Functional Genomics Final Exam

How siRNAs were linked with DNA methylation and H3K9me2

Looked for overlap in differentially regulated targets of pol-iv
Looked for siRNA abundance, found a vastly decreased number of 24nt reads in pol-iv mutants
Regions that have pol-iv siRNA production are also found to have methylation, and there is an increase in H3K9me2 we find them increased in pol-iv regulated regions

Mononucleosome

Consists of 8 histone protein subunits (2 of each): H2A, H2B, H3, and H4

How many base pairs around one nucleosome

149 bp

Linker

The region of DNA between the nucleosomes - where the DNA is "open for business"

ATAC-Seq assays purpose

Assays for Transposase Accessible Chromatin
Tells us where open regions of chromatin is - more reads corresponds to open regions

ATAC-Seq assays steps

1. Isolate nuclei with chromatin intact
2. Expose to Tn5 transposase (barcodes and primers loaded on transposase)
3. Isolate transposed fragments and amplify using primers
4. Sequence and identify accessible regions

Chromosome Conformation Capture (aka HiC) purpose

Used to determine physical interactions between distant genes (the actual sequences)
ie genetic interaction

Higher order DNA interactions

Open regions of chromatin on different chromosomes will sometimes interact (heat map data)

Chromosome territories

Chromosomes will generally occupy specific positions or territories within the nucleus

Cis regulatory sequences

Physically adjacent to the sequences that they regulate. They are in the DNA sequence.

Core promoter

Within 100bp of TSS. Associated proteins are RNA Pol-II, TFs, and TATA binding proteins

Proximal promoter

Between 100bp to 3kb of TSS. Associated proteins are TFs.

Enhancers

Can be upstream or downstream, and can be very distant from the gene that they regulate. Can look at where enhancers are via chromosome conformation capture/HiC

Trans regulatory sequences

Far acting factors, usually proteins. Whatever binds to the core promoter (RNA Pol-II, general TFs, TATA binding proteins)

ChIP-seq steps

1. Fragment the DNA
2. Use an antibody targeted to a specific protein so that we only precipitate DNA associated with that specific protein
3. Go through PCR with illumina adaptors and sequence
4. Align reads to genome :)

ChIP seq control

Fragment with no immunopurification, amplify as usual. Some sequences might occur more frequently or be easier to fragment, so it's important to be able to differentiate between where a protein is found vs high fragments

ChIP seq visual interpretation

Peaks in the graph are where you have the TF binding to the DNA

2 Methods to determine direct transcriptional regulatory interactions

1. Genetics: Look at gene expression in WT and mutant
2. Inducible transcription factor (Time course as TF is activated.
RNA Seq at 0min, 15min, 1hour, etc - ID target genes that change expression at these time points)
For both: Look at ChIP data, determine which regions have changes in transcription. TF binding event is associated with regulation

Where to TFs bind in relation to the TSS in yeast

Upstream

Single regulator

A single TF binding site in the promoter

Repetitive motifs

Two or more binding sites of the same TF in the region

Multiple regulators

Different TF binding sites within a single promoter

Co-Occurring regulators

Always see a pair of TFs in a promoter - ie two TFs will always work together or co-occur

Condition invariant

TF binds to same target regions regardless of the environment

Condition enabled

TF will have no binding sites under 1st condition, will bind to target under 2nd condition (a change in condition will enable binding)

Condition expanded

TF will have few binding sites under 1st condition, will bind to many more targets under 2nd condition (a change in condition will enable the TF to bind to more sites in addition to the sites it can already bind to)

Condition altered

TF will have binding sites under 1st condition, and will bind to a subset of those sites plus more different sites under the 2nd condition

High Occupancy Target region (HOT region)

A region of chromatin where almost every TF is known to bind to

Enhanceosome

Multiple TFs bound to contiguous cis regulatory sequences, protein protein interactions form a complex, and the whole complex regulates transcription

What method can identify an enhanceosome

Use immunoprecipitation/chip for a single protein you know interacts, run a western blot and see what you also pulled

Consensus sequence

The average binding site for a transcription factor/regulatory element, the sequence we would expect the TF to bind to

Position weight matrix (PWM)

Represent both frequency of a given nucleotide at a given position, and the weight of letters in a position can represent probability of a given nucleotide at a given site/position (counts vs bits)

SELEX Assay purpose

Used to identify and isolate nucleic acid sequences that have high affinity and specificity for a particular target sequence

SELEX Assay steps

1. Pool of synthetic oligos, longer than 32bp but at center of the sequence, the 32 bp is randomized
2. GST-tagged trxn factor
3. Incubate your TF (GST-tagged) with resin or brads with glutathione
4. Add DNA to mixture
5. Wash complex
6. Carry out PCR to enrich for oligos that bind to TF
7. Elute this DNA
8. Repeat process several times (steps 3-8 over and over which is why its called systematic evolution)
9. Sequence resulting DNA
10. Data analysis - identify the TF consensus sequence or position weight matrix(PWM)

Protein Binding Microarray assay purpose

To identify TF binding specificity for TFs from yeast, worm, mouse, and human

Protein Binding Microarray assay steps

1. Get substrate(glass or nylon) that DNA can adhere to
2. On substrate it has 44,000 spots which have sequence of DNA which are divided up amongst 1,408,576 10-mers which designed to represent MAX diversity of all possible 10-mers for G,C,T,A in the universe which is why its called universal array
3. Produce copy of 10mers with fluorophore labeled nucleotides
4. GST protein fusion to TF DNA binding domain
5. Apply GST protein fusion to protein binding microarray which contains probes with fluorophore
6. Image and look for lack of light (can only screen one protein at a time :/ )

E-score (she incorrectly referred to it as E-value)

Represents binding affinity of a TF for a target (high score means higher binding affinity ex. 0.5 and they usually have a color to distinguish)

How are protein binding microarrays used to determine the differences and similarities in transcription factor DNA binding domains of a given family

The columns are TF binding domains and clustered according to TF binding preference
Each row is a different 8mer and are hierarchically clustered according to similarity in sequence
Groups of TF that display similar binding preferences will have a position weight matrix calculated for that group
Those groups of TF that have similar binding preferences will have similarities to one another and through this we can see the differences between them (a heatmap)

How was this finding "transcription factors can bind to sites of multiple affinity" made?

By graphing IrF4 (8-mer E-score) vs IrF5 (8-mer E score) where each dot represents over 1 million seq that present on micro-array
If they had an exact affinity, we would expect a straight line. Instead we see difference where in some cases IrF4 is higher, some where IrF5 is higher, and some where they have equivalent affinity
This leads where within a family they can have high affinity shared between most TFs of that family but can also have a second site of high affinity not necessarily the same same site as another TF within the family

Steps of a Yeast One Hybrid Assay

1. Introduce bait into yeast
2. Test for autoactivation
3. Test for DNA-protin interactions
4. Record results

Y1H Assay Bait

DNA sequence of interest (promoter with HIS 3 or LAC Z)

Y1H Assay Prey

TF you're testing for DNA binding (TF bind to GAL4 AD)

Y1H Assay Reporter

Gene whose expression used as a readout for bait-prey interactions (ex: LAC Z and HIS 3)

Y1H Assay purpose

To test for protein interactions with DNA (Protein:DNA interactions)

False Negative

an interaction that does not take place in an assay but which does occur in an organism

What could cause a false negative?

many proteins bind in complex. These arrays only permit introduction of a single protein so if a TF requires a partner to bind (heterodimer) or a complex; then it will not bind

False Positive

an interaction that takes place in an assay but which does NOT take place in an organism

What can cause a false positive?

huge amount of proteins added to assays, TFs may act in a dose-dependent manner; only bind to a particular concentration

Causes of either a false positive or false negative

1. DNA can be synthetic or not in chromatized context
2. Protein - fusion protein or only part of a protein; protein folding may influence fxn

Binding of a transcription factor

Binding of a trxn factor is physical interaction between trxn factor and specific DNA sequence usually within promoter or enhancer regions of a gene which is essential for the trxn factor to exert regulatory effects but binding alone does not confirm that the gene's expressed is being regulated

Regulation of expression levels

refers to control of gene expression levels (activation or repression) mediated by TF. not only involves binding of the TF to DNA but also its interaction with other regulatory proteins and the transcriptional machinery affecting trxn.

What can be used to determine binding of TFs

Yeast one-hybrid identifies proteins that can bind to a specific DNA sequence in yeast but doesn't show the regulatory effect in its native context

What can be used to determine functional outcome of TF binding

reporter gene assays can be used to measure the functional outcome of TF binding on gene expression levels, indicating whether the TF can activate or repress trxn

Why are protein-protein interactions within a cell important

we can get a sense of how proteome is organized, its fxn, and regulation of cellular machinery

Bait construct Y2H Assay

protein of interest + GAL4 DBD

Prey Y2H Assay

protein + GAL4 AD

Yeast strain used in yeast assays (Y1H and Y2H)

Yeast is without HIS 3(HIS3 autotroph) has GAL4 binding sequence + HIS3 and GAL4 binding sequence + LACZ

Guilt by association (Y2H analysis)

Proteins that are found together

Advantages of Y2H assay

Sensitivity
Flexibility
Large # of variable inserts can be examined at once

Disadvantages of Y2H assay

Labor and material intensive
Need efficient and elaborate pooling and deconvolution schemes
False positives and negatives

Y2H False Negatvie

Real Interactions are missed

Y2H False positive

Non-relevant interaction/overexpression and Autoactivation

Steps in TAP Tagging

1. Bait Protein - Protein, CaBP, TEV protease cleavage site, Protein A
2. Introduce construct into cells (transformation)
3. Bait protein forms a complex with other cellular proteins
4. Cell extract, pass it over a column of IgG beads
5. Cleave with TEV protease
6. Trypsin digestion of the protein complex
7. Mass spectrometry analysis

Purpose of TAP Tagging

To determine proteins within a protein complex

Compare Y2H Assay to TAP Tagging

Uses a bait protein to determine protein protein interaction, but TAP can ID proteins in complex

Examples of criticla signaling proteins which are membrane bound

Chemoattractant receptors(G protein-coupled receptors), receptor tyrosine kinases, plant receptor like kinases, TLRs

Steps of a split ubiquitin assay

1. Make a bait with POI+C-terminus ubiquitin + TF(DBD+AD)
2. Make prey with PY+N-terminus ubiquitin
3. Make reporter gene with LexA-HIS3 and LexA-LACZ in yeast lacking HIS3(autotroph)
4. Add bait alone to test for autoautoativation
5. Add no bait or prey for negative control
6. Introduce prey and bait to yeast (if interactions between prey and bait the ubiquitin will be reconstructed releasing TF which translocates into nucleus and yay growth

guess what

yeast sex

Why is a ubiquitin split assay needed

to test protein-protein interactions with proteins that localize outside of the nucleus (membranous proteins, vesicle proteins, organelle specific proteins, etc)

Ubiquitin false negative

no interactions occur in the assay, but interactions do occur in vivo

Ubiquitin false positive

you get an interaction in your assay that does not occur in vivo

Ubiquitin false negative caused by

Bc interaction conditions aren't favorable (Ph, cofactors, reductants, oxidants, etc)
Fusion proteins may also remove the tertiary structure needed for interaction

Ubiquitin false positive caused by

Conditions may not be typical for bait and prey
These two proteins may never actually be in the same cell at the same time

How to mutate different components of the genome

Knock them out or use a cis-regulator to destroy the TF binding site in a promoter or enhancer (necessity) by introducing a copy of this CRE in a new place(sufficient) and repeat elements by recreating chromatin architecture

Ways to knock out a gene

crispr cas 9, transposons [tap tag], chemical/physical mutagenesis, homologous recombination with a template, classical breeding of a homozygous mutant, use a temp sensitive mutant, mutations in binding protein by abolishing binding and increase affinity of binding, increasing/decreasing transcription rate to determine interaction w/RNA polymerase II, abolish interaction/make interaction take place all the time to determine

What is important about whole genome mutant collection

Gives information for better understanding of genes and their function, if you have a mutant library can sequence/assay genetic elements and see which differ between your chosen mutant and wild type

Targeted genome collection

know where every gene is and the means to target annotation to that gene(CRISPR-Cas9)

Unbiased genome collection

randomly only mutate the genome and by tracking phenotypes you can determine when you have reached "saturation" and multiple alleles of each gene and map based cloning to identify the location of your mutation

Yeast Mutant Collections

Collection of yeast with a bunch of genes knocked out, identified essential genes because when those genes (each specific mutant was barcoded, sig-tag mutagenesis) were knocked out the yeast died

Signature tagged mutagenesis

1. Knockout the gene (in yeast) using homologous recombination with a barcode and the necessary adaptors for illumina sequencing
2. Each deletion mutant contains and unique barcode
3. Grow all mutants in a mixed population and quantify growth
4. Amplify barcode sequence everything to find mutations that cause slow growth (Bc these are genes that are highly impacting organismal function)

Guilt by association coupled with phenotype clustering to further enrich determination of gene function

We see genetic elements in the same cell at the same time/ we see them together so we think they might be interacting

how were small RNAs were used to knock down the majority of the genes in the genome and examine resulting phenotypes

You introduce dsRNA into c elegans via injection, soaking, feeding, or transgenesis, those dsRNAs are then processed into siRNAs and silence the genes they target, you can then imply whatever phenotypic change in the worm is due to that gene, and gain better insight into that gene's function

Difference between haploid and diploid organisms in large-scale mutagenesis

Having to worry about redundancy
If you knockout a gene in a haploid if the gene causes a phenotypic change it will show/ be present because there is not another copy of the gene to rescue the mutant (easier as you don't have to do as much crossing)
However, if you knockout a gene in a diploid, you still have a second copy (maybe more with WGDs) that can replace the function of the knockout gene (redundancy) so you may not see a phenotypic change despite having a successful knockout (need to do more crossing/breeding to get an effective knockout)

Transposon mediated insertional mutagenesis

Unbiased mutant screen
Use engineered transposons with a left and right border
Within gametes, artificially induce activation of transposase in the presence of the transposon (Causes a jump, or random insertion into genome)

Gene redundancy

If gene had a local duplication (one after another) and had a homozygous recessive mutation in first gene, if the second gene was depressed in the same time and place then the second gene can take on the function of the first

Synthetic lethal mutations

Mutating both genes performing a redundant action will cause a synthetic lethal mutation

PAM

protospacer adjacent motif, must flank the gene for cas9 to alter it

Cas9

endonuclease with 2 domains that makes a double stranded cut (RuvC like and HNH)

sgRNA

small guide RNA, hybridizes/pairs with DNA target sequence/ guides Cas9 to our gene of interest

NHEJ use

create knockouts and deletions within a gene

More tareted mutations can be done by

Introducing a ssDNA repair template that the cell can use to repair the double stranded break through homologous recombination

Dead Cas9

cas9 but it has the endonuclease domains removed
(Typically fused to another enzyme that can alter the genetic material (methyltransferase, etc))