Epigenetic Marks and Chromatin Modifications

• Transmission of heritable information is not encoded by the DNA sequence

Epigenetic Marks and Chromatin Modifications

• H2A is modified by S122 phosphorylation by Mec1 and Tel1, and erased by PPH3
• H2A is modified by S129 phosphorylation by Mec1 and Tel1, and erased by PPH3
• H2B is modified by S10 phosphorylation by Ste20
• H2B is modified by K11 acetylation and erased by Hos3
• H2B is modified by K123 ubiquitination by Rad6/Bre1, and erased by Ubp10 and Ubp8
• H3 is modified by K4 methylation by Set1/COMPASS ,and erased by Jhd2
• H3 is modified by K36 methylation by Set2, and erased by Jhd1
• H3 is modified by K79 methylation by Dot1
• H3 is modified by T45 phosphorylation by AKT1, 2
• H3 is modified by K9 acetylation by Gcn5
• H3 is modified by K14 acetylation by Sas3, and erased by Hos3
• H3 is modified by K18 acetylation by Gcn5
• H3 is modified by K56 acetylation by Asf1, Rtt109, and erased by Hst3 and Hst4
• H4 is modified by K16 acetylation by Sas2, and erased by Sir2

Epigenetic Marks Communication

• Histone protein 1(HP1), adds methyl groups on H3-K9
• DNA Methyltransferases(DMT), add 5-methylcytosine(5mC)
• Methyl-CpG-binding domain proteins MeCP recognizes the DNA methylation
• Histone deacetylase complex(HDAC), deacetylates acetyl groups(Ac)

Heritable Epigenetic States

• Heritable (somatic) states have epigenetic barriers
• Cells with specific transcription factors are precursors
• Cells with developmentally induced epigenomes induce germline reprogramming into neurons and muscle fibers

DNA Synthesis Key Characteristics

• DNA synthesis proceeds from right to left on one strand
• DNA synthesis proceeds from left to right on the other strand
• DNA synthesis is always 5' to 3'
• The two template strands are antiparallel

Continuous and Discontinuous Replication

• On the lower template strand, DNA synthesis proceeds continuously in the 5' to 3' direction
• The synthesis proceeds in the same direction as that of unwinding
• On the upper template strand, DNA synthesis begins at the fork and proceeds in the opposite direction of unwinding, soon running out of the template
• DNA synthesis restarts on the upper strand at the fork, each time proceeding away from it
• DNA synthesis on this strand is discontinuous, with short fragments called Okazaki fragments

DNA Polymerase Activity

• Alpha(α) polymerase initiates nuclear DNA synthesis and DNA repair and has primase activity
• Delta(δ) polymerase synthesizes the lagging strand of nuclear DNA, is involved in DNA repair, and performs translesion DNA synthesis
• Epsilon(ε) polymerase synthesizes the leading strand

Eukaryotic vs Bacterial Replication

• Eukaryotic DNA is complexed with histone proteins in nucleosomes
• Nucleosomes are reassembled quickly following replication
• Creating nucleosomes requires: disruption of original nucleosomes on the parental DNA, redistribution of preexisting histones on the new DNA and the addition of newly synthesized histones
• Reconstitution of higher order chromatin structures

Budding Yeast for Studying DNA Replication

• Budding yeasts are a model organism used to study DNA replication and replication-coupled chromatin re-assembly
• Humans have a life cycle of 15-20 years; S. cerevisiae has a life cycle of 90 minutes
• Humans have a complexity of 10^11cells; S. cerevisiae has a complexity of 1 cell
• Humans have a genome size of 6x10^9 bases; S. cerevisiae has a genome size of 12x10^6 bases
• Humans have origins of replication that are not defined; S. cerevisiae has well defined consensus element
• Both Humans and S. cerevisiae have heterochromatin and euchromatin
• Yeast is easy to grow and manipulate, has practically no ethical concerns in experiments, and smells good

S. cerevisiae Replication

• Budding yeasts (S. cerevisiae) origins have a defined sequence called Autonomously Replicating Sequence (ARS)
• The core element of ARS is ACS (ARS Consensus Sequence) 5-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3]
• ACS binds the Origin Recognition Complex (ORC)
• There are 12000 ACS in the yeast genome
• Only 400-600 fire in each S-phase

Yeast Origin Auxiliary Elements

• B1 is an AT-rich short element and secondary site for ORC binding
• B2 is the site where DNA is unwound by the MCM helicase
• B3 binds a protein called Abf1
• Abf1 has multiple functions, acting as an activator/repressor of transcription or a heterochromatin factor, depending on binding position

Yeast Origins and Chromatin

• ACS generates nucleosome-free region and facilitates the association of pre-initiation factors
• Nucleosome-free regions at RNA polymerase II promoters in eukaryotes are similar
• ORC binding induces positioning of nucleosomes adjacent to the ACS

Eukaryotic Origins

• Origins in most other eukaryotes don't have a consensus sequence
• The more complex the genome, the larger the number of origins
• In mammals, 30000-50000 origins fire in each somatic cell with a higher number in early embryonal development
• There are more potential origins per cell-than needed in both yeast and humans
• Normally, one out of five potential origins within a replicon fires in each cell cycle
• Origins in both yeast and humans bind ORC and recruit pre-initiation factors

Hierarchy of Origin Firing

• S. cerevisiae: 400-600 origins fire in each cell cycle
• Active origins in S. cerevisiae are close to the centromere and nearby active genes
• Transcription supports euchromatin structures and influences the firing of origins
• Many dormant origins are close to the telomere, and heterochromatin is abundant in the sub-telomeric loci
• Heterochromatin has a negative effect on origin activity
• Dormant origins stimulate the formation of heterochromatin
• Mammals: 30000-60000 origins fire in each cell cycle
• Origin-bound factors can sense chromatin and determine the activity of the origin

Pre-Replication Complex Assembly

• Licensing of origins occurs in the G1 phase of the cell cycle
• Pre-replicative complexes (pre-RC) are assembled on origins
• A significant number of potential origins are "licensed", meaning that they are loaded with ORC and MCM in G1
• Only a subset of the "licensed" origins fire in S-phase

Initiation of DNA Replication in Eukaryotes

• ORC binds to replication origins in a low CDK state
• Cdc6 and Cdt1 load MCM helicase, licensing replication
• DDK creates Pre-RC, forming the complete CMG helicase complex
• Helicase activation occurs followed by poymerase recruitment, after which DNA replication begins

Origin Firing

• RIF1 recruits Protein Phosphatase 1 (PP1)
• PP1 counters the activity of DDK and prevents the activation of MCM.
• RIF1 and PP1 regulate the elongation step of replication

ORC Complex

• The Origin Recognition Complex (ORC) plays a key role in selecting active origins
• The ORC structure is highly conserved between S. cerevisiae, S. pombe and metazoan
• It consists of 6 subunits: Orc1 and Orc2, with DNA in the center

Hierarchy of Origin Firing by ORC

• ACS generates nucleosome-free region and facilitates the association of pre-initiation factors
• Nucleosome-free regions at RNA polymerase II promoters in eukaryotes are similar
• ORC binding induces positioning of nucleosomes adjacent to the ACS

Replication Elongation

• New DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs)
• The 3'-OH group of the last nucleotide on the strand attacks the 5'-phosphate group of the incoming dNTP during replication
• A phosphodiester bond forms between the two nucleotides
• Two phosphates are cleaved off

Replisome Complex

• The replisome is an enormous fast-moving complex
• GCM helicase promotes MCM/CDC45 and GINS
• Topoisomerase reverses the supercoiling of DNA caused by MCM
• DNA pol ε synthesizes the leading strand
• DNA pol δ synthesizes the lagging strand
• DNA pol α synthesizes the lagging strand
• DNA ligase ligates the lagging strand
• RP-A is a single stranded DNA binding protein on the lagging strand
• PCNA associates with both strands
• PCNA loader loads the lagging strand
• PCNA unloader unloads the lagging strand

Elongation Roadblocks and Consequences

• Elongation pauses upon encountering roadblocks during DNA replication, causing pausing of the forks
• Road blocks Proteins that are tightly-bound to DNA, secondary structures on DNA (G4 quadruplexes), gene promoters (including tRNA gene promoters), transcribing RNA polymerases
• Consequences involve FPC (Fork Protection Complex) activity: leading to a higher risk of distortion of the fork and collapse
• FPC stabilizes the fork, sending a signal to checkpoints to arrest the cell cycle and helps remove the barrier, resuming elongation

Fork Pausing

• At fork barriers there is an accumulation of positive supercoiling ahead and negative supercoiling behind the fork
• Top1 and Top2 slow down forks by direct inhibition of CMG helicase and prevent barrier-disrupting DNA topology
• Mrc1-Tof1-Csm3 proteins build FPC in S. cerevisiae and Claspin-Tipin-Csm3 build it in humans
• Several factors reduce fork pausing, removing the impediment to elongation
• Rrm3 is a cork-screw DNA helicase in S. cerevisiae that unwinds DNA and removes tightly-bound proteins, with homologous helicases in mammals believed to have a similar function
• Pif1 and another DNA helicase acts on secondary DNA structures

Paused Fork Activity

• A complex recruits DDK to the stalled fork and phosphorylates MCM and Tof1
• Rrm3 helicase removes tightly bound proteins

Detecting Paused Forks

• ChIP with replisome factors (DNA polymerases, PCNA, MCM) in rrm3Δ cells
• Molecular combing stretches long DNA molecules
• Newly synthesized DNA in vivo is labeled by exposing the cells to an artificial precursor (BrdU instead of Thymine)
• An antibody against BrdU is used to identify the length of the newly synthesized DNA

Measuring Fork Speed

• Incubate cells with a 5 min short burst with labeled precursor of DNA with modified dNTP: BrdU, EdU
• The precursors are incorporated in DNA followed with excess dTTP
• DNA is prepared from each time point and combed
• The molecules are stained with Syber green
• The burst of incorporation of the labeled precursor is detected

Gel Electrophoresis

• Two-dimensional gel electrophoresis allows direct detection of DNA replication and recombination intermediates in preparations of total genomic DNA
• This technique identifies replication origins in the yeast genome and is based on the mobility of linear and branched DNA molecules in agarose gels
• Low-voltage and a low-percentage agarose gel favors separation of molecules by their mass during the 1st dimension
• A higher voltage, a higher percentage agarose gel, and the presence of ethidium bromide significantly delays the migration of branched structures during the 2nd dimension

Chromatin Components Replication

• Certain histone and DNA modifications (epigenetic marks) are always associated with heterochromatin
• Other histone and DNA modifications are always associated with euchromatin
• In metazoan, in all cells of a given tissue, the state of a gene (active or silent) and the modifications of histones and DNA are always the same
• Tissues are built by millions of cells that have replicated DNA many times

Heritable Chromatin

• Heritability of chromatin structure warrants uniform tissue-specific gene expression in all cells of the tissue
• Setting active genes for the tissue during development silences other genes
• A mechanism for faithful transmission of histone marks and DNA methylation during DNA replication
• Tissue specific epigenetic landscape

Histone Transmissions

• Histone transmissions involve interplay between histone chaperones, readers and writers
• There are no free histones
• Histones are either in a nucleosome or associated with histone chaperones
• During elongation, chaperones accompany disassembled “old” histones, ferry them behind the fork and match them with the “new” histones that arrive from the cytoplasm
• Histone codes on the “old” histones are read by “readers”, that recruit “writers” that eventually write the same histone code on the “new” histones
• The examples in the previous two slides are for H3K9Me3 and H3K27Me3. Similar mechanisms are believed to operate for other epigenetic marks
• H3/H4 can transfer as tetramers or split dimers
• After the transfer, nucleosomes are assembled

H3/H4 Hystones

• MCM is a Histone H3/H4 chaperone
• MCM2, CTF4 and DNA pol α directs H3/H4 tetramers to the lagging strand
• DNA pol & directs H3/H4 tetramers to the leading strand
• Symmetrical distribution is achieved by these opposing activities

Epigenetic Mark Loss

• With the loss of epigenetic marks, chromatin road blocks occur during elongation and the reassembly of chromatin
• Replication fork pauses/ slows down at a tightly bound protein barrier (supply)
• CAF-I assembles tetramers of H3/H4 only from new H3/H4 histones

Rrm3 Effect

• Failure of RRM3 to relieve the block prolongs the deposition of "new" histones with paused forks
• Replication forks that pause or slow down at a tightly bound protein barrier experiences this loss

DNA Methylation Transmission

• There is a general model for the transmission of DNA methylation during DNA replication: DNMT and MBDs

• A family of Methylated DNA Binding Proteins (MBD1, MBD2, MBD3) utilise hemithylated DNA

• MBD1, MBD2, MBD3 are reader proteins that direct DNMTs to the newly synthesized DNA strand

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Lecture Summary:

• Epigenetic marks are copied and maintained during DNA replication
• Histone chaperones and DNMT play central roles in the transmission of epigenetic marks
• ncRNA together with siRNA machinery, PIWI proteins and rixosome maintain heterochromatin based on the existing epigenetic marks (H3K9Me3, H3K27Me3)
• Pausing replication forks pre-disposes loss of epigenetic marks and acquiring new epigenetic state
• Cis-elements (silencers, antisilencers, promoters, TADS) work together with DNA replication to control paused fork outcome
• There is ability to transmit chromatin that marks to progeny
• Take into account histone chaperones, as their activity is within the FPC
• There is flexibility in the chromatin, which can affect cell fate transitions