding RNA

ChIP-Seq: _________ is fixed in cells, then _________ by sonication and _______ digestion treated (DNA bound to protein is resistant to DNase digestion). Enrich for the protein epitope of interest using a specific ______. Crosslinks are reversed using ________ and heat. DNA is then prepared for analysis by sequencing, array hybridization, or PCR a) Fill in the blanks b) define protein epitope

a) Chromatin, fragmented, DNase, antibody, proteinase K b) a specific region on a protein that is recognized and bound by an antibody

ChIRP-Seq: Biotinylated _______ oligos are designed to the _______ of interest. These oligos are then hybridized to chromatin that has been _________-crosslinked in intact cells and then fragmented by _________. Oligo-bound RNA and its associated DNA are isolated with _________ beads, and DNA is then purified and sequenced.

antisense, ncRNA (non-coding RNA), gluteraldehyde, sonication, streptavidin

When doing chip-seq, the antibodies need to be ________

specific

there is over ________ human TFs

1000

Match the following a) Basic domains b) Helix-turn-helix domains c) immunoglubulin fold d) beta-sheet binding to DNA e) zinc-coordinating DNA-binding domains

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

There is a lot of ______ in the TFs and how they bind

diversity

The _______ is a general binding domain for the TBP

TATA box

Match the following a) Heterosynergy b) Homosynergy c) Homotypic cooperativity d) Heterotypic cooperativity

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

Define a) Heterosynergy b) homosynergy

a) the cooperative interactions btw different types of transcription factors b) cooperative interactions btw identical TFs

Define a) homotypic cooperativity b) heterotypic cooperativity

a) the cooperative binding of identical TF to their DNA-binding sites enhancing their affinity b) cooperative binding of different transcription factors to regulatory sequences leading to enhanced/repressed transcription

Distinguish homosynergy vs homotypic cooperativity

Synergy refers to the interaction btw identical proteins. While typic refers to the binding of those identical proteins to the DNA (the act of those proteins being bound increases the affinity for the complex to bind to the DNA)

Which of the following dictates the transcriptional response the most a) number of TFs b) type of TF c) number of binding sites d) type of binding site

T or F - synergy involves protein-protein interactions

T or F - Synergy involves protein-DNA interactions

F - its protein-proteins interactions instead

Transcription factors can interact as homo or hetero _______ or in tandem a) monomers b) dimers c) trimers d) tetradimer

Which of the following TFs has a high affinity heterotypic interaction based on the image? Select all that apply a) AP-1 + AP-2 b) USF + NF-Y c) ZNF143+CTCF d) GABP + ELF1 e) PU.1 + REST

b to d have a high affinity

Which of the following TF have a homotypic interaction? Select all that apply a) GABP b) CTCF c) MYC d) ELK4

a and b

MYC and MAX for the most part do not contain _________dimers (homo/hetero)

homo

a) NF-Y – NF-Y: _______dimers (homo/hetero) bind on the ______ (same/opposite/both) strand and the distance between motifs is about _______ nucleotides b) NF-Y -- UA2: _______dimers (homo/hetero) bind on the _______ (same/opposite/both) strand and there are about _____ nucleotides between motifs. c) NF-Y – YY1: _______dimers (homo/hetero) bind ________ (same/opposite/both) strands equally, and does not seem to be sequence specific (they are all over the place).

a) homo, same, 15-20 b) hetero, same, 5 c) hetero, both

This is an example of a _____ map

heat

NF-Y – NF-Y: homodimers bind on the same strand, and the distance between motifs is about 15-20 nucleotides. What do the 15-20 nucleotides refer to here

It is stating that these homodimer binding sites on the DNA strand are 15-20 nucleotides apart

a) which ones are general TFs b) which ones are sequence specific TFs c) Which ones are we interested in?

a) the ones NOT in the red box b) the ones in the red box c) the sequence specific because they are important for driving GOI

TFSS?

sequence specific txn factor

TFNS?

non specfiic txn factor

ChromStr?

chromatin strucure factors

ChromRem?

chromatin remodelling factors

HISase

histone methyltransferases

Pol3F

polymerase 3 assocated factors

a) What are genecards b) Give 4 examples of gene cards

a) bioinformatic database that provides detailed info on human genes b) CHD2, SMC3, SETDB1, SMARCA4

Match the following genecards (CHD2, SMC3, SETDB1, SMARCA4) to their definition a) structural maintenance of chromosome 3, is a component of the multimeric cohesin complex that holds together sister chromatids during mitosis, enabling proper chromosome segregation. b) alter gene expression possibly by modification of chromatin structure, thus altering access of the transcriptional apparatus to its chromosomal DNA template. c) a histone methyltransferase that regulates histone methylation, gene silencing, and transcriptional repression. d) have helicase and ATPase activities and are thought to regulate the transcription of certain genes by altering the chromatin structure around those genes.

a) SMC3 b) CHD2 c) SETDB1 d) SMARCA4

_______ is another type of genecard site that only contains TFs

Factorbook

Match the following a) MCF7 and NHLF b) LHCN, HSMM or H1ES c) TBX2

a) green b) pink c) blue

What does the green line indicate

binding site