Topic 2: Nucleosomes, Chromatin and Chromosome Structure

How is DNA organized in Eukaryotes?

• associated with proteins and organized into linear chromosomes and enclosed in a nucleus

What is Chromatin?

• given region of DNA with its associated proteins on a chromosome

What shapes can a chromosome be?

linear or circular

How is DNA organized in a prokaryote?

• one circular chromosome (critical for life) and other small circular DNA called plasmids in the cytoplasm (advantageous genes for competition)

What needs to happen to chromosomes during mitosis/meiosis?

• need to be fully duplicated (DNA replication) and properly transmitted to each daughter cell

What are origins of replication?

DNA sequences along chromosome which initiate DNA replication

What are centromeres?

DNA sequences required for correct segregation of chromosomes by directing formation of the kinetochore protein complex in which the mitotic spindle attaches

What are telomeres?

DNA sequences located at the ends of the chromosome that prevent degradation and allow proper replication of the chromosomal ends

What happens to segregation if the chromosome did not have a centromere or two centromeres?

No centromeres → random segregation of chromosomes

Two centromeres → chromosome breakage

What is the ploidy of eukaryotes?

• Majority of eukaryotic cells are diploid (two copies of each chromosome = homologous chromosomes: maternal and paternal)

• Gametes are haploid

Why are some eukaryotes polyploid, such as large protists??

• really large cell that floats around, so needs a higher rate of transcription and translation

What is the eukaryotic cell cycle?

Ordered set of processes by which one cell grows and divides into two daughter cells

• need to fully replicate DNA and organelles and properly segregate them into daughter cells

What are the steps of the cell cycle?

1. G1 and G2 (gap phase): synthesis of macromolecules (proteins, RNA, metabolites)

2. S phase: DNA replication (replicated chromosome = sister chromatids)

3. M phase (mitosis): Nuclear division/chromosome segregation

4. Cytokinesis: cell division

5. G₀: Resting phase (most of our cells are here, except hair, blood, skin, etc)

What is mitosis? How do homologous chromosomes act?

Proliferation of cells where the genome content is identical in the parental cell and the two daughter cells

• Homologous chromosomes act independently throughout mitosis

What is meiosis?

Creation of gametes where the number of homologous pair of chromosomes is reduced from 2 in the parental cell to one in the four daughter cells.

How is genomic content unique in four daughter cells following meiosis?

• genes are the same, but allelic combinations differ due to recombination after homologous chromosomes align in prophase I

What are the reasons for DNA organized as chromosomes?

1. chromosomes compact DNA so that it can fit into the cell/nucleus (total length of diploid human DNA = 2m; nucleus = 10-15um, compaction factor = 10,000X)

2. Chromosomal structure protects DNA from damage (naked DNA is unstable and prone to degradation by nucleases, fragmentation and oxidative damage

3. Chromosomes can easily separate and transmit to each daughter cell during cell division (compact structure prevents fragmentation from mechanic forced during chromosome segregation)

What makes up half of the mass of chromatin/chromosomes?

Proteins

What are the most abundant chromatin proteins?

Histones, which form nucleosomes for DNA compaction

How were nucleosomes discovered?

• chromosomes treated with a sequence-nonspecific nuclease: Micrococcal nuclease (MNase)

• Mnase cleaves protein-free DNA sequences (linker DNA) rapidly and protein-associated sequences poorly (due to wrapping)

What does light digestion indicate?

Light digestion produces DNA fragments of 160 - 220 bp

• MNase cuts some linkers

• Multiple bands, evenly spaced due to the presence of mononucleosomes, dinucleosomes, trinucleosomes, etc.

What does heavy digestion indicate?

Heavy digestion produces a single band of 147 bp

• MNase cuts most linkers; primarily mononucleosomal DNA is left

• Mostly chromatin is reduced to mononucleosome-sized DNA fragments

Why is nucleosome arrays important?

→ strong evidence for the regular, repeating nature of nucelosomes

What is a nucleosome composed of?

Two subunits of each of the core histones: H2A, H2B, H3, H4 to form an octamer

What histone is not a core histone?

Histone H1, one per nucleosome or not present

Are nucleosomes conserved?

Yes, they are 98% identical in all eukaryotes with 146 bp/ nucleosome

What are the characteristics of a histone?

Small (11-21 kDa), highly basic proteins containing lysine and arginine residues to interact with negatively charged backbone

What does each nucleosome contain?

• 146 bp of DNA wrapped in left-handed solenoidal supercoil

• 1.65X revolutions/nucleosome

What do nucleosomes look like on an electron micrograph? Is all linker DNA the same length?

• Nucleosomes look like beads on a string with linker DNA between nucleosomes

• Linker DNA length is variable

What does each core histone have?

Histone-fold domain and N-terminal tails

What is the histone-fold domain?

• three alpha helical regions separated by two short loops involved in formation of heterodimers of H3-H4 and H2A-H2B

What does the formation of an octamer require? How would you test this?

• requires DNA

• To test, you could reconstitute histones in vitro with and without DNA.

  • compare what complexes form under both conditions using size-exclusion chromatography, electrophoresis, etc.

  • If octamers only form when DNA is present, then DNA is required

  • After SEC, an octamer would give a single at 110 kDa whose fraction contains all four histones in equal amounts on SDS-PAGE

  • Multiple peaks and fractions showing only H3/H4 or only H2A/H2B on SDS PAGE indicates that no octamers formed.

What happens if there is no DNA or DNA present?

No DNA: formation of H3-H4 heterotetramers and H2A-H2B heterodimers but NO octamer assembly

DNA: H3₂-H4₂ tetramer binds DNA and then recruits two H2A-H2B heterodomers

Where do the N-terminal tails go when octamer is formed?

extend away from nucleosome

What is the histone fold domain involved in and what does it contain?

involved in both histone-histone and histone-DNA interactions

• each histone-fold dimer contains three DNA binding sites

Where does the histone fold contact DNA?

Hydrogen bonds at the sugar-phosphate backbone and minor groove (not bases) because it binds DNA in a non-specific manner

What promotes contact between histones and DNA? What has opposite effect?

Two or more consecutive A-T pairs (A tracts) causes bending and narrowing of the minor groove - promote

Opposite - G-C tracts found at non-bending DNA regions not facing nucleosome

What nucleosome positioning is favored along the chromosome?

regions with roughly 10 bp spacing of A-T tracts

What are chacteristics of N-terminal extensions?

Flexible and disordered that can dynamically change conformation, and is not required for DNA-histone octamer interaction

What do the N-terminal tails do?

Form internucleosomal contacts to organize adjacent nucleosomes into higher-ordered chromatin structures since DNA wrapping around nucleosomes only compacts DNA by 6-7 fold

What do N-terminal tails undergo?

Posttranslational modifications (phosphorylation, acetylation, methylation) at K, R , S, T and Y amino acids that influence contacts to control chromatin structure and gene expression

How does internucleosomal contacts effect DNA replication and recombination?

• tighter contacts by histone tails = less accessible DNA to TF and other proteins

Is DNA packing along the chromosome uniform? What regions exist?

No, it is not

• Euchromatin regions: lower DNA compaction = genes actively expressed

• Heterochromatin: high DNA compaction = genes silenced

  • Constitutive heterochromatin: always highly compacted

  • Facultative heterochromatin: switches to euchromatin depending on cell type

How many binding sites are on H1?

1) Linker DNA at one end of nucleosome

2) Central region of DNA wrapped around the nucleosome

What does binding H1 to linker DNA do?

Increases the length of DNA wrapped tightly around the nucleosome and a more defined angle of DNA entry and exit from the nucleosome

What does addition of H1 induce?

Induces a more compact structure of nucleosomes in a zig zag pattern versus in absence of H1 (beads on a string = 10 nm)

What are the nucleosomes eventually condensed into?

Compact filament with a width of 30 nm (30- to 40-fold compaction)

What is the formation of 30 nm filament dependent on?

Histone tails forming internucleosomal contacts

What is solenoid (one-start helix) model?

Nucleosomes form a superhelix containing roughly 6 nucleosomes/turn, face of nucleosome face each other, linker DNA circles but does not pass the central axis (donut hole)

What is the zigzag (two-start helix) model?

Nucleosomes form zigzag pattern with two stacks, linker DNA passes through central axis connecting two nucleosomes on opposite sides of the filament

Can both structures (solenoidal and zigzag) form in the same chromatin filament?

Yes, as it is a dynamic process and can change back and forth

What are the major components of the chromosomal scaffold? What attaches to it?

• SMC proteins and Topo II

• 30 nm fiber forms loops that are tethered at their base to a proteinaceous chromosomal scaffold

How is the chromosomal scaffold exposed?

• removal of histones and incubation in low salt buffer (hypotonic) causing chromosome swelling

What is topo II hypothesized to do for chromosome structures?

• prevent entanglements and torsional stress of 30 nm fiber loops

• hold loops together at the base during chromosome condensation

What are SMC (structural maintenance of chromosome) proteins?

• highly conserved key regulators of chromosome structure (also found in prokaryotes)

What do SMC proteins contain?

• Head domains → At both C- and N-terminal ends and contains a functional ATPase when fused

• Hinge domain → connected to each head domain by an alpha-helical coil-coiled domain and required for dimerization

• Kleisin family proteins → binds to both head domains to fuse them and form a ring

What are the heterodimer SMC proteins?

• SMC1-SMC3 (Cohesin) → Keeps sister chromatids together from DNA replication to onset of anaphase

• SMC2-SMC4 (Condensin) → Compaction of chromosomes at mitotic entry by inducing loops and positive supercoils

• SMC5-SMC6 → Functions in DNA repair

What degrades what in early anaphase?

• Seperase degrades cohesins in order to pull sister chromatids apart

How are chromosomes condensed?

• SMC2-SMC4 Condensins position along chromosome and hinge domain binds to DNA

• ATP binding fuses head domains and supercoiled DNA loop extrusions are created

• Condensins are arranged in a rosette-like appearance and condenses the DNA

How is a bacterial chromosome compacted?

Into a nucleoid structure: A rosette of - supercoiled DNA mega loops that extend from a proteinaceous scaffold

What are bacterial scaffold proteins?

• Nucleosome associated proteins (NAPs) such as HU which are functionally analogous to histones

• HU interacts with Topo I to induce - supercoiling in DNA

• Bacterial SMCs cause loop extrusions to form DNA mega-loops

What is loop structure and number influenced by?

• its dynamic and influenced by amount of transcription

Can nucleosomes be moved?

Yes, they can either be ejected or repositioned by sliding

What complexes disrupt contacts between DNA and nucleosomes?

• Chromatin remodeling complexes have an ATPase domain that uses ATP energy to disrupt contacts and kick off or loosen to slide

What are the three main classes of chromatin remodeling complexes?

• SWI-SNF (transcriptional activation → Gene expression ON)

• ISWI and Mi2/NURD (both transcriptional repression → Gene expression OFF))

What is the sliding mechanism?

• SWI-SNF pulls DNA, forming a loop that detaches from the nucleosome. The loop moves around the nucleosome until it reaches the linker DNA

What is immunoprecipitation?

• The isolation of a protein species from cellular extracts by a specific-binding antibody and separation of the complex from the remaining proteins by precipitation

What is the first step for immunoprecipitation?

1) Lyse cells by sonication, bead beating and detergents to allow antibody access to target proteins in whole cell extracts (WCE)

What is chromatin immunoprecipitation?

Isolation of DNA sequences bound to chromatin proteins such as histones, SMC’s, transcription factors, and DNA polymerases

• Anti H4 Ab (recognizes H4)

What is ChIP/Chip Seq?

Performing chromatin immunoprecipitation of nucleosomes and isolate DNA fragments bound to nucleosome

How is the location of DNA fragments in genome mapped?

• Sequencing or flourescently labelling the fragments and hybridizing to a tilting microarray (probes are every 20bp on a chromosome

What do the identified genome sequences tell you? The genome sequences not identified?

Identified sequences are bound to nucleosomes, while unidentified sequences are devoid of nucleosomes

What are the steps for ChIP/Chip Seq?

1) Cross-link nucleosomes to DNA within chromatin by addition of formaldehyde

2) Digest DNA with nuclease (only want nucleosomal DNA)

3) Immunoprecipitate nucleosome-DNA complex with histone antibody → unbound DNA washed away

4) Nucleosome-DNA cross-links reversed, histones removed

5) ChIP-Seq = identify DNA by sequencing OR ChIP-Chip = label DNA with fluorescent group, anneal and probe microarray

• peaks = high nucleosome activity

Why do promoters have low nucleosome occupancy?

• Transcription initiation requires access to DNA by transcription factors and RNA polymerase

What is the relationship between nucleosome occupancy and gene expression?

• Housekeeping genes (needed on for basic cellular processes) = low nucleosome occupancy, always accessible, constant expression.

• Kinase/regulated genes (regulatory) = higher nucleosome occupancy at rest, require remodeling for activation, expression is more dynamic.

Why are mitochondrial DNA devoid of nucleosomes?

They are from bacteria, so mitochondria lack histones. Instead, mtDNA is organized into nucleoids with non-histone proteins

What do histone variants do?

Alter nucleosomal function

What is H3.3?

H3 variant → maintains transcriptionally active open state in promoter region by preventing H1 incorporation in chromatin

What is CENPA?

H3 variant → incorporated in nucleosomes in the centromere; contains a N-terminal tail that recruits kinetochore proteins to the centromere

What is H2AX?

H2 variant → incorporates in nucleosomes at DSBs, is rapidly phosphorylated (gamma-H2AX) and recruits DNA repair enzymes (DNA damage sensor)

What is the MacroH2A Histone variant involved with?

→ in females, gene expression of one of the X chromosomes is inactivated by heterochromatin formation (Barr body - dosage compensation)

• incorporation of the histone H2A variant macroH2A in nucleosomes and coating of noncoding Xist RNA on the entire inactive X chromosome

Is X inactivation ordered and not inherited? How does this explain calico cats?

No, X inactivation is random and heritable

→ Calico cats are heterozygous females (black and orange patches depend on which X was inactivated; white color is autosomal)

What are the four posttranslational modifications (PTMs) of histone tails?

1) acetylation of lysine

2) Histone deacetylases (HDACs)

3) Methylation of lysine and arginine

4) Phosphorylation of serine. threonine and tyrosine

What is the acetylation of lysine?

Acetyl group added by histone acetyltransferases (HATs) nuetralizes the positive charge of lysine and unfolding of the 30 nm fiber, as well as nucleosome stability and eviction (eg. H4-K16AC)

What are histone deacetylases (HDACs)?

Catalyzes opposite reaction from HATs

• acetyl group removed

What is methylation of lysine and arginine?

Histone methyltransferases can add mono, di or trimethyl groups to activate or repress gene transcription; opposite is catalyzed by histone demethylases

What is phosphorylation of serine, threonine and tyrosine?

Involved in chromosome condensation in mitosis (H3), DNA damage (gamma-H2AX) and transcriptional regulation (H3-S10 can promote H3-K14)

What adds and removes PTM?

Histone writers: add PTMs to histones (HATS, HMT)

Histone erasers: remove PTMs on histones (HDAC, HDM)

What is the function of histone readers?

→ protein that recognizes and binds to PTMs on histones and forms a complex with other proteins to regulate chromatin structure and gene transcription

What do readers with bromodomains do?

→ bind to acetylated histones and recruit HATs, TFs and remodelers to promote an open chromatin state (near promoter etc. to turn transcription on)

What do readers with chromodomains do?

→ bind to methylated histones and recruit heterochromaton-binding proteins, remodelers, HDACs to promote closed or opened chromatin states depending on the methyl group and on which amino acid

What do histone readers recruit?

→ writers/erasers to propagate/spread the PTM to neighboring histones for localized chromatin closing/opening

What is epigenetics? What are some examples?

→ changes in gene expression that occur without changes in DNA sequence

→ Histone PTMs (acetylation, methylation, phosphorylation), chromatin remodelling, DNA methylation

Are epigenetic marks permanent or transient over a lifespan?

• Can be both

What are epigenetic diseases? What do therapeutics target?

→ aberrant gene expression in response to environment or altered physiology

→ therapeutics target histone writers, readers and erasers because if gene is expressed abnormally high, but sequence is normal, we will try to condense that area and repress transcription

What is the histone code?

Complex combination of PTMs on different histones that regulate gene expression by recruiting a specific protein complex and producing a specific chromatin state (short and long range)

Are histone PTMs like a cascade?

Yes, the combination of histone PTMs can involve a specific order/pathway whereby one PTM will induce another PTM with different protein complexes recognizing each PTM

What is epigenomics? How is it important?

→ identifies all histone PTMs in the genome to decipher the histone code using ChIP-Chip/Seq with antibodies that recognize specific histone PTMs (ex. H3-K16Ac)

→ Global changes in epigenetic marks will enhance our understanding of chromatin structure and regulation of gene expression in cellular processes, growth and development and disease states

How are parental nucleosomes split?

→ randomly split in the two-daughter duplex DNA where nucleosome density is reduced by half (semi conservative)