NONG LAM UNIVERSITY HO CHI MINH CITY

Faculty of Biological Sciences

CHROMATIN CONFORMATION CAPTURE

AND TOPOLOGICALLY ASSOCIATING DOMAINS

by GROUP 4
Thu Duc City, 30th October 2023
 Group members

Nguyễn Tuấn Kiệt Phan Hoàng Kim

MSSV: 22126075 MSSV: 22126077
Content, powerpoint Content, presentation
● The organization of chromatin fibers in
somatic cell nuclei have followed from the
development of a technique called 3C
(chromosome conformation capture) and its
many derivatives, including Hi-C.

● In Hi-C, cells are treated with the fixative

formaldehyde, which nonspecifically crosslinks
proteins to the DNA.
● After cleaving the DNA with a restriction
endonuclease, the ends are labeled with
a biotinylated nucleotide and then ligated
together.

● These sequences may be very far apart

on the chromosomal DNA or even on
different chromosomes.
 ● This analysis reveals two levels of chromatin
organization: TADs and compartments.

● TAD is a region of the chromosome - usually

spanning 100,000 to 1,000,000 base pairs.

● Hi-C maps also show longer-range interactions

known as compartments.

Figure 8.13. Hi-C reveals the presence of both very

long-range compartments and TADs in chromosomes.
 https://www.researchgate.net/figure/Schematic-structure-of-CCCTC-binding-factor-CTCF-and-its-known-interacting-partners_fig1_349868956

● CTCF (CCCTC binding factor), a protein found at TAD boundaries, associates

with insulators that separate regions with active and inactive genes.

● CTCF binding in the insulator can physically block the DNA from being
methylated, providing another defense against the spread of heterochromatin.
● CTCF can recruit a ring shaped complex called cohesin, which is a key
architectural factor in chromosomes.

● Defects in the cohesin loading machinery cause Cornelia de Lange

syndrome.

https://ern-ithaca.eu/wp-content/uploads/2020/12/Deardorff_CdLS_general_GeneRev2020.pdf
● CTCF and cohesin are thought to function
together to bring regulatory elements
together with genes, this association with
a mobile DNA element -> one factor that
contributed to humans developing
complex patterns of gene regulation.

● Clustering of loops in TADs may provide a

mechanism to coordinate the regulation of
gene expression, possibly, DNA
replication. https://www.frontiersin.org/files/Articles/522878/fgene-11-
00338-HTML/image_m/fgene-11-00338-g002.jpg
 https://www.researchgate.net/figure/The-b-LCR-HS2-region-showing-
the-positions-of-the-three-SNPs-and-the-microsatellite_fig1_232717676

● Locus control regions (LCRs) were identified, they could

influence the transcriptional activity of cloned DNA
sequences in transgenic animals.

● When genes are introduced into cultured cells, they

normally insert at random into the chromosomes.
● LCRs are DNA sequences that permit the
expressing of transgenes no matter where
they are inserted into the chromosomes.

● LCRs typically consist of clusters of multiple

short 150 to 300 base pair regions.

● LCRs work by physically associating with a

gene, forming a loop in the chromatin and
establishing an active chromatin hub that
turns on its expression.
Organization of Mitotic Chromosomes
● When cells divide, the chromatin is
reorganized, forming mitotic chromosomes that
can be segregated efficiently to daughter cells.

● Classic hierarchical coiling models suggested

that the 30-nm chromatin fiber coils on itself,
reaching larger diameters and higher degrees
of compaction.

● Chromatin fiber coiling is an important feature

of mitotic chromosome formation.
 ● Chromatin loops containing 15,000 to
100,000 base pairs provide the structural
basis for large scale chromatin compaction
in mitotic chromosomes.

Figure 8.14 C. Chromatin loops

● TADs disappear from chromosomes as cells
enter mitosis and are replaced by uniform
distribution of approximately 80,000 to 120,000
base pair loops.

● Key proteins become concentrated along the

axial regions of the chromosome arms,
stabilize the overall structure.
● Although much less ordered than polytene
chromosomes, the arms of typical diploid
mitotic chromosomes nonetheless have a
reproducible substructure.

● If mammalian chromosomes from prometa-

phase of mitosis are subjected to a staining
procedure called G-banding, up to 2000
discrete bands are observed.
● Although the structural basis for the bands is not
known, the pattern is highly reproducible.

● Cytogeneticists used these highly reproducible

banding patterns for many years to identify individual
human chromosomes.

● The quasi-reproducible higher-order structure of

mitotic chromosomes is also seen when specific
DNA sequences marked by hybridization.
 Role
of Nonhistone Proteins
in Chromosome Architecture
● Mitotic chromosomes are composed of DNA, histones, and
nonhistone proteins have almost equal mass.

● Nonhistone proteins might contribute to mitotic chromosome

structure.
 ● If the DNA wasn’t digested, loops of DNA
protruded from the protein. The protein remnant
was called the chromosome scaffold - it
looked like a structural backbone for the
chromosome.

● Chromosome scaffolds contain several proteins

with essential roles in the structure and
maintenance of mitotic chromosomes.
Figure 8.16 C. Scaffold
● Condensin has a complex role in establishing the
architecture of mitotic chromosomes.

● Condensin I regulates the timing of chromosome

condensation, has an essential role in changing the
genome organization.

● Condensin II apparently drives the compaction of the

chromosome loops along the sister chromatid axes.

● When condensin binds to naked DNA in a test tube, it

can use the energy of ATP hydrolysis to supercoil the
DNA.
● Cohesin is the second major SMC-containing
protein complex of mitotic chromosomes.

● Cohesin assembles on chromosomes during DNA

replication and is recruited to regions of
heterochromatin by HP1.

● Cohesin also has an important role in regulating

gene expression during interphase, by stabilizing
chromatin loops that assemble active chromatin
hubs.
● DNA topoisomerase IIα- an enzyme that
alters DNA topology by passing one
double-helix strand through another.

● Topoisomerase IIα is very dynamic in

vivo, moving on and off chromosomes in
a time frame of seconds.
● Topoisomerase II is also required for
replicated sister chromatids to separate
from one another during mitotic anaphase.

● More than 4000 proteins found in mitotic

chromosomes, only the histones, and fewer
than 20 nonhistone proteins are known to
have a role in mitotic chromosome
formation.
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