(Cours 7) Genome Structure: Chromatin and Nucleosomes

• Module 3.1 pertains to genome maintenance, focusing on genome structure, chromatin, and nucleosomes.
• Topics covered encompass genomic sequences, chromosome diversity, chromosome duplication and segregation, nucleosomes, chromatin hierarchical structures, nucleosome assembly, and the regulation of chromatin structure.

Chromosome Structure

• Chromosomes can be circular, like in bacteria, or linear
• Bacterial genomes are mostly circular, can be linear.
• Circular bacterial chromosomes undergo topoisomerase action after DNA replication for separating daughter DNA molecules, preventing concatenation.
• Eukaryotic genomes invariably have linear chromosomes, numbering 2 to 50.
• Each cell maintains a constant chromosome number
• Genome size correlates with organism complexity, the correlation is not perfect
• Bacteria possess one chromosome copy per cell
• Eukaryotes are mostly diploid
• Eukaryote homologous chromosome copies exist and are located in the nucleus

Eukaryote Gene Density

• Eukaryote gene density is lower and more variable, when compared to prokaryotes.
• Most bacterial chromosomes are made of coding sequences (genes coding for proteins/RNAs)
• Short non-coding sequences regulate gene transcription
• More complex organisms tend to have lower gene density.
• The length of eukaryote genes increases with complexity
• The size of the proteins they encode do not increase with gene length
• Eukaryote coding sequences (exons) are interrupted by non-coding sequences (introns)

Introns and Repetitive Sequences

• A rise in the number of introns and the amount of repetitive sequences is observed for senior eukaryotes
• The human genome is composed of 60% intergenic DNA
• Intergenic DNA contains over 70% of interspersed repeated sequences (100-1000 bp from transposable elements).
• Intergenic DNA contains 25% of unique sequences, like regulatory regions for transcription and microRNAs.
• Intergenic DNA contains 3% of microsatellite DNA (short tandem repeats below 13 bp)
• Only 1.5% of the human genome is composed of the code

Gene Sequences

• 40% of the human genome is occupied by gene sequences, made up of 95% include things like:
  • Introns
  • Non-translated regions
  • Gene fragments
  • Non-functioning pseudogenes

Pseudogenes

• Pseudogenes are the result of a retroviral reverse transcriptase copying functional gene mRNA into double-stranded DNA (cDNA).
• The resulting cDNA is integrated into the genome as a pseudogene
• Pseudogenes don't have regulatory sequences and aren't expressed

Chromatin and Visibility

• Eukaryote chromosomes consist of DNA, RNA, and proteins, creating a complex called chromatin.
• Chromosomes are visible only during chromosome segregation (mitosis or meiosis)
• During the interphase, DNA is transcribed and duplicated, therefore, chromosomes stay diffused and non-visible

Chromatin Under the Microscope

• In interphase, chromatin is diffused
• During the M phase, it's compact
• Chromatin fibers are either relatively free and thin (needed for replication) or thicker

Nucleosome Structure

• Nucleosomes are the building blocks of chromosomes.
• A nucleosome includes a histone octamer, surrounded by DNA
• DNA wraps around the octamer 1.65 times in a left helix.
• The nucleosome's core DNA is 147 bp in all eukaryotes
• The linker DNA that separates nucleosomes varies in length (20-60 bp) between species.
• Nucleosomal DNA is about 6x more compact than naked DNA

Nucleosome Lengths and Composition

• The average distance between nucleosomes is determined via partial digestion with micrococcal nuclease.
• Each nucleosome consists of five histones
• Histones are among evolutionarily conserved proteins
• Each nucleosome has two copies each of H2A, H2B, H3, and H4 (core histones) and one H1 (linker histone)
• Histones are small proteins, abundant in basic amino acid residues
• Histones undergo post-translational modifications, like methylation, acetylation, and phosphorylation of specific residues modifies their charges and DNA interactions
• Post-translational modifications influence gene expression and cell differentiation and can be reversed

Histones and Acetylation

• Histone lysine residue acetylation modulates their interaction with DNA.
• It occurs through charge modification
• Acetylation prevents binding to negatively charged DNA
• Histones have a conserved structural domain made of three alpha helices.
• This domain allows heterdimer assembly in a "head-to-tail" structure from momomers

Histone Tails

• Each histone in the octamer has a N-terminal tail that is accessible and unstructured once the nucleosome forms.
• Peptidases digest histone N-terminal tails without altering the DNA's association with the nucleosome core.
• The nucleosome presents a double symmetry axis (dyad)

H3-H4 Tetramer

• The H3-H4 tetramer's histone-fold interacts with 60 bp at the center of the 147 bp DNA.
• The N-terminal end of H3 interacts with 13 bp from both ends of the nucleosomal DNA at both the entrance and exit of the nucleosome

H2A-H2B Dimer

• Each H2A-H2B dimer binds 30 bp on either side of the H3-H4 tetramer's 60 bp.
• There are 14 points of contact between the nucleosomal DNA's minor groove and the histone octamer
• Interactions involve about 40 hydrogen bonds, mostly with phosphodiester backbones.
• Only seven bonds involve bases in the minor groove
• No specific DNA sequence binding occurs.
• Histone N-terminal tails guide left-handed DNA winding around the octamer.
• Left-handed winding causes negative supercoils
• Supercoiling is the over- or under-winding of a DNA strand

Topoisomerase and Nucleosomes

• Topoisomerase is needed for nucleosome assembly on a closed circular DNA covalently.
• (Plasmid DNA presents an artificial situation)

Chromatin Types

• Two Chromatin Types Includes:
  • Euchromatin: loosely packed/open structure and associated with high gene expression.
  • Heterochromatin: more compacted, exhibits low gene expression, and is typically located on nuclear periphery.

Histone H1

• H1 binds to:
  • Internucleosomal DNA
  • Core of the nucleosome
• The addition of H1 protects 20 bp more
• The addition of histone H1 result in the formating of the 30nm fiber

Higher-Order Chromatin

• The addition of histone H1 leads to 30 nm fiber formation, representing the second compaction level.
• There are two models for 30 nm fiber structures:
  • Solenoid forms a superhelix with about 6 nucleosome per turn
  • Zigzag is dependable on the linker size

Histones and 30nm Fibers

• The N-terminal ends of histones are essential to stabilize the 30 nm fiber.
• The interaction between the N-terminal end of H4 and a negatively charged portion on the histone-fold is key to maintain structural integrity
• More compaction of the of a 30 nm fiber forms the third level of compaction
• Transversal section of human metaphase chromosomes show association of loop-formed DNA with a central nuclear matrix.

Nuclear Matrix Protiens

• Nuclear matrix contains multiple:
• Type II topoisomerase
• SMC proteins (Structural Maintenance of Chromosome).

Chromatin Modifications

• Higher-order chromatin structure involves a 300 nm fiber
• Histone variants alter nucleosome functions
• CENP-A replaces histone at the centromere level, its longer N-terminal tail helping kinetochore component binding
• H2A.X, a widespread H2A variant, becomes phosphorylated upon chromosomal DNA double-strand breaks.

Nucleosome assembly

• Nucleosome assemble immediately after DNA replication
• Assembly follows an order which is :
  • H3-H4 are assembled
  • H2A-H2B dimers are assebmeld next
  • Finally the H1 gets assembled
• Interaction between DNA and histones causes
• Nucleosome assmebly necessitaties the action of chaperone interactions

Chaperone Actions on Histones

• Chapperone have negative changs that help mask repulsion that occurs in histones
• CAF-1 - 4 - H3-H4 - Yes
• HIRA - 4 - H3-H4 - No
• RCAF - 1 - H3-H4 - No
• NAP-1 - 1 - H2A-H2B -No
• CAF-1 is recruited as a level of novely synthated DNAn through anderactions wiht PCNA (is a machine to replicate ADN )

Protein HP1

• Regulatory chromatin structure is regulated through chromatinienne and structures are reciganized through specific complxes
• Protein HP1 recognises lysine and condense to condense chromatin

Nucleosome Movement

• Regulation of chromatin structures requries nucleoisomes to be realaxed to proteins such trancriton factors and to get acces to speicfic DNA sequences
• Interactions between ADN and histone octamer are dynmic
• ADN must be released to be intermittant for spontaneious acces to proetiens
• Nucleomosal exponas extremites like position 1 and 174 to ligaion with protieins but rarelly the ocnsitstutio of 73

Remodeling Complexes

• Remondeling complex faciltaties the movements of nucleosimmes
• Sliding is a remodel that slides ADn arround histotnes octamer
• Exchange causes some remodelcs to inject and transfter a nuclseomse
• Some can faciltate that damer exchange can facilitate h2a/h2b byh3.ex /h2b

ATP usage on remodeling

• "remodelage" use ATP(translocase), to sliding,
• This can brise interaction with the histrones until 5 points of contacts
• Interactions between ADN and histone can reform and adn to be aocated to nuclear

Nuclesosmes postitioning

• 2 proetins will bind to ADN to have positions withi les sthat 150 pb and a free protein
• Attachment of a protein liaosn from adn will favorise the assembly through nucleosomes
• Ligaison that connectADN the to that curbed

Modifications of histones

• Lys postions and initiation can be associsated with a new expression
• Lys at potions 5 and 12 that identify new synthases
• These modifications form new coded histones
• Lys 4, 36,79 on H3 causes association in genes that expresseon
• lys 9 causes reprression of a transciption
• addition causes postve chargfe between hisotens and causes more essitenail to fibres forming
• Addition of groupement reduces the postive change from quenue which brises interaciotn