Chromosome Biology Flashcards
Genome architecture in eukaryotic cells
Morphology of diving cells differs significantly from interphase cells
Interphase and mitosis structure change
Highly dynamic and regulated process
Mistakes/different in diseases
Physical organisation of the genome
Sequence (2001 - first draft of human genome), epigenetics (modifications of DNA and histones), structure beyond double helix (DNA spatial organisation), dynamicity (response to stimuli and DNA status)
Knowledge about the genome sequence alone is not enough
Chromatin at different zoomed views
In vivo and vitro different
eg in lab (vitro) smallest (regions of DNA double helix < beads on string form of chromatin < chromatin fibre of packed nucleosides < chrimatin fibre folded into loops < entire multitude chromosome)
In vivo - chrimatin fibre of packed nucleosomes probably doesn’t exist
Nucleosome is the basic unit of chromatin structure
October of 8 histones
Histone H2A, H2B, H3, H4 (all x2)
Shared throughout all eukaryotes - evolutionary (old)
Nucleosome is formed from histones and DNA
DNA - acid
Nucleosome - basic
So bind to eachother, DNA wraps around histone octomer (almost twice)
Histone proteins - Histone fold domain (responsible for forming octomer, interacts with other histones in hand shake fold) and N-terminal tail (regulation)
Post translational modifications (PTMs) of histones play key regulatory role
Unstructured Histone N terminal tail
Regulation and Histone code - major epigenetic characteristics in eukaryotic cells (leaves marks on Histone tails for post translational modifications eg methylation, phosphorylation, ubiquitation, acetylation)
Histone code example
Specific patterns of post translational modifications to histones act like a molecular code recognised and used by non Histone proteins to regulate specific chromatin functions
Eg Histone H3 - K9 M = heterochromatij formation and gene silencing
K4 M + K9 A = gene expression
K27 M = gene silencing, poly comb repressive complex
K = lysine
M = trimethylayed
A = acytelated
How trimethylation if Histone H3 Lysine 9 silences transcription
Histone methyl transferase (HMT) SUV38H1 methylated H3K9
Chromodomain of HP1 recognises H3K9me3 and binds to it
More HMT attracted, so greater silencing signal on H3
HP1 spreads along a long domain of chromatin and become transcriptonally silent
Chromatin boundary elements will isolate this domain from the “open” chromatin and so silence transcription of that area. Silencing will start and stop at boundary elements
What does Histone code need to work?
Writers - proteins/enzymes that’ll put mark on (communication with outside world to put mark on eg SUV39H1)
Readers - (sometimes different from effectors) HP1
Binders/effectors - HP1 hererochromatirasation
Erasers - sometimes need to remove silencing marks due to different times and conditions, marks leads to opening also need to be removed sometimes
Misregulated Histone code is correlated with diseases
Cancer - up or down regulation of writers eg acetyl-transferases or HATs
FIND ANOTHER or specific
Examples of other epigenetic mechanisms
DNA methylation - silencing of transcription of domain of DNA so chromatin closed
Histone code
RNA based mechanisms - growing feild, related to DNA methylation
Histone variant replacement - function in some situations and replaced in others eg CENTA (centumeric protein A) SPECIFIC TO centromeres
We do not know exactly how Nucleosome a form higher Ofer structure
Idea: 11nm fibre of beads on a string for of chromatin with linker DNA and Nucleosome (DNA AND HISTONES), Nucleosome contains ~ 200 nucleotide pairs of DNA
3 features help chromatin to fold and maintain higher order structures
1) non Histone proteins bind chromatin (affecting structure of chromatin)
2) linker histones (H1) - (bind to linker DNA between nucleosomes regulates compaction, more H1 = more compaction but different variants do different things) - do not contain Histone golf and less conserved
3) tails of core histones - interact with DNA, other nucleosomes around and non Histone proteins (ESSENTIAL IN HIGHER ORDER CHROMATIN FORMATION)
How do higher order structures correspond to the organisation of chromatin in interphase nucleus?
Initially - microscopy (nuclear pores - communication and regulation of gene expression, nucleolus - , euchromatin - open and transcription ally active, herterochromatin - dense and stuck to envelope and abundant at periphery, much less active transcriptional pov)
Why is how the genome packed in nuclei important?
Organisation of chromatin affects all functions of DNA, including maintenance of accessibility and gene expression
Cell fate influenced by genome organisation - cell differentiation and pluripotency
Pathological states eg cancer related to aberrant regulation of genomic structures
Genomic architecture changes dramatically during cell cycle
Mitosis - chromatin condensed, nuclear envelope and pore dissociation, ejection of transcription factors and chromatin binding proteins, disruption of laminate associated domains
g1 - permissive for differentiation genes, pre PC assembly (prep for s phase) chromatin opens
S - early s phase: early origins fire Histone synthesis, late s phase: late origins fire, Histone synthesis inhibition (duplicate all chromosomes)
G2 - Histone biogenesis inhibited, Nucleosomes mature (prep for mitosis)
What do we currently know about the genome architecture in interphase cells
HI-C - new experimental method, new discoveries
Chromosome territories
Technique to paint chromosomes
Multi-colour FISH (spectral karyotyping) helps visualise entire chromosomes
Chromosomes in interphase cells do not occupy random spaces, occupy defined spaces. Helps to see if there’s any mistakes eg in cancer
Metaphors chromosomes random and overlap
What we know about chromosomal territories
1) after decondesation occupy defined and non random areas in interphase nuclei
2) high gene density chromosomes are located inside nuclei, gener poor chromosomes close to nuclear periphery
This also correlates with transcriptional activity
3) arrangement is conserved across different species (evolutionary conservation)
4) arrangement not found in early embryos
5) position in nucleus depends on cells type and may change over time eg transcriptional activity - may loop out of home territories
FISH labelling - two loci red, chromosomes green, activation of locus moved towards middle BUT longer time scale and sometimes may require passing through mitosis
NOT FULLY UNDERSTOOD - movement of territory within cells
Don’t know if transcription follows movement of DNA, or DNA movement occurs because of transcription
Correlation, don’t have good understanding of mechanism
Mitosis to interphase transition (chromosome structures)
Mouse embryonic stem cells, single cell resolution:
Immediately after cell division chromosomes de condense and change shape from rod-like to spherical
Equally condensed regions unfold to more than
What lies “below” chromosomal territories?
Smallest to biggest so eg compartments within territories
Nucleosomes scale: epigenetic modifications - nucleosomes
Supranucleosomal scale: Intra TAD dynamics - chromatin loops
Inter TAD dynamics - b compartments and a compartments
Nuclear scale: Nuclear positioning - chromosome territories
Chromosomal compartment: A compartment
Active transcriptionally
Chromosomal compartment: B compartment
Not active transcriptionally
Hi-C technique to map chromatin interactions
Evolved from 3C method.
Used to study spatial organisation of entire genomes
Study at different resolutions- whole genome to several kb
Hi-C - map genomic interactions on global scale
Possible to apply method to analyse interactions in single cell
Cross link DNA, cut with restriction enzyme, fill ends and mark with biotin, ligate, purify and shear DNA:pull down biotin, sequence using pared ends
Example of how hi-c mapping is visualised
Diagonal is mirror image
Triangle - TAD
Darker red = shorter range interaction
Lighter/further = longer range interaction
Sub- TAD = triangle within triangle
Hierarchical organisation of genome
Hi-c permits genome wide resolution detection of pair wise contacts between genomic loci
Zoom in - look at more and more details
1mb > 100kb > 10kb
Cell organisation in a particular cell depends on
Organism, cell type (tissue), stage of development, cell cycle, current physiological status (eg from stimuli)
Eg m. Musculus E14 ESC = 2200 TADs, cortex 1518 TADS. FLIES 10x smaller because genome size smaller
Lots of variability in spatial genome organisation
New compartmentalisation of chromatin after fertilisation
Mouse model - pool embryos
Oocytes: homogenous chromatin folding lacks TADs & other structural features
Zygote: highly diminished higher order structure, 2 sets of parental chromosomes are spatially separated and display distinct compartmentalisation pattern. Slow establishment of higher structures until 8 cell stage
Chromatin compaction in preimplantation embryos can partially proceed in absence of zygotes transcription and multi level hierarchy’s process
Every cell division get closer to compartments and organisation - due to differentiation?
Molecular level of TADs and chromatin loops
Individual fragments of DNA looping out and clearly defined by structures sitting at the base of the loop
Made of two things: cohesin (protein complex), CTCF (protein)
BUT not all loop and TAD borders are marked by CTCF and cohesin
Methods for genome mapping
Organisation lvl: chromosome territories - 3D fish
Compartments - super resolution fish, electron microscopy
Topologically associating domains, chromatin loops, nano domains, functional loops - super resolution fish
Nucleosome clutches - super resolution fish, electron microscopy
CTCF
Boundaries of compartments, TADS and loop domains are enriched for the binding of CTCF, 11 zinc finger, sequence specific DNA binding protein
Cohesin complex involved in defining loop boundaries (anchors)
Loop extrusion model
Single/double loop of cohesin, loop fed through cohesin, local regions of chromosome are kept close together, CTCF protein will stop extrusion of DNA it’s bound to but only if pointing in correct direction, finished loop will have two CTCF proteins and cohesin at its base
Energy used for movement of DNA unknown. maybe ATP but no proof, condensin similar model uses ATP
Modes of transcription factors action on 3D genome organisation
Direct oligermerisation
Cofactor oligomerisation
Condensate formation
Interactions with loop extenders
Chromatin modifications: Histone modifications, DNA methylation
Interaction with nuclear landmarks
Protein RNA interactions
Higher order chromatin structures in differing dendritic cells
Differentiation of dendritic cells:
Lymphoid primed multi potent progenitor (LMPP) HISTONE ACETYLATION (lysine 27 on Histone h3)
Monocyte dendritic cell progenitor (MDP) COMPARTMENT CHANGE (b>a)
Common dendritic cell progenitor (CDP) INCREASE INTRA TAD INTERACTION AND GENE INDUCTION
Dendritic cell (DC)
Higher order structures dynamically respond to changes in chromatin
Stimulus - activation or silence = response at lvl of genome organisation to the stimulus
Higher order chromatin structures and sister chromatids (after DNA replication)
No sister chromatid interactions
Interactions within the same sister chromatid
Interaction between 2 sister chromatically (don’t wonder far from eachother)
What keeps sister chromatically close?
Cohesive cohesin
Cohesin can mediate interactions both within (extrusion of DNA loops) and between sister chromatids (sister chromatids)
Proposed model of centromeric sister chromatid confirmation where closely spaced binding by cohesin and condensin molecules mediate tight and aligned interactions of the sister chromatids at the centromere - transcriptional regulation
Proposed model of sister chromatid interactions
Extruding cohesin for Intra sister interactions and inter sister interactions formed by cohesive cohesin
Loops of different sizes made within sisters by extruding cohesij with loop sizes 10-50kb
Inter sister interactions ~ 35kb apart and occur between sites that can be offset by 5 - 25 kb
Only happens after s phase (not in g1 where there’s only 1 strand of dna)
Compartments
Groups of topologically associating domains (TADs). Either contain actively expressed genes (a) or mostly inactive (b). TADs can come in or out - active process - dynamic
Topologically associating domains (TADs)
Medium sided genomic regions (100kb to 2 mb) that interact only weakly with neighbouring regions but strongly within themselves
TADs share replication timing features
Loops
Created by interactions between 2 small genomic regions typically separated between 100-750 kb
LADs - laminate associated domains
Bound to inner nuclear membrane, chromatin regions may be both repressed or active
NADs - nucleolus associated domains
Bound to nucleoli inside nuclei
Transcriptional factories
Aggregation of RNA polymerase containing multi protein complexes
Poly comb bodies
Accumulation of poly comb containing regions of chromatin involved in silencing, characterised by similar PTMs eg H3K27Me3
Other structures of transcriptional regulation
Cajal bodies, nuclear speckles
Chromatin is highly dynamic
Loops, transcriptional factories, formation of nuclear bodies and association of chromatin with nuclear laminate and nucleolus may contribute cell-to-cell variability of chromatin spatial organisation
Structures in nuclei have different stability/activity eg speckles least stable, nucleolus - LADs very stable
Will reflect transcriptional activity
Linopathies and nuclear envelopathies
Disease caused by defects in nuclear envelope structure and/or function due to mutated or not properly modified proteins
Hutchinson-gilford progeria syndrome (HGPS)
Causes premature aging
Do not know but genes bound to nuclear laminar messed up/dissociated (LADs and changes in chromatin activity)
Nucleus “spread out”
Mitotic chromosome structure
Different from interphase chromatin
(Normal chromosome 2 chromatids, centromere, telomere)
Each chromatid contains single dna molecule
Diploid so 2 copies
Early chromosome structure micrographs
Existence of “scaffold” and DNA loops
(Removal of histones from mitotic human chromosome - purification, 2 meters of DNA)
1970s study suggesting looping of chromatin in mitotic chromosomes
Ulrich laemmli - centrally located scaffold provides support for loops of chromatin, bind to protein network via scaffold associated regions(SARS)
Scaffold composed of topoisomerase2, condensin and AT hook architectural proteins (bind to DNA DIRECTLY)
Later- concerns about biological relevance of scaffolding structures observed in EM
What are the molecular features of the structure of mitotic chromosomes:
Organisation of mitotic chromosome (2013)
Lots of old ideas still hold true eg DNA loops
Highly compartmentalised organisation of chromatin restricted to interphase - mitotic compartments and TADs disappear, mitotic pattern is very similar in different cell types
Chromosomes characterised by the longitude assembly of loop baes running along length of chromosome
Loop size for human chromosome models ~80kb and positioning of loop bases seems random
2 step model for chromosome condensation 1:linear array of continuous chromatin loops formed 2: longitudinal compression takes place
Hi-c data for genome organisation on different stages of cell cycle: Organisation of mitotic chromosome (2013)
Synchronised cells, so mix of cells in different stages and then synchronised to purify by arresting in certain stage
Found that: chromosome 21 - g1,g2,s,m
G1,g2,s have complicated structures and TADs
M all absent - no compartments and TADs (flat compared to others)
Different cell lines show very similar structure of mitotic chromosomes although interphase organisation of chromatin may differ a lot between different cell types
Hi-C
Model obtained: 1 step) consecutive loops, cylinder oval geometry and linear organisation
Compaction model (2013)
Stages:
1) Linear compaction - loops are formed
2) axial compaction - loops are shortened and fit within small volume of mitotic chromosome
Interphase to mitotic cell transition belief
Interphase cell when genome in nucleus, mitotic no nucleus, change from heirarchal structure (cohesin and CTCF) to different sizes of loops (random, condensin)
Proteins involved in mitotic chromosomes structure
Condensin 1 (SMC2&4)
Condensin 2 (SMC2&4)
Topoisomerase 2A
Cohesin and codensins = same family of protein complexes
Defined by 2 components:
1) SMC proteins
2) non SMC subunits
SMC proteins
Structural maintenance of chromosome (proteins) (ATPases)
Single molecule - 2 heads (walker donains) on n and c terminals, hydrolyse ATP
In between coiled-coil with hinge in the middle
Always a dimer (2 SMC proteins dimerise via hinge domain, conserved)
Eg smc1&3 in humans same as psm1&3 in yeast (cohesins)
Cap-e & cap-c in humans same in frogs (condensin)
SMC proteins + non SMC components for multi protein complexes, important for different functions related to genome maintenance
1)cohesin, condensin, SMC5/6 = genome organisation during interphase, mitosis and meiosis, regulation of transcription and repair of DNA
2) ring like structures mostly to embrace DNA molecules
3) functions over lap (in vitro studies)
4) ATP hydrolysis helps dimerisation, DNA binding & maybe allows the movement of protein along DNA
5) non SMC subunits of SMC proteins = regulators of multiple processes eg loading of complexes onto DNA
Cohesin
1) essential for major nuclear functions related to DNA organisation - chromatin loops and TAD formation
2) loading and off loading chromatin regulated by Wapl (release from DNA) and sororin (binding to chromatin)
3) responsible for sister chromatin cohesion established in a phase (acetylation of SMC3 necessary for stability of cohesion)
4) interphase role - DNA repair, positioning of congestion’s along genome regulates transcription (maybe due to loops and TADs)
5)shortly before and during mitosis cohesin is removed from chromosomes in 2 steps, needed for chromosome segregation, most removed early before mitosis, small amount left bound to centromeres until just before anaphase
How does cohesin complex interact with DNA
Different ideas - not fully known, can’t assume 1 binding is in all organisms
Current model - one complex of cohesin, DNA goes through centre
(PHOTO)
How is cohesin complex loaded onto chromatin
Scc2 & Scc4 are loading factors
Acetylation of cohesin so it can bind to DNA
After DNA replication cohesin holds sister chromatids together
How is cohesin complex removed from chromatin
2 steps
1) prophase pathway (mitotic kinases phosphorylate targets and protein Wapl) - cohesin on the arms of chromosomes
2) metaphase/seperase pathway (APC/C and seperase) - digest component of cohesin sitting at centromeric region
So separated by the time anaphase begins
Condensins
SMC2 & 4
1) Involved in chromosome condensation during early prophase
2) May play role in interphase but dk
3) Complex has ability of sliding along DNA, atp dependent
4) Condensin complexes play different roles in chromosome condensation
Alternative ways condensin may work to condense chromosome
1) random so mesh of loops
2) loops via loop extrusion (CURRENT IDEA)
Consensin 1 and 2 localise to chromosomes differently
Antibody immuno fluorescent staining,
Immuno fluorescent anti G (condensin 1) anti D3 (condensin 2),
anti g/D3 - g green, D3 orange, not a lot of yellow so not a lot of overlap
Quantitative super resolution imaging shows exact localisation and no. Of condensin 1 & 2 complex within motor chromosomes
Condensin 1 : condensin 2
4:1
Condensin 1 = mini loops(fingers) (peripheral of chromosome)
Condensin 2 = big loop (palm) (more central of chromosome)
Condensin 1 & 2 are involved in mitotic chromosome formation
1) condensin 2 associates with chromatin in prophase, condensin 1 is cytoplasmic so can only interact with chromosomes after bucked envelope breakdown
2) condensin 2 and not - is required for chromosome condensation in early prophase
3) condensin 1 is needed for complete dissociation of cohesin from chromosomes arms, chromosome shortening and normal timing of progression through prometaphase and metaphase. Condensin 2 lvls dispensable for these processes
Topiisomerase 2
Enzymes which introduce transient DNA breaks to relax supercoiled DNA and remove catenaries (cut, move across and ligate)
Play role in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability
Deregulation of it can cause neurodegenerative diseases, immune diseases and cancer
May use RNA as substrate as well
Topo 2 present
Proper chromosomal organisation
Equal genome partitioning, genome integrity
Topo 2 depleted
Defective sister chromatid resolution, defective chromosome compaction
Unequal genome partitioning, failed cell division (tetraplody, DNA breaks, micro nuclei formation, loss of genome integrity, chromosome rearrangements
Topoisomerase 2 - what do we know?
Topo 2 localise to central axis of mitotic chromosomes
Suggested: component of chromosome scaffold, unclear
Chromosome condensation - topo 2 shortens chromatids (axial compaction)
Resolving of sister chromatids (efficient and unperturbed chromosome separation)
Likely functions regulated differently in different species
Untangling function of topo 2 affected by condensin 1
Topo 2 has tile in preservation of chromosome compaction (2020)
Study: introduce “degrom” - special fragment of protein that leads to degradation of protein
Removal of topo 2 prior to mitosis did not affect prophase timing of initiation of chromosome condensation. Instead prevents chromatin condensation in prometaphase, extends the length of prometaphase and causes cells to exit mitosis without chromosome segregation occurring
Leads to bunuckeation, giant mononucleation, multinucleation
2020 study of role of topo 2
Arrest cells through purifying
Reach certain stage and then degrade protein, lead to loss of protein, see the cell cycle after
Essential for maintenance of mitotic chromosomes
Proposed steps in folding of chromatin fibre into mitotic chromosome
11nm fibre
liner looping
(Protein complexes will serve as bases for chromatin loops)
axial compression
lateral compression
Steps of mitotic chromosome condensation and role of major structural regulators
G2 - cohesin interphase chromatin
Early prophase LINEAR LOOPING - load condensin 2
Late prophase AXIAL COMPRESSION- topo 2, condensin 2 on, loss of cohesin
Metaphase LATERAL COMPRESSION - condensin 1
Current view on mitotic chromosome formation
“Pathway for mitotic formation 2018”
Achieving high lvl of synchrony key to understanding architecture of genome changes upon mitotic entry
DAPI, Hi-C, TADs
Different stages of cell cycle eg 4 stages of prometaphase
TADs and compartments disappear over time in cell cycle (g2 to end of prometaphase)
Conclusion: compaction of chromatin which is dependent upon presence of condensin 2&1 complexes
Prometaphase: 80kb inner loops nested within 400kb outer loops
Spiral staircase condensin scaffold
Staircase scaffold
During loop extrusion individual condensins that translocate DNA will eventually bump into eachother
Loops associated with each complex are arranged into helical spiral
Condensins line the longitudinal axis of chromosome around which loops radiate
Mitotic bookmarking
Cells lose structure in terms of chromatin organisation, but factors help cells identify itself with certain tissue or transcriptional profile
1) mitotic bookmarking by transcription factors - many degraded and displaced before mitosis but individual (bookmarking) factors that will remain and recruit drive transcription more efficiently
If genome is remodelled during reach cell division, how is the cellular memory preserved
1) Mitotic bookmarking
2) preservation of Histone modifications through mitosis
3) mitotic transcription
preservation of Histone modifications through mitosis
Histone code helps in mitotic bookmarking, keeps memory from 1 cell generation to another
Bookmarking active genes, active histones PTMs maintained or increases at promoters or enhancers
Bookmarking repressed genes, repressive Histone PTMs maintained and reinforced
Mitotic transcription
Transcription maintained at basal levels during mitosis
Interphase - presence of enhancer-promoter loops, efficient transcription
Mitosis- loss of enhancer-promoter loops, low level enhancer independent transcription (thousands of genes)
Post mitotic gene activation
Cascade of gene reactivation from low levels seen in mitotic cells
Timing of reactivation of different classes different: initial genes relating to rebuilding cell and later genes relating to specialised functions in somatic cells. Pluripotency associated genes are first to be reactivated in stem cells
Mitosis specific Histone modifications (mitotic Histone code) examples:
Histones H3Thr3ph (close to n terminus)
Enzyme: haspin kinase
Effect: recruitment of chromosomal passenger complex (aurora b complex) to inner centromeres
Histone H3Ser10ph
Enzyme: aurora b kinase (adds mark)
Effect: displacement of HP1 from chromosome arms, recruitment of condensin complex to chromatin
DNA attached to microtubules
Microtubules same place as cenp-a (centromere)
Microtubules attach to kinetichores
Centromeres
Fragment of DNA on chromosome
Chromatid - centromere - kinetichore - Microtubules
Centromere important because it dictates where the kinetochore forms
Centromeres present throughout whole cell cycle
Mitotic only - kinetochores and Microtubules
Structure and composition of centromeres differ between species
Yeast - point centromere- DNA precisely sequenced/defined, bind cenp-a attached to one Microtubule 125bp
S.Pombe - 35-110 kbp. Number of Microtubules bound to each 2-4
Human - number of Microtubules bound to each centromere ~20
What is the role centromeric DNA
Centromeres are defined epigenetic ally and not by DNA sequence
Centromeric chromatin is assembled on centromeric DNA. Centromeric DNA = template where centromeres are built
Centromeric transcripts are important for centromere establishment and function
Centromeric DNA is methylated
Vertebrate centromeric DNA contains CENP-B boxes that bind centromeric protein CENP-B, functionally not fully understood
Point centromeres require well defined sequence for centromeres to work eg s.cerevisae
Regional centromeres established epigenetic ally, specific DNA sequence insufficient and not required for the establishment of centromeres eg humans
Example of DNA sequences composing point centromeres of s.cerevisiae and regional centromeres of other model species
A.cerevisiae 125bp - CDE I,II &III
Patterns eg humans alpha satelites
Mouse major and minor satelites
Different origin and arrangement of DNA sequences in different centromeres
Eg satDNA - nearly identical on all centromeres
Chromosome specific satDNA subfamilies
satDNA intermingled with retrotransposons
Retrotransponoson based centromeres
Repeat based and reappear free centromeres
Holicentric chromosomes
SO DNA SEQUENCES NOT EVOLUTIONARY CONSERVED but function conserved
Monocentric vs holocentric centromeres
Monocentromere - normal human so centromeres in the middle, pulled apart from that middle but
Holocentromwre - centromeres all around chromatid, pull apart from different areas
But same role
Epigenetic establishment of centromeres relies on the presence of centromer specific Histone H3 variant called CENP-A
Critical for formation of centromeres
CENP-A with loop insert in Histone fold area
Human HFD is only 62% identical with canonical varients so not as conserved as normal histones
Centromere function and kinetochore assembly
What is special about CENP-A
CENP-A nucleosomes may assemble on any DNA, in vivo centromeres preferentially established on repetitive sequences
Unique motifs with sequence of CENP-A allow for binding of proteins that do not bind to other H3 variants
Regulation of loading of newly synthesised CENP-A into chromatin is special: CENP-A-specific Histone chaperones and cell cycle timing different from canonical histones
Some structural features of nucleosomes with incorporated CENP-A are different from other Histone variants
Chromatin built of CENP-A-containing nucleosomes is different from chromatin formed from H3 containing nucleosomes
When are histones loaded onto DNA
S phase - on newly synthesised DNA
Important parts of Histone for loading and maintenance of new CENP-A
1) insertion - CENP-A targeting domain (CATD)
2) CENP-C binding
Experimentally: took CATD, put into any other Histone, that Histone behaves like CENP-A
Loading of CENP-A
S phase normal histones
CENP-A loaded late M/early G1 phase
Certain amount of CENP-A in late g1, s phase diluted by 2, half maximal CENP-A occupancy at M phase, loaded fully at next g1, but again diluted in s phase
What we need for new histones to be loaded into Nucleosome: M18[BP1] alpha, beta and bp1
MIS18 complex = life sensing, mark when HJURP will bring new CENP-A. MIS18 binds to CENP-C
Chaperone: HJURP (physically loads CENPa to replace Histone h3 in Nucleosome)
Kinases regulating this activity: CDK (inhibits loading of CENP-A so negative regulator), PLK1 (mitotic kinase, positive regulator)
Other proteins: RSF1 & MagRacGAP - maintenance of CENP-A on chromatin & cytokinesis
CENP-A bioinformatics as a dimer with Histone 4 to replace some h3 and h4
MIS18 marks where loading will take place
Loading of new CENP-A problems
Do not want more than one centromere per chromatid
New centromere forms I’m correct place, old centromere needs to be removed (done through ubiquitination)
Centromeric chromatin is different from canonical euchromatij or heterochromatin
CENP-A containing Nucleosome arrays are more condensed
DNA at the nucleosomes entry/exit sites is less constrained
Centromeric chromatin is enriched in other core Histone variants eg H2AZ (on average more H2AZ at centromeric chromatin than others)
Centromeric chromatin exhibits Histone modifications pattern distinct from euchromating and heterochromatin
Binding of linked histones to CENP-A containing nucleosomes is hindered
Centromere specific set of proteins called constituatuvr centromere associated network (CCAN) binds human centromeric chromatin
Transcription of centromeric chromatin is necessary for proper establishment of centromeres, kinetochore assembly and chromosome segregation in mitosis
Long non coding RNAs also play a role in centromeric bio (binding of non coding RNA important for establishment of centromeric chromatic, CENP-C binds to these)
CCAN
Set of special proteins that’ll bind to centromeres (only - most not all)
CCAN details overview
Constitutive centromere associated network
Assembly of proteins and complexes located at CANP-A containing nucleosomes
Part of centromeric chromatin supporting CENP-A incorporation and kinetochore formation
CCAN sub complexes eg CENP L & N, CENP S & X, CENP T and W
CENP-C and CENP-N bind CENP-A directly (necessary for retention of CENP-A at centromeres
CCAN subcomplezds associate with centromeric chromatin
High degree of interdependence among CCAN components
Some CCAN subunits play similar roles in maintenance of centromeric chromatin and liner to hire assembly and chromosome segregation
Multiple functions of CCAN components
KMN recruitment - CENP - T,W,S,X but also c, H, I, K,m
Chromosomes congression - OPQUR but also HIKM
CENP A Nucleosome binding - CLN
CENPT complex may form a Nucleosome structure (resembles core Nucleosome structure) bind DNA in similar way
CCAN STRUCTURE
KMN recruitment - CENP - T,W,S,X but also c, H, I, K,m
Chromosomes congression - OPQUR but also HIKM
CENP A Nucleosome binding - CLN
CENPT complex may form a Nucleosome structure (resembles core Nucleosome structure) bind DNA in similar way
CCAN DETAILS
CCAN components present at centromeres throughout cell cycle
CENP-C most upstream and important
Organisms eg drosophila melanogaster and c Elagans which don not have CCAN but have CENP-C
Functions of CENP-C
CENP-A loading via MIS18 complex binding
CENP-A maintenance at centromeric chromatin
(2 different CENP-A binding sites)
CCAN formation and maintenance
Binding of centromeric RNAs (important for centromere and kinetochore activities)
Kinetochore formation during mitosis
When are kinetochores and centromeres present
Centromeres: throughout cell cycle
Kinetochores: mitosis
Major functions of kinetochores
Generate stable Microtubule attachment attachment needed for correct chromosome segregation during prophase
Involved in harnessing the forces needed for chromosome movement
Specialised kinetichore components responsible for detection and repair of incorrect Microtubule attachment
Kinetochore assembly
CENP-C Microtubule built on
Centromere to kinetochore: important kinases CDK1 and aurora B
Complicated structure made of many proteins
KMN network - core kinetochore super complex
KNL1/Spc105
Mis12/MIND
Ndc80
Protein network directly linking centromeric chromatin with Microtubules
Structural core - helps bind other proteins to kinetochore
2 independent assembly pathways: CENP-C and CENP-T
Assembly regulated in cell cycle:KMN network binds centromeres during mitosis only
CENP-C driven KMN network
Mis12 made of 4 components and binds to CENP-C n terminus
Other end platform for binding of KNL1 (unstructured other than where bound to MIS12) and NDC80 complex (4 components: SPC24 & 25, NUF2, coil coiled arm)
NDC80/HEC1 will directly bind Microtubules during mitosis
Can bind one KMN network complex on each CENP-C molecule
Mis12 complex
4 proteins: NSL1, DSN1, MIS12, PMF1
2 heads independent: regulation, closest to CENP-C
Head 2: Long unstructured fragment of DSM1- free to interact with other proteins
CENP-C will bind to head 1, “tie around head”, chromosome can be moved via this
Interphase to mitosis transition: binding between head 2 and 1 so no binding of CENP-C as DSN1 tail blocks site, DSN1 tail is phosphorylated by aurora B and no longer binds head 1, head 1 free to interact with CENP-C
CENP-T pathway
Max 3 KMN network binding
Can recruit Ndc80 complex without Mis12 as CENP-T may bind to it directly
Regulation of CENP-C and CENP-T
CENP-T Regulated via phosphorylation by CDK1 mitotic kinase
CENP-C regulated by aurora B (mitotic kinase)
No activity before mitosis
CENP-c bound to CENP-a containing Nucleosome (CDK1 independent)
CDK1 will phosphorylate residues on tail of CENP-T (CENP-T makes own Nucleosome like structure) CDK1 creates 3 sites for KMN network complexes or NDC80c of Mis12c
KMN NETWORK SUMMARY
3 complexes: KNL1, Mis12, Ndc80 - core kinetochore (KMN network)
Recruitment of KMN complexes regulated by mitotic kinases aurora B and CDK1/cyclin B
Assembly of KMN network on centromeres occurs 2 independent competitive pathways: CENP-C and CENP-T
1 Ndc80 complex via CENP-C vs 3 via CENP-T
KMN network provides direct link between centromeres and Microtubules of mitotic spindle bound by Ndc80 complex
KMN network (esp KNL1/spc105) is platform for many other proteins to bind to
KNL1/spc105
Major interaction platform for many kinetochore proteins - recruitment of proteins involved in SAC activity
Can bind Microtubule at n terminus unknown why
Error/correction
Spindle assembly checkpoint (SAC)
Delay the anaphase onset until last kinetochore properly attached to Microtubules/ mitotic spindle
If Ndc80 complex is not bound to Microtubule it is sensed by Mps1 which theough phosphorylation of KNL1 will inform other spindle checkpoint components - pauses and recruits eg BubR1 complex
Proper attchement, spindle assembly checkpoint gets silenced and no longer inhibits anaphase onset by removing KNL1 phosphorylations and so BubR1 complex removed from kitechore
What other proteins are present at kinetochores?
Spindle assemble checkpoint (SAC) components
Kinesics and other motor proteins - Microtubule dynamicity and transport along Microtubules
Major cell cycle regulators - kinases eg CDK, phosphates eg PP1, PP2A
Other regulatory proteins, mainly regulators of mitotic spindle eg CLASP family proteins
Microtubules polymerise and depolymerise constantly - quickly and slowly so lots of proteins to affect stability of Microtubules
100-200 different proteins at different cell cycle phase to centromeres or kinetochores
Most complex machinery
Role:establishment of centromeres and regulation of Microtubule binding and activities of mitotic spindle
Role of kinetochore: molecular mechanism for proper chromosome segregation
How are kinetichore la organised 3D
Most Nucleosome in centromeric regions are H3 containing Nucleosome
CENP-A minority
Linear - interspersed so reacts CENPa and h3, in 3D very CENP-A close to eachother, face external side of each chromatid where kinetochore will be formed later
Evolution of centromeres and kinetochores
Differences at protein level (kinetochore proteins)
KMN network - not many species have same proteins but conserved at structural level, protein sequence level poorly conserved
Human most complicated - 2 pathways unlike drosophila that only have CENP-A
Many insects lack CENP-A
Hollow centromeric insects eg butterflies and moths
Most have Ndc80
Centromeric and kinetochore component my may be localised to defined areas within or around centromeres of mitotic chromosomes
Centromere: CCAN, MIS18 complex, HJURP
Kinetochores: KMN network, kinases, phosphatases, motor proteins and SAC components
Inner centromer: aurora B comped, cohesin
