Exam 2

molecular genetics

three ways to measure 3D chromatin interactions

3C

Hi-C

DNA FISH

3C

method of measuring chromatin interactions

sequencing based method that looks to see if two regions within a gene are in contact with each other

start by designing DNA primers for the 2 regions

Hi-C

method of measuring chromatin interactions

expands on 3C

measures the entire genome to see where ALL regions have interactions with each other

challenges: use lots of data so not high resolution

DNA FISH

method of measuring chromatin interactions

use fluorescence to see if 2 regions within a gene are interacting with each other

low throughput BUT HIGH resolution

what is chromatin accessibility

ability for transcription machinery, TFs, modifying factors, etc to access the DNA/chromatin

affects gene expression throughout the cell

factors that determine chromatin accessibility

nucleosome positioning

covalent modifications of the nistones/nucleosomes

post translational modifications

DNA methylation

heterochromatin

tightly packed chromatin

too dense

cannot access the genes

silence

euchromatin

less dense DNA → chromatin in accessible

expressed genes

two ways to measure chromatin accessibility

ATAC-seq

DNase-seq

ATAC-seq

method of measuring chromatin accessibility

Assays for Transposase Accessible Chromatin

uses a Tn5 transposase: this is a protein that inserts its own DNA into a sequence

Tn5 will insert its DNA/adaptors anywhere along the DNA where there are gaps/spaces → fragements the DNA strand

sequence the fragments after PCR amplification to analyze them

DNase-seq

method of measuring chromatin accessibility

uses DNase enzyme → will cut anywhere along the DNA where the strand is accessible

you select fragments that you want to sequence

ChIP-seq (chromatin immunoprecipitation)

method of measuring histone modifications → can see exactly where those modifications occur within the genome

steps:

• crosslink histones/proteins to DNA with formaldehyde

• will sonicate the DNA to fragment

• use antibodies that bind specifically to modifications to “pull down” and allow fragments with modifications to associate with the DNA

ChIP-seq overall steps

fragment DNA

add antibody that will detect specific marks/modifications within the fragments

isolate all the antibody bound fragments

sequence and find locations within the genome

enhancers

regulatory elements that regulate genes from a distance

act independently

often found in NFR and H3K4me1

ex: enhancers can cause DNA looping to influence expression

cell type specific; BUT many enhancers can regulate the same gene in many different tissues

promoters

specific DNA sequence near a TSS that allows for RNA poly to bind and initiate transcription

most are associate with CpG islands (sites of DNA methylation that control histone/gene expression)

many genes can have multiple alternative promoters that allow for manufacturing of different protein lengths/product length outcomes

where/what are active promoters often found

nucleosome free regions

histone acetylation

H3K4me3

what are repressed promoters often found with

H3K27me3

lack a NFR

what are bivalent promoters

promoters that have an active and repressive mark

primer to begin transcribing once activated

TATA box

specific region that is found in a promoter that helps with the recruitment of RNA Poly II

promoter vs enhancer methylation

promoters: found with me3 = trimethylation = only at active promoters

enhancers: found with me1 = monomethylation

transcription factors

proteins that bind to DNA that will influence gene expression

have major developmental, evolution, disease related roles

what are TFs classified by

DNA-binding motifs → determine specificity for location/sequence on gene

how are TFs organized

hierarchical and control cell identity differentiation

changes in TF number/type will have downstream effects

transcription

DNA being transcribed into mRNA

what does prokaryotic transcription rely on

lac operon

what is the lac operon/genes

• operon found in prokaryotes that is an example of transcription

• controls 3 genes from metabolising lactose

  • lac Z Y A

steps of lac operon

• lactose enters cells and is converted to allolactose

• allolactose bind to lacI repressor and causes it to release from operator

• CAP can then bind to promoter when glucose is low to increase transcription of the 3 genes in the operon downstream

how to prevent lac operon

lacI repressor stays attached to operator region in front of operon

this prevents RNA poly II from binding + transcribing

what is ground state of prokaryotic transcription

• nonrestrictive

• RNA Poly can bind directly to DNA

• always on unless repressed

what is ground state of eukaryotic transcription

• restrictive

• NEED activators to allow for transcription

• always OFF unless it is turned on/activated

three stages in eukaryotic transcription

• initiation

• elongation

• termination

what do eukaryotes use to transcribe

RNA Pol II

initiation step in eukaryotic transcription

TFs are recruited, bind to promoter, RNA Pol II can then bind to

elongation in eukaryotic transcription

• uses RNA Pol II that will transcribe and then pause 25-50 bp downstream of TSS

• recruitment of p-TEFb will unpause RNA Pol II

• transcription of gene can resume after unpausing

termination in eukaryotic transcription

two models

• allosteric:

  • the polyA tail is encountered → conformational change → termination just happens

• torpedo model:

  • exonuclease will degrade the RNA strand

  • exonuclease eventually reaches the RNA Pol II

  • termination occurs

what is alternative splicing and what are the results

• a form of post-transcriptional regulation

• splicing variants that allow for different isoforms of a gene

what are different examples of regions included in alternative splicing

varying introns, donor/acceptor sites, skipping exons, alternative exons

what regulates alternative splicing

sequence elements, epigenetic features, polymerase speed

what is exon-intron boundary

• the splice junction in alternative splicing

• marked by conserved sequences

what are the intron boundaries for alternative splicing

• start: GT sequence (splice donor site)

• end: AG sequence (splice acceptor site)

what are three stages of DNA replication

• initiation

• elongation

• termination

what method is DNA replication

semiconservative - daughter strands have one parental + one new strand

initiation in DNA replication

• DNA double helix is opened at origin of replication

• DNA starts to unwind via helicase to create replication bubble and replication fork

• primase will synthesize RNA primer

• Pol will then be able to bind to RNA primer to allow for transcription

elongation in DNA replication

• DNA polymerase synthesizes 5’ → 3’ ??

• leading strand is continuous and lagging strand creates Okazaki fragments - each fragment needs its own RNA primer

• once RNA primers are removed and replaced with DNA, ligase will seal the fragments together

termination in DNA replication

• replication forks meet, replication stops, ligase will seal strands

examples of DNA replication factors

• ssBinding proteins

• helicase (MCM complex)

• topoisomerase

• primase

• DNA poly

• PCNA - helps to hold DNA Pol on strand to extend elongation/synthysis times

• DNA ligase

where are Okazaki fragments found + average length

• found on lagging strand in DNA replication

• ~ 100-200nt long in eukaryotes

Okazaki fragment maturation - LOOK UP

connect via:

• Pol strand will run underneath adjacent fragment to create a flap

• FEN1 will cut flap

• ligase will seal nicks

Messelson-Stahl Experiment

what is chromatin inheritance

process that cells pass on the epigenetic modifications/info that have occurred within the chromatin/cell

what does chromatin consist of

• chromatin = DNA + histone proteins + modifications

• carries different chemical modifications that will determine silencing/active genes

what happens if chromatin inheritance does not occur

• the modifications/epigenetic changes are NOT restored

• cells lose expression patterns and cell identity is lost

steps in chromatin inheritance

in replication

• old histones are recycled and randomly distributed to varying daughter DNA molecules

• new daughter strand will have some old recyled and new histone modifications

new histones added

epigenetic marks are copied to new chromatin/histones

• old histones are templates

• via DNMT1 which is a maintenance DNA methyltransferase that will restore the methylation patters

what is differentiation

process that cells become specialized to perform specific roles and functions throughout an organism

what controls diferentiation

• chemical signals (different TFs, signaling pathways, etc)

• determined by TF hierarchical set up that pushes cells towards a specific cell type

what does differentiation cause/activate

• activates cell-type specific genes

• causes activation/silencing of certain promoters/enhancers based on cell types

• varies in histone modifications, methylation, chromatin conformation, 3D organization

overview of developmental process

• cell proliferation: multiplication of cells

• migration: movement from one location to another within an organism

• differentiation: cell specialization and distinction of roles

• apoptosis: controlled cell death

what is epigenetics

• the study of chemical modifications that alter the gene activity of a cell without changing the DNA sequences

• influences what genes are active/silence

what does epigenetics include

the gene sequence AND all the proteins, modifications, packaging that occurs in DNA

• DNA, RNA, proteins, any modifications

can change throughout growth and development

what is chromatin

• the organized form of DNA within the nucleus

• composed of: DNA and histones that form octamers to create nucleosomes

two forms of chromatin

• heterochromatin

• euchromatin

what are histones

what are nucleosomes

overview of 4 forms of post-transcriptional modifications

• 5’ guanine cap

• 3’ polyA tail

• RNA/alternative splicing

• RNA editing

function of 5’ guanine cap

• protects against degradation of exonucleases

• aids in nuclear export to the cytoplasm

function of 3’ polyA tail

• increases RNA stability

• protects against degradation

function of RNA/alternative splicing

• creates different isoforms of a gene

• one gene = ability for multiple protein products

examples of post-translational modifications

• RNA folding/binding: regulates translation, stability, influences if translation occurs

• phosphorylation

• glycosylation

• acetylation

• methylation

• ubiquitination

what are three categories of histone/epigenetic regulation modifiers

• writers

• erasers

• readers

writers

• one form of histone/epigenetic regulation modifiers

• will add groups

• ex: HMTs, HATs

erasers

• one form of histone/epigenetic regulation modifiers

• will remove groups

• ex: HDM, HDACs

readers

• one form of histone/epigenetic regulation modifiers

• bind to specific modified residues to initiate something

• ex: can enhance TF binding, recruitment, and increase/decrease transcription levels

HAT - ADD FUNCTION/EFFECTS

• histone acetyl transferases

• type of epigenetic writer

• typically add acetyl group to a lysine residue on histone tail

HMT ADD FUNCTION/EFFECTS

• histone methyl transferases

• type of epigenetic writer

HDAC ADD FUNCTION/EFFECTS

• histone deacetylases

• type of epigenetic eraser

• remove acetyl groups

• DNA wraps more tightly around histones

• causes transcription to decrease

HDM

• histone demethylases

• remove methyl groups from histones

• type of epigenetic eraser

acetyltransferase ADD FUNCTION/EFFECTS

• enzyme

• adds acetyl groups

• typically causes histones to bind to DNA LESS tightly

• chromatin becomes more open = euchromatin

• allows for increase in transcription

methyltransferase ADD FUNCTION/EFFECTS

• adds methyl groups

• activates OR repress

• ex:

  • H3K4me3 - often found at active promoters and increases transcription

  • H3K9me3 - associated with heterochromatin and silencing

what are CpG islands

• main sites where DNA methylation is found - FUELS epigenetic memory

• method to maintain methylation through DNA replication

where are CpG islands commonly found

• in front of promoters - allows for regulation of a gene sequence

• active genes - NOT methylated

example of CpG methylation

• CGCGCG

• cytosine methylated is written as 5mC

  • MUCH more common in repressed regions = heterochromatin

two main categories of control of DNA methylation in humans

• maintenance (DNMT1)

• de novo (DNMT3 A/B)

maintenance methylation

• via DNMT1

• maintains the DNA methylation in DNA by adding the methylation mark to the complementary strand

de novo methylation

• via DNMT3 A/B

• when there is no existing methylation, this enzyme will bind to DNA regions and initiate methylation

DNA demethylation in eukaryotes

• no good erasers to remove methylation marks

2 methods of DNA demethylation

• passive dilution

  • stops the DNMT1 from copying the methylation to complementary strand

• oxidative demethylation - active

  • relies on TET enzymes that will remove the methylation mark

what are nucleosome free regions (NFR)

• regions along a gene where no nucleosomes are found

• often 150bp long

what are NFRs often associated with

active promoters - allows space/accessibility for TFs and RNA Pol to bind

two methods to detect NFR

• ATAC-seq

• DNase

what are topologically associated domains (TAD)

• domains that are more likely to associate with THEMSELVES than the surrounding regions

• ~500kb regions that are found to play structural roles and can be conserved across a species

• related to chromatin regulation at 3D level

activity of TADs

• repressed or activation is dependent on surrounding NFR and chromatin density

• active TAC = interior of nucleus

• repressed TAD = often found in exterior/periphery of nucleus

what is euchromatin

• lose form of chromatin

• histones have decreased associated with the DNA

• MORE accessible and active

• these are the expressed genes

• tails are acetylated

what is heterochromatin

• dense/tightly packed form of chromatin

• histones have increased association with the DNA

• LESS assessable

• repressed and silences gene transcription

what is a motif

• a small repeating sequence within a protein or DNA that leads to recognition

• ex: TATA box - found within promoters and signals for TSS

what is origin licensing

• licensing refers to loading the MCM helicase so the origin of replication can fire and start replication in S phase

• assembly of pre replication complex

• polymerase and other replication factors are recruited

when does origin licensing occur

late M early G1 phase

what does license mean

replication machinery is loaded BUT DNA synthesis has not begun yet

three steps in replication origins activation

• origin licensing (M/G1)

• origin firing (early S phase)

• replication elongation (S phase)

what are replication origins

• site where DNA replication begins

• prokaryotic: most contain one RO

• eukaryotic: have many RO due to being more complex

origin licensing step

• first step in activating replication origins

• recognition sequence binds to DNA and recruits any proteins needed to assist in replication

• occurs in late M/early G1 phase