0 Posted 2021-11-27Updated 2024-01-11Notes / Class / Tulane / Biochemistry25 minutes read (About 3749 words)

Histone Modifications in Transcriptional Regulation

ChromatinStructure

Chromatins

Heterochromatin: Highly condensed nonexpressing DNA
Euchromatin: Less condensed, transcriptionally active DNA

Histone Modifications


© Geoffrey P. Dann, 2017

This model depicts the lengths of the histone tails (dotted lines),which are not visible on the crystal structure of a nucleosome.

acK: acetyl lysine
meR: methyl arginine
meK: methyl lysine
PS: phosphoryl serine
uK: ubiquitinated lysine

Histone Code

“Specific modifications of histones evoke certain chromatin-based functions, and these modifications act sequentially or in combination to generate unique biological outcomes.”

Wikipedia – Gene transcription is regulated by chemical modification to histone proteins.

Histone Acetyltransferases (HATs)

HATs acetylase histone lysine side chains.
Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysine residues in histones. Zinc is involved (not shown)

Structure of the HAT Domain from Tetrahymena thermophila


© PDBID=1QSN

The enzyme is deeply clefted.
The histone H3 peptide KSTGGK14APRKQ (yellow) and coenzyme A (pink) are bound at the deep cleft.

Catalysis appears to involve water-mediated proton extraction from the substrate lysine by a glutamic acid general base.

Acetylated Lys Recognition by Bromodomains


© PDBID=1EQF

Bromodomains specifically bind acetylated lysine residues on histones.
Structure of double bromodomain of human TAF1, a subunit of transcription factor TFIID (PDB code 1eqf)
Two binding sites are separated by ~25Å, ideal for two acetyl-Lys residues separated by 7-8 residues.
Histone H4 has Lys at positions 5, 8, 12, 16
Fully acetylated H4 binds tighter to double than single bromodomains (5/12 or 8/16 pairs).

Role of TAF1 Double Bromodomain

TAF1 double bromodomain targets TFIID to promoters
A refined proposal for transcription initiation
1. A HAT-containing coactivator binds to upstream DNA- binding protein (an activator)
2. The HAT acetylates nearby histone tails
3. TFIID is recruited to the site via the binding of the TAF1 double bromodomain to the acetyl-Lys residues of histones.
4. Followed by recruitment of other initiation factors and RNAP-II for transcription initiation.

Histone Deacetylases (HDACs)

18 HDAC enzymes that deacetylate acetyl lysine substrates including histones.

Histone deacetylase family (zinc-dependent)
Sirtuin family (NAD-dependent)

Reaction mechanism:

Bound Zn mediates the nucleophilic attack of water on acetylated lysine, forming a tetrahedral oxyanion intermediate.
The carbon–nitrogen bond of the intermediate is then broken and the nitrogen accepts a proton from His-Asp.
The acetate and lysine products are formed.

Sirtuins


© William Giblin, 2016

NAD⁺-dependent reaction

Nucleophilic addition to form a C1′- O-alkylamidate intermediate and free nicotinamide.
The 2′-hydroxy group of the NAD+ ribose attacks the C1′-O- alkylamidate to form the 1′,2′-cyclic intermediate.
The formation of deacetylated lysine and 2′-O-acetyl-ADP ribose.

Thus, nicotinamide, a deacetylated lysine-containing histone, and 2′- O-acetyl-ADP ribose are the final reaction products.

Histone Methylation

Methylation at Lys and Arg of H3 and H4 tends to silence the genes, inducing heterochromatin formation
However, trimethylated Lys4 of H3 is associated with active genes!!!
Histone methyltransferases (HMTs) use S- adenosylmethionine (SAM) as a methyl donor
The HMT enzymes have a SET domain containing the catalytic site for methylation.

Structure of SET7/9 (PDB code 1o9s)


© Hao-Bo Guo, 2007

SET7/9 mono-methylates Lys4 of H3.
SAM (SAH) and the Lys4-containing peptide bind to opposite sites of the protein.
The Lys4 side chain is inserted through a narrow channel and positioned for methylation by SAM.

Methylated Histone Recognition


© Kuo, 2012

Chromodomains and plant homeodomain (PHD) fingers bind to mono-, di- or tri-methylated Lys by an aromatic cage.

Extral Reading: Epigenetic Regulation Mediated by Histone Marks

Consequence of Methylated Histone Recognition

• The chromodomain of heterochromatin protein 1 (HP1) binds to methylated H3 Lys9, contributing to gene silencing.
• Bound HP1 recruits the HMT Suv39h, methylating nearby histone lysines (H3K9), thereby recruiting more HP1 and forming HP1
complexes.
• This mechanism explains how heterochromatin spread to silence neighboring genes.

Histone Demethylases


© Susanne Marije Kooistra, 2012

LSD1 & LSD2 demethylases
• Flavin adenine dinucleotide (FAD)-dependent amine oxidation
• Demethylate mono- and dimethylated substrates
The JMJC family
• Dioxygenases dependent on Fe(II) and α-ketoglutarate
• Demethylates mono-, di- and trimethylated substrates

The reaction lose two protons, one from the carbon and another from the (+) nitrogen, requiring the protonated nitrogen.

Understanding the demethylation reaction from the LSD1 Structure


© Sang-AhKim, 2020

The LSD1 is bound to a 21 amino acid H3 peptide with a mutation of Lys4 to Met (green).
Modeling predicts that methylated Lys4 binds in a solvent inaccessible area in front of the FAD cofactor (electron extraction).

Understanding the demethylation reaction from the JMJD3 Structure


© SanchariBasu Mallik, 2016

The JMJD3 is bound to a H3 peptide with a trimethylated Lys27 (yellow).
The cofactor analog N-oxalylglycine NOG (not α-ketoglutarate) and nickel (not iron) are used to prevent the reaction.

The methylated Lys27 side chain inserts deep into the catalytic pocket, close to the cofactor analog (pink) and nickel (green) for demethylation reaction.

Chromatin Writers, Readers and Erasers


Source Unknow

Inhibitors of DNA synthesis and their specificities

AZT and DDI: reverse transcriptase, chain terminate

nuceoside analogues of guanosine: phosphorylated by a viral kinase

Polymerase processivity Enhanced by a Clamp

β clamp in bacteria
PCNA in eukayotes
loaded onto dsDNA on calmp

Clamp loader couples ATP Hydrolysis to Clamp Loading

Sliding clamp + ATP: open and bind DNA; ATP → ADP, DNA releases

Biological advantages of DNA

Provides stable, yet mutable storage of genetic information. Metabolic stability and availability of repair mechanisms ensure long-term survival under a variety of physiological conditions.
Serves as a template for accurate and adaptable replication of genomes. This biological role ensures the transmission of important physiological functions for multiple generations.
Serves as a template for the expression and regulation of genetic information. This biological role facilitates adaptation of genomes to changing physiological environments.
Amenable to diversification (i.e., can evolve). Mutation and genetic recombination (DNA rearrangements) are the two major driving forces in the evolution of genomic diversity and adaptation in nature.

DNA ligases

Eukaryotic DAN ligase use ATP rather than NADP

initiation od DNA replication in E. coli

origin region (OriC) ~245bp

Duplex unwinding by DNA helicase

Exposed ssDNA allows SSB and helic …

Initiation: OriC-DnaA intercation → Entry of SSB and DnaB (hexlicase) → ENtry of promase → RNA primers are synthesized and the elongation complexes for 2 DNA replication forks are assembled

Elongation:

Prokaryotic DNA replication

A typical DNA replicsome

synthesis of leading strand are coupled with lagging strand by clamploader complex.

The e.coli Pol III holoenzyme …

Pols enyzme subunit factors

Eukaryotic DNA replication:

Pro: no cell cycle and start every time

Eu: onece anc only once in S phase; highly regulated

G1: Preparing
Checkpoint: end of the S phase

Time is variatd

Eukaryotic chromaomse have multiple origins…

Pro: single initial site
Euk: multiple initial site

Eukaryotic replication initiation is hihgly oreder… well regulated and complex…

Helicase (Mcm2-7)
Sld2/3 phosphorylated by CDK

Super Current Biochemical Reconstitution of Regulated replication origin firing

Termination in prokaryotes

Ter sites, 2 converging new ds circular DNA molecules separated from each other by recombination and with assistance of

Cell are constantly exposed to agents that can cause damage to their genome

Repair, tolerant, destroy the cell

Types of mutation

Transition: AT → G/C
Transversion: A/T → C/G
Frameshift: GGGGGG → GGGGG

Base alteration

Oxidation:
8-oxodG(antl)-dC(antf)
8-oxodG(synl)-dA(antf)

The rotation of the bound which cause the shift of the side chain and attribute difference hydrogen bonds. 3 to 2, C to A

Alkylation; Cross-links

Exogenous Damage: Exposure to ultraviolet radiation

Absorbed by adjacent pyrimidine bind and form double bond and cause mutation or lethal during replication/transcription

Repair

Base Excision Repair

Single Nucleotide

Nucleotide Excision Repair

oligonucleotide

Mismatch Excision Repair

mismatched base

Base Excision Repair

how recognize
AP site
Donw string/ leading string doesn’t matter
ligates involved

Nucleotide excission repair (NER)

recognize Enzymes (XPC)
recruit other factors
unwinding, mutation is exposed
Some protein was recruited and mistake was cut / rescued.

Recognition → resynthesis

Xeroderma pigmentosum (XP)

Accelerated skin cancer; mortality likely due to metastatic melanoma or squamous cell carcinoma

MMR pathway - E. Coli

MutS, MutL, MutH, ATP
MutH: cut the strand (down/leading)

methelation is critical here

RNA polymerases

Transcription using DNA to RAN
- Bacterial: ON enzyme for all of RNA synthesis
- Eukaryotic: At leased Three I II III
Strangs 1:
- Template;
- Non-coding;
- Anti-sense:
- Completed strand
Strands 2:
- Sense strand: coding strange of the DNA
- Non-completed strand
Bacterial promoter consensus sequences
- UP Element; -35; -10; RNA stra +1
- -35; -10: promoters (similar to regulatories in Eukaryotic.)

Expression controlled by interactions of promoter elements with RNA polymerase and specific repressor/Activators

Bacterial RNAP

Holoenyme versis core enzyme
Core subunits: α₂ββ’ω (400kDa)
Holoenzyme: core subunits plus factors
…

Five σ factirs in E. coli gene expression

Exp: (not important)
- σ 70: Most genes
- σ 32: Heat schock proteins
Trascription Elongation
- RANP trancks along the DNA template, synthesizes mRNA in te 5’ to 3’ direction and unwinds and rewinds the DNA as it reads.
- Transcription elongation causing DNA supercoiling, relaxed by topisomerases
Termination
- Transcription Terminattion
  - A series of 4 - 10 base
  - Sequence depended: CGGCGCTTTTTT (CG rich region; AT rich region)
- ρ factor-dependent
  - The amino-termina domain of ρ factor binds to hte RNA sequence of ρ utilization site
  - the carboxy-terminal domain of ρ hexameric ring closes
  - Ring closure propels ρ moving close to RNAP
  - ρ dis-engages RNA and RNAP from DNA

Eukaryotic Transcriotion

Three types of Nuclears RNAPs
- I: rRNA (28S, 18S)
- II: mRNA; snRNAs; miRNAs
- III: tRNAs; 5S rRNA; snRNA U6 7S RNA; other stable short RNAs
Transcription
- Subunit compositions of nueclear RNAPs
  - β’; β; αI; αII; ω
  - All yeast RNAP has five subunits similar to Bacteria
- RNAP II
  - Yeast RNAP II strcture resembling bacterial RNAPs
    - A vrab claw-like strcuture
- RNA biosynthesis
  - The clamp of the Rpb1 subunit moves down to trap DAN between the two claw
  - Unwind DNA at the active site
  - Wall of Rpb2 kinks the template by 90° out of the active site
  - One vase of the template points at the active site
  - This base is paired with the ribonucleotide
- RNA translocation
- Transcription promoters
  - Class I, II, II to RAN poly I, II, III
- Mammalian Gene and proximal promoter (Class II)
  - Proximal region (-200 ~ -30), within 200 bp of transcription start; similar to the -10; -35 in the bacteria’s
  1. TATA Box (TATAAA)
  - Recruits TATA binding protein …
  1. Initiator region (Inr)
  2. Other activation elements
- EPromoter and Activator proteins
  1. Transcriptional Activators: DNA binding domain; activation domain;
  2. Activator functions: Chromatin remodeling (acetylation-HAT); Mediator facilitates PIC assembly
- Repressor
  - Bind UAS.enhancers → displace activators
  - Prevent mediator → no PIC formation
  - Attracts HDACs and HMTs → heterochromatin
- PIC Assmbled
  - mediator facilitates TBP and TFIIB binding promoter
  - Other basal TFs and Pol II bind
  - phosphorylation of the CTD of Pol II by TFIIH → Transcription initiation
- Six transcription factors for clas II promoters (Not important)
- ==Phospho-code on RNAP II CTD
  - TFIH: Phosphate the Ser5 would initiated the transcription activity
  - P-TEFb: phospholate Ser2 to maintain the elongation state
- Non-canonical functions of transcription
  - CENP-A defines centromeric chromatin
- RNA…
- ChiP-Seq: Determine protein distributed on chromatins
- RNA-Seq: Expression profiles

Transcription inhibition as a therapeutic target for cancer

mRANs of many oncogenes
Transformed → RNA-directed agents: different sensitivity.

Alpha-amanitin: from mushroom
- directed binding to RNAP, adn block the bridge to repress the conformation change

Bacterial Transcription

Eukaryotic transcription

RNAP II CTD phospho-code

Non-canonical functions of transcription

Assays to study transcription

posttranscriptional Modification of Eukaryotic RNAs

Role of 5’ cap
- Ribosomal recogintion during translation
Cap structure
7-methylguanosin (m⁷G)
joined to the mRNA first nucleotide
Via a 5’-5’ tri-P brifge
Involved Enzymes
- RNA riphosphatase removing the γ-P of the mRNA’s 5’ site
- mRNA guanlyltransferase (a capping enzyme) adding GMP. (Take GMP to the site)
- Guanine-7-methyltranserase (second capping enzyme) methylating guanine
- Capping enzymes bind to RNAP-II, which will switch RAN synthesis initiation to elongation ()

posttransicriptional Modification of Eukaryotic RANs

Poly A tails; ~250 nt
- Cleavage and polyadenylation specificity factor (CPASF), cleaving up to 35 nt past the AAUAAA sequence
Poly A polymerase generate poly A tail using ATP. (termination signals given to RNA-polymerase)
CPSF binds to RNAP-II, coupling polyadenylation to transcription termination.
Poly A tail binds to poly-binding protein, protecting from degradation, increasing mRNA stability.

Exons and Introns

Precurso mRANs (pre-mRNAs) are processsed by the excision of introns and the splicing (joing) of exons.
Exon splicing in Two-Stage Reactions
- Invariant Sequences for splice junction
  - GU at the intron’s 5’
  - AG at teh intron’s 3
    - (graph), the number under the NT means the ration you are supposed to see them on the Intron
  - A branch point near the 3’ splice site
  - Free G, not paire to the intron…
  1. A2’-5’ P-diester bind between the tinron A (OH²’) and the intron at teh 5’ splice site, forming a “lariat” structure.
  2. The Exon 1 OH³’ group at the 5’ splice site from a 3’-5’ P-diester bind with the Exon 2’ at the 3’ splice site, releasing the intron with the free OH³’ group.
  3. The intron keeps the lariat structure.
  - Note: Splicing proceeds w/o free energy lose, Cleavage of one P-diseter bond and formation of a new bond.

Spliceosome-aided RNA splicing

Splicing could be focilited by the protein Splicesome.60S spliceosome particle containing five small nuclear RNAs + Ribonucleoproteins (U 1~6, no 3)

U1 recognizes the 5’ splice junction
U2 recognizes the branch point (intron A)
The binding of U4-U5-U6 forms spliceosome.
RAN cleavage at the 5’ splice site
RNA cleavage at the 3’ splice site
Intron …

Self-Splicing RNA

Group II Intron
- Exson 1 and Exon II aligned together
- the giant rondant was splicesd by themselves
Group I intron:
- not Intron A to initiated the reaction
- G , GTP, GDP, GMP… as the starting site
1. The 3’-OH of …

Summary of spliceosome-aided Splicing and Self-Splicing

In goup I introns, aguanosine cofactor (G) tat is not part of the RNA chain associates with the active site. The 3’-hydroxyl group of this G attacks the 5’ splice sit;
- The reaction is similar to those involving the 2 hydroxyl groups of branch sites as in group II introns and RNA inrons spliced in spliceosomes.
The subsequent trans-e…

Interactions of RNAP-II with capping enzyme, splicing and CPSF

Figure 16-5

Aminoacyl-tRAN Synthetase

Attach amino acids to tRNAs
Two step reactions
- Amino acid activation by the reaction with ATP
  (Aminoacyl-tRNA syntheiase)
- Formation of an aminoacyl-tRNA

Aminoacyl-tRAN SYnthetases

The synthetase enzymes have an elongated shape.
Binding the anticodon of tRNA near the one end of the enzyme.
Binding the A.a. acceptor stem of tRNA near th other end

Proofreading by Aminoacyl-tRAN Synthetase

Binding to the amino acid substrate pocket of tRNA Synthetase that consists of the zinc ion.L-Group: Megan, Ryan, Ka

Threonine binds at this pocket b/c it can coordinate Zn²⁺ using it NH₂ and OH groups
tRNA^thr is tRNA for threonin.
- Attaching ThR to tRNA^thr makes Thr-tRNA^thr (tRNA is correctly charged)
- Attaching Ser to tRNA^thr makes Ser-tRNA^thr (tRAN is mischarged)

Acetylation

definition
histone modification

Acetylation by HATs
Recognition by bromodomains
Deacetylation by lysine deacytelases (HDACs adn Sirtuins)
- cofactors: Zinx, NAD
Methylation by histone methyltransefrases (HMTs)
- The donor: S-adenosylmethionine (SAM)
- SET domains in SAM
Recognition by chromodomains and plant homeodomain (PHD) fingers
- Aromatci cage: to recognize
- Chroodomain-containing heterochromatin protein 1 (HP1) function: regulated transcription
Demethylation
- Lysine-specific histone demethylases (LSD1 and LSD2)-FAD-dependent
- Jumonji C-domain (JMJC) family members - F2a and α-ketoglutarate-dependent

Chromatin structure

Chrmosome → chromatin fiber → Beads on a string DNA wound on nucleosomes → Histones + Doble helix

Heteochromatin: Highly condensed nonexpressing DNA. (PS: Canot be transcripted because transcript machinary can’t recognize and bind)
Euchromatin: Less condensed, Transcriptionally active DAN

H2A, H2B, H3, H4

Histone Modification

Histone was circled in the center of the DNA, but with tail out of the stracture and cann’t be visualized by cristal structure. But they are the key site for the Methylating or Acetylating

acKL acetyl lysine
meR:
meK:
PS: phosphoryl serine
…

Histone Code

Histone modification is the signals of transcription on/off.

Histone Acetyltraseferases (HATs)

Histone + Me-CoA → CoA + Me-Histone

Structure of the HAT domain from Tetrahymena thermophila

The enzyme is deeply clefted
The histone H3 peptide KSTGGK¹⁴APRK! and coenzyme A are bound at the deep cleft.

Catalysis appears to involve water-mediated proton extraction from the substrate lysine by a glutamic acid general base.

Acetylated Lys recognition by Bromodomains

Bromodomains specifically bind acetylated lysine residues on histones.
- A deep hydrophobic pocket (hole) accommodates the acetyl-Lys side chain (PS, the hole recognize the acetyl-Lys specificity)
TAF1: has two brpmodomains and dipart from each with ~25Å, seperated by 7 ~8 residues.
A subunit of transcription factor TFIID (PDBid=1eqf)
…
…

Role of TAF1 double Bromodomain

TAF1 double bromodomain targets TFIID to promoters
A refined proposal for transcription initiation
1. A AHT-cotaining coactivator binds to upstream DAN binding protein (An activator)
2. The HAT acetylates nearby histone tails
3. TFIID is recruited to the site via the binding of the TAF1 double bromodomain to the acetyl-Lys residues of histones.
4. followed by recruitment of other initiation factors and RNAP-II for transcription initiation.

Histone Deacetylases (HDACs)

18 HDAC enzymes that deacetylate acetyl lysine substrates including histones.
- Histone deacetylase …
- …

Reaction mechanism:
- Bound Zn²⁺ mediates the nucleophilic attack of water on acetylated lysine, forming a tetrahedral oxyanion intermidiate
- The carbon-…

Situins

NAD⁺-dependent reaction

nucleophilic addition to form a C1’-O-alkylamidate intermediate and free nicotinamide.
Teh 2’hydroxy group of the NAD⁺ ribose attacks the C1’-O-alkylamidate to form the 1’,2’cyclic intermediate
The formation of deacetylasted lysine and 2’-O-acetyl-ADP ribose.

Thus, nicotinamide, a deacetylated lysine-containing histone, and 2’-O-acetyl-ADP

Histone Methylation

Methylation at Lys and Arg of H3 and H4 tends to silence the genes, inducing heterochromatin formation
However, trimetheylated Lys4 of H3 is associated with active genes

(methylation can add one to 3 methyl in a time) (give a exampel)

Histone methyltransferases (HMTs) use S-adenosylmethionine (SAM) as a methy donor
The HMT enzymes have a SET domain containing the catalytic site for methylation.

Structure of SET7/9 (PDB=1o9s)

SET7/9 mono-methylates Lys4 of H3

SAM (SAH) adn the Lys4-containing peptide bind to oppsite sites of the protein
The lys4 side chain is inserted through a narrow channel and positioned for methylation by SAM.

Methylated Histone Recognition

Chromodomains and plant homeodomain (PHD) fingers bind to mono, di- or tri-methylated Lys by an aromatic cage.

Fits in the shape,
lots of electron to nutralize the positive charge of the methyl group???

Consequence of Methylated hisone Recognition

The chromodomain heterochromatin protein 1 (HP1) bidns to methylated H3 Ly9, contributing to gene silencing,
Bound HP1 recruitsthe HMT Suv39h, methylating nearby histone lysines (H3K9), thereby recruiting more HP1 and forming HP1 complexes.
This mechanism explains how heterochromatin spread to silence neighboring genes.
HP1 clust prefer to form a tide complex and so, silence the DNA transcription.

Histone Demethylases

LSD1 & LSD2 demethylases

Flacin adenine dinucleotide (FAD)- dependent amine oxidation
Demethylate mono- and dimethylated substrates

The JMJC family

Dioxygenases dependent on Fe²⁺ and α-ketoglutarate
Demethylates mono-, di- and trimethylated substrates

Focuse on the Chemical reaction

Understanding the demethylation reaction from the LSD1 Structure

The LSD1 is bound to a 21 amino acid H2 peptide with a mutation of Lys4 to Met.

Modeling predicts that methylated Lys3 binds in a solvent inaccessible area in front of the FAD cofactor (electron extraction).

Understanding … JMJD3 structure

THe JMJD3 is bound to a H3 pep with a trimetrhylated Lys27

The cofactor analog N-oxylayglycine NOG not α-ketoglutarate and nickel not iron are used to prevetn teh reaction
The methylated Lys27 side chain insertes deep into the catalytic pocked, close to the cofactor analog and nickel for demethylation reaction.

Chromatin Writers, Readers and Erases

brif adn = =

Recombination

homologous reconbination, need small similar sequnces
Site-specific reconbination, nees specific DNA sequence
ranspositions, no Specific DNA
Non-homologous DNA end-joining (), specific proteins that repair dsDNA breaks

Homologous reconbination

Required for accurate chomosome segregation
repairing and tolerating DNA damage
Recovery of stalled or broken replication forks
Defects in recombination can contribute to genome instability and cancer predispositions

Biological roles

Genetic reassortments in gametogenesis
Repair of DNA damages
Repair replication forks
laterl DNA transfer between ceels
therOther, deletions, inversions, translocations, etc.

How HR produces deletions, insertions and inversions

Incersion:
DNA fold in a revised direction, The DNA form a arch bridge and at the foot of the bridge connected
Deletion and insertion
DNA fold in a same direction. A circle formed and the cross point was connected together and the circle was deleted. (Insertion happens the similar way.)

Translocation that casue cancers

Chronic myeloid leukemia
BCR/ABL mRNA
p210 fusion protein

ABL are concertive hyregulated RTK, BRC is a higly active promoter which drive the high expression of the ABL which resbonsible to growth and differentiation.