Transcript Il codice genetico è a triplette e degenerato

Colinearità tra POSIZIONE dei geni sul cromosoma ed
ESPRESSIONE lungo l'asse antero-posteriore dell'embrione
Drosophila
Mammifero
Drosophila
Mammifero
The alpha and beta-globin loci
Capturing Chromosome Conformation
Job Dekker, et al. Science 295, 1306 (2002)
We describe an approach to detect the frequency of interaction between any two
genomic loci.
Generation of a matrix of interaction frequencies between sites on the same or different
chromosomes reveals their relative spatial disposition and provides information about
the physical properties of the chromatin fiber.
This methodology can be applied to the spatial organization of entire genomes in
organisms from bacteria to human.
Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features
of chromosome organization within the nucleus and dynamic changes in that
organization during meiosis.
We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that
chromatin is highly flexible throughout.
Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit
different conformations, and a population-average 3D model of chro- mosome III could
be determined.
Chromosome III emerges as a contorted ring.
Chromosome Conformation Capture (3C)
Analysis of the structure of chromosome III
during interphase - II
Population-average 3D model of chromosome III,
drawn with Truespace software. The model was
calculated using the set of 78 distances shown in (C).
The numbers correspond to the positions shown in Fig.
1C.
The AT-rich region in the right arm (positions 6 to 9) is
indicated in green, the GC-rich domains (position 2 to 6
and 9 to 12) are indicated in red, and the subtelomeric
Genome Res. 2006 Oct;16(10):1299
Chromosome Conformation Capture Carbon Copy (5C): A
massively parallel solution for mapping interactions between
genomic elements. J. Dostie, ... and Job Dekker
Physical interactions between genetic elements located throughout the genome play
important roles in gene regulation and can be identified with the Chromosome
Conformation Capture (3C) methodology. 3C converts physical chromatin interactions
into specific ligation products, which are quantified individually by PCR.
Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs
microarrays or quantitative DNA sequencing using 454-technology as detection
methods.
We applied 5C to analyze a 400-kb region containing the human b-globin locus and a
100-kb conserved gene desert region. We validated 5C by detection of several
previously identified looping interactions in the b-globin locus.
We also identified a new looping interaction in K562 cells between the b-globin Locus
Control Region and the g–d-globin intergenic region. Interestingly, this region has been
implicated in the control of developmental globin gene switching.
5C should be widely applicable for large-scale mapping of cis- and trans- interaction
networks of genomic elements and for the study of higher-order chromosome structure.
3C “Carbon Copy” (5C)
Analysis of the human b-globin locus and
development of 5C - I
Analysis of the human b-globin locus and
development of 5C - III
Science 2009 October 9; 326: 289–293
Comprehensive mapping of long range interactions reveals folding
principles of the human genome.
E. Lieberman-Aiden, N. L. van Berkum5,........, and J. Dekker5
We describe Hi-C, a method that probes the three-dimensional architecture of whole
genomes by coupling proximity-based ligation with massively parallel sequencing.
We constructed spatial proximity maps of the human genome with Hi-C at a resolution of
1Mb. These maps confirm the presence of chromosome territories and the spatial
proximity of small, gene rich chromosomes.
We identified an additional level of genome organization that is characterized by the
spatial segregation of open and closed chromatin to form two genome-wide
compartments.
At the megabase scale, the chromatin conformation is consistent with a fractal globule, a
knot-free conformation that enables maximally dense packing while preserving the ability
to easily fold and unfold any genomic locus.
The fractal globule is distinct from the more commonly used globular equilibrium model.
Our results demonstrate the power of Hi-C to map the dynamic conformations of whole
genomes.
Hi-C, a method that probes the three-dimensional
architecture of whole genomes
Cut with
restriction
enzyme
Fill ends
and mark
with biotin
Ligate
blunt ends
Crosslink
DNA
Purify and
shear DNA
Sequence
paired-ends
Pull down
biotin
Hi-C contact matrices and sub-matrices
Hi-C library from a karyotypically normal human lymphoblastoid cell line
sequenced on two lanes of an Illumina Genome Analyzer:
•
•
8.4 million read pairs could be uniquely aligned to the human genome sequence
6.7 million correspond to long-range contacts between segments >20 Kb apart.
Hind III
Hind III (biol. repeat)
r = 0.990
p<10−300
Nco I
r = 0.814
p<10−300
•
A genome-wide contact matrix M was constructed by dividing the genome into 1 Mb
regions (‘loci’) and defining the matrix entry mij to be the number of ligation products
between locus i and locus j (SOM).
•
Each pixel represents all interactions between a 1Mb locus and another 1Mb locus;
intensity corresponds to the total number of reads (0-50). Tick marks every 10Mb.
The presence and organization of
chromosome territories
In(s) = average
intrachromosomal contact
probability for pairs of loci
separated by a genomic
distance s
Even at distances greater than 200 Mb, In(s) is always much
greater than the average contact probability between different
chromosomes
This implies the existence of chromosome territories.
The presence and organization of
chromosome territories - II
Observed/expected
number of
interchromosomal
contacts between all pairs
of chromosomes.
Red indicates enrichment,
and blue indicates
depletion (up to twofold).
Small, gene-rich
chromosomes tend to
interact more with one
another.
HUMAN CHROMOSOMES
HUMAN CHROMOSOMES
Map of chromosome 14 at 1Mb resolution
(A) The map of chromosome 14 at 1Mb
resolution exhibits substructure in the form
of an intense diagonal and a constellation of
large blocks
The Observed/expected matrix (B) shows
loci with either more (red) or less (blue)
interactions than would be expected given
their genomic distance (range: 0.2 – 5).
Correlation matrix for Chr 14
(C) Correlation (red: 1, blue: −1)
between
the
intrachromosomal
interaction profiles of every pair of 1 Mb
loci along chromosome 14. The plaid
pattern indicates the presence of two
compartments within the chromosome.
(D) Interchromosomal correlation map
for chromosomes 14 and 20 (red:
0.25, blue: -0.25). Each compartment
on chromosome 14 has a counterpart
on chromosome 20 with a very similar
genome-wide interaction pattern.
The nucleus is segregated into two compartments
corresponding to open and closed chromatin
L3 (blue) was consistently closer to
L1 (green) than to L2 (red), despite
the fact that L2 lies between L1 and
L3 in the primary sequence of the
genome.
L2 (red) was consistently closer to
L4 (green) than to L3 (blue).
Correlation map of chromosome 14 at a
resolution of 100kb
The principal component (eigenvector) correlates with the
distribution of genes and with features of open chromatin.
A 31Mb window from the chromosome 14
The indicated region (yellow dashes) alternates between the open and closed in
compartment in GM06990 (top, eigenvector and heatmap), but is predominantly open in
K562 (bottom, eigenvector and heatmap).
The change in compartmentalization corresponds to a shift in chromatin state (DNAseI).
The local packing of chromatin is consistent
with the behavior of a fractal globule - I
(A) Contact probability as a function
of genomic distance, averaged
across the genome (blue) shows a
power law scaling between 500kb
and 7Mb (shaded region) with a slope
of −1.08 (fit shown in cyan).
(B)
Simulation
results
for
equilibrium (red) and fractal (blue)
globules. The slope for the fractal
globule closely resembles the slope
we observed in the genome.
Equilibrium versus Fractal globules
UNFOLDED POLYMER
Coloration corresponds to distance from one endpoint,
ranging from blue to cyan, green, yellow, orange, and red
FOLDED POLYMER
An equilibrium globule.
The structure is highly entangled;
loci that are nearby along the
contour (similar color) need not
be nearby in 3D.
A fractal globule.
Nearby loci along the contour
tend to be nearby in 3D, leading
to monochromatic blocks both on
the surface and in cross-section.
The structure lacks knots.
Cross-section view
Cosa sono i “Frattali”
• Un frattale è un oggetto geometrico in cui una struttura
di base contiene al suo interno diverse ripetizioni, in
piccolo, di se stessa.
• Una conseguenza è che l’oggetto non cambia aspetto
anche se visto con una lente d’ingrandimento.
• Questa caratteristica è spesso chiamata autosimilarità.
• Il termine frattale fu introdotto da Benoît Mandelbrot e,
come il termine “frazione”, deriva dal latino fractus
(spezzato).
• Le immagini frattali sono considerate dalla matematica
oggetti di dimensione frazionaria.
L’insieme di Mandelbrot
• I frattali compaiono spesso nello studio dei
sistemi dinamici e nelle teorie del caos.
• Sono spesso descritti da equazioni
semplici ma ricorsive (algoritmi).
• L’equazione che descrive l’insieme di
Mandelbrot è la seguente:
L'insieme di Mandelbrot visto con una
lente di ingrandimento sempre più potente
ha sempre lo stesso aspetto.
Genome architecture at three scales
Two compartments,
corresponding to
open and closed
chromatin, spatially
partition the genome.
Chromosomes (blue,
cyan, green) occupy
distinct territories.
Individual
chromosomes weave
back-and-forth
between the open
and closed chromatin
compartments.
At the scale of single
megabases, the
chromosome
consists of a series
of fractal globules.
Un articolo scientifico “animato” sulla Hi-C
Il filmato corrisponde a un articolo scientifico pubblicato sulla rivista online JOVE (Journal of Virtual Experments),
che riporta gli esperimenti di HiC da cui deriva il modello di organizzazione della cromatina eucariotica a “globulo
frattale”.
Il video completo può essere scaricato da:::(Dekker&al.)