Cohesin mediates transcriptional

1
Cohesin mediates transcriptional insulation by
CCCTC-binding factor Kerstin S. Wendt1, Keisuke
Yoshida2, Takehiko Itoh3, Masashige Bando, Birgit
Koch, Erika Schirghuber,Shuichi Tsutsumi,
Genta Nagae, Ko Ishihara, Tsuyoshi Mishiro,
Kazuhide Yahata, Fumio Imamoto,Hiroyuki
Aburatani, Mitsuyoshi Nakao, Naoko
Imamoto, Kazuhiro Maeshima, Katsuhiko Shirahige
Jan-Michael Peters
Presented by Li-Chen Wu 2-22-2008
2
A model of the cohesin complex
Jan-Michael Peters ECR 312(2006)

• Replicated DNA molecules are physically
  connected by cohesin complexes from the time of
  their synthesis in S-phase until they are
  segregated during anaphase of the subsequent
  mitosis or meiosis.
• 2. Cohesin is composed of four core
  subunitsSMC1,SMC3,SCC1 and SCC3.
• It contains two large ATPases of the
  structural maintenance of chromosomes (SMC)
  family, called
• Smc1 and Smc3. These proteins form
  heterodimers via a hinge domain and contain
  intra-molecular
• coiled-coil domains that are 50 nm in
  length. The N and C- terminal of Smc1 and Smc3
  assemble into
• globular ATPase domains that are joined
  together by the a-kleisin subunit
  Scc1/Mcd1/Rad21, resulting
• in the formation of a tripartite molecular
  ring. Scc1 in turn binds to Scc3, a protein that
  exists as two
• isoforms in somatic vertebrate cells,
  called SA1 and SA2. It has been proposed that the
  cohesin ring
• encircles both sister chromatids and
  thereby holds them together.
• 3. Cohesin is essential for the biorientation of
  chromosomes on the mitotic or miotic spindle,
  during G2
• phase of cell cycle to allow repair of DAN
  double-strand breaks by homologous recombination.
  In
• addition, it is also contributes to gene
  regulation, chromatin structure and development.

3
Introduction
Cornelia de Lange syndrome (CdLS)
CdLS is a disorder that results in several
physical and developmental abnormalities. It is
characterized by growth and mental
retardation, craniofacial anomalies, Limb
differences and microcephaly. It occur in 1 in
10,000 to 1 in 30,000 live births and is found
equally in males and females.
Roberts/SC phocomelia syndrom (RBS/SC)
Phocomelia syndrome (PS) is a rare birth defect
that causes severe birth defects, especially of
the upper limbs. The bones of the arms, and in
some cases other appendages, may be extremely
shortened and even absent. The fingers of the
hands may be fused. 80 patients with PS also
have facial and microcephaly abnormalities.. This
disorder, may be genetically transmitted within
families as an autosomal recessive trait or may
be the result of spontaneous (sporadic) changes
in the gene.
4
CTCF
J Pathol 2007 211 261268
1. CCCTC-binding factor (CTCF) is a widely
expressed 11-zinc finger (ZF) nuclear
protein originally identified as a transcription
factor that binds to the avian and
mammalian MYC promoters. 2. CTCF involvement in
multiple aspects of epigenetic regulation,
including regulation of genomic imprinting
and X-chromosome inactivation. 3. CTCF was also
found to play a role in regulation of noncoding
transcription and establishing local
chromatin structure at the repetitive elements in
mammalian genomes. Emerging evidence also
points to the role of CTCF deregulation in
the epigenetic imbalance in cancer.
5
Figure 1. Cohesin is expressed in postmitotic
cells
SCC1 staining was observed in the nuclei of
neurons by IFM from sections of mouse brain
cortex.
6
Figure 2. Identification of cohesin- and
CTCF-binding sites in the human genome by
ChIP-chip with ENCODE arrays
Vertical axes show the MAT score (log scale),
which reflects the fold-enrichment of the
ChIP-chip samples. Regions in which signals
were significantly enriched are coloured in
orange with red flags.
Chromosome 5
27 cohesin sites were confirmed by SCC1 and SMC3
ChIP sample with qPCRs.
7
Most identified binding sites are
cohesin-specific. Most SCC1 sites(82) were
identical in the G1 and G2 phase. Most SCC1
sites were also found in diploid hTERT RPE-1 and
immortalized B cells.
8
Cohesin co-localizes with CTCF in the human genome
S3.
Distribution of cohesin (SCC1) and CTCF binding
sites within the human genome
S4c.
9
S6.
Nuclear staining in interphase and cytoplasmic
staining from prometaphase until anaphase shown
that cohesin and CTCF antibodies yielded similar
IFM.
10
CTCF is required for the positioning of cohesin
on DNA
The abundance of cohesin sites was reduced after
SCC2 depletion, the same was true after CTCF
depletion.
In ChIP-qPCR experiments also found a 50
reduction in CTCF binding after cohesin depletion.
11
S8a. Similar SCC1 signals in chromatin pellets in
control and CTCF-depleted HeLa cells. S8b-d.
qIFM confirmed that chromatin-bound cohesin
levels were not reduced after CTCF depletion, and
CTCF on chromatin was also not reduced by SCC1 or
SCC2 depletion.
CTCF is not required for loading of cohesin onto
DNA, they associated with DNA independently of
each other.
12
If cohesin is required for the insulator function
of CTCF ?
13
Table 3.
Genes within 25 kb of cohesin sites had a higher
tendency to be upregulated than downregulated.
Consistent with an insulator function for
cohesin/CTCF sites.
Many genes whose transcription was altered in G2
phase after depletion of SCC1 or CTCF by RNAi in
HeLa cells. ---- Cohesin and CTCF may regulate
genes near their binding sites in similar
ways.
14
If cohesin depletion influences the ability of
CTCF sites to insulate a gene from a distant
enhancer?
15
Figure 3. Cohesin is required for the insulator
function of the H19 ICR
Cohesin co-localizes with CTCF at the H19/IGF2
locus.
CTCF depletion increased luciferase expression
similar effects for SCC1 and SMC3 depletion
in HeLa cells.
The reporter constructs that were used in
enhancer -blocking assays.
The insulator function of the H19 ICR requires
cohesin binding to DNA, but not the establishment
of cohesion.
16
Like CTCF, is cohesin specifically bound to the
maternal H19 ICR?
H19 is only transcribed from the maternal allele,
IGF2 gene is only transcribed from the paternal
allele. This imprinting depends on CTCF, When
CTCF bind to the H19 ICR on the maternal allele.
H19 will be activated. In contrast, on paternal
allele, the CTCF binding is blocked by
methylation on CpG sequences, the distal
enhancer will activate IGF2 but cannot
stimulate H19 transcription.
17
b. Chromosome-specific fluorescence in situ
hybridization (FISH) confirmed a single copy
of human chromosome 11 in A911M-2 and
A9111p-1 cells. c. RTPCR indicated that
specific transcripts KCNQ1OT1, KCNQ1 and H19
are still expressed in an imprinted
fashion. d. The ChIP-qPCR indicated that CTCF
and SMC3 antibodies could isolate the human
H19 ICR only from maternal specific hybrid
cells. e. Re-ChIP assays shown cohesin and
CTCF are bound to the same DNA molecules.
Cohesin co-localizes with CTCF on the maternal
allele of the H19 ICR.
18
Figure 5. Cohesin controls transcription at the
H19/IGF2 locus.
a. After depletion, IGF2 and H19 are imprinted
in HeLa cells in both CTCF and
SCC1-dependent manner.
b,c. FACS analysis shown that SCC1 and
CTCF depletion altered H19 and IGF
trancrip levels in similar way for cells
synchronized in the G1 phase.
Cohesin is required for imprinting at the
H19/IGF2 locus.
19
Conclusion
1. Cohesin has an important role at CTCF-binding
sites, which may function as transcriptional
insulators or boundary elements in
vertebrate genomes. 2. Hypomorphic mutations in
cohesion genes may therefore cause defects
in insulators and boundaries that could lead to
transcriptional and developmental
abnormalities. 3. Cohesin has important
transcriptional roles during the G1 phase
and in postmitotic cells, provided a potential
explanation for why vertebrate cells remove
the bulk of cohesin from chromosome arms in
prophase and reload it onto DNA in telophase
instead of destroying most cohesin in
metaphase, as occurs in yeast. 4. Except the
essential role of cohesin in cohesion, The
cohesin complexes are also contribute to
gene regulation, chromatin structure and
development.