Download X-inactivation

Chromosomal basis of heredity
RNDr. Z.Polívková
Lecture No135 – Course:Cell structure
History of chromosomal study:
 1903 - Sutton and Boveri – chromosomes are related to heredity
 1923 - Painter – chromosomal number = 48 = uncorrect
 1956 - Tjio and Levan – chromosome number in man = 46
 1959 - Lejeune et al.- 1st chromosomal abnormality = trisomy 21 in
patients with Down syndrome

Jacobs and Strong - 47, XXY karyotype in males with
Klinefelter syndrome

Ford et al. - monosomy X in females with Turner syndrome
 1960 - Patau et al. – trisomy 13 in patients with Patau syndrome

Edwards et al. – trisomy 18 in patients with Edwards syndrome
 1966 – Steel, Breg – determination of chromosome constitution from
amniotic fluid
 Cytogenetics – study of chromosomes
 Clinical cytogenetics- study of chromosomal
abnormalities
Ultrastructure of chromosomes
 DNA
 Histones – basic proteins
H1,H2A, H2B,H3,H4
 Non histones proteins – neutral or slightly acidic
The whole length DNA 2 m
Human genome contains 20-25 000 structural genes
= protein coding genes
= small fraction of the genome (1.5%)
Organization of chromatin in interphase
Nucleosome
 DNA double helix + histone core
 Histone core = octamere of two copies of H2A, H2B, H3, H4
 DNA double helix is winded around the histone core,
 spacer segment of DNA between two nucleosomes is free
or associated with H1 histone (appearance of beads on a
string)
= condensation to 1/10 of native DNA length
 String of nucleosomes is coiled into solenoid
(6 nucleosomes in each turn)
= fundamental unit of chromatin fiber
Condensation of chromatin into chromosomes
 Solenoid is packed into loops attached to the
nonhistone protein scaffold (Laemli loops) =
chromosome in prophase – 1/3000 of native length
 Chromosome in metaphase = nonhistone protein
scaffold with loops is coiled into spiral structure of
chromatids
 Many steps of coiling = DNA is shortened to 1/10000
of its native length
Human chromosomes – morphology
Chrom. metacentric
submetacentric
acrocentric
p
telomere
centromere
q
chromatids
p = short arm
q = long arm
NOR = nucleolus organizer region (rRNA genes)
satellite
sat. stalk (NOR)
Centromeres
Centromere (primary constriction) = DNA + histones (mostly
α-satellite DNA = large numbers of short tandemly repeated
sequences)
Kinetochore = complex proteinaceous structure at centromere
– mediates attachment of spindle microtubules and chromosome
movement in metaphase and anaphase
Centromere malfunction → nondisjunction (error in distribution
of chromosomes during division)
Nucleolus organizer
Nucleolus - located in nucleus – not bounded by membrane
= site of transcription and processing of rRNAs, site of assembly of rRNA
and proteins into two ribosomal subunits (subunits join to form
cytoplasmic ribosomes)
nucleoli disappear during mitosis, formed at telophase at specific sites of
acrocentric chromosomes (satellite stalks of chromosomes Nos
13,14,15,21,22 = nucleolus organizer region (NOR)
nucleoli - tendency to fuse together – satellite association
NORs contain tandemly repetitive ribosomal RNA gene clusters
variability in the length of this region (number of rRNA genes on
each acrocentric is variable (10 -100 copies)
Telomeres
 tandemly repeated TTAGGG/CCCTAA sequences
(several thousand times)
 protects chromosome ends from degradation and from
fusions
(telomeric DNA is packed to loops and asociated with
proteins – i.e. protected from exonucleases that attact free
ends of DNA )
 essential role in pairing of homologs in meiosis
 association of telomeres with nuclear envelope
Replication of telomeres:
 enzyme telomerase (= ribonucleoprotein complex = reverse
transcriptase – synthesizing DNA from RNA template)
 telomerase is abundant in embryonic and cancer cells, expresed
highly in stem cells
low, almost undetectable activity in somatic cells
 reduction of telomere length after each round of replication →
cellular aging, or senescene (Hayflick limit – cell dies after
certain number of cell division - due to the shortening of
chromosomal telomeres to critical length)
 cancer cells are immortal (high level of telomerase activity)
Mouse telocentric
chromosomes
FISH with telomeric probes
Human chromosomes:
22 pairs of autosomes
1 pair of gonosomes (heterochromosomes)
 Karyotype: man 46, XY, woman 46, XX
Chromatin – consist of:
basic proteins (histones), DNA,
nonhistone proteins, small amount RNA
Euchromatin (active form of chromatine, transcribed)
 despiralized in interphase
 spiralized in mitosis
 contains structural genes
Heterochromatin

repetitive sequences (in constitutive-stable heterochromatin)

not transcribed into mRNA (=inactive)

partially folded in interphase

late replicating

tendency to form condensed clumps adjacent to nuclear membrane

Constitutive (stable) – at centromeres of all chromosomes,
blocks of heterochromatin on 1q, 9q,16q, Yq (Y chromatin),
tandemly repeated sequences (satellite DNA)
length variability of heterochromatic parts - origin by unequal crossing-over
 Facultative (reversible) - structurally euchromatin, but
behaves as heterochromatin (potentially transcribable
sequences that are specifically inactivated
 = one of two X chromosomes in women = genetically
inactive, late replicating (replication at the end of S phase)
= X chromatin (sex chromatin = Barr body)
Karyotype 46,XX – G bands
Karyotype 46,XY – G bands
Heterochromatin
1. Richness in satellite DNA (= tandemly repeated sequences) - in
constitutive heterochromatin
2. Stability: constitutive heterochromatin is stable, facultative is reversible
(inactive X is reactivated before meiosis)
3. Staining: constitutive heterochromatin is strongly stained by C-band
technique
C-banding = specific staining of heterochromatic parts (=strong
denaturation of all euchromatic parts – pale, resistant heterochromatin
is darkly stained)
4. Polymorphism : constitutive heterochromatin is polymorphic in size
and localization (instability of satellite DNA) – without phenotypic effect
Properties of heterochromatin:
1. condensation (both constitutive and facultative)
2. late replicating (both constitutive and facultative,
inactive X replicates at the end of S phase)
3. methylation (on cytosines)
4. histones in heterochromatin are hypoacetylated
(hyperacetylated histones are in active chromatin)
Histone modification, DNA methylation
and chromosome condensation
Histone acetylation removes positive charge of histones – thus reducing
the force of attraction with DNA = open chromatin (active)
Deacetylation of histones restores positive charge leading to close
attraction betwen histones and DNA (condensed chromatin
structure – inactive – not accesible for transcription factors)
Numerous transcription factors have either activity Histon Acetyl Transferase
(HAT) – in activation of transcription
or Histon De-Acetylases (HDAC)- in repression of transcription
HDAC = multiprotein complex - contains methyl cytosin binding proteins
(MeCP1, MeCP2) - selectively bind methylated DNA
HDAC is targeted to methylated DNA (CpG)
Other histone modifications: phosphorylation, methylation – chrom.
condensation
5. histones in heterochromatin are methylated on lysine
- methylation of histones creates binding site for heterochromatic
protein HP1 – role in organisation of heterochromatin
6. Heterochromatin is transcriptionally inactive
constitutive heterochromatin does not contain any genes
facultative: genes are not usually transcribed
7. Heterochromatin does not participate in genetic recombination
polymorphism of heterochromatic regions - difficulties in
homologous pairing
8. Tendency to agregate during interphase
agregation of short arms of acrocentrics – nucleolus organiser
region=NOR
9. Role of nuclear RNA in formation of facultative heterochromatin –
X inactivation (mRNA – product of gene XIST)
Function of heterochromatin:
1. Heterochromatin and euchromatin occupy different domains.
heterochromatin is on periphery of nucleus attached to nuclear
membrane
Active chromatin – central position in nucleus, it allows
maximal efficiency of replication and transcription
2. Centromeric heterochromatin - role in centromeric function –
in cohesion of sister chromatids and normal disjunction of
chromatids
3. Role in epigenetic regulation of gene expression
during differentiation: probably certain active genes are
transported into heterochromatic domain to become inactive
X-inactivation – Lyon´s hypothesis (Lyon 1961)
 only one X is active in somatic cells of all mammalian
females, second one (or all others) is inactive
(methylated) = condensed in interphase = stained as
X-chromatin = Barr body (described by Barr and Bertram in 1949)
 inactivation begins in early embryonic development
(probably at 1000- to 2000- cell stage)
 inactivation is random (according to parental origin of X)
 inactivation is stable in all daughter cells (descendant of any
cell that underwent X-inactivation)
 woman = mosaic of cell with inactive paternal and
maternal X chromosomes
 inactivation is not complete – some genes on X chrom.
escape inactivation
 in oogenesis both X are active – reactivation before meiosis
 structurally abnormal X - nonrandom inactivation
- chromosomal abnormality (rearrangement) balanced
(no material additional, no material missing) –
preferentially normal X is inactive
- chromosomal abnormality unbalanced (gain or loss of
genetic material) – abnormal X is inactive
Nonrandom inactivation in case of chromosomal
abnormalities = consequence of selection
Barr body
X inactivation
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
XM XP
 X-inactivation centre on Xq133, gene XIST X-
inactivation controlled by XIST mRNA - expressed
only on the inactive chromosome
 Screening method for sex determination - sex
chromatin examination in some sports disciplines
Unbalanced aberration - terminal deletion Xp
Nonrandom X inactivation (detected by BUDr method):
unbalanced aberration - terminal deletion of Xp
The abnormal X chromosome is inactive - late replicating (pale) in
all cells tested
Unbalanced aberration –ring chromosome X
Nonrandom X inactivation (detected by BUDr method):
unbalanced aberration – ring chromosome X
The abnormal X chromosome is inactive - late replicating (pale) in all
cells tested
Balanced aberration – reciprocal X/A translocation
Nonrandom X inactivation (detected by BUDr method):
Balanced aberration – X/autosomal reciprocal translocation
Normal X is inactive - late replicating (pale) in all cells tested
Thompson &Thompson: Genetics in medicine,7th ed.
Chapter 2: The human genome and chromosomal basis of
heredity, parts: Organization of human chromosomes, Human
karyotype
Chapter 6(part): X chromosome inactivation
+ informations from presentation
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