Download The human genome and the chromosomal basis of heredity

The human genome and the
chromosomal basis of heredity
• The genome
• The chromosome
 Mitosis
 Meiosis
• 3D
Thierry Voet
([email protected])
Grown body = 100 trillion somatic cells
DNA:
*46 chromosomes
• linear
• ~3.1 x 109 bp : autosomes + X + Y
• … genes?
*Mitochondrial DNA
• circular
• 16.6 x 103 bp
• 37 genes
Number mtDNA-copies is cell type dependent
+
Only maternal inheritance !
Transposons
• Retrotransposons (copy-and-paste)
20%
4.6%
4%
3%
• Transposons (cut-and-paste | inactive)
Model of L1- / Aluretrotransposition
TPRT:
Target-site primed reverse transcription
Integration in TTTT|A (preference for AT-rich regions)
1/100 are full lengths (genome-wide average = 900bp)
80-100 full-lengths L1 (n=6000) not active
Gene mutation for L1-insertion
LINE-1 products used for retrotransposition of SINEs,
mRNAs (-> processed pseudogenen) and retrogenes
Processed pseudogenes: defect copy of a gene consisting
only of exons (no introns, no promoter sequences)
retrotransposition
Retrogene: integration of cDNA at a promoter + selection
Testis-specific expression of intron-less retrogenes
(copies of genes from the X-chr)
Basic unit of a chromosome ?
DNA is not naked in the cell
(nucleosome)
Linker DNA
(10 bp – 60 bp)
DNA is not naked in the cell
basic unit of chromosomes =
nucleosome
Linker DNA
(10 bp – 60 bp)
DNA is not naked in the cell
H1
basic unit of chromosomes =
nucleosome
Linker DNA
(10 bp – 60 bp)
DNA is not naked in the cell
H1
basic unit of chromosomes =
nucleosome
Linker DNA
(10 bp – 60 bp)
DNA is not naked in the cell
H1
basic unit of chromosomes =
nucleosome
Linker DNA
(10 bp – 60 bp)
Chromatin (structural) modifications play key
roles in DNA-related processes
A. Histone modifications:
Chromatin modifications
are important
for DNA-related
1. Histone-modifying
enzymes (N/C-tail
posttranslational
modification)
processes:
-DNA-transcription (gene expression/repression)
Histone code
-DNA-repair
-DNA-replication
-DNA-compaction
-chromosome segregation…
2. ATP-dependent chromatin-remodelling enzymes
3. Histone-variants
B. DNA modification: DNA CpG methylation
Chromosomes ensure transport and integrity of genetic
information
Sister chromatids
Kinetochore
Microtubules
Functional domains
Origins of replication
- replication of the genetic information
once per cell cycle
Telomeres
- protect against degradation, fusion and
recombination
- complete end replication
- chromosome movements
- subtelomeric gene expression
Centromere
- correct segregation
(capture microtubules)
- chromosome movements
(1) Centromere
A
B
Constitutive proteins are permanently associated with the centromere even during
interphase, whereas facultative proteins are recruited only during mitosis to assemble the full
kinetochore.
Centromeric DNA:
Alpha-satellite or alphoid DNA at normal human chromosomes
Centromeric DNA:
Alpha-satellite or alphoid DNA at normal human chromosomes
Centromeric DNA:
Alpha-satellite or alphoid DNA at normal human chromosomes
One alphoid higher order repeat can be :
- specific for one chromomosome
- occurring on different chromosomes
Different alphoid higher order repeats can be co-existing on the same chromosome
Alpha-satellite DNA is not sufficient nor
necessary for centromere function
A. Pseudo-di-centric chromosomes
B. Neo-centromeres without alpha-satellite DNA
Centromere function is epigenetically regulated
Centromere DNA elements
Histone H3 variant CENP-A is the best candidate to
carry the epigenetic centromere mark
Localised centromeres
Diffuse
centromeres
FEBS Letters 582 (2008) 1950–1959
CENP-A is found at active centromeres
Current Opinion in Cell Biology 2008, 20:91–100
Achieving ordered chromatin structure at the centromere
Current Opinion in Cell Biology 2008, 20:91–100
Molecular players of functional centromeres
Chromosomes ensure transport and integrity of genetic
information
Sister chromatids
Kinetochore
Microtubules
Functional domains
Origins of replication
- replication of the genetic information
once per cell cycle
Telomeres
- protect against degradation, fusion and
recombination
- complete end replication
- chromosome movements
- subtelomeric gene expression
Centromere
- correct segregation
(capture microtubules)
- chromosome movements
Nucleoprotein complex: (TTAGGG)2500 + shelterin
TRF1
TRF2
Rap1
TIN2
TPP1
POT1
Telomeric Repeat-Binding Factor 1
Telomeric Repeat-Binding Factor 2
Telomeric Repeat Binding Factor 2, Interacting Protein
TRF1-Interacting Nuclear Factor 2
TIN2 And POT1-Interacting Protein
Protection Of Telomeres 1
Nucleoprotein complex: (TTAGGG)2500 + shelterin
Chromosomes ensure transport and integrity of genetic
information
Sister chromatids
Kinetochore
Microtubules
Functional domains
Origins of replication
- replication of the genetic information
once per cell cycle
Telomeres
- protect against degradation, fusion and
recombination
- complete end replication
- chromosome movements
- subtelomeric gene expression
Centromere
- correct segregation
(capture microtubules)
- chromosome movements
Origins of DNA-replication
ORC (multiprotein origin of replication complex)
Chromosomal segregation during mitosis
Haploid cell:
• n (# different chrs = 23; chromosome set)
• C (DNA-content) = ~ 3.5 pg
Diploid cell:
• 2n
• 2C
Nulliploid cells / Polyploid cells
interfaze
Metaphase
Prometaphase
Anaphase
Telophase
Prophase
Cytokinesis
Interphase
DNA
tubulin
Metaphase
Prometaphase
Anaphase
Telophase
Prophase
Cytokinesis
Interphase
Metaphase
Prometaphase
Anaphase
Telophase
Prophase
Cytokinesis
Interphase
Foutieve vasthechting van microtubuli aan kinetochoren komt tijdens de
prometafase meer voor, maar moet geremedieerd worden alvorens anafase start
Molecular glue between replicated DNA = cohesin complex
Prophase
Sister chromatids
of a chromosome
‘molecular glue’ between replicated DNA-molecules (sister
chromatids)
Smc1: Structural maintenance of chromosomes protein 1
Smc3: Structural maintenance of chromosomes protein 3
Scc1: Sister chromatid cohesion protein 1
Scc3: Sister chromatid cohesion protein 3
Loss of sister chromatid cohesion during mitosis
Prophase
Metaphase
‘prophase
cycle’
Sister chromatids
of a chromosome
Primary constriction
‘molecular glue’ between replicated DNA-molecules (sister
chromatids)
Smc1: Structural maintenance of chromosomes protein 1
Smc3: Structural maintenance of chromosomes protein 3
Scc1: Sister chromatid cohesion protein 1
Scc3: Sister chromatid cohesion protein 3
Prometaphase
Metaphase
Loss of sister chromatid cohesion during mitosis
Prophase
Metaphase
‘prophase
cycle’
Sister chromatids
of a chromosome
Primary constriction
‘molecular glue’ between replicated DNA-molecules (sister
chromatids)
Smc1: Structural maintenance of chromosomes protein 1
Smc3: Structural maintenance of chromosomes protein 3
Scc1: Sister chromatid cohesion protein 1
Scc3: Sister chromatid cohesion protein 3
Loss of sister chromatid cohesion during mitosis
Prophase
Metaphase
‘prophase
cycle’
Anaphase
‘separase’
‘point of no return’
‘molecular glue’ between replicated DNA-molecules (sister
chromatids)
Smc1: Structural maintenance of chromosomes protein 1
Smc3: Structural maintenance of chromosomes protein 3
Scc1: Sister chromatid cohesion protein 1
Scc3: Sister chromatid cohesion protein 3
Loss of sister chromatid cohesion during mitosis
Pro(meta)phase
Metaphase
Cohesin complex
Metaphase
Anaphase
Loss of sister chromatid cohesion during mitosis
Pro(meta)phase
Metaphase
Metaphase
Anaphase
Loss of sister chromatid cohesion during mitosis
Pro(meta)phase
Metaphase
Metaphase
Anaphase
Physical dislocation of Sgo-PP2A allows
phosphorylation and separase-mediated
cleavage of centromeric cohesin
Chromosomal segregation during meiosis
Meiosis: ‘to reduce’
Diploid somatic cell (2n)
Chromosomes replicated once
meiosis I: reductional division
- separation of homologous autosomes
- separation of sex chromosomes
meiosis II: equational division
Haploid gametes
4 x (n)
separation of sister chromatids
(~mitosis)
Meiosis: ‘to reduce’
Diploid somatic cell (2n)
Chromosomes replicated once
Crossovers
-> genetic diversity
-> correct homologue segregation
meiosis I: reductional division
- separation of homologous autosomes
- separation of sex chromosomes
meiosis II: equational division
Haploid gametes
4 x (n)
separation of sister chromatids
(~mitosis)
Meiosis: ‘to reduce’
Diploid somatic cell (2n)
Chromosomes replicated once
Crossovers
-> genetic diversity
-> correct homologue segregation
meiosis I: reductional division
- separation of homologous autosomes
- separation of sex chromosomes
Random assortment: 223 = 8388608
meiosis II: equational division
Haploid gametes
4 x (n)
separation of sister chromatids
(~mitosis)
Tijdens profase I van meiose I worden chiasmata (genetische crossovers) gevormd. Profase I duurt
bijgevolg lang en kan in verschillende stadia worden ingedeeld afhankelijk van de ‘nucleus
architectuur’: homologe chromosomen aligneren, paren, gaan in synapsis en recombineren. Het
DNA wordt hiertoe ondermeer opzettelijk beschadigd tijdens leptonema. De chromosomen zullen
eveneens condenseren.
1.Leptonema
2.Zygonema
3.Pachynema
4.Diplonema
SC
DSB
Genetic crossover
5.Diakinesis
Bouquet formation
DSB
Tijdens leptonema van profase I zullen meiotische cellen opzettelijk hun DNA beschadigen
(creatie van dubbelstrengige DNA-breuken (DSB)) door middel van het SPO11 proteïne. De
breuken zullen bij voorkeur hersteld worden door een intacte DNA-matrijs te gebruiken van het
homoloog chromosoom.
Sister chromatids of the
maternal homologue
Meiotic cohesin complex
(SMC1β / SMC3 / REC8 / STAG3
SMC1α / SMC3 / REC8 / STAG3
SMC1β / SMC3 / RAD21 / SA1/2)
DSB
SPO11
Tijdens leptonema van profase I zullen meiotische cellen opzettelijk hun DNA beschadigen
(creatie van dubbelstrengige DNA-breuken (DSB)) door middel van het SPO11 proteïne. De
breuken zullen bij voorkeur hersteld worden door een intacte DNA-matrijs te gebruiken van het
homoloog chromosoom.
Sister chromatids of the
maternal homologue
Meiotic cohesin complex
(SMC1β / SMC3 / REC8 / STAG3
SMC1α / SMC3 / REC8 / STAG3
SMC1β / SMC3 / RAD21 / SA1/2)
DSB
SPO11
meiosis
Sister chromatids of the
paternal homologue
Homologous Recombination
(generation of genetic crossovers)
Spo11
RPA
Rad51
Dmc1
Maternal chromatid
Szostak model
Paternal chromatid
Recombination nodules
Mlh1
Mlh3
Crossover ± Gene conversion
>200 DSBs are generated <-> ~1 crossover per chromosome arm (~ sex)
Homologous Recombination
(non-crossovers)
Spo11
Maternal chromatid
Paternal chromatid
Gene conversion
Synthesis-dependent strand annealing
Majority of >200 DSBs is repaired as a non-crossover
Tussen leptonema en pachynema zullen deze dubbelstrengige breuken hersteld worden als
crossovers en non-crossovers. De homologe chromosomen zullen hiertoe aligneren, paren en
in synapsis gaan (vorming van synaptonemal complex tussen homologe chromosomen).
Sister chromatids of the
maternal homologue
Synaptonemal
complex
Sister chromatids of the
paternal homologue
Meiotic cohesin complex
(SMC1β / SMC3 / REC8 / STAG3
SMC1α / SMC3 / REC8 / STAG3
SMC1β / SMC3 / RAD21 / SA1/2)
DSB
SPO11
meiosis
Axial/lateral element
(SCP2 / SCP3)
Transverse filaments
(SCP1)
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(γH2AX (marker for DSB)– SCP3 - DNA staining)
Leptonema
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Rad51 – SCP3 - DNA staining)
Early leptonema
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Rad51 – SCP3 - DNA staining)
Late leptonema - Early zygonema
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Rad51 – SCP3 - DNA staining)
Late zygonema
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Rad51 – SCP3 - DNA staining)
Early-Pachynema
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Mlh1 (merker voor genetische crossover) – SCP3 - DNA staining)
Mid-Pachynema
Cross-over interference
h.PAR:
2.6Mb Xp/Yp
0.32Mb Xq/Yq
Analysis of prophase I :
DSB formation and repair process / Homologue pairing and synapsis
(Rad51 – SCP3 - DNA staining)
Diplonema
Analysis of metaphase I :
20 bivalents in normal mouse meiosis
(FISH Y – X – DNA staining)
Chiasmata
Homologous Recombination
(generation of genetic crossovers)
Spo11
RPA
Rad51
Dmc1
Maternal chromatid
Szostak model
Paternal chromatid
Mlh1
Mlh3
Crossover ± Gene conversion
Homologous Recombination
(generation of genetic crossovers)
Maternal chromatid
Maternal chromatid
Replicated
maternal
homologue
Szostak model
Paternal chromatid
Paternal chromatid
Replicated
paternal
homologue
Crossover ± Gene conversion
Profase
Premeiotische S-fase
First meiotic division
separase
Cohesin rings at the chromosomal arms are opened,
but remain intact at the centromeres
Second meiotic division
separase
Cohesin rings at centromeres are opened
Physical dislocation of Sgo-PP2A allows
phosphorylation and separase-mediated
cleavage of centromeric cohesin
Differences between
spermatogenesis and
oogenesis
Mitosis
DNAsynthesis
Meiosis I
Meiosis II
Continuous from puberty till death.
4 spermatids per primary spermatocyte.
Discontinuous from fetal life till menopause.
1 definitive oocyte per primary oocyte possible.
More genetic crossovers.
Less “strict”.
Recombination is not random across chromosomes and differs
according to sex
Physical map
Genetic map
Sex-averaged recombination rate: 1.22cM per Mb
(0.88cM / Mb in male // 1.55 cM / Mb in female) <> PAR
The human genome and the
chromosomal basis of heredity
• The genome
• The chromosome
 Mitosis
 Meiosis
• 3D
Thierry Voet
([email protected])
Individual chromosomes occupy distinct
chromosome territories in the interphase nucleus
DAPI
18
19
Positioning of chromosomes in the nucleus is not exact, but also not random
(centromeres in the G1-phase nucleus at the periphery;
Non-overlapping territories; gene-poor <-> gene-rich)