6450–6458 Nucleic Acids Research, 2008, Vol. 36, No.

20 Published online 14 October 2008

doi:10.1093/nar/gkn688

The Cohesin loading factor NIPBL recruits

histone deacetylases to mediate local
chromatin modifications
Philipp Jahnke1, Weizhen Xu1,2, Manuela Wülling3, Melanie Albrecht1,
Heinz Gabriel4, Gabriele Gillessen-Kaesbach1, and Frank J. Kaiser1,*
1
Institut für Humangenetik, Universität zu Lübeck, 23538 Lübeck, Germany, 2Institute of Cell Biology,
College of Medicine, Zhejiang University, China, 3Zentrum für medizinische Biotechnologie, Arbeitsgruppe

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Entwicklungsbiologie, 45141 Essen and 4Zentrum für Medizinische Genetik, 49076 Osnabrück, Germany

Received September 4, 2008; Revised September 22, 2008; Accepted September 24, 2008

ABSTRACT This association, called sister chromatid cohesion (SCC),

depends on Cohesin, a highly conserved complex of pro-
Cornelia de Lange Syndrome (CdLS) is a rare con- teins. This complex consists of at least four subunits, the
genital malformation disorder. About half of the structural maintenance of chromosomes (SMC) proteins
patients with CdLS carry mutations in the NIPBL SMC1 and SMC3, a member of the kleisin protein family
gene encoding the NIPBL protein, a subunit of the RAD21 and SCC3 (1). Cohesins are thought to form a
Cohesin loading complex. Recent studies show ring structure that surrounds the replicated sister chroma-
association of Cohesin with chromatin-remodeling tids, with SMC1 and SMC3 creating a V-shaped hetero-
complexes, either by establishing cohesion or by dimer, bridged by RAD21 (2,3). SCC3 binds to this
recruiting Cohesin to specific chromosome loca- complex via a C-terminal domain of RAD21. The associa-
tions. In yeast two-hybrid assays, we identified an tion of Cohesin complexes with chromatin appears to be
interaction of NIPBL with the histone deacetylases -1 facilitated by two additional proteins, SCC2 and SCC4.
and -3. These interactions were confirmed in mam- Recently, a growing number of Cohesins and its regu-
latory proteins have been associated with human develop-
malian cells by coimmunoprecipitation and a critical
mental disorders. Mutations in ESCO2, which encodes a
region for interaction was defined to a stretch of 163 factor essential for the establishment of SCC, result in
amino acids of a highly conserved region of NIPBL, Roberts syndrome and SC phocomelia (4). In addition,
which is mutated in patients with CdLS. Utilizing about 50% of patients with Cornelia de Lange syndrome
reporter gene assays, we could show that NIPBL (CdLS) have mutations in the NIPBL gene, the human
fused to the GAL4-DNA-binding domain (GAL4- homolog of the Drosophila Nipped-B and yeast Scc2
DBD) represses promoter activity via the recruit- gene (5,6). Recently, the first missense mutations in
ment of histone deacetylases. Interestingly, this SMC1A and SMC3 were identified in patients with mild
effect is dramatically reduced by both NIPBL mis- variants of CdLS (7,8).
sense mutations identified in CdLS and by chemical Although Cohesins were initially identified for their role
inhibition of the histone deacetylases. Our data are in SCC, Drosphila Nipped-B was isolated as a protein that
facilitates transcriptional regulation by remote enhancer
the first to indicate a molecular and functional con-
sequences (9). Whether SCC2-like proteins have indepen-
nection of NIPBL with chromatin-remodeling pro- dent functions in cohesion and transcription, or whether
cesses via the direct interaction with histone these functions are interconnected remains unclear.
deacetylases. Furthermore, increasing evidence indicates that SCC
correlates with chromatin remodeling. The Cohesin’s
SMC1, SMC3 and Scc1/RAD21 have been found to
INTRODUCTION copurify with a chromatin-remodeling complex contain-
Following replication, sister chromatids are attached to ing the ATPase SNF2h, presumably due to a direct inter-
each other prior to segregation in mitosis and meiosis. action between Cohesins and chromatin-remodeling

*To whom correspondence should be addressed. Tel: +49 451 5002623; Fax: +49 451 5004861; Email: frank.kaiser@uk-sh.de

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors

ß 2008 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Nucleic Acids Research, 2008, Vol. 36, No. 20 6451

complexes (10). Additional links are suggested by muta- was added. After a defined incubation period (t) reaction
tions of the chromatin-remodeling factors RSC and was stopped by the addition of 1 M Na2CO3. The solu-
INO80 that result in defects in SCC (11–13). tions were cleared of insoluble material by centrifugation
To understand the molecular mechanisms underlying and the OD was measured at 420 nm. The b-galactosidase
CdLS, we used a yeast two-hybrid screen to identify bind- activity (U) was calculated by the following equation:
ing proteins of NIPBL. We identified a specific interaction U = 1000
OD400/(t
V
OD600). All b-galactosidase
of NIPBL with the histone deacetylases-1 and -3 (HDAC1 activities (U) listed represent the result of at least six inde-
and HDAC3) that was verified by coimmunoprecipitation pendent yeast transformants.
in eukaryotic cells. Luciferase reporter gene assays and
chromatin-immunoprecipitation analyses support the Immunoprecipitations
model that NIPBL may initiate chromatin-remodeling
processes through the recruitment of these HDACs. A volume containing 1 mg of total protein extracts from
HeLa cells was dissolved in 1 ml of incubation buffer

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Furthermore, we could show that missense mutations
identified in patients with CdLS influence the interaction (20 mM HEPES pH 7.9, 75 mM KCl, 2.5 mM MgCl2,
of NIPBL with histone deacetylases and result in a 1 mM DTT, 0.1% NP-40, 0.5 mM phenylmethylsulfonyl
decreased activity of this functional interaction. fluoride, 1 mM Na3VO4) and Proteinase Inhibitor
Cocktail (Roche). The solutions were precleared with
30 ml of preequilibrated Protein G PLUS/Protein A-Agar
MATERIAL AND METHODS Suspension (Calbiochem, Darmstadt, Germany) for
Yeast two-hybrid assay 60 min. The supernatant was then incubated with 10 ml
of anti-delangin (anti-NIPBL) antibody (Absea, Beijing,
Two overlapping fragments (fragment N, aa 1117–1899 China) for 100 min and 60 ml of preequilibrated A/G-sus-
and fragment C, aa 1838–2597) representing the highly pension slurry for 12–16 h. The loaded suspensions were
conserved region of NIPBL were used as yeast two- precipitated, washed three to five times with incubation
hybrid baits. These fragments were PCR-amplified (pri- buffer and resuspended in SDS-gel loading buffer
mers available upon request) from a marathon-ready [62 mM Tris pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol,
human fetal brain cDNA library (Clontech-Takara, 5% (v/v) 2-mercaptoethanol, 0.005% (w/v) bromophenol
Saint-Germain-en-Laye, France). Fragments were inserted blue]. All incubations were carried out at 48C with con-
into the Matchmaker GAL4 Two-Hybrid Sytem 3 stant motion using an end-over-end rotor. Precipitates
(Clontech-Takara) pGBKT7 plasmid. Yeast cells were were analyzed by SDS–PAGE and western blotting
transformed according to the Matchmaker 3 manual and using the anti-delangin (Absea), anti-HDAC1 (Santa
bait expression was confirmed by western blotting using Cruz) and anti-HDAC3 antibodies (Santa Cruz).
an anti-GAL4-DBD antibody (Santa Cruz, CA, USA).
Human chondrocyte and human ovary cDNA prey
libraries were screened according to the manufacturer’s Reporter gene assays
protocol (Matchmaker 3, Clontech-Takara). Plasmids NIPBL fragment 4 was inserted into a pcDNA-GAL4-
from clones obtained under stringent selection were DBD expression plasmid to obtain the GAL4-NIPBL
isolated and sequenced. Identity was determined using fusion construct. Full-length HDAC1 was amplified and
BlastN and BlastP homology searches. All constructs inserted into pcDNA3.1. HDAC3 and HDAC6 expression
used were verified by sequencing (PE Applied Biosystems, plasmids were obtained from InvivoGen. The GAL4-
Darmstadt, Germany). TATA-Luc and the GAL4-tk-Luc reporters have been
described (14,15).
b-Galactosidase assay Transient transfection assays of COS-7 and CHO cells
Liquid b-galactosidase assays were performed as per the were performed in 96 half-area well plates (Corning,
manufacturer (Matchmaker 3, Clontech). Briefly, NIPBL- Munich, Germany) with Lipofectamine2000 reagent
subfragments 4–8 were generated from fragment C and (Invitrogen, Karlsruhe, Germany) according to the man-
inserted into the pGBKT7 plasmid. Full-length HDAC1, ufacturer’s instruction. The phRG-TK Renilla luciferase
HDAC3 and HDAC6 were amplified from human fetal expression vector (Promega, Mannheim, Germany) was
brain cDNA (HDAC1) or full-length ORFs containing used as a transfection control. Activity of Firefly and
plasmids (InvivoGen, San Diego, USA), restriction sites Renilla luciferase was measured after 48 h incubation
added by PCR and inserted into the pGADT7 plasmid with the Dual Luciferase Reporter Assay System
(Clontech-Takara). Yeast cells (AH109) were cotrans- (Promega) in a GeniosPro Luminometer (Tecan,
formed with the NIPBL-fragments 4–8 and all HDAC Crailsheim, Germany). All measurements were performed
containing plasmids, respectively. Proper expression was in triplicates. Relative luciferase activity was determined
verified by western blotting using specific anti-GAL4- as rate of the average firefly:renilla luciferase activity. To
DBD and anti-GAL4-AD antibodies (Santa Cruz). The inhibit histone deacetylases, cells were treated with 100
overnight cultures were measured at 600 nm, cells were nM Trichostatin A (Merck) and 5 mM Sodium butyrate
harvested and resuspended in buffer Z (60 mM, (Sigma, Taufkirchen Germany), respectively for 24 h
Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM prior to measurement. DMSO was used as negative con-
MgSO4, 50 mM 2-mercaptoethanol, pH 7.0). An aliquot trol for Trichostatin A treated cells. All assays had a mini-
(V) was taken and o-nitrophenyl-b-D-galactopyranoside mum of six replicates.
6452 Nucleic Acids Research, 2008, Vol. 36, No. 20

Chromatin immunoprecipitation assays (encoding aa 1838–2000) and 5 (aa 1838–2380) interact

with HDAC1 as well as HDAC3, whereas no interactions
As adapted from Wei et al. (18), COS7 cells were
with fragments 6–8 (6: aa 2000–2380; 7: 2000–2598; 8: aa
transfected with GAL4-tk-reporter and either the
2200–2597) were detectable. Thus, the HDAC-interacting
NIPBL-fragment4 GAL4-DBD or the empty GAL4-
region of the NIPBL protein could be narrowed down to
DBD expression plasmids as described. Chromatin immu-
163 aa (1838–2000). This domain includes the highly con-
noprecipitation (ChIP) assays were performed according to
served HEAT repeats H2 and H3. In addition, we ana-
the manufacturer’s recommendation (Upstate Biotechnol-
lyzed the interaction of NIPBL with HDAC6, which was
ogy, Lake Placid, NY, USA). Proteins were cross-linked to
not identified in our yeast two-hybrid screens. HDAC6 did
DNA by adding formaldehyde to a 1% final concentration.
not interact with the NIPBL-fragments.
Chromatin was incubated with anti-acetylated histone 3,
anti-HDAC1 or anti-HDAC3 antibodies (Upstate Bio- Missense mutations affect the interaction of NIPBL
technology; Millipore, Schwalbach, Germany; Santa with histone deacetylases

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Cruz) overnight at 48C. Precipitated immunecomplexes
were treated with proteinase K, and DNA was purified In separate studies, we identified a novel de novo missense
by phenol/chloroform extraction for PCR detection with NIPBL mutation (R1895T) predicted to result in an
primers flanking the tk promoter region of the GAL4- amino acid exchange within the critical region of NIPBL
tk-luc reporter (50 -agcgtcttgtcattggcg-30 and 50 -ttaagcgg interaction with HDAC1 and HDAC3. Another missense
gtcgctgcag-30 ). Amplified fragments (102 bp) were analyzed mutation (c.5566A>G; p.R1856G) within this region was
on a 1% agarose gel. An SV40 promoter fragment was recently described by Selicorni et al. (17). Both patients
amplified as an internal control (130 bp; 50 -ttagtcagcaac show classical CdLS facial features and growth but no
caggtg-30 and 50 - gttaggggcgggactatg-30 ). limb anomalies. We used site-directed mutagenesis to
create NIPBL-expression plasmids including the amino
acid exchanges R1895T and R1856G, respectively. To
RESULTS test whether these mutations have any influence on the
binding capacity of NIPBL to HDAC1 and HDAC3, we
Identification of HDAC1 and HDAC3 as NIPBL binding performed b-galactosidase assays. Equal expression levels
proteins of the NIPBL and HDAC fusion proteins were monitored
The Cohesin complex consists of multiple protein subu- by western blotting (Figure 1D). While these mutations
nits. In order to identify direct binding proteins of NIPBL, do not significantly alter the binding of NIPBL to
we used the GAL4-Matchmaker III yeast two-hybrid HDAC1, a 2-fold decrease in b-galactosidase activity sug-
system (Clontech). In silico analyzes of the NIPBL protein gests a reduction in NIPBL interaction with HDAC3
predict multiple HEAT-repeats, a glutamine-rich region in (Figure 1D).
the N-terminal part and a bipartite nuclear localization NIPBL forms stable complexes with endogenous HDAC1
signal (Figure 1A) (5,6,16). Furthermore, at least two dif- and HDAC3 in mammalian cells
ferent isoforms of NIPBL exist in humans; a small isoform
[2697 amino acids (aa) and a calculated molecular weight Since the above assays were performed in transformed
(mw) of 304 kDa], and a large isoform (2804 aa and mw yeast cells, we analyzed whether endogenous NIPBL pro-
316 kDa). Because of the large protein size, we were tein interacts with HDAC-1 and -3 in mammalian cell
unable to express full-length NIPBL in yeast cells and lines. Because HeLa cells express detectable amounts of
created two overlapping fragments (fragment N and frag- NIPBL (18), we used a monoclonal anti-NIPBL antibody
ment C). The latter includes the C-terminal half of to a C-terminal motif of NIPBL (18) for immunoprecipi-
NIPBL, which is highly conserved during evolution tation. Western blotting of the anti-NIPBL precipitates
(Figure 1A and B). Both fragments were used to screen with anti-HDAC1 and anti-HDAC3-specific antibodies
human chondrocyte and ovary libraries. Using fragment C confirms interaction of endogenous NIPBL and HDAC-1
as bait, we isolated more than 250 clones encoding 56 and -3 (Figure 2). No HDAC1- or HDAC3-specific
putative NIPBL-binding proteins, which are currently signal was detected in control samples using A/G sephar-
under investigation. Five of these clones encoded three ose loaded with anti-Ig antibodies for precipitation.
overlapping fragments of the histone deacetylases 1
(HDAC1). Three clones coded for the entire open reading NIPBL-mediated recruitment of histone deacetylases
frame of the histone deacetylases 3 (HDAC3), two of modifies promoter activity
which are independent and only differ in the length of To further investigate a functional association of NIPBL
the 30 untranslated region (UTR) (Table 1). with histone deacetylases, we utilized luciferase reporter
To confirm our yeast two-hybrid results and to narrow gene assays. To accomplish this, the minimal HDAC-
down the interaction region within NIPBL, we performed interacting region of NIPBL, fragment 4, was fused to
liquid b-galactosidase assays using fragment C and five the GAL4-DBD containing plasmid to obtain a GAL4-
additional constructs (fragments 4–8) encoding overlap- DBD-NIPBL fusion construct. This fusion protein is able
ping parts of fragment C (Figure 1B). As prey constructs, to bind to the promoter regions of reporter plasmids con-
we used the full-length open reading frames (ORFs) of taining GAL4-binding sites 50 to the luciferase cDNA
HDAC1 and HDAC3, each fused to the GAL4-activating under the control of the TATA box (TATA-Luc) or the
domain (GAL4-AD). As shown in Figure 1C, fragments 4 tk promoter, respectively. Baseline luciferase activities of
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Figure 1. Predicted structure of the entire NIPBL protein and all fragments used. (A) The two described isoforms (A and B) of NIPBL differ in the
very C-terminal part of the protein only. The domains predicted by in silico analyses are indicated, including the five HEAT repeats (H1–H5), the
Glutamine (Gln)-rich region (418–462), the bipartite nuclear localization signal [NLS; 1108–1124 and the HP1-interacting motif (22)]. The two
missense mutations R1856G and R1895T are indicated by asterisks. (B) Fragments ‘N’ and ‘C’ were fused to the GAL4-DNA-binding domain and
used as baits in yeast two-hybrid assays. Fragments 4–8 represent overlapping sub-fragments of fragment C and were used in b-galactosidase assays
to narrow down the smallest region necessary for the interaction of NIPBL with HDAC1 and HDAC3. The expression of all constructs was verified
by the use of an anti-GAL4-DBD antibody. (C) Fragment 4 (columns 1–3) as well as fragment 5 (columns 4–6) interact with HDAC1 (columns 1
and 4) and HDAC3 (columns 2 and 5), whereas no interactions with fragments 6–8 were detectable (data not shown). As additional control,
interaction of HDAC6 with any of the fragments used could be excluded (columns 3 and 6). (D) The two human missense mutations R1856G and
R1895T were generated by site-directed mutagenesis and the binding capacities to HDAC1 and HDAC3 were analyzed and compared to the wild-
type protein, respectively. While both amino acid substitutions do not alter the binding capacities of NIPBL to HDAC1 (compare columns 1 with 2
and 3), the interaction with HDAC3 resulted in a reduction of b-galactosidase activity to 50% for both mutations compared to the wild-type
construct (columns 4–6). Equal expressions of the HDAC1- and HDAC3-GAL4-AD fusion constructs were verified by western blotting.
6454 Nucleic Acids Research, 2008, Vol. 36, No. 20

Table 1. Clones encoding HDAC1/HDAC3 identified by yeast two- To analyze whether the missense mutations R1895T
hybrid and R1856G within the HDAC-interacting region of
NIPBL have an effect on NIPBL-mediated repression,
Clone Protein Insert (position bp) cDNA library
we generated the constructs GAL4-DBD-R1895T and
1 HDAC 1 0
236 –3 UTR + 26 Chondrocyte GAL4-DBD-R1856G. These constructs were used to
2 HDAC 1 236  30 UTR + 26 Chondrocyte transfect different cell lines carrying the TATA-Luc
3 HDAC 1 3 – 1417 Chondrocyte or tK promoter containing reporter plasmid. While
4 HDAC 1 53 – 1440 Ovary R1895G only slightly decreases NIPBL-mediated repres-
5 HDAC 1 53 – 1440 Ovary
6 HDAC 3 4 – 30 UTR + 13 Chondrocyte sion (55.4–64.9%), R1856T has a more dramatic effect
7 HDAC 3 4 – 30 UTR + 13 Chondrocyte (55.5–83.8%; Figure 3C). Equal expression of NIPBL
8 HDAC 3 1 – 30 UTR + 56 Chondrocyte constructs was verified by western blotting of extracts
with an anti-GAL4-DBD antibody (Figure 3C).
The clones encoding HDAC1/HDAC3 are numbered 1–8. The encod-

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ing regions of the isolated plasmids are listed by base pair position in
the ORF of HDAC1/HDAC3. The minus () defines the position in The activity of NIPBL is sensitive to chemical inhibition
the 50 UTR, 30 UTR positions are defined with plus (+). of histone deacetylases
The direct binding of NIPBL with HDAC1 and HDAC3
as shown in our coimmunoprecipitations and the tran-
scriptional repression demonstrated in the luciferase
reporter assays, strongly suggest a functional correlation
of NIPBL and histone deacetylation mechanisms. To
further characterize whether the NIPBL-mediated repres-
sion was dependent upon histone deacetylase activity, we
utilized sodium butyrate (SoBu) and trichostatin A (TSA),
chemical inhibitors of histone deacetylases. Both inhibi-
tors have been shown to result in hyperacetylation of his-
tones H3 and H4, and result in enhanced promoter
activities (19). In their presence, the activity of the
GAL4-DBD-NIPBL fusion construct was nearly abol-
ished (Figure 3D), providing further support between
the function of NIPBL and histone deacetylation.
Figure 2. NIPBL coimmunoprecipitates with HDAC1 and HDAC3 in Notably, TSA- or SoBu-treatment in the presence of the
HeLa cells. (A) Expression of NIPBL was detected with an anti-NIPBL
antibody in extracts of HeLa cells (lane 1). The anti-NIPBL antibody empty GAL4-DBD construct, demonstrate only a mini-
was used for immunoprecipitation of NIPBL from HeLa extracts and mal increase in reporter gene activity, suggesting the effect
specific precipitation of NIPBL was monitored (lane 2). (B) HDAC3 is directly on the transcriptional repression by NIPBL,
was shown with an anti-HDAC3 antibody in HeLa cell extracts (lane rather than on global up-regulation of transcription.
1). The anti-NIPBL antibody and a control anti-IG antibody were used
for immunoprecipitation (IP), respectively. HDAC3 was found to be
coprecipitated with NIPBL (lane 2), whereas no HDAC3-specific signal NIPBL-mediated recruitment of endogenous HDACs
could be detected in the control IP (lane 4). Noncoprecipitated HDAC3 induces histone deacetylation
was visible in the supernatant (lane 3). (C) Proper expression of
HDAC1 was monitored in HeLa cell extracts (lane 1). HDAC1 could To verify that the promoter activity of the GAL4-DBD-
only be identified in the anti-NIPBL precipitates (lane 2), while no NIPBL fusion is a direct consequence of NIPBL-mediated
HDAC1-specific signal was detectable in the IPs using the anti-IG anti-
body (lane 4). Nonprecipitated HDAC1 could be found in the super- recruitment of endogenous HDAC1 and HDAC3, ChIP
natants of the IPs (lane 3). assays were conducted. COS7 cells were cotransfected
with tK reporter and the GAL4-DBD-NIPBL construct.
mammalian COS7 and CHO cell lines transfected with Following formaldehyde cross-linking, anti-HDAC1 and
TATA-Luc or the tk promoter plasmids were arbitrarily anti-HDAC3 antibodies were used for immunoprecipita-
set as 100% (Figure 3A, column 1). Notably, cotransfec- tion. Purified DNA fragments were amplified by PCR and
tion with NIPBL results in a concentration-dependent analyzed in electrophoresis (Figure 4A). A tK promoter-
decrease of reporter activity (Figure 3A, columns 2–4). specific fragment of 130 bp was identified in both anti-
To elucidate whether this NIPBL-mediated trans-repres- HDAC1 and anti-HDAC3 precipitates, indicating an
sional activity may be due to the recruitment of endogen- interaction of both endogenous histone deacetylases with
ous histone deacetylases, we cotransfected HDAC1 and the tK promoter (Figure 4A, upper panel, lanes 2 and 4).
HDAC3 expression plasmids. Both HDACs-1 and -3 In contrast, no signal was detectable in precipitates of
result in additional decrease of the promoter activity, cells transfected with an empty GAL4-DBD plasmid
whereas cotransfection with HDAC6 shows no functional (Figure 4A, upper panel, lanes 1 and 3). Furthermore,
effect (Figure 3B). In control samples, overexpression of HDAC1 and HDAC3 do not interact with the SV40 pro-
each histone deacetylase (HDAC-1, -3 or -6) in the moter, which regulates the expression of the GAL4-DBD-
absence of the GAL4-DBD-NIPBL construct has no NIPBL construct (Figure 4, lower panel). These data
significant influence on reporter activities (Figure 3B, col- clearly support a role for NIPBL-mediated recruitment
umns 4, 6 and 8). of endogenous histone deacetylases.
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Figure 3. NIPBL-mediated recruitment of HDACs induces transcriptional repression. (A) The HDAC-interacting region of NIPBL, fragment 4, was
fused to GAL4-DBD to obtain a GAL4-DBD-NIPBL-frag4 construct (NIPBL-frag4). Cells carrying a luciferase reporter with GAL4-consensus
binding sequences 50 to a LUC reporter gene were transfected with increasing amounts of NIPBL-frag4. The luciferase activity of cells transfected
with an empty GAL4-DBD plasmid was set as 100% (column 1). NIPBL-frag4 reduces the reporter activity in a concentration-dependent manner
(compare columns 1 with 2–4). (B) Cotransfections with HDAC1- as well as HDAC3-encoding plasmids enhance the transcriptional repression
mediated by NIPBL-frag4 (columns 3 and 5), while transfection with HDAC6 does not alter the activity (column 7). As control, the effects of the
individual HDAC on the reporter (HDAC1, 3, 6 and columns 4, 6, 8) were monitored in the absence of NIPBL. (C) Whereas missense mutation
R1895T only slightly decreases the NIPBL-mediated activity (column 3), mutation R1856G severely reduces the activity (column 4). Equal expression
of wild-type and mutant constructs was monitored by the use of an anti-GAL4-DBD antibody in western blots. (D) The effect of TSA and sodium-
butyrate (SoBu) on the trans-repressive activity of the NIPBL-frag4 construct was assessed in COS7 cells. Columns 1–3 show the activities of cells
tansfected with the empty GAL4-DBD plasmid. Here, TSA and SoBu only very slightly increase the activities. Columns 4–6 represent the results of
cells transfected with the NIPBL construct. While the transfection with NIPBL-frag4 decreases reporter gene activity down to 50% in nontreated
cells (column 4), treatments with TSA and SoBu, respectively, almost completely abolish the trans-repressional effect of NIPBL (columns 5 and 6).
Experiments were done at least in triplicates and the standard deviations are indicated by error bars.

To further analyze if these interactions have an effect on GAL4-DBD plasmid (Figure 4B, lane 6). The acetylation
chromatin acetylation, we used an assay (20), where chro- status of the SV40 promoter that regulates the expression
matin is precipitated with an antibody that specifically of the NIPBL construct was monitored in parallel (lower
recognizes acetylated lysines-9 and -14 of histone H3. panel of Figure 4B). As expected, since no GAL4-binding
The precipitated DNA fragments were PCR-amplified sites are present in this vector, H3 acetylation is present,
and analyzed by agarose gel electrophoresis. An expected regardless of the presence or absence of the GAL4-DBD-
130 bp tK promoter-specific fragment can be amplified NIPBL construct (Figure 4B, lanes 5 and 6).
from control plasmid DNA (Figure 4B, lane 2) and
from chromatin prior to precipitations (Figure 4B, lanes
3 and 4). Interestingly, no PCR product is seen for the DISCUSSION
precipitates of cells transfected with the GAL4-DBD- NIPBL is a member of the highly conserved Scc2-protein
NIPBL construct, indicating a specific deacetylation of family. These proteins, required for the loading of Cohesin
the tK promoter (lane 5). However, the tk promoter onto chromatin, have been implicated in double-strand
PCR product was detectable in controls using the empty DNA repair and in the regulation of gene expression.
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Figure 4. Chromatin-immunoprecipitation (ChIP) assays. (A) The NIPBL-mediated recruitment of HDAC1 (lane 2) and HDAC3 (lane 4) was shown
by specific PCR analyses of the purified anti-HDAC1- and anti-HDAC3-precipitates, whereas no signal was detectable in the precipitates of cells
transfected with the empty GAL4-DBD plasmid (lanes 1 and 3). As control, the association of HDAC1 and HDAC3 with the SV40 promoter was
excluded (lower panel). (B) ChIP assays were used to demonstrate histone deacetylation. The histone deacetylation occurs as the result of the GAL4-
DBD-NIPBL (+) expression (lane 5), but not if the GAL4-DBD is expressed alone () (lane 6). The upper panel of the figure shows a GAL4-tK
promoter specific fragment of 130 bp. In the bottom part, a 120 bp PCR product indicating acetylation of the SV40 promoter is shown. Water
(lane 1), plasmid DNA (lane 2) as well as input DNA before immunoprecipitation (lanes 3 and 4) were used as controls. As additional control a
nonspecific antibody was used for precipitation (a-IG; lanes 7 and 8).

Although both isoforms described for the human NIPBL separated by 39 amino acids and are localized within
are fairly large, only a few functional domains have been (R1856G) or adjacent (R1895T) to HEAT-repeat 2.
predicted by in silico analyses. These include a putative In luciferase reporter gene assays we show that this
bipartite nuclear localization signal, a glutamine-rich region of NIPBL when fused to the GAL4-DBD modifies
region, an HP1-interacting motif and several HEAT reporter gene expression. Using ChIP assays we show that
repeats, which have been implicated in protein–protein this NIPBL-mediated activity is due to direct interaction
interactions (21). These HEAT repeats are located with endogenous histone deacetylases. Furthermore,
within the C-terminal part of NIPBL, spanning about cotransfection of HDAC1 and HDAC3 result in a further
amino acids 1300–2500, which is highly conserved in dif- decrease of promoter activity, whereas HDAC6, which
ferent species and expected to be functionally significant. does not directly associates with the NIPBL fragment,
Moreover, a majority of CdLS-associated missense muta- does not modify the reporter gene expression significantly.
tions map to this domain. Interestingly, the two missense mutations analyzed have
In a yeast two-hybrid assay to identfiy NIPBL-binding similar effects on HDAC1 and HDAC3 binding affinities,
proteins, we isolated the histone deacetylases, HDAC1 but exert different functional activities. Mutation R1895T
and HDAC3, important cofactors that regulate chromatin only slightly alters this function, whereas mutant R1856G
structure by deacetylating histone proteins. Through the significantly decreases the activity mediated by NIPBL.
use of truncated NIPBL constructs, we refined the critical Lastly, chemical inhibition of histone deacetylation by
region of NIPBL interaction with HDAC1 and HDAC3 treatment of the cell cultures with TSA or SoBu almost
to a stretch of 163 aa (1838–2000), which contains two of completely abolishes the NIPBL-mediated activity. These
the five conserved HEAT repeats. As the use of partial data are the first to clearly point to a direct functional
fragments of NIPBL could result in altered protein bind- connection of the mammalian SCC2-like protein NIPBL
ing, we analyzed the interaction of full-length NIPBL with the recruitment of histone deacetylating enzymes.
with HDAC1 and HDAC3. In immunoprecipitation
assays, we were able to verify the interaction of the endo- Histone acetylation, chromatin remodeling and Cohesin
genous NIPBL with both HDAC1 and HDAC3 in Although the interplay of chromatin structure and histone
mammalian cells. modification with Cohesin has been frequently described,
To assess relevance of the critical region for the inter- the precise function is poorly understood. It has been
action of NIPBL with HDAC1 and HDAC3 to CdLS, we hypothesized that chromatin structure may play an impor-
tested two newly identified NIPBL missense mutations. tant role in determining whether, and where, Cohesin
One of these we recently identified as a de novo mutation binds to chromosomes in eukaryotic cells (22).
(R1895T) and the other was recently described by It is known that histone modifications affect the recruit-
Selicorni and colleagues (R1856G). Both patients have ment of Cohesin to particular chromosomal loci (23). In
classical CdLS phenotype without limb abnormalities our ChIP analyses, we could show that aa 1838–2000 of
(17). Each mutation results in decreased binding of NIPBL are able to initiate the deacetylation of lysine 9 of
NIPBL to HDAC3, whereas the interaction with histone 3 (H3K9), which would allow its methylation. The
HDAC1 seems to be unaffected. Both mutations are heterochromatin binding protein 1 (HP1), which was also
 Nucleic Acids Research, 2008, Vol. 36, No. 20 6457

described to bind NIPBL (24), recognizes methylated transcribed regions and overlap with RNA polymerase II.
lysine 9 on histone H3 (H3K9) and deletion of Swi6, the According to these results, high transcriptional activity
yeast ortholog of HP1, causes a loss of Cohesin binding does not seem to prevent Cohesin binding (36). The colo-
(25). This is consistent with earlier observations that the calization of Nipped-B with Cohesin supports the idea
interaction between the Scc3 Cohesin subunit with Swi6 that it dynamically regulates binding of Cohesin and sug-
is needed to recruit Cohesin to centromeric regions in gests additional mechanisms by which Cohesin might
yeast (23,26). On the other hand, it was shown very affect transcription (36). Although we have identified
recently, that the Suv39h-HP1 pathway is not essential NIPBL-mediated recruitment of histone deacetylases to
for the enrichment of Cohesin at centromeres in mammals chromatin, which results in local alterations of the chro-
(27). These observations clearly indicate differences in the matin structure, the precise function of this interaction is
functional connection of HP1/Swi6 and Cohesins in yeast still unknown. The numerous genomic loci where Nipped-
and mammalian cells. B has been identified suggest a more general role in tran-

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In addition to the above literature, it has been shown scriptional regulation. Whether NIPBL directly regulates
that diminishing HDACs by TSA-treatment or mutation specific promoter activities or may participate in creating
promotes abnormal sister chromatid separation. Sister a platform for the recruitment of specific transcription
chromatids do not separate when cells enter mitosis with (co-) factors needs to be analyzed in future experiments.
hyperacetylated histones. Thus, the presence of acetylated In this report, we show for the first time a direct inter-
histones in mitosis induces both aberrant chromosome action and functional consequence of histone deacetylat-
numbers (i.e. aneuploidy) and defects in chromosome ing enzymes with the Cohesin-loading protein NIPBL.
structure (28). Recently, Kimata and colleagues (29) Although the precise function of this interplay is still
described TSA-hypersensitivity of a mutant Mis4, a unknown, our data presented here give new insight into
Cohesin loading adherin protein. TSA-treatment of mis4 the molecular pathology of CdLS and further contribute
mutant cells results in decreasing Cohesin-binding to chro- to the hypothesis that it is likely not the result of defects in
matin in the chromosome arm regions. Furthermore, the cohesion; but rather, it is likely due to changes on chro-
correlation of various histone modifications and Cohesin matin structure and effects on gene expression.
is the subject of a continuously growing number of
publications. In human cells, a chromatin-remodeling
complex containing the ATPase SNF2h was found to ACKNOWLEDGEMENTS
copurify with RAD21 and SMC proteins, presumably We are thankful to Matthew Deardorff (Division of
due to a direct interaction between Cohesin and this com- Genetics, Department of Pediatrics, the University of
plex (10). Mutations in the yeast chromatin-remodeling Pennsylavnia, USA) and Stefan Weger (Institut für
complex RSC cause modest defects in sister-chromatid Infektionsmedizin, Charité Campus Benjamin Franklin
cohesion (11,12). Furthermore, deletions of Rsc2, encod- in Berlin, Germany), for the critical and fruitful discussion
ing a further alternative variant of the RSC complex, of the article.
significantly reduces SCC by reducing the amount of
chromosome-associated Cohesins (30).
FUNDING
The function of NIPBL in regulating gene expression Medical Faculty of Lübeck (E15-2008).
Although Scc2-like proteins are necessary to recruit the
Conflict of interest statement. None declared.
Cohesin complex to chromosomes, patients with CdLS
usually do not show significant cohesion defects (31,32),
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