Lecture #3 (Transcription Factors and Chromatin)

Question 1

Chromatin (Overall)

Answer 1

Chromatin = Things co-associated with DNA
- When you spin down DNA here are proteins and RNA that come with it (weight of proteins = weight of DNA)

Chromatin = DNA + RNA + Histoen proteins + Non-histone proteins
- Chromatin = Nucleosomes + Histone proteins

Question 2

Two types of proteins that come with DNAT6

Answer 2

1. Histones - Form the fundamental units of chromatin organization
   - Basic positivley charged proteins
   - There are 5 histone proteins
2. Non-histone proteins
   - Acidic proteins
   - There are thousands on non-histone proteins (DNA/RNA polymerase, DNA binding proteins etc.)

Have as much histone and non-histone proteins by weight

Question 3

Epigenome

Answer 3

Really just another term for chromatin

Two components of epigenome:
1. Transcription factors/Regulatory DNA elements
2. Chromatin

Question 4

Genome + Epigenome

Answer 4

Genome + epigenome = work together to make organisms

Genome + Epigenome = olecular blue print for everything (Ex. Growth + adaptation + differentiation)
- Also affects hormonal signals between organs

There is NOTHING in biology that is not affected by gene expression via epigenome

Question 5

Mutations in epigenome

Answer 5

Mutations = affect DNA and RNA in epigenome –> Leads to diseases

Have many pathologies related to dyfunction of the epigenome

Question 6

Nucleosome

Answer 6

Nucleosome = fundemental unit of chromatin

Question 7

Conservation of Histone proteins

Answer 7

Histone proteins = most conserved proteins on planet
- H4 is the most conserved of the histones

Histones = small –> 100-120 Amino Acids –> H4 in mammal vs. plants has 1 Amino aciid difference (VERY conserved)
- Other histoes have more divergene but stil consevred

Conservation = allows you to study histones in model organsims –> the principles will apply to other organsims inclduing humans evcause have similar proteins

Question 8

Underlying prinsiples between organisms

Answer 8

Underlying principles of cell and tissue differentiation in Eukaryotes

Example - principals of turning on genes in humans is model organisms is not veyr different bery turning on genes in humans
- Can pick which organism is easiest to study in and traslate to humans

Question 9

Human Genotype Expression prohect (GTEx)

Answer 9

Mapped expression of gene gene in every cell in human body

Take homes:
1. Human genome has 20,000 protein coding genes
2. Human genome has 10,000-13,000 non-coding RNAs
3. In any cell type only HALF the genome is expressed (a little abive half) –> ONLY 10,000-13,000 of protein coding genes is expressed ; 7,000-10,000 of the protein coding genes are silenced
- Need to make sur ethe right genes are silences or expression( (Different in different cell types)
4. 8,000 of the expressed protein coding genes are core acitivies (ubiquitus) - what every cell needs to survive and replicate
5. 2,000-5,000 of teh PCGs are preferntailly expressed in certain cell types (expressed ebeyrwhere but enhanced in some cells and not others)
6. 200 of the PCGs are tissue specific
- Want to look at the 200 master proteins to look at cell type and cell fate

Question 10

Ubiqitous genes in genome

Answer 10

House keeping genes = ubiqitous genes = ALWAYS ON

How do they get truned on?
- Example 0 Insulin gene –> HAVE Transcription factors that are specific to be expressed in that cell type

Question 11

What regulates Eukaryotic Transcription Factors

Answer 11

Eukaryotic trasnscription is regulated by proximal and distal promoters enhancer DNA elements
- Instruction for intiating transcription are near transcripton strat site AND can be far away

Enhancers = located away from the promoters (1kb-1MB)
- Enhancers are genetically validating as transcriptional control elements –> muttaions od enhancers affects gene expression
- Enhancers = can be upstream or downstream of the promoter –> feed and give infomration to trasncrtion start site where RNA polymerase binds
- Enhancers = orientation-independent (doesn’t matter if at 5’ or 3’ end)
- Ehnacers = regulate a target that is far away
- Enhancers = enhance trasmcription
- Enhancers = just as important as promoters
- Can be affected by envirnmnetal signal (give complexity to gene regulation)

Question 12

Regulatory DNA Elements

Answer 12

Regulatpry DNA elements = short DNA sequences (10-20 BP) –> Elements will be recognized by transcrition factors
- Transcription factor protens recognize 10-20 BP
- More BP in sequence = more specific because fewer probaility those sequence will exist genome wide) –> TF have evoloved to recognize specfic sequences by making them longer (Ex. recognition sites are longer than RE)

MOst Transcription factors recognzie 100-1,000 sites but have some that only recognize 1 site

Question 13

Heat shock protein Transcription factors

Answer 13

Heat shock proteins = encodes protein chaparones to help proteome not denature at a high temperture –> have a set of proteins and each portein coding –> each protein has an upsteram regualtpry elements (15 nucleotde lement) that is unique that binds to the transcriotion fcator and swicthes on the sets of genes because they have the same sequence on the upstrea regulatory

WATCH VIDEO

Question 14

Promoters

Answer 14

Promoters = where transcription begins

Promoters and enhancers have TF binding site

Question 15

Discovery of promoters/ehnacers

Answer 15

Discover promoters and enhancers done by knocking out promoters/enhacers

Can do systemic delation –> detect enhnacer –> see if teh gene stops working or decrease in expression
- Find enhmacers/promoters by knockout

Question 16

Enhancer DNA elememts

Answer 16

Enahcer = DNA elements that are similar to elemenst at promoter –> bind to TF that may or may not be shared with TF that bind to the promoter (may or may not be same TF)

IN IMAGE - Ehnacer fartheer awya have different TF

Seqwunece specific TF are different from geenratal trascription fcators
- Gentral transction factors = found at the promoter

DNA and protein DNA intercatioons that need to talk to get RNA polymerase to promoter and commucate that it is time to move

Question 17

Model for enhancer-promoter interaction

Answer 17

Model for how enhancers can work depsite being far away from the promoter = enhancer-promoter intercation by looping faciliated by cohesin ring (physical proximity)

Before - thought the single oozes down the chromatin fibers

NOW - think DNA can bend allowing the enhancer to contact the promoter lock together with cohesin ring
- IF mutate cohesin protein –> destroys the ring strcuture = impairs enhnacer-promoter communication (Doesn’t REALLY prove that the model is right)
- Promoter recruits RNA pilymerase –> Polymerase can lod onto the promoter

STILL DEBATE - people think it could be indirect protein cluster (condestae) betwen the enhancer and promoters instead of direct contact

Question 18

Evidence that things are DNA elements are close to each other

Answer 18

Evidenece that DNA sequences that are far away from each other linearly are close spatially - Uses chromain cpature

Chromatin cpature - take cells –> cross link DNA using formadheye (cross links teh lysine residues) –> digest uncessary things away; cross linkages keeps the two fragments connected –> ligate DNA (if two things are close then they will ligate) –> Sequence –> Can see DNA is ligated to something that should be far away to know thas omething brought them together to be able to ligate

Question 19

Issue with Chromatin Conformation Capture

Answer 19

Issue = measuring ligation NOT contact
- Looking at genome that shows TADs (TADs = regions that are ligatable)

In Chart - Peak of the trainge shows ligation –> Means that the places at teh two bottom corners of he trainge (regions far away on chrosmome) are interacting at the top point of traingle
- Interpre this a the DNA sequences are close BUT really they are just ligatable

THe sequences COULD be far apart and still be ligatable –> because they could be brought together by a cytoskeata protein (If thinsg are far away and moving randomly then you get a certain low ligation frequencey BUT of there is a cytosklatal cable between the two then they will be connected and can have a higehr ligation frequnecey
- Connected far apart but still have impored ligation (Flow in HiC)

Question 20

2nd way to see 3D chromosomal interactions

Answer 20

Use Genome Archtecture Mapping (GAM) –> high resolution sectioning of the cell and part of the nucleus

Microdisect individual slides –> Put slices in well for genome analsyis –> measure co-segregation frequencey of two parts of the genome

GAM often condirems HiC results (main results between GAM and HiC are similar)

GAM = has single cell sensitivity (1 nucleus at a time) Vs. popultion cell in test tube for ligation reaction in HiC

Can detect mltiple interactions in 1 section (Ex. 10 things comthing togetehr) vs. HiC only see intercation between 2 things

Can detect interaction of super ehnacers and actived genes as triplets across Mbp distance (see enhnaer and proxbmity genes)

Question 21

Issue with GAM

Answer 21

Resolution - issue with how thin you can slice

Have a proxmity limit of 220 nm BUT a nucleosome if 10nm –> Means you could have 22 nucleosomes in each slice

Question 22

Super enhancers

Answer 22

A subset of human genes are regulated by super-enhancers (Common in pluripotencey genes and oncogenes)

Idea of what a super enhancer is:
1. Super-enhancer is its own thing
- Ex. Cell cylcle assocated genes + tumor supresser genes + oncogemes = regulated by 10-20 kb super enhnacer
- Ehnacer = only a few 100 BP (Super enhnacer is longer)
2. Super-enhancers are clusters of stnadrad enhnacers

Question 23

What type of genes use superenhnacers

Answer 23

Very important genes use super-enhnacers to regulate genetic ectivity (Ex. oncogenes)
- Super-enhnacer = has signators of chromatin + have RNA pol there + enriched for mediators

Example 2 - Glbulin or insulin genes that are more simple (might not have super enhnacer?)

THINGS that recevive more signla s= need exrtra regulatpru circut = have many things that affect one promoyer = have kb (super-ehnacer) regulating that 1 promoter
- Regulation is not just 1-3 elements BUT it is a large cluster of elments over 10kb (ALL 10 kb is important)
- Ex. pluri=potencey gene = repsonding to a lot of signals = needs a lot of enhnacers

Question 24

Promters + Enhancer DNA elements

Answer 24

Promoter and Enhancer DNA elements interact with sequence specifc Transcrtion factors to recruit general trnascription fcators + mediators + RNA polymerase 2

Question 25

Promoter

Answer 25

Promoter = part of the gene where transcrion strats - Usually only have 1 transcription start site BUT can have multiple (seoerated by 10s of BP) - Start = where RNA Polymerase is initiated Image: - Have nuleosomes at the end (downstream) of promoter - Have RNA polymerase 2 at start site Underlying DNA sequence of promoter = 70-90 BP (DNA that includes transcriptoon start site - common feature to eveyr gene)

Question 26

Complex at the promoter

Answer 26

Form transcrioton factor 2D complex --> recsuites RNA polymerase along with mediator - 60 proteins involoved TF that bind to enhancers and to proximal sites birngs RNA pol to promoters with high speficity = have domains that recruit conetrate co-regulators and the mediator (comunciates infomration to get thinsg started)

Question 27

Core promoter

Answer 27

Includes: 1. TATA box (-30 from start site) - Most common feature - TATA protein binds to this 2. INR (-2-+4) 3. DPE (Distal promter element)(+32) Where general transcription factors and RNA Pol 2 binds ALL together - core promoter = 60-70 BP) - Not EVERY core promoter has all of the elements but most do Core promoter = commanility for ALL 20,000 genes (10,000 ubiqitous genes expressed in every cell have the same core promoter)

Question 28

What distiguishes between promoters

Answer 28

Transcription fcator binding sites that are upsteram of the core sequences distiguish between promoters Genral transcription factors are DIFEFRENT from sequence specific transcrtion factors - general transcription factors recognize the core promoter + do not recognize DNA the same way at the sequence specific TF Sequence specific transcription factors bind to enhancers + proximal sequnece (upsteram of core promoter) - Not THAT specifc (binds to 1000s of genes BUT more specific) - Combination of TF that ind to enhnacer/proximal sites that brings RNA polymerase to promoter with high specificty

Question 29

Discovery of GTF and SSTF

Answer 29

Discivered by biochemistry and then proven by genetics Start with biochemsirty using "grind and find" --> use radioactive nucelotode and DNA transcript --> grind cell and see what happens when you put ribonucleatides (does it match protein) TF cam out of biochem BUT then verify with genetics (mostly with yeats because of saturation) - Yeast geneticiss confirmed biochemistry findings

Question 30

Sequence specific transcription factors

Answer 30

Sequence specifc transcrion factors = include master developmental regulators and progarmming fcators TF can take the differentiated cell (ex. take a fibroblast) and can reprogram the cell to make a different differentated state by chnaging the TF - Ex. Mylb B = Fibroblast --> muscle OR CLEP takes B cell --> Macrophage OR OSKN factors reprogram cells to pluripoetent stem cells IF you know how the master genes work = can d a lot of reprodgraming

Question 31

How many sequence specific TF do humans have?

Answer 31

Humans have 1600 sequence specific transcription factors --> MEANS of the 20,000 genes only 8% if eh genome is dedicated for sequence specifc TF recgining promoter (proximal?) and enhacer elememts - ONLY 200 genes are tissue specififc (master regulaters) - Ex. firboblast or B cel specfic

Question 32

Issue with OSKN story (Inducing pluripotent stem cells)

Answer 32

Issue = frequencey of repreogramming is small In a popultion with 1 million cells <1% is reprogramed - Measn that reorgnization of the entire epigenome must be complicated

Question 33

Sequence Specific TF + DNA binding

Answer 33

Sequence specifc TF have DNA binding and other modular fucntion domains Sequence specific TF = modular protein --> MEANS you can chop up and mix modules ALL Sequence specifci TF have: 1. DNA binding domain (reading sequence) - recognizes the sequence logos 2. Transcription activation domain (must bring transcription machinery down to the DNA) IF the TF responds to signals THEN needs a ligand binidng domain (Ex. If respnds to estrogen THEN needs domain that binds to estrogen) - When the ligand binds its exposes teh DNA binding domain or transactivating domain The different domains (modules) that amke teh Transcription factor dimerize or trimerize etc --> expands (can recognzie more BP) - One modile recognzies 5 BP --> trimer has 3 modules = can recognize 15 BP - Example - Heat shock TF = trimer that recognizes 15 BP but eah module recognizes 5 BP

Question 34

Doing a selection fir DNA sequence that TF bind to

Answer 34

Logos = DNA sequence that are most often found when a transcrtion factor binds - Example - SP1 binds to C rich elements - Logo = binding site of TF - Sequence specific TF = all look for short DNA sequence THEN look if the 1600 sequence specifci TF have domain characteristics --> Creates families Chart - Looking at restricted region of DNA within an enhancer orpromter hat the Sequence specifc TF recognize

Question 35

Prevelant DNA binding domain

Answer 35

Zinc Finger - MOst prevelant DNA biding domain (engages with DNA promoter) - TF can have multiple zinc fingers (Ex. CTCF has 11 zinc fingers) ; proteins that binds to 1 site on genome have many zinc fingers - Zinc fingers can also read RNA and protein - Longers BP recogniztion sequnce = more unisque so it can be better recgnized by zinc fingers Cystine-Histodnine that cooredenates zine ion - zinc finger is coordinated by zinc Zinc fineger reaches to the major groove of DNA and recognized by AMno acid side chains and DNA bases = read the bases (HOW it reachs DNA code) 60% of the 1600 Seqeunec sepcifc TF are zinc finger proteins

Question 36

Transcription fcator transactvating domains

Answer 36

Trnascription factor binds to promoters and enhnacers --> the transactivating domain on the TF willl recruit compoenents of regulatory machinery or transcription machinery

Question 37

Structured motifs of TF

Answer 37

There are a limited number of structured motifs that Transcription fcators use to engange DNA promoters C2H2 (Cytosine-Histodine) zinc finger = most domient THEN heleix turn helix then helix loop helix (Zipper domain) ALSO have Zip B Zip = Lucine Zipper DNA binding domain

Question 38

DNA binding domains of TF

Answer 38

DNA binding domains include: 1. Zinc Finger 2. bZip - 2 alpha heliceies (each helix goes into major groove on opposite sides) - Ex. GCN in yeast or AP1 in humans - Long helix intecat with another long helix thorugh coil coil inreractions --> presents to DNA binding domwinas --> reaches to major groove of target 3. bHLH 4. HTH families (helix turn helix --> helix goes to the major grove) IMage - different colros = different recogniztion helix --> goes into major helix and reads the bases (have different orientation of the recognition helix in major groove) - Helix reads the DNA by inserting into he major groove at different orientations - Main element of specificty = to read sequence on major groove

Question 39

Zinc Finger proteins (depth)

Answer 39

Zinc finger proteins = comrpise multiple copies of a small beta-beta-alpha domain stabilized by a zinc ataom - Zinc cordinates finger Zinc finger protein = Type of TF that has zinc fingers (I THINK) Each molecule in he protein function independentley and recognzies 3-4 BP on DNA Select residues in each domain are repsonsible for intetacting with DNA (rest provide a structual scafold) Read DNA sequence depend on which side chain is used (fingers insert to major groove and read off different DNA bases )

Question 40

Promoter and enhnacer DNA elements binding to TF

Answer 40

Promoter and enhnacer DNA elements bind to gene specfic TFs that recsuit generat Transcription factors + mediator + RNA plymerase 2 to the PIC General transcrtion factor = forms the prei-initiation complex (PIC) - General TF go to the promoter and recruit and position RNA pilymerase to start transcription PIC has: 1. RNA polymerase 2 2. Mediator 3. TF2D (big complex) - Include TATA biding Protein (TBP)

Question 41

Protein for intiation

Answer 41

Uses ~60 polypeptides for intiaion Proteins are conserved in yeast + humans + plants

Question 42

TF2D multi-protei complex

Answer 42

Left Blue circle = Shwos teh TATA box --> TBP proteins binds to teh TATA motif Right - TAF6 + TAF2 + INR + DPE --> porteins that recognize core promoter

Question 43

Making guides for CRIPSR

Answer 43

Whe making gRNA for CRIPSR = use Phage polymerase T7 Phae polymerase T7 = has 1 SU - Phage doesn't need many things --> just wants to make more phage --> has T7 polymerase with 1 SU (more simple) - Recognzies T7 promoter on DNA (reason why need T7 promoter to make gRNA) Phage vs. Metazoans - metazonas have 1-- proteins to get RNA polymerase started

Question 44

How do you get all 100 proteins onto the promoter

Answer 44

Using Transcription fcators Transcripting fcators recognize DNA sequenxes unique to gene families --> TF recruits the machinery (means you have to touch it at the big complexes) TF recruits the mediator

Question 45

Mediator

Answer 45

Have core head + middle + tail THEN ALSO have enzymatic compeoenets Mediator = helps get all 100 proteins onto promotr Image: - Blue circles = proteins tha touch meidator compeonet (have ones that bind nuclear erecptors or estrgen rectprs or bind to ELK1) - P5 binds to Med 6 - Grey = binds to nuclear receptors and P53 Have groups of proteins that bind to places on mediators

Question 46

RNA polymerase Phosphorylation

Answer 46

RNA polymerase has C terminal tail that is heavily phosphorylated Phosphorylation is needed for iniation + elongation of transcription To get phosphorylation = binds to mediator -- mediator has kinase that phosphorlyates the tail of polymerase

Question 47

Cryo EM of PIC-Mediator supercomplex

Answer 47

Question 48

Affect of nucleosoomes on transcription

Answer 48

In Eukaryotes - Chromatin nucleomes block the transcriptional machinery Histones and nucleosomes on DNA = block DNA ---> transcription machinery cant get in --> need to get rid of nucleosomes - Cells have machinery to move nucleosomes out of the way

Question 49

Moving nucleosomes out of the way

Answer 49

Nucleosomes block polymerase = they need to be kicked off or pushed aside --> have remodeling enzymes - Remodeling enzymes = mobilize histones + mobilize the nucleosomes to move out of the way - Remodleing enzyms are recruited by Sequence specifc TF --> remodeling enzumes move nucleoesomes --> have space that can be filled by transcription machinery Start - Have Dynamic nucleosome array 1. PTF recruits remodelers + modiferes 2. Have Nucleosome depleted region + H2Az incorpoation histone modifications 3. PIC recruitment 3. Pol 2 intiation --> pol 2 pasue 4. STF triggers --> Pol 2 elongation --> pol 2 reintiation END - Generation of chromatin accessibility by nucleosome remodeling helps PIC recruitment

Question 50

Past thoughts on nucleosomes

Answer 50

Past - Nucleosomes were considered artifacts 1973 - Two groups looked at chromtin in fixed cells under a microscope --> got cabels - Took the chromatin and put it into water with no salt = chromatin explodes from the nucleus (because when have no salt there is no charge repulsion = chromatin coms out of the nucleus) THEN olins = showed partcils of chromatin (saw 'beads on string') Luger = crystilzied chromatin 1991 - crystilzied hostone octomer without DNA and solved atomic structure

Question 51

Histone structure

Answer 51

Overall - Octomer - 4 histone proteins (form kernel of nucloesome) - 5th hostone proteon = linker protein (inker between 2 nucleosomes) - Nucleomes = contain 8 histone proteins (2 of each of teh 4 core histone proteins) Have 4 histone proteins (small proteins ; 100 AA) - Each histone protein has different genes (ALL lysine rich BUT the sequence is not idetotical) - Would not say that histone proteins are related based on sequnece BUT based on secondary structure they are related

Question 52

Histone protein

Answer 52

4 Histone proteins = H2A, H2B, H3, H4 H2A = lysine rich ; H3/H4 = argiine rich 4 core histone proteins form dimers: 1. H2A and H2B form dimer 2. H3 and H4 form a dimer AND H3 and H4 dimers form tetromer

Question 53

Key element of octomer

Answer 53

Histone fold - fundemental way that you create intercation between proteins - Histone fold intercation = creates octomer Histone fold = Helix --> Loop --> helix --> loop --> helix - Histoen fold = handshape motif (long helicies in center form coil coil intercatinos) - H3 and H4 form coil coil inteactions ; H2A and H2B form coil coil interactions Image: - Left = histone fold - shows long helices in coil coil intercation - Right - imagrnay path that DNA might take on surface of octomer (predicted because that is wehre many lysine residues are) ; DNA is in left handed super helix (writhe of DNA is left handed on nuceosome superhelix) ; doted lines show the unstructed histone tails that contain histone modification (Ex. acylation)

Question 54

Interaction between 2 dimers

Answer 54

2 dimers an form a 4 helix bundle --> 4 helicies come together hrough interhelical intercation In histones - H3 and H4 and H2A and H2B stick together because of 4 helix bundles - The 2 dimers of H3 and H4 for a tetromer -AND 2 H2A and H2B come together to form the rest of the octomer

Question 55

NUcleosome core partcile

Answer 55

145 BP of DNA on octomer (forms Nucleosome core partcile) - Nucleosme includes linker between DNA and NCPs DNA is wraped 1.7 left-handed superhelical truns (each turn is 80 BP) - Left handed because proteins tell the DNA how to wrap (guided by histone octomer) Dyad axis of symetry - Psudosymetru because DNA on the left and right side might not be the same) Disk like (11nm diamter and 5 nm height) - has 2 faces Linker DNA between NCPs (histone partciles) is varaible (10-80 BP) --> linkers are essential Core histones = essiental for organisms - Genetic shows histones are essnetial - Have multiple histone genes 30% of histone = unstructured histone tails - Cyrtsal structre gives 70% of protein complex)

Question 56

Answer 56

Nucleosome core particle Blue = H3 and H4 dimers forming tetramer - 4 helix bundle = where arrow us Long green helix = coil coil intercation Dotted lines = histone tails (where modifctaions are) - Non-strictured histine pairs (intrinsic domain) Ends of helix and loops poiint towards DNA (close to the minor groove)

Question 57

Blockage of DNA on histone

Answer 57

50% of DNA surface is occluded by histone octamer (DNA pointing owards histone is blocked) - DNA surface that is available to be touched by zinc fingers = facing inwards = blcoked Lose 50% of the information when wrapped aorund histones - Sequence specific compenents need to shift out --> done by shift 5 BP on the nucelosome --> causes what was facing in to now face out because the pitch is 10 OTHER 50% is curved and facing out

Question 58

Inside Nucleosome cut in half

Answer 58

Image - see 4 helix bundle H3-H3 = 4 helix bundle that forms the H3:H4 tetromer H4-H2B = 4 helix budle that assmebles H2A:H2B dimers on the H3:H4 tetromer

Question 59

Arginine side chain

Answer 59

ARgine = positivley chaged --> they are at loops and paired ends of histone folds (end of helix) Have arginine at al superhelical 1/2 locatons = stciked into the minor groove and attaches DNA onto the surface of the histone Arginiene = allows you to pack DNA onto histone - Done in a non-specifc way Have 14 arhinies that poke into the minor groove = brings DNA onto histone octomer (attach and bends DNA over histone)

Question 60

Positions on nucleosomes

Answer 60

Have 14 positions: 0 = at the middle of the nuceosome --> AS go down the wrap there are 10 BP Major grooves = superhelical locations (+ in one direction and - in other direction) - 10 BP down the wrap on major groove facing inwards = superhelix 1 --> ANOTHER 10 BP is suepr helix 2 etc. Minor groove = between 0 and 1 (have o.5) - 1/2 positions = going from major groove to minor groove Minor groove comes close to the histones 14 times (because each time have 7)

Question 61

Does DNA want to bend around Octomer

Answer 61

DNA does NOT want to bend around octomer 145 BP if DNA is not felxible = need ALL ariginine to get teh DNA to bend over the histone octomer

Question 62

Core Octomer Feature

Answer 62

Have Acid patch on ecah NCP surface - Has hydrophoic character - Acid patch contour can fit many interacting DNA motifs (Ex. loop or hairpin) Red patch = aciidc patch --> have many prteins that interact with histones that interact with acidic patch

Question 63

Quesions about histones

Answer 63

1. Without DNA wraooed around it the hostone octomer itself is not stable in soltion --> WHY - Answer - electrstartc reuplsuion between histone lysine and arghinne residues AND weak hydrophobic intercation with 4 helix buncles 2. Despite instability in solution conditions ca favor histone octamer reconstitution in vitro - What conditions? - Answer - Denatires core histnes are renatured by 2M NaCl by salt gradient dialysis (Salt nuetrolizes charge repulsion THEN use dialyos to remove salt THEN only have DNA and DNA takes over salt) 3. DNA on nuceolsomes is bent which is different from the normal stiff rod - WHY? - Answer - electrostatic repulsion along phsophodieter backbone (phosphates don't want to be next to each other) + maximal DNA base stacking interactions (bent DNA loses base stacking interacions)

Question 64

What do you need when DNA is in solution

Answer 64

Need salts when DNA is in solution - because the phosphates don't want to be next to each other

Question 65

Chromatin accesibility paradign for transcrtional control

Answer 65

Question 66

Biogensis of chromatin accesibility

Answer 66

Biogensis of chromatin accesibility uses ATP ATP driven remodelers recuirted by pioneer Transcription factors Remodelers = grab nucleosomes and move it relative to DNA - Have families based on characteristic ATPases (Use ATP hydrolysis to move nucleosomes on DNA)

Question 67

ATP driven chromatin remodeling enzymes

Answer 67

ATP driven chromatin remodeling enzymes mobilize nucleosomes

Question 68

Families of ATP dependent chromatin remodlers

Answer 68

Have 4 families of ATP dependent chromatin remodelers

Question 69

Movement of nucleosome remodelers

Answer 69

Nucleosome remodelers = have distict genic locations and activities Movment = NOT random Want to move the nucleosoes away from promoters/enhancers --> have psuhers tha moce away from enhnacers Family that will put back to the enhnacers = pullers BOTH co-exist in nucelus

Question 70

Movement of nucleosomes

Answer 70

Nucleosomes = ALL jiggling --> caused by ATP dependent molecules MOvemmet = creates transient window of oppertunity so tha the transcription machinery can come in and functiion Have competitio bteween moving nucleosomes and transcription machinery that is waitingig for an open target to assmeble

Question 71

Nucleosome mobilization by DNA

Answer 71

Nucleosome mobilization by DNA twist or bulge IDea for how DNA is moved over a histone octomer Hinge Enzymes have 2 DNA binding domains (has DBD and Tr) --> moves from state 1 to state 2 by pushing the DNA (psuhing makes a microbulge or twist on nucleosome surface) --> propegates around

Question 72

Modes of Transcriotion factors

Answer 72

Have Diverse modes of Transcription fcator interactions with nucleosomes - Nucleosome consecutive affinity purification of a nucleosome library shows majority of 220 TFs have less access to DNA Paper did a systematic analysis fo how DNA binidng molecules recignize DNA if it is on nucleosome - TF can see 5 BP that are exposed ; TXB2 (homobox TF) can reconize and bind nucleosome --> orineted bind on the exposed major groove --> binds and deastabilizes Paper FOUND TF prferntially bind close to the end of DNA or to perioidic positions on solevent xposed side ; some bind to xis ; others apnd two gyres at specific sites - TF binidng typically destabilizes DNA

Question 73

Pioneer TF

Answer 73

Pioneer factor sbind on nucleosomes = catylyze arrial of remodling enzymes (IF nucleosome is bound BUT TF is bound then there is no bare DNA and you still need remodelling enzymes) Pioneer TF = bind to nucleomes = increase nucleosome accesivility for non-pioneer TFs and for the transcription machinery - Binary classifciation depending on nucleosome affinity alone is not neccesarily predictive of pioneering activity PTFs = collaborate with ATP-dirven chroatin remodeliers to mobilize nucleomes at promoters and enhancers PTFs = have distctict interating domains that recruit remodeliers

Question 74

GAGA factor

Answer 74

GAGA factor (GAF) = recuirts chromatin remodelers

Question 75

Histone tails location

Answer 75

Histone tails = poke out of nucleosome H3 and H2B tails emerge between minor grooves aligned bytween two DNA gyryes H2A and H4 tails protrude fro each NCP face Part of the tail is sturctured before leaves

Question 76

Histone tails

Answer 76

Core histone tails = 15-37 residues Tails = protease sensitive Have Low complexity domain/Intrinsically Disordered region Tail sequences = highly basic + unique to each histone + evolutionarily conserved - Lots of positive Lys + Arg residues Tails have tansient intercations with nucleosomal DNA (fuzzy conformational ensebles) - Fold back on DNA because + charged Tails infleunec - DNA breathing (H3) at edge of nuclseome + Nucleosome stability + inter-nucleosome association Tails have many post translational modifications that alter sturcture-function - Ex. S/T phosphate + K-Ac = weakns DNA binding + increases tail acesibility to epigenetic readers Tails = sites for protein/enzyme binding (Ex. target of epigentic readers + writers + Earasers)

Question 77

Histone tails + DNA

Answer 77

Histone tails collapse on DNA Image - Shows tail that is unstractured colapsing onto DNA - Because tail is felxible = ca't get strictire = ONLY have prbabilities of how might work - Histone tails collapsing onot DNA might stabilize nucleosome

Question 78

Stimulation of Histone dynamics

Answer 78

To make video - Take bond energy and the computer can show microsecond of the dynamics of the protein - ALL baed on physics and chemistry Video shows - DNA end unwrapping + DNA strand translation register + DNA twsit + DNA deformation + Histone helix movment + Histone tail rearrangment

Question 79

Histone tail Contacts

Answer 79

Models of histone tail contacts in various chromatin states and regulatpry effects of fuzzy tail DNA intreactions - Confirmational dynamic tails can regulate chromatin structure + partner protein interaction (tails cause intercation between nucleosome + protein) + DNA accesibility - Fast dynamics of histone tails allows for greater accsibility to Post translational modifications - Faster transitions between intra nuclseosome tail collapse-extension and higher order nucleosome arrangment (Tail intreaction could bring nucleosomes togetehr in cluser in higher ordeer folded cofigurations) - Tails confer proerties onto nucleosome array - Tails can interact with RNA polymerase

Question 80

Histone Modifications at Gene locations

Answer 80

Discovering the enzymes that modify histone tailes (Ex. Acytlyase enzymes) - Used biochemistry --> THEN sequenced --> FOUND GCM5 catalytic SU that is a well known for transcription in yeast Linked histone acylation to GCM5 (know it is involoed BU do not know mechansim) - Genetics tells you that it is involoved ; Biochem tell you the mechanims Image: - List = shos modifications - Have some histone modification at the promter of the gene bod and some modifications at the end of the gene body - Modifying histone adds information to chromatin template that tells proteins the begining/middle/end o genes --> provides signal for process

Question 81

Histone modifying enzymes

Answer 81

Writters - Add histone modifications Readers - Recognzie the modification Earasers - Remove the hiostone modifcation Histone code - signals for nucleosome stability + modifying/de-modifying enzymes + effector function

Question 82

Importance of histone modification

Answer 82

Histone modifcation = improtant --> many diseases have impaired histone modifications

Question 83

H3K4me3 and H3K3me3

Answer 83

H3K4me3 and H3K9me3 methylation have opposing functions (1 is activation and 1 is repression?)

Question 84

Histone tail acetylation

Answer 84

Histone tail acetylation destabilizes intra- and inter-nucleosome associations that comes at the clustering at nucleosomes

Question 85

Protein effector domains

Answer 85

Question 86

Histone modification cross talk

Answer 86

Histone modification cross-talk one PTM can positively or negatively affect another PTM Acetylated Lysine will affect the lysine at the next position because proteins can recognize multiple amino acids = have cross talk

Question 87

Interconnected mechanisms of epigenetic regulation (What is involoved in epigenetic regulation)

Answer 87

What is involoved in epigenetic regulation: 1. Mechanical properties of promoter and enhancer DNAs (Stiffness of DNA) 2. Specific recognition promoter and enhnacer DNAs (Sequence sepecifc TF) 3. Chromatin accesibilitu at promoters and enhnacers by ATP enzymes (ATP driven chromatin remodeling enzymes) 4. Covalent modifcations of DNA and histones (DNA and histone modification enzmes) - Modifications on DNA itself = also part of epigenome

Question 88

Issue with textbooks

Answer 88

Textbook models features of hiearchal folding of chromatin fibers from nucleosome array to solenoid to metaphase chromosome - REALITY - have DNA --> Nucleosome BU anything solenoid and up is imagination - Soilenoid = 6 nuclseosomoes wrapped in helical configuration condensed by H1 linker histone WHY??? - Because in Cryo-EM you get what you put in --> MEANS the solenoids and ribbnes seen in Cryo EM is semiartifcial - Need to get natie xhromatin in cell (NOT aryificial sequence) and see how the native chromatin folds beyond he single nucleosome

Question 89

DNA folding and gene regulation

Answer 89

Is folding of DNA part of regulation of gene expression HiC and GAM approaches = designed to reveal aspects of higher order nucleosome folding

Question 90

What does Cryo-EM show

Answer 90

Cyro-EM shows zigzag in two strat helix model for 30 nm chromatin fiber BUT there is NO evidnece for soleoid - Cryo EM gave another model Text on slide (HE did not realk go over): - 12 x 601 reconstituted chromatosomes (nucleosome with H1) with 187 bp repeats --> give tetranucleosomal unit - Four nucleosomes zigzag back and forth with interveneing straight linker - Histone H1 bound to dyad (axis) + entery + exits sites of linker - Asymetric H1 location and self-association creates twist between stacked nuclesomes - Consistent with zigzag two strat helix model - X-ray crystal structure is similar ISSUE - STILL NO EVIDNVE FOR SOILENOID Soilenoid

Question 91

Left handed two start helix

Answer 91

Don't know if Left handed two start helix exists in cell

Question 92

EM tomography Results

Answer 92

EM tomorgraphy shows NO evidnce of thw two start twisted helix in celluar chromatin AND no evidence of Solenoid - IF you want to know how the nucleosome organsims you need a method that follows the DNA or follows the histones to see where they go down BUT at the level of whole nucleosomes you can't follow the DNA easily Experiment (follwoing DNA): CromeEMT texhnique on fixed cells uses a flourescent DNA -specifc dey DRAQ5 to gnerate ROS + polymerize DAB to make ectron dense region + Somium stains chromatin for EM cintrast - Uses fixed cells NOT recnstituted nucleosomes - RESULTS - reveals chromatin as disordered 5-24 nm diameter granular chain - Found that in interphase and mitotic chromtic the granular chain is packed at different CVC densities (Difefrent Chromatin volme concetration) - In cryoEM it is hard to seperate DNA and protein = stained with DNA dye (DRAQ5)

Question 93

EM tomography Results (DEPTH)

Answer 93

Image - see nuclear pore + Nuclear memebrane + Chromatin in interior (Shows chromatin ultra strcutre and 3D organization of genome in nucleus) FOUND - NO slenoids or two start twisted helix --> INSTESD see structually disordered 5-24 nm granukar chain with different particle arrangments + conformations + densitoes + 3D motifs - No ribbon - Thin partciles = nucleosomes --> near nuclear memebrane (heterochromatin) see a higheer density (BUT still have lots of space between + have RNA there so know things can get between) ; In interior the clusters are less dense but stll there - Shows Cryo-EM is NOT refelctive of the cell - DONT SEE A PATTERN Issue = lose resolution in chemistry - following the electron dense material depsited by DAq5 florophor you not REALLY following DNA (Follwoing shell of DNA)

Question 94

Chromatin Volume Concetrations

Answer 94

Chromatin volme concetrations percept interphase chromatin volume per 120 nm cube - CVC = how dense is this Reserachers doing EM tomorgraphy used CVC - found high concentration in nuclear lamina and low concentration in interior - Interphase CVC = 30% ; mitotoc chromsome = 50%

Question 95

ChromEMT of human mitotic chromosomes

Answer 95

Even in mitotioc chromsome (thought of very dense) had high cnebtration BUT the maximum concetration is 50% on mitotc chromsome - Means have 50% empty space

Question 96

What should ChromEMt reveal

Answer 96

ChromEMT should reveal ultrastructure of chromatin fibers, megabase domains, mitotic chromosomes in serial slices - Super-resolution histone fluorescence imaging in fixed cells also shows disordered granular chains (Consistent with ChromEMT) Trying to do all genes

Question 97

Future Question

Answer 97

Consider how to reconcile divergent models of chromatin compaction of nucleosome arrays derived from: 1. Cryo-EM & X-ray crystallography of reconstituted ‘601’ high affinity nucleosome arrays 2. ChromEMT and Super-resolution florescence of fixed cells ISSUE: 601 (Cryo-EM and X-ray crystallography of reconstituted 601) - sequence selected by proteins for binding nucleosomes with higehst affinity but it is not natural - If have 601 array = get ribbones and solinoids - NO 601 in cell = no ribbones