the human genome and the chromosomal basis of heredity · the human genome and the chromosomal...

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 !

• Retrotransposons (copy-and-paste)

• Transposons (cut-and-paste | inactive)

4.6%4%

3%

20%

Transposons

TPRT:Target-site primed reverse transcription

Model of L1- / Alu-retrotransposition

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 ?

Linker DNA(10 bp – 60 bp)

DNA is not naked in the cell(nucleosome)

basic unit of chromosomes =nucleosome


DNA is not naked in the cell

basic unit of chromosomes =nucleosome


H1

DNA is not naked in the cell

1. Histone-modifying enzymes (N/C-tail posttranslational modification)

2. ATP-dependent chromatin-remodelling enzymes3. Histone-variants

Histone code

Chromatin (structural) modifications play key roles in DNA-related processes

A. Histone modifications:

B. DNA modification: DNA CpG methylation

Chromatin modifications are important for DNA-relatedprocesses:

-DNA-transcription (gene expression/repression)-DNA-repair-DNA-replication-DNA-compaction-chromosome segregation…

- correct segregation(capture microtubules)- chromosome movements

Centromere

- protect against degradation, fusion and recombination- complete end replication- chromosome movements- subtelomeric gene expression

Telomeres

- replication of the genetic information once per cell cycle

Origins of replication

Sister chromatids

Microtubules

Kinetochore

Functional domains

Chromosomes ensure transport and integrity of geneticinformation

Constitutive proteins are permanently associated with the centromere even during interphase, whereas facultative proteins are recruited only during mitosis to assemble the full kinetochore.

(1) Centromere

BA

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

Centromere function is epigenetically regulated

B. Neo-centromeres without alpha-satellite DNA

Centromere DNA elements

Localised centromeres

Diffuse centromeres

FEBS Letters 582 (2008) 1950–1959

Histone H3 variant CENP-A is the best candidate to carry the epigenetic centromere mark

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


Centromere


Telomeres



Sister chromatids

Microtubules

Kinetochore

Functional domains


Nucleoprotein complex: (TTAGGG)2500 + shelterin

TRF1 Telomeric Repeat-Binding Factor 1TRF2 Telomeric Repeat-Binding Factor 2Rap1 Telomeric Repeat Binding Factor 2, Interacting ProteinTIN2 TRF1-Interacting Nuclear Factor 2TPP1 TIN2 And POT1-Interacting ProteinPOT1 Protection Of Telomeres 1

Nucleoprotein complex: (TTAGGG)2500 + shelterin


Centromere


Telomeres



Sister chromatids

Microtubules

Kinetochore

Functional domains


ORC (multiprotein origin of replication complex)

Origins of DNA-replication

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

Interphase

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

DNAtubulin

Interphase

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

Interphase

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

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

‘molecular glue’ between replicated DNA-molecules (sister chromatids)Smc1: Structural maintenance of chromosomes protein 1Smc3: Structural maintenance of chromosomes protein 3Scc1: Sister chromatid cohesion protein 1Scc3: Sister chromatid cohesion protein 3

Sister chromatids of a chromosome

Loss of sister chromatid cohesion during mitosis

Metaphase

‘prophasecycle’



Prophase

Primary constriction

Prometaphase Metaphase


Metaphase

‘prophasecycle’



Prophase

Primary constriction


‘prophasecycle’

‘separase’

‘point of no return’

MetaphaseProphase Anaphase



Pro(meta)phase

Metaphase

Metaphase

Anaphase

Cohesin complex

Metaphase

Metaphase

Anaphase

Pro(meta)phase


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)

Haploid gametes4 x (n)

meiosis I: reductional division

- separation of homologous autosomes- separation of sex chromosomes

meiosis II: equational division

separation of sister chromatids(~mitosis)

Chromosomes replicated once








Chromosomes replicated onceCrossovers -> genetic diversity

-> correct homologue segregation








Chromosomes replicated once

Random assortment: 223 = 8388608

Crossovers -> genetic diversity-> correct homologue segregation

2.Zygonema 4.Diplonema

Bouquet formation

3.Pachynema

Tijdens profase I van meiose I worden chiasmata (genetische crossovers) gevormd. Profase I duurtbijgevolg lang en kan in verschillende stadia worden ingedeeld afhankelijk van de ‘nucleusarchitectuur’: homologe chromosomen aligneren, paren, gaan in synapsis en recombineren. HetDNA wordt hiertoe ondermeer opzettelijk beschadigd tijdens leptonema. De chromosomen zulleneveneens condenseren.

1.Leptonema

DSB

SC

Genetic crossover

DSB

5.Diakinesis

Sister chromatids of the maternal homologue

Meiotic cohesin complex(SMC1β / SMC3 / REC8 / STAG3SMC1α / SMC3 / REC8 / STAG3SMC1β / 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.



DSB

SPO11

Sister chromatids of the paternal homologue

meiosis

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.

Mlh1Mlh3

Spo11

RPARad51Dmc1

Homologous Recombination(generation of genetic crossovers)

Maternal chromatid

Paternal chromatid

Szostak model

Crossover ± Gene conversion

Recombination nodules

>200 DSBs are generated <-> ~1 crossover per chromosome arm (~ sex)

Synthesis-dependent strand annealing

Majority of >200 DSBs is repaired as a non-crossover

Spo11

Homologous Recombination(non-crossovers)

Paternal chromatid

Maternal chromatid

Gene conversion



DSB

SPO11

Sister chromatids of the paternal homologue

meiosis

Synaptonemal complex

Transverse filaments (SCP1)

Axial/lateral element (SCP2 / SCP3)

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).

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

Late leptonema - Early zygonema


Late zygonema


Early-Pachynema


Mid-Pachynema

Analysis of prophase I :DSB formation and repair process / Homologue pairing and synapsis(Mlh1 (merker voor genetische crossover) – SCP3 - DNA staining)

Cross-over interference

h.PAR:2.6Mb Xp/Yp0.32Mb Xq/Yq

Diplonema


Analysis of metaphase I :20 bivalents in normal mouse meiosis(FISH Y – X – DNA staining)

Chiasmata

Mlh1Mlh3

Spo11RPARad51Dmc1


Maternal chromatid

Paternal chromatid

Szostak model



Maternal chromatid

Paternal chromatid

Szostak model


Maternal chromatid Replicated maternal homologue

Paternal chromatid

Replicated paternal homologue

Premeiotische S-faseProfase

separase

Cohesin rings at the chromosomal arms are opened, but remain intact at the centromeres

First meiotic division

separase

Cohesin rings at centromeres are opened

Second meiotic division

Physical dislocation of Sgo-PP2A allows phosphorylation and separase-mediated

cleavage of centromeric cohesin

Differences between spermatogenesis and

oogenesis

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”.

Mitosis

DNA-synthesis

Meiosis I

Meiosis II

Recombination is not random across chromosomes and differs according to sex

Sex-averaged recombination rate: 1.22cM per Mb(0.88cM / Mb in male // 1.55 cM / Mb in female) <> PAR

Physical mapGenetic map

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

DAPI1819

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)

the human genome and the chromosomal basis of heredity · the human genome and the chromosomal...

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