The human genome and the chromosomal basis of heredity The genome The chromosome Mitosis Meiosis 3D Thierry Voet (Thierry.Voet@med.kuleuven.be)
Grown body = 100 trillion somatic cells DNA: *46 chromosomes linear ~3.1 x 10 9 bp : autosomes + X + Y genes? *Mitochondrial DNA circular 16.6 x 10 3 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: 1. Histone-modifying Chromatin modifications enzymes (N/C-tail are important posttranslational for DNA-related modification) processes: -DNA-transcription (gene expression/repression) -DNA-repair -DNA-replication -DNA-compaction -chromosome segregation 2. ATP-dependent chromatin-remodelling enzymes 3. Histone-variants Histone code B. DNA modification: DNA CpG methylation
Chromosomes ensure transport and integrity of genetic information Sister chromatids Microtubules Kinetochore 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 Microtubules Kinetochore 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 Telomeric Repeat-Binding Factor 1 TRF2 Telomeric Repeat-Binding Factor 2 Rap1 Telomeric Repeat Binding Factor 2, Interacting Protein TIN2 TRF1-Interacting Nuclear Factor 2 TPP1 TIN2 And POT1-Interacting Protein POT1 Protection Of Telomeres 1
Nucleoprotein complex: (TTAGGG) 2500 + shelterin
Chromosomes ensure transport and integrity of genetic information Sister chromatids Microtubules Kinetochore 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 Primary constriction 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
Prometaphase Metaphase
Loss of sister chromatid cohesion during mitosis Prophase Metaphase prophase cycle Primary constriction 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 Anaphase prophase cycle 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 Cohesin complex Metaphase 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: 2 23 = 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 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
Homologous Recombination (generation of genetic crossovers) RPA Rad51 Dmc1 Spo11 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) Axial/lateral element (SCP2 / SCP3) DSB Transverse filaments (SCP1) SPO11 meiosis
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) RPA Rad51 Dmc1 Spo11 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 (Thierry.Voet@med.kuleuven.be)
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)