- Email: [email protected]
Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression
Biochemical and Biophysical Research Communications 356 (2007) 773–779 www.elsevier.com/locate/ybbrc
Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression Johann Bo¨hm
b
a,1
, Frank J. Kaiser b,1, Wiktor Borozdin Ju¨rgen Kohlhase a,d,*
a,d
, Reinhard Depping c,
a Institut fu¨r Humangenetik und Anthropologie, Universita¨t Freiburg, Freiburg, Germany Institut fu¨r Humangenetik, Universita¨tsklinikum Schleswig-Holstein, Campus Lu¨beck, Germany c Institut fu¨r Physiologie, Universita¨t zu Lu¨beck, Lu¨beck, Germany d Praxis fu¨r Humangenetik, Heinrich-von-Stephan-Str. 5, 79100 Freiburg, Germany
Received 6 March 2007 Available online 19 March 2007
Abstract Loss of function mutations in SALL4 cause Okihiro syndrome, an autosomal dominant disorder characterised by radial ray malformations associated with Duane anomaly. In zebrafish and mouse Sall4 interacts with TBX5 during limb and heart development and plays a crucial role for embryonic stem (ES) cell pluripotency. Here we report the nuclear interaction of murine Sall4 with Cyclin D1, one of the main regulators of G1 to S phase transition in cell cycle, verified by yeast two-hybrid assay, co-immunoprecipitation and intracellular co-localisation. Furthermore, using luciferase reporter gene assays we demonstrate that Sall4 operates as a transcriptional repressor located to heterochromatin and that this activity is modulated by Cyclin D1. 2007 Elsevier Inc. All rights reserved. Keywords: SALL4; Okihiro syndrome; Zinc finger; Nuclear localization; Cyclin D1; Cell cycle regulation; Transcriptional activity
SALL4 is one out of five human genes related to spalt (sal) of Drosophila melanogaster [1–5]. The mouse Sall4 protein [6] is likewise encoded by four exons and shares a homology of 74% with SALL4. SALL4 mutations cause Okihiro/Duane-Radial Ray syndrome (DRRS, OMIM 607323), an autosomal dominant disorder characterised by radial ray defects and Duane anomaly [1,3], but may include anal, renal, cardiac, ear and foot malformations, hearing loss, postnatal growth retardation and facial asymmetry [7–9]. The SALL4 gene product contains an amino terminal C2HC motif and three highly conserved, evenly distributed C2H2 double zinc finger domains. A single C2H2 motif is attached to the second domain [7]. All but one reported SALL4 mutations lead to preterminal stop codons [7,10]. These truncating mutations as well as larger
deletions involving SALL4 provided evidence of haploinsufficiency as the underlying pathogenic mechanism in Okihiro syndrome [11]. Sall4 controls Fgf10 expression in a synergistic manner together with Tbx5 in the forelimbs or with Tbx4 in the hindlimbs via direct effects on the Fgf10 promoter, whereas the effect of Sall4 and Tbx1 co-expression on the Fgf10 promoter is additive [12]. In the heart, Sall4 interacts with Tbx5 and activates Gja5, and on the other hand it interferes with Tbx5-dependent activation of Nppa [12]. Sall4 also interacts with Nanog, suggesting an important function of this complex in the regulatory networks of embryonic stem cells [13]. Recently Sall4 was shown to be an essential transcription factor required for the early development of inner cell mass derived cell lineages [14].
* Corresponding author. Address: Praxis fu¨r Humangenetik, Heinrichvon-Stephan-Str. 5, 79100 Freiburg, Germany. Fax: +49 761 896454 9. E-mail address: [email protected] (J. Kohlhase). 1 These authors contributed equally to this work.
Materials and methods
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.050
Synthesis of cDNA. Total RNA from adult mouse testis was prepared with TRIzol reagent and RNase-free DNase (Invitrogen, Carlsbad, CA).
774
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen) and oligo(dT) priming (Promega, Madison, WI). Yeast-two-hybrid assay. The complete coding sequence of murine Sall4 transcript variant A was used as a bait (Matchmaker Two-Hybrid System, BD Biosciences, Mountain View, CA). The fragment was amplified from murine testis cDNA and cloned into pGBKT7. The sequence was verified by automated sequencing (ABI PRISM 3100 Genetic Analyser, Applied Biosystems, Darmstadt, Germany) and the expressed protein was monitored by SDS–PAGE and Western blotting using monoclonal anti-c-Myc antibody (BD Biosciences). We screened against a mouse 11-day Embryo entire cDNA Library. After screening, clones were selected for auxotrophy on SD/LEU-HIS-TRP-ADE/X-Gal plates. Interaction partners were identified by sequencing and homology searches by BlastN. Co-immunoprecipitation. The coding region of the murine Sall4 was cloned into a FLAG expression plasmid (pFLAG-N3, derived from pEGFP-N3, BD Biosciences). The full-length Ccnd1 CDS, was cloned into the expression vector pCMV-Myc (BD Biosciences). Co-immunoprecipitation and subsequent Western blot analysis were carried out in COS-7 cells as described before [15]. Cell culture. Cell cultures were grown in minimal essential medium (MEM), 10% Fetal Calf Serum. 100 lg/ml pen/strep, 1· MEM nonessential amino acids (all purchased from Invitrogen) and maintained in a humidified environment with 5% carbon dioxide. Intracellular localisation. Transient transfection of HeLa, MCF-7, and COS-7 cells on coverslips (5 · 104 cells) was carried out as described before [15]. Intracellular localisation of the fusion proteins was visualized by confocal laser scanning microscopy. b-Galactosidase assay. Yeast AH109 cells were co-transfected with Ccnd1 (pACT2) and overlapping Sall4 fragments (pGBKT7). b-Galactosidase assay was performed as decribed elsewhere [15]. Reporter gene assays. Sall4 fragments were cloned in-frame to the GAL4 DNA-binding domain into the pM2 vector (BD Biosciences). As reporter construct, pGAL45tkLUC was used, which contains the luciferase gene under the control of the thymidine kinase (tk) promoter with four upstream GAL4-binding sites. Transient transfection of COS-7 cells was performed as described before [16]. Using 500 ng pGAL45tkLUC expression vector and 500 ng of different Sall4 (pM2) constructs to determine the repression domain of Sall4. For studies analysing the ability of Cyclin D1 to modulate Sall4 transcriptional repression, 500 ng pGAL45tkLUC, 200 ng of Sall4 (pM2) and 200 ng Ccnd1 (pM2) expression vectors were used.
Results Identification of potential Sall4 interacting proteins Screening a mouse embryonic prey library (11 dpc), we have isolated 34 clones encoding 32 different putative Sall4 interacting proteins. Two independent clones contained the entire ORF of Cyclin D1 (Ccnd1), the other clones are currently under investigation (data not shown). To confirm our data and to narrow down the Cyclin D1 interaction domain within the Sall4 protein, we generated overlapping truncated Sall4 constructs (Fig. 1A) and performed liquid b-galactosidase assays using ONPG as a substrate. Fragment 1 encoded aa 1–342 and contained a glutamine rich region as well as the single zinc finger domain residing in the amino terminal part of the protein. Fragment 2 (aa 705–1100) beared a large N-terminal truncation but still includes the carboxy-terminal double zinc finger. Fragments 3 (aa 1–756) and 4 (aa 305–1100) represented truncated, overlapping proteins missing the car-
boxy- or aminoterminal parts, respectively. In addition, Sall4 variants B and C (623 and 278 amino acids, respectively) were tested for interaction with Cyclin D1. The accurate expression of each construct was examined by Western blot analysis (Fig. 1B). Only AH109 cells cotransfected with Sall4 constructs bearing exons 3 and 4 and Ccnd1 demonstrated b-galactosidase activity. Consequently, we could narrow down the Cyclin D1 binding region within the Sall4 protein to the C-terminal 238 amino acids, which include the carboxy-terminal double zinc finger domain. Sall4 and Cyclin D1 interact in eukaryotic cells For co-immunoprecipitation studies, extracts of cotransfected COS-7 cells, co-transfected with Ccnd1-c-Myc and Sall4-FLAG were incubated with the M2 anti-FLAG agarose and protein complexes were precipitated and analysed by Western blot (Fig. 2). As controls, COS-7 cells were co-transfected with each construct and the respective empty vector. The Cyclin D1-c-Myc protein could only be identified in the precipitates of cells co-expressing Sall4 and Ccnd1, whereas the extracts of cells co-transfected with the empty FLAG plasmid did not show any specific Cyclin D1c-Myc signal (Fig. 2D). Our results clearly indicate that Sall4 and Cyclin D1 are able to form stable complexes in eukaryotic cells. Sall4 and Cyclin D1 co-localise in the nucleus Sall1/SALL1 proteins present a high degree of sequence similarity compared to the Sall4/SALL4 proteins and show specific nuclear distribution that is associated with heterochromatin localisation [17,18]. Because of this similarity we wanted to analyse the intracellular localisation of Sall4 as well as the localisation of its interaction with Cyclin D1, which was previously shown to be a nuclear protein [19]. We therefore co-transfected COS-7 cells with Sall4-FLAG and Ccnd1-GFP constructs and analysed the intracellular distribution of all proteins by confocal laser scanning microscopy (CLSM). Similar to the localisation of Sall1/SALL1, strong Sall4 signals were detected in the nuclei of the analysed cells, appearing in numerous dot-like structures. Interestingly, the distribution of Cyclin D1 proteins was almost identical, showing a distinct and homogeneous arrangement in the nucleus. An overlay of Sall4 and Cyclin D1 signals demonstrated a co-localisation of both proteins, suggesting a specific function of these protein complexes within the nucleus (Fig. 3C). To further elucidate these intranuclear dot-like structures formed by Sall4 and Cyclin D1, we performed additional co-localisation studies in MCF-7 cells and HeLa cells using antibodies against heterochromatin protein 1 (HP1) and PML bodies. HP1 is a conserved nonhistone chromosomal protein involved in heterochromatin formation and gene silencing [20]. PML nuclear bodies have been associated with various nuclear functions [21].
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
775
Fig. 1. (A) Structure Sall4 constructs used for yeast two-hybrid and b-galactosidase assays. All constructs result in proteins fused to the GAL4 DNAbinding domain. Zinc fingers are depicted as black rhombuses. (B) Expression of the Sall4 transcript variants and the truncated constructs monitored by Western blot analysis. (C) b-Galactosidase assays to narrow down the Cyclin D1-binding region. Expression of the reporter gene lacZ is activated in case of interaction between both proteins. Only constructs containing the regions encoded by exons 3 and 4 show interaction with Cyclin D1.
We could show that Sall4 does not co-localise with the PML bodies (Fig. 3A), but Sall4 staining overlaps with the HP1 signal (Fig. 3B), indicating an association of Sall4/SALL4 with heterochromatin. Sall4 operates as a transcriptional repressor The heterochromatic localisation of Sall1/SALL1 was previously shown to be associated with transcriptional repression activity [17,18]. We therefore wanted to elucidate if Sall4/SALL4 may also act as a repressor of transcription. We fused the full length ORFs of Sall4/SALL4 to the GAL4 DNA-BD and analysed their transcriptional activities in luciferase reporter gene assays by co-transfection with the pGAL45tkLUC reporter. Interestingly, we could identify a strong repressional activity of SALL4/ Sall4. To narrow down the repression-domain we generated several overlapping fragments of the human SALL4 (1–670, 1–960, 1–3159, 680–2334, and 2440–3076). The main repression domain could be located within the amino terminal part of the protein (aa 1–320, aa 1–223), being
capable of repressing transcription down to 10.3% in average compared to the control vector (Fig. 4A, columns 3 and 4). Constructs lacking the N-terminus and encoding amino acids 226–778 and 813–1025 exhibited a weaker repression activity (39.5% and 57.3%, respectively as compared to the empty pM2 vector (Fig. 4A, columns 5 and 6). It became apparent that a second repression domain resides within the carboxy terminal part of the protein. Transfecting COS-7 cells with the pGAL45tkLUC vector and a gradient amount of the Sall4 resulted in a linear and reciprocally proportional correlation between cellular Sall4 concentration and transcriptional repression activity (data not shown). Because Sall4 was shown to act as transcriptional repressor and its association with Cyclin D1 at heterochromatic region, we wanted to analyse the ability of Cyclin D1 to influence the repressing activity of Sall4. For this purpose we co-transfected COS-7 cells with Sall4-pM2, Ccnd1-pM2 and the pGAL45tkLUC reporter (Fig. 4B). The luciferase activity of cells transfected with pGAL45tkLUC and the empty pM2 vector was set to
776
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
A
B
Expression
1
2
3
4
1
130 Sall4-Flag pFlag Ccnd1-c-Myc c-Myc
C
IPs
2 anti-FLAG
130 + + -
1
+ +
2
+ + -
+ +
3
4
55 35
Ccnd1-c-Myc + c-Myc Sall4-Flag + pFlag -
D
Sall4-Flag Ccnd1-c-Myc c-Myc
+ + -
+ +
1
2
3
4
55 anti-c-Myc
35 + +
+ + -
+ +
Ccnd1-c-Myc c-Myc Sall4-Flag pFlag
+ + -
+ +
+ + -
+ +
Fig. 2. COS-7 cells were transiently transfected with Sall4-FLAG and Ccnd1-c-Myc constructs. (A) Shows the expression of the Sall4-FLAG constructs whereas (B) verifies the precipitation of the Sall4-FLAG protein. (C) Represents the expression of Ccnd1-c-Myc and (D) shows the co-precipitated Cyclin D1-c-Myc Protein only in extracts of cells co-transfected with Ccnd1-c-Myc and Sall4-FLAG. As controls, constructs were co-transfected with the respective empty vector.
Fig. 3. (A) Intracellular localisation of Sall4 and PML bodies. Sall4-GFP (green) was transfected into MCF-7 cells. Sall4 could be shown to appear in several dot-like structures within the nucleus of every analysed cell. Endogenous PML bodies (red), forming distinct structures in the nucleus, were shown not to overlap with the distribution pattern of Sall4. The nuclei, stained with DAPI, appear in blue. (B) The Sall4 signal (green) was shown to overlap with the pattern of endogenous HP1 (red). (C) Co-localisation of Sall4 and Cyclin D1. The subcellular distribution of the proteins was analysed in COS-7 cells. Sall4-FLAG (red) was detected in the nucleus of the analysed cells, appearing in dot-like structures of variable size, Cyclin D1-GFP (green) displayed a nuclear distribution pattern similar to Sall4. Overlay showed a co-localisation of Sall4 and Cyclin D1 in distinct dots within the nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
100%. Sall4-pM2 decreased the luciferase activity down to 46% and Cyclin D1-pM2 down to 66%. Interestingly, cotransfection Sall4-pM2 and Ccnd1-pM2 almost abolished
the luciferase activity. These findings clearly indicate a synergistic function of Sall4 and Cyclin D1 in transcriptional repression.
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
Rel. luciferase activity
A 120 100 80 60 40 20
G + AL 68 45 0- tk 23 LU 34 C GA + L 23 45 40 tk -3 LU 07 C 6
G + AL pM 45 2 tkL UC GA + L Sa 45 ll4 tk LU C GA + L 1- 45 67 tk 0 LU C GA + L 1- 45 96 tk 0 LU C
0
Rel. luciferase activity
B 120 100 80 60 40 20
-L + C uc + cnd Sall 1-p 4-p M2 M2
c+ -Lu TK
TK
M2 1-p Cc
Sa TK
-Lu
c+
-Lu TK
nd
ll4
c+
-pM
pM
2
2
0
Fig. 4. (A) Overlapping Sall4 constructs fused to the sequence encoding the GAL4 DNA-binding domain were transiently co-transfected with the pGAL45tkLUC vector into COS-7 cells. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty pM2 vector (set to 100%). Fragments containing amino acids 1– 320 (nucleotides 1–960) were shown to exhibit strong repression activity. The C-terminal part of the protein affects reporter gene expression level in a weaker fashion, suggesting that two distinct domains may work in concert. (B) Sall4-pM2 and Cyclin D1-pM2 independently repress expression of the reporter gene (columns 2 and 3). Co-transfection of both constructs results in synergistic repression of reporter gene expression (column 4).
Discussion SALL4 is currently the only gene associated with Okihiro/DRRS or acro-renal-ocular syndrome [7]. At least 90% of patients with classical Okihiro syndrome show mutations or deletions in the SALL4 locus. Nevertheless, in some clear cases of Okihiro syndrome we did not find mutations or deletions, raising the possibility that the phenotype is due to mutations in genes encoding putative binding partners of SALL4. Hence we have intended to identify proteins, which bind to the murine homologue Sall4 in order to clarify its biological function and to elucidate the pathogenesis of Okihiro syndrome. In a yeast two-hybrid assay, using the entire coding sequence of Sall4 as bait, we screened a mouse embryonal cDNA library for Sall4-binding candidates and obtained 34 different putative interacting proteins. Remarkably, we did not detect an interaction with any other member of the Sall protein family despite previous
777
findings showing homo- and heterodimerisation via the conserved glutamine-rich region in the N-terminal part of the proteins [22–24]. Although we achieved high transfection efficiencies in our two-hybrid assay, it is possible that we did not cover a representative amount of all Sall cDNAs. Analyzing all 34 clones, we identified two independent clones carrying the full-length ORF of Cyclin D1 (Ccnd1), which contained different parts of the 3 0 UTR, starting from 63 and 15 from ATG, respectively. Using extracts of transiently transfected COS-7 cells for co-immunoprecipitation assays, we were able to verify that Sall4 and Cyclin D1 do interact in mammalian systems. By liquid yeast bgalactosidase assays we could narrow down the region for interaction with Cyclin D1 within the C-terminus of Sall4, which encloses a double zinc finger domain. Truncated proteins lacking this carboxyterminal part of the protein do not bind to Cyclin D1. Interestingly, all but one reported SALL4 mutations in patients with Okihiro syndrome lead to preterminal stop codons within or 5 0 of exon 3 and result in proteins lacking the Cyclin D1 interaction domain [7]. However, it remains to be proven if truncated transcripts are—as expected—subject to nonsense mediated mRNA decay or if truncated and wildtype proteins have comparable stability. The Cyclin D1 protein is known to display a diversity of functions, which can either be dependent or independent on the association with kinases (CDK, Cyclin-dependent kinases) [25]. Phosphorylation and thus inactivation of retinoblastoma protein through the formation of a holoenzyme containing Cyclin D1 and CDKs accelerates G1 progression and is required for the entry to S phase. It has been demonstrated that Cyclin D1 interacts with a large number of transcription factors and cofactors/activators. Binding of histone acetylases, histone deacetylases and chromatin remodeling proteins results in modifications of the chromatin structure of genes involved in regulation of cell differentiation and proliferation. Our analysis of the intracellular localisation of Sall4 and Cyclin D1 revealed that Sall4, similar to SALL1, is distributed within small dot-like structures. As shown for SALL1, co-localisation studies with a heterochromatic marker (heterochromatin binding protein 1 (HP1)) confirmed that these structures are heterochromatic [18]. The association of Sall4 and Cyclin D1 within heterochromatic structures gave reason for the hypothesis that Sall4 acts as transcriptional repressor, whereby Cyclin D1 may modulate this function. By means of reporter gene assays, we could show that murine and human Sall4/SALL4 are able to act as transcriptional repressors. The main repressing domain of SALL4 is located within the amino terminal part of the protein and a second domain is located in the carboxyterminal region. Interestingly, similar results were also obtained for SALL1, which harbours one repression domain in its amino terminal region and a second one in the central part of the protein [26]. The mechanism determining whether Sall4/SALL4 act as a transcriptional activator or
778
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
repressor have to be elucidated, as Sall4 has previously been shown to act as an activator of murine Fgf10 [12]. It is known that transcription factors are usually part of multi-protein complexes. The composition of these structures determinates the specific function by recruiting cofactors, which initiate repression or activation of the respective target gene. In luciferase reporter gene assays, we analysed the potential ability of Cyclin D1 in modulating the Sall4 transcriptional activity. Since no Sall4 target genes for transcriptional repression are known, we generated Sall4-pM2 and Ccnd1-pM2 constructs, resulting in GAL4 DNA-BD fusion proteins, and a reporter gene under the control of the tk promoter and repetitive GAL4 binding sequences. Sall4 reduced the reporter gene activity down to 46% and Cyclin D1 decreased luciferase activity down to 66% as compared to the positive control. Cyclin D1 has been shown to interact with a wide variety of transcription factors probably leading to a direct or indirect effect on reporter gene expression. Co-transfection of Sall4 and Cyclin D1 dramatically decreased reporter gene expression, which clearly indicates a synergistic effect of both proteins. Remarkably, SALL4 and CCND1 are both downstream targets of the canonical Wnt signaling pathway [16,27], and both were shown to be overexpressed in a multitude of tumor types [3,28]. Our data provide new insights in the biological function of Sall4 and may help to understand abundant expression of SALL4 in diverse cancer cell lines. It remains to be clarified, how the interrupted interaction between Sall4 and Cyclin D1 caused by nonsense or frameshift mutations in the SALL4 gene may contribute to the pathogenesis of the Okihiro syndrome. Acknowledgments The authors thank Bernhard Horsthemke and Beate Albrecht for their support. This work was supported by the Deutsche Forschungsgemeinschaft (Grant DFG KO1850/6-2,3 to J.K.). References [1] R. Al-Baradie, K. Yamada, C. St Hilaire, W.M. Chan, C. Andrews, N. McIntosh, M. Nakano, E.J. Martonyi, W.R. Raymond, S. Okumura, M.M. Okihiro, E.C. Engle, Duane Radial Ray Syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family, Am. J. Hum. Genet. 71 (2002) 1195–1199. [2] J. Kohlhase, S. Hausmann, G. Stojmenovic, C. Dixkens, K. Bink, W. Schulz-Schaeffer, M. Altmann, W. Engel, SALL3, a new member of the human spalt-like gene family, maps to 18q23, Genomics 62 (1999) 216–222. [3] J. Kohlhase, M. Heinrich, L. Schubert, M. Liebers, A. Kispert, F. Laccone, P. Turnpenny, R.M. Winter, W. Reardon, Okihiro syndrome is caused by SALL4 mutations, Hum. Mol. Genet. 11 (2002) 2979–2987. [4] J. Kohlhase, A. Ko¨hler, H. Ja¨ckle, W. Engel, R. Stick, Molecular cloning of a SALL1-related pseudogene and mapping to chromosome Xp11.2, Cytogenet. Cell Genet. 84 (1999) 31–34.
[5] J. Kohlhase, R. Schuh, G. Dowe, R.P. Ku¨hnlein, H. Ja¨ckle, B. Schroeder, W. Schulz-Schaeffer, H.A. Kretzschmar, A. Ko¨hler, U. Mu¨ller, M. Raab-Vetter, E. Burkhardt, W. Engel, R. Stick, Isolation, characterization, and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene spalt, Genomics 38 (1996) 291–298. [6] J. Kohlhase, M. Heinrich, M. Liebers, L. Froehlich Archangelo, W. Reardon, A. Kispert, Cloning and expression analysis of Sall4, the murine homologue of the gene mutated in Okihiro syndrome, Cytogenet. Genome Res. 98 (2002) 274–277. [7] J. Kohlhase, D. Chitayat, D. Kotzot, S. Ceylaner, U.G. Froster, S. Fuchs, T. Montgomery, B. Rosler, SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders, Hum. Mutat. 26 (2005) 176–183. [8] J. Kohlhase, L. Schubert, M. Liebers, A. Rauch, K. Becker, S.N. Mohammed, R. Newbury-Ecob, W. Reardon, Mutations at the SALL4 locus on chromosome 20 result in a range of clinically overlapping phenotypes, including Okihiro syndrome, Holt–Oram syndrome, acro-renal-ocular syndrome, and patients previously reported to represent thalidomide embryopathy, J. Med. Genet. 40 (2003) 473–478. [9] P. Terhal, B. Rosler, J. Kohlhase, A family with features overlapping Okihiro syndrome, hemifacial microsomia and isolated Duane anomaly caused by a novel SALL4 mutation, Am. J. Med. Genet. A 140 (2006) 222–226. [10] J. Miertus, W. Borozdin, V. Frecer, G. Tonini, S. Bertok, A. Amoroso, S. Miertus, J. Kohlhase, A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome, Hum. Genet. 119 (2006) 154–161. [11] W. Borozdin, D. Boehm, M. Leipoldt, C. Wilhelm, W. Reardon, J. ¨ . Giray, Clayton-Smith, K. Becker, H. Mu¨hlendyck, R. Winter, O F. Silan, J. Kohlhase, SALL4 deletions are a common cause of Okihiro and acro-renal-ocular syndromes and confirm haploinsufficiency as the pathogenetic mechanism, J. Med. Genet. 41 (2004) e113. [12] K. Koshiba-Takeuchi, J.K. Takeuchi, E.P. Arruda, I.S. Kathiriya, R. Mo, C.C. Hui, D. Srivastava, B.G. Bruneau, Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart, Nat. Genet. 38 (2006) 175–183. [13] Q. Wu, X. Chen, J. Zhang, Y.H. Loh, T.Y. Low, W. Zhang, W. Zhang, S.K. Sze, B. Lim, H.H. Ng, Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells, J. Biol. Chem. 281 (2006) 24090–24094. [14] U. Elling, C. Klasen, T. Eisenberger, K. Anlag, M. Treier, Murine inner cell mass-derived lineages depend on Sall4 function, Proc. Natl. Acad. Sci. USA 103 (2006) 16319–16324. [15] F.J. Kaiser, T. Moroy, G.T. Chang, B. Horsthemke, H.J. Ludecke, The RING finger protein RNF4, a co-regulator of transcription, interacts with the TRPS1 transcription factor, J. Biol. Chem. 278 (2003) 38780–38785. [16] J. Bo¨hm, C. Sustmann, C. Wilhelm, J. Kohlhase, SALL4 is directly activated by TCF/LEF in the canonical Wnt signaling pathway, Biochem. Biophys. Res. Commun. 348 (2006) 898–907. [17] S. McLeskey Kiefer, B. McDill, J. Yang, M. Rauchman, Murine Sall1 represses transcription by recruiting a histone deacetylase complex, J. Biol. Chem. 277 (2002) 14869–14876. [18] C. Netzer, L. Rieger, A. Brero, C.-D. Zhang, M. Hinzke, J. Kohlhase, S.K. Bohlander, SALL1, the gene mutated in Townes-Brocks syndrome, encodes a transcriptional repressor which interacts with TRF1/PIN2 and localises to pericentromeric heterochromatin, Hum. Mol. Genet. 10 (2001) 3017–3024. [19] V. Baldin, J. Lukas, M.J. Marcote, M. Pagano, G. Draetta, Cyclin D1 is a nuclear protein required for cell cycle progression in G1, Genes Dev. 7 (1993) 812–821. [20] T.C. James, S.C. Elgin, Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene, Mol. Cell. Biol. 6 (1986) 3862–3872.
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779 [21] S. Zhong, S. Muller, S. Ronchetti, P.S. Freemont, A. Dejean, P.P. Pandolfi, Role of SUMO-1-modified PML in nuclear body formation, Blood 95 (2000) 2748–2752. [22] S. McLeskey Kiefer, K.K. Ohlemiller, J. Yang, B.W. McDill, J. Kohlhase, M. Rauchman, Expression of a truncated Sall1 transcriptional repressor is responsible for Townes–Brocks syndrome birth defects, Hum. Mol. Genet. 12 (2003) 2221–2227. [23] M. Sakaki-Yumoto, C. Kobayashi, A. Sato, S. Fujimura, Y. Matsumoto, M. Takasato, T. Kodama, H. Aburatani, M. Asashima, N. Yoshida, R. Nishinakamura, The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development, Development 133 (2006) 3005–3013. [24] D. Sweetman, T. Smith, E.R. Farrell, A. Chantry, A. Mu¨nsterberg, The conserved glutamine rich region of chick csal1 and csal3
[25]
[26]
[27]
[28]
779
mediates protein interactions with other spalt family members. Implications for Townes–Brocks syndrome, J. Biol. Chem. 278 (2003) 6560–6566. M. Fu, C. Wang, Z. Li, T. Sakamaki, R.G. Pestell, Minireview: Cyclin D1: normal and abnormal functions, Endocrinology 145 (2004) 5439–5447. C. Netzer, S. Bohlander, M. Hinzke, Y. Chen, J. Kohlhase, Defining the heterochromatin localisation and repression domains of SALL1, Biochim. Biophys. Acta 1762 (2006) 386–391. M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D’Amico, R. Pestell, A. Ben-Ze’ev, The cyclin D1 gene is a target of the betacatenin/LEF-1 pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 5522– 5527. J.A. Diehl, Cycling to cancer with cyclin D1, Cancer Biol. Ther. 1 (2002) 226–231.
Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression Johann Bo¨hm
b
a,1
, Frank J. Kaiser b,1, Wiktor Borozdin Ju¨rgen Kohlhase a,d,*
a,d
, Reinhard Depping c,
a Institut fu¨r Humangenetik und Anthropologie, Universita¨t Freiburg, Freiburg, Germany Institut fu¨r Humangenetik, Universita¨tsklinikum Schleswig-Holstein, Campus Lu¨beck, Germany c Institut fu¨r Physiologie, Universita¨t zu Lu¨beck, Lu¨beck, Germany d Praxis fu¨r Humangenetik, Heinrich-von-Stephan-Str. 5, 79100 Freiburg, Germany
Received 6 March 2007 Available online 19 March 2007
Abstract Loss of function mutations in SALL4 cause Okihiro syndrome, an autosomal dominant disorder characterised by radial ray malformations associated with Duane anomaly. In zebrafish and mouse Sall4 interacts with TBX5 during limb and heart development and plays a crucial role for embryonic stem (ES) cell pluripotency. Here we report the nuclear interaction of murine Sall4 with Cyclin D1, one of the main regulators of G1 to S phase transition in cell cycle, verified by yeast two-hybrid assay, co-immunoprecipitation and intracellular co-localisation. Furthermore, using luciferase reporter gene assays we demonstrate that Sall4 operates as a transcriptional repressor located to heterochromatin and that this activity is modulated by Cyclin D1. 2007 Elsevier Inc. All rights reserved. Keywords: SALL4; Okihiro syndrome; Zinc finger; Nuclear localization; Cyclin D1; Cell cycle regulation; Transcriptional activity
SALL4 is one out of five human genes related to spalt (sal) of Drosophila melanogaster [1–5]. The mouse Sall4 protein [6] is likewise encoded by four exons and shares a homology of 74% with SALL4. SALL4 mutations cause Okihiro/Duane-Radial Ray syndrome (DRRS, OMIM 607323), an autosomal dominant disorder characterised by radial ray defects and Duane anomaly [1,3], but may include anal, renal, cardiac, ear and foot malformations, hearing loss, postnatal growth retardation and facial asymmetry [7–9]. The SALL4 gene product contains an amino terminal C2HC motif and three highly conserved, evenly distributed C2H2 double zinc finger domains. A single C2H2 motif is attached to the second domain [7]. All but one reported SALL4 mutations lead to preterminal stop codons [7,10]. These truncating mutations as well as larger
deletions involving SALL4 provided evidence of haploinsufficiency as the underlying pathogenic mechanism in Okihiro syndrome [11]. Sall4 controls Fgf10 expression in a synergistic manner together with Tbx5 in the forelimbs or with Tbx4 in the hindlimbs via direct effects on the Fgf10 promoter, whereas the effect of Sall4 and Tbx1 co-expression on the Fgf10 promoter is additive [12]. In the heart, Sall4 interacts with Tbx5 and activates Gja5, and on the other hand it interferes with Tbx5-dependent activation of Nppa [12]. Sall4 also interacts with Nanog, suggesting an important function of this complex in the regulatory networks of embryonic stem cells [13]. Recently Sall4 was shown to be an essential transcription factor required for the early development of inner cell mass derived cell lineages [14].
* Corresponding author. Address: Praxis fu¨r Humangenetik, Heinrichvon-Stephan-Str. 5, 79100 Freiburg, Germany. Fax: +49 761 896454 9. E-mail address: [email protected] (J. Kohlhase). 1 These authors contributed equally to this work.
Materials and methods
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.050
Synthesis of cDNA. Total RNA from adult mouse testis was prepared with TRIzol reagent and RNase-free DNase (Invitrogen, Carlsbad, CA).
774
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen) and oligo(dT) priming (Promega, Madison, WI). Yeast-two-hybrid assay. The complete coding sequence of murine Sall4 transcript variant A was used as a bait (Matchmaker Two-Hybrid System, BD Biosciences, Mountain View, CA). The fragment was amplified from murine testis cDNA and cloned into pGBKT7. The sequence was verified by automated sequencing (ABI PRISM 3100 Genetic Analyser, Applied Biosystems, Darmstadt, Germany) and the expressed protein was monitored by SDS–PAGE and Western blotting using monoclonal anti-c-Myc antibody (BD Biosciences). We screened against a mouse 11-day Embryo entire cDNA Library. After screening, clones were selected for auxotrophy on SD/LEU-HIS-TRP-ADE/X-Gal plates. Interaction partners were identified by sequencing and homology searches by BlastN. Co-immunoprecipitation. The coding region of the murine Sall4 was cloned into a FLAG expression plasmid (pFLAG-N3, derived from pEGFP-N3, BD Biosciences). The full-length Ccnd1 CDS, was cloned into the expression vector pCMV-Myc (BD Biosciences). Co-immunoprecipitation and subsequent Western blot analysis were carried out in COS-7 cells as described before [15]. Cell culture. Cell cultures were grown in minimal essential medium (MEM), 10% Fetal Calf Serum. 100 lg/ml pen/strep, 1· MEM nonessential amino acids (all purchased from Invitrogen) and maintained in a humidified environment with 5% carbon dioxide. Intracellular localisation. Transient transfection of HeLa, MCF-7, and COS-7 cells on coverslips (5 · 104 cells) was carried out as described before [15]. Intracellular localisation of the fusion proteins was visualized by confocal laser scanning microscopy. b-Galactosidase assay. Yeast AH109 cells were co-transfected with Ccnd1 (pACT2) and overlapping Sall4 fragments (pGBKT7). b-Galactosidase assay was performed as decribed elsewhere [15]. Reporter gene assays. Sall4 fragments were cloned in-frame to the GAL4 DNA-binding domain into the pM2 vector (BD Biosciences). As reporter construct, pGAL45tkLUC was used, which contains the luciferase gene under the control of the thymidine kinase (tk) promoter with four upstream GAL4-binding sites. Transient transfection of COS-7 cells was performed as described before [16]. Using 500 ng pGAL45tkLUC expression vector and 500 ng of different Sall4 (pM2) constructs to determine the repression domain of Sall4. For studies analysing the ability of Cyclin D1 to modulate Sall4 transcriptional repression, 500 ng pGAL45tkLUC, 200 ng of Sall4 (pM2) and 200 ng Ccnd1 (pM2) expression vectors were used.
Results Identification of potential Sall4 interacting proteins Screening a mouse embryonic prey library (11 dpc), we have isolated 34 clones encoding 32 different putative Sall4 interacting proteins. Two independent clones contained the entire ORF of Cyclin D1 (Ccnd1), the other clones are currently under investigation (data not shown). To confirm our data and to narrow down the Cyclin D1 interaction domain within the Sall4 protein, we generated overlapping truncated Sall4 constructs (Fig. 1A) and performed liquid b-galactosidase assays using ONPG as a substrate. Fragment 1 encoded aa 1–342 and contained a glutamine rich region as well as the single zinc finger domain residing in the amino terminal part of the protein. Fragment 2 (aa 705–1100) beared a large N-terminal truncation but still includes the carboxy-terminal double zinc finger. Fragments 3 (aa 1–756) and 4 (aa 305–1100) represented truncated, overlapping proteins missing the car-
boxy- or aminoterminal parts, respectively. In addition, Sall4 variants B and C (623 and 278 amino acids, respectively) were tested for interaction with Cyclin D1. The accurate expression of each construct was examined by Western blot analysis (Fig. 1B). Only AH109 cells cotransfected with Sall4 constructs bearing exons 3 and 4 and Ccnd1 demonstrated b-galactosidase activity. Consequently, we could narrow down the Cyclin D1 binding region within the Sall4 protein to the C-terminal 238 amino acids, which include the carboxy-terminal double zinc finger domain. Sall4 and Cyclin D1 interact in eukaryotic cells For co-immunoprecipitation studies, extracts of cotransfected COS-7 cells, co-transfected with Ccnd1-c-Myc and Sall4-FLAG were incubated with the M2 anti-FLAG agarose and protein complexes were precipitated and analysed by Western blot (Fig. 2). As controls, COS-7 cells were co-transfected with each construct and the respective empty vector. The Cyclin D1-c-Myc protein could only be identified in the precipitates of cells co-expressing Sall4 and Ccnd1, whereas the extracts of cells co-transfected with the empty FLAG plasmid did not show any specific Cyclin D1c-Myc signal (Fig. 2D). Our results clearly indicate that Sall4 and Cyclin D1 are able to form stable complexes in eukaryotic cells. Sall4 and Cyclin D1 co-localise in the nucleus Sall1/SALL1 proteins present a high degree of sequence similarity compared to the Sall4/SALL4 proteins and show specific nuclear distribution that is associated with heterochromatin localisation [17,18]. Because of this similarity we wanted to analyse the intracellular localisation of Sall4 as well as the localisation of its interaction with Cyclin D1, which was previously shown to be a nuclear protein [19]. We therefore co-transfected COS-7 cells with Sall4-FLAG and Ccnd1-GFP constructs and analysed the intracellular distribution of all proteins by confocal laser scanning microscopy (CLSM). Similar to the localisation of Sall1/SALL1, strong Sall4 signals were detected in the nuclei of the analysed cells, appearing in numerous dot-like structures. Interestingly, the distribution of Cyclin D1 proteins was almost identical, showing a distinct and homogeneous arrangement in the nucleus. An overlay of Sall4 and Cyclin D1 signals demonstrated a co-localisation of both proteins, suggesting a specific function of these protein complexes within the nucleus (Fig. 3C). To further elucidate these intranuclear dot-like structures formed by Sall4 and Cyclin D1, we performed additional co-localisation studies in MCF-7 cells and HeLa cells using antibodies against heterochromatin protein 1 (HP1) and PML bodies. HP1 is a conserved nonhistone chromosomal protein involved in heterochromatin formation and gene silencing [20]. PML nuclear bodies have been associated with various nuclear functions [21].
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
775
Fig. 1. (A) Structure Sall4 constructs used for yeast two-hybrid and b-galactosidase assays. All constructs result in proteins fused to the GAL4 DNAbinding domain. Zinc fingers are depicted as black rhombuses. (B) Expression of the Sall4 transcript variants and the truncated constructs monitored by Western blot analysis. (C) b-Galactosidase assays to narrow down the Cyclin D1-binding region. Expression of the reporter gene lacZ is activated in case of interaction between both proteins. Only constructs containing the regions encoded by exons 3 and 4 show interaction with Cyclin D1.
We could show that Sall4 does not co-localise with the PML bodies (Fig. 3A), but Sall4 staining overlaps with the HP1 signal (Fig. 3B), indicating an association of Sall4/SALL4 with heterochromatin. Sall4 operates as a transcriptional repressor The heterochromatic localisation of Sall1/SALL1 was previously shown to be associated with transcriptional repression activity [17,18]. We therefore wanted to elucidate if Sall4/SALL4 may also act as a repressor of transcription. We fused the full length ORFs of Sall4/SALL4 to the GAL4 DNA-BD and analysed their transcriptional activities in luciferase reporter gene assays by co-transfection with the pGAL45tkLUC reporter. Interestingly, we could identify a strong repressional activity of SALL4/ Sall4. To narrow down the repression-domain we generated several overlapping fragments of the human SALL4 (1–670, 1–960, 1–3159, 680–2334, and 2440–3076). The main repression domain could be located within the amino terminal part of the protein (aa 1–320, aa 1–223), being
capable of repressing transcription down to 10.3% in average compared to the control vector (Fig. 4A, columns 3 and 4). Constructs lacking the N-terminus and encoding amino acids 226–778 and 813–1025 exhibited a weaker repression activity (39.5% and 57.3%, respectively as compared to the empty pM2 vector (Fig. 4A, columns 5 and 6). It became apparent that a second repression domain resides within the carboxy terminal part of the protein. Transfecting COS-7 cells with the pGAL45tkLUC vector and a gradient amount of the Sall4 resulted in a linear and reciprocally proportional correlation between cellular Sall4 concentration and transcriptional repression activity (data not shown). Because Sall4 was shown to act as transcriptional repressor and its association with Cyclin D1 at heterochromatic region, we wanted to analyse the ability of Cyclin D1 to influence the repressing activity of Sall4. For this purpose we co-transfected COS-7 cells with Sall4-pM2, Ccnd1-pM2 and the pGAL45tkLUC reporter (Fig. 4B). The luciferase activity of cells transfected with pGAL45tkLUC and the empty pM2 vector was set to
776
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
A
B
Expression
1
2
3
4
1
130 Sall4-Flag pFlag Ccnd1-c-Myc c-Myc
C
IPs
2 anti-FLAG
130 + + -
1
+ +
2
+ + -
+ +
3
4
55 35
Ccnd1-c-Myc + c-Myc Sall4-Flag + pFlag -
D
Sall4-Flag Ccnd1-c-Myc c-Myc
+ + -
+ +
1
2
3
4
55 anti-c-Myc
35 + +
+ + -
+ +
Ccnd1-c-Myc c-Myc Sall4-Flag pFlag
+ + -
+ +
+ + -
+ +
Fig. 2. COS-7 cells were transiently transfected with Sall4-FLAG and Ccnd1-c-Myc constructs. (A) Shows the expression of the Sall4-FLAG constructs whereas (B) verifies the precipitation of the Sall4-FLAG protein. (C) Represents the expression of Ccnd1-c-Myc and (D) shows the co-precipitated Cyclin D1-c-Myc Protein only in extracts of cells co-transfected with Ccnd1-c-Myc and Sall4-FLAG. As controls, constructs were co-transfected with the respective empty vector.
Fig. 3. (A) Intracellular localisation of Sall4 and PML bodies. Sall4-GFP (green) was transfected into MCF-7 cells. Sall4 could be shown to appear in several dot-like structures within the nucleus of every analysed cell. Endogenous PML bodies (red), forming distinct structures in the nucleus, were shown not to overlap with the distribution pattern of Sall4. The nuclei, stained with DAPI, appear in blue. (B) The Sall4 signal (green) was shown to overlap with the pattern of endogenous HP1 (red). (C) Co-localisation of Sall4 and Cyclin D1. The subcellular distribution of the proteins was analysed in COS-7 cells. Sall4-FLAG (red) was detected in the nucleus of the analysed cells, appearing in dot-like structures of variable size, Cyclin D1-GFP (green) displayed a nuclear distribution pattern similar to Sall4. Overlay showed a co-localisation of Sall4 and Cyclin D1 in distinct dots within the nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
100%. Sall4-pM2 decreased the luciferase activity down to 46% and Cyclin D1-pM2 down to 66%. Interestingly, cotransfection Sall4-pM2 and Ccnd1-pM2 almost abolished
the luciferase activity. These findings clearly indicate a synergistic function of Sall4 and Cyclin D1 in transcriptional repression.
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
Rel. luciferase activity
A 120 100 80 60 40 20
G + AL 68 45 0- tk 23 LU 34 C GA + L 23 45 40 tk -3 LU 07 C 6
G + AL pM 45 2 tkL UC GA + L Sa 45 ll4 tk LU C GA + L 1- 45 67 tk 0 LU C GA + L 1- 45 96 tk 0 LU C
0
Rel. luciferase activity
B 120 100 80 60 40 20
-L + C uc + cnd Sall 1-p 4-p M2 M2
c+ -Lu TK
TK
M2 1-p Cc
Sa TK
-Lu
c+
-Lu TK
nd
ll4
c+
-pM
pM
2
2
0
Fig. 4. (A) Overlapping Sall4 constructs fused to the sequence encoding the GAL4 DNA-binding domain were transiently co-transfected with the pGAL45tkLUC vector into COS-7 cells. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty pM2 vector (set to 100%). Fragments containing amino acids 1– 320 (nucleotides 1–960) were shown to exhibit strong repression activity. The C-terminal part of the protein affects reporter gene expression level in a weaker fashion, suggesting that two distinct domains may work in concert. (B) Sall4-pM2 and Cyclin D1-pM2 independently repress expression of the reporter gene (columns 2 and 3). Co-transfection of both constructs results in synergistic repression of reporter gene expression (column 4).
Discussion SALL4 is currently the only gene associated with Okihiro/DRRS or acro-renal-ocular syndrome [7]. At least 90% of patients with classical Okihiro syndrome show mutations or deletions in the SALL4 locus. Nevertheless, in some clear cases of Okihiro syndrome we did not find mutations or deletions, raising the possibility that the phenotype is due to mutations in genes encoding putative binding partners of SALL4. Hence we have intended to identify proteins, which bind to the murine homologue Sall4 in order to clarify its biological function and to elucidate the pathogenesis of Okihiro syndrome. In a yeast two-hybrid assay, using the entire coding sequence of Sall4 as bait, we screened a mouse embryonal cDNA library for Sall4-binding candidates and obtained 34 different putative interacting proteins. Remarkably, we did not detect an interaction with any other member of the Sall protein family despite previous
777
findings showing homo- and heterodimerisation via the conserved glutamine-rich region in the N-terminal part of the proteins [22–24]. Although we achieved high transfection efficiencies in our two-hybrid assay, it is possible that we did not cover a representative amount of all Sall cDNAs. Analyzing all 34 clones, we identified two independent clones carrying the full-length ORF of Cyclin D1 (Ccnd1), which contained different parts of the 3 0 UTR, starting from 63 and 15 from ATG, respectively. Using extracts of transiently transfected COS-7 cells for co-immunoprecipitation assays, we were able to verify that Sall4 and Cyclin D1 do interact in mammalian systems. By liquid yeast bgalactosidase assays we could narrow down the region for interaction with Cyclin D1 within the C-terminus of Sall4, which encloses a double zinc finger domain. Truncated proteins lacking this carboxyterminal part of the protein do not bind to Cyclin D1. Interestingly, all but one reported SALL4 mutations in patients with Okihiro syndrome lead to preterminal stop codons within or 5 0 of exon 3 and result in proteins lacking the Cyclin D1 interaction domain [7]. However, it remains to be proven if truncated transcripts are—as expected—subject to nonsense mediated mRNA decay or if truncated and wildtype proteins have comparable stability. The Cyclin D1 protein is known to display a diversity of functions, which can either be dependent or independent on the association with kinases (CDK, Cyclin-dependent kinases) [25]. Phosphorylation and thus inactivation of retinoblastoma protein through the formation of a holoenzyme containing Cyclin D1 and CDKs accelerates G1 progression and is required for the entry to S phase. It has been demonstrated that Cyclin D1 interacts with a large number of transcription factors and cofactors/activators. Binding of histone acetylases, histone deacetylases and chromatin remodeling proteins results in modifications of the chromatin structure of genes involved in regulation of cell differentiation and proliferation. Our analysis of the intracellular localisation of Sall4 and Cyclin D1 revealed that Sall4, similar to SALL1, is distributed within small dot-like structures. As shown for SALL1, co-localisation studies with a heterochromatic marker (heterochromatin binding protein 1 (HP1)) confirmed that these structures are heterochromatic [18]. The association of Sall4 and Cyclin D1 within heterochromatic structures gave reason for the hypothesis that Sall4 acts as transcriptional repressor, whereby Cyclin D1 may modulate this function. By means of reporter gene assays, we could show that murine and human Sall4/SALL4 are able to act as transcriptional repressors. The main repressing domain of SALL4 is located within the amino terminal part of the protein and a second domain is located in the carboxyterminal region. Interestingly, similar results were also obtained for SALL1, which harbours one repression domain in its amino terminal region and a second one in the central part of the protein [26]. The mechanism determining whether Sall4/SALL4 act as a transcriptional activator or
778
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779
repressor have to be elucidated, as Sall4 has previously been shown to act as an activator of murine Fgf10 [12]. It is known that transcription factors are usually part of multi-protein complexes. The composition of these structures determinates the specific function by recruiting cofactors, which initiate repression or activation of the respective target gene. In luciferase reporter gene assays, we analysed the potential ability of Cyclin D1 in modulating the Sall4 transcriptional activity. Since no Sall4 target genes for transcriptional repression are known, we generated Sall4-pM2 and Ccnd1-pM2 constructs, resulting in GAL4 DNA-BD fusion proteins, and a reporter gene under the control of the tk promoter and repetitive GAL4 binding sequences. Sall4 reduced the reporter gene activity down to 46% and Cyclin D1 decreased luciferase activity down to 66% as compared to the positive control. Cyclin D1 has been shown to interact with a wide variety of transcription factors probably leading to a direct or indirect effect on reporter gene expression. Co-transfection of Sall4 and Cyclin D1 dramatically decreased reporter gene expression, which clearly indicates a synergistic effect of both proteins. Remarkably, SALL4 and CCND1 are both downstream targets of the canonical Wnt signaling pathway [16,27], and both were shown to be overexpressed in a multitude of tumor types [3,28]. Our data provide new insights in the biological function of Sall4 and may help to understand abundant expression of SALL4 in diverse cancer cell lines. It remains to be clarified, how the interrupted interaction between Sall4 and Cyclin D1 caused by nonsense or frameshift mutations in the SALL4 gene may contribute to the pathogenesis of the Okihiro syndrome. Acknowledgments The authors thank Bernhard Horsthemke and Beate Albrecht for their support. This work was supported by the Deutsche Forschungsgemeinschaft (Grant DFG KO1850/6-2,3 to J.K.). References [1] R. Al-Baradie, K. Yamada, C. St Hilaire, W.M. Chan, C. Andrews, N. McIntosh, M. Nakano, E.J. Martonyi, W.R. Raymond, S. Okumura, M.M. Okihiro, E.C. Engle, Duane Radial Ray Syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family, Am. J. Hum. Genet. 71 (2002) 1195–1199. [2] J. Kohlhase, S. Hausmann, G. Stojmenovic, C. Dixkens, K. Bink, W. Schulz-Schaeffer, M. Altmann, W. Engel, SALL3, a new member of the human spalt-like gene family, maps to 18q23, Genomics 62 (1999) 216–222. [3] J. Kohlhase, M. Heinrich, L. Schubert, M. Liebers, A. Kispert, F. Laccone, P. Turnpenny, R.M. Winter, W. Reardon, Okihiro syndrome is caused by SALL4 mutations, Hum. Mol. Genet. 11 (2002) 2979–2987. [4] J. Kohlhase, A. Ko¨hler, H. Ja¨ckle, W. Engel, R. Stick, Molecular cloning of a SALL1-related pseudogene and mapping to chromosome Xp11.2, Cytogenet. Cell Genet. 84 (1999) 31–34.
[5] J. Kohlhase, R. Schuh, G. Dowe, R.P. Ku¨hnlein, H. Ja¨ckle, B. Schroeder, W. Schulz-Schaeffer, H.A. Kretzschmar, A. Ko¨hler, U. Mu¨ller, M. Raab-Vetter, E. Burkhardt, W. Engel, R. Stick, Isolation, characterization, and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene spalt, Genomics 38 (1996) 291–298. [6] J. Kohlhase, M. Heinrich, M. Liebers, L. Froehlich Archangelo, W. Reardon, A. Kispert, Cloning and expression analysis of Sall4, the murine homologue of the gene mutated in Okihiro syndrome, Cytogenet. Genome Res. 98 (2002) 274–277. [7] J. Kohlhase, D. Chitayat, D. Kotzot, S. Ceylaner, U.G. Froster, S. Fuchs, T. Montgomery, B. Rosler, SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders, Hum. Mutat. 26 (2005) 176–183. [8] J. Kohlhase, L. Schubert, M. Liebers, A. Rauch, K. Becker, S.N. Mohammed, R. Newbury-Ecob, W. Reardon, Mutations at the SALL4 locus on chromosome 20 result in a range of clinically overlapping phenotypes, including Okihiro syndrome, Holt–Oram syndrome, acro-renal-ocular syndrome, and patients previously reported to represent thalidomide embryopathy, J. Med. Genet. 40 (2003) 473–478. [9] P. Terhal, B. Rosler, J. Kohlhase, A family with features overlapping Okihiro syndrome, hemifacial microsomia and isolated Duane anomaly caused by a novel SALL4 mutation, Am. J. Med. Genet. A 140 (2006) 222–226. [10] J. Miertus, W. Borozdin, V. Frecer, G. Tonini, S. Bertok, A. Amoroso, S. Miertus, J. Kohlhase, A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome, Hum. Genet. 119 (2006) 154–161. [11] W. Borozdin, D. Boehm, M. Leipoldt, C. Wilhelm, W. Reardon, J. ¨ . Giray, Clayton-Smith, K. Becker, H. Mu¨hlendyck, R. Winter, O F. Silan, J. Kohlhase, SALL4 deletions are a common cause of Okihiro and acro-renal-ocular syndromes and confirm haploinsufficiency as the pathogenetic mechanism, J. Med. Genet. 41 (2004) e113. [12] K. Koshiba-Takeuchi, J.K. Takeuchi, E.P. Arruda, I.S. Kathiriya, R. Mo, C.C. Hui, D. Srivastava, B.G. Bruneau, Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart, Nat. Genet. 38 (2006) 175–183. [13] Q. Wu, X. Chen, J. Zhang, Y.H. Loh, T.Y. Low, W. Zhang, W. Zhang, S.K. Sze, B. Lim, H.H. Ng, Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells, J. Biol. Chem. 281 (2006) 24090–24094. [14] U. Elling, C. Klasen, T. Eisenberger, K. Anlag, M. Treier, Murine inner cell mass-derived lineages depend on Sall4 function, Proc. Natl. Acad. Sci. USA 103 (2006) 16319–16324. [15] F.J. Kaiser, T. Moroy, G.T. Chang, B. Horsthemke, H.J. Ludecke, The RING finger protein RNF4, a co-regulator of transcription, interacts with the TRPS1 transcription factor, J. Biol. Chem. 278 (2003) 38780–38785. [16] J. Bo¨hm, C. Sustmann, C. Wilhelm, J. Kohlhase, SALL4 is directly activated by TCF/LEF in the canonical Wnt signaling pathway, Biochem. Biophys. Res. Commun. 348 (2006) 898–907. [17] S. McLeskey Kiefer, B. McDill, J. Yang, M. Rauchman, Murine Sall1 represses transcription by recruiting a histone deacetylase complex, J. Biol. Chem. 277 (2002) 14869–14876. [18] C. Netzer, L. Rieger, A. Brero, C.-D. Zhang, M. Hinzke, J. Kohlhase, S.K. Bohlander, SALL1, the gene mutated in Townes-Brocks syndrome, encodes a transcriptional repressor which interacts with TRF1/PIN2 and localises to pericentromeric heterochromatin, Hum. Mol. Genet. 10 (2001) 3017–3024. [19] V. Baldin, J. Lukas, M.J. Marcote, M. Pagano, G. Draetta, Cyclin D1 is a nuclear protein required for cell cycle progression in G1, Genes Dev. 7 (1993) 812–821. [20] T.C. James, S.C. Elgin, Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene, Mol. Cell. Biol. 6 (1986) 3862–3872.
J. Bo¨hm et al. / Biochemical and Biophysical Research Communications 356 (2007) 773–779 [21] S. Zhong, S. Muller, S. Ronchetti, P.S. Freemont, A. Dejean, P.P. Pandolfi, Role of SUMO-1-modified PML in nuclear body formation, Blood 95 (2000) 2748–2752. [22] S. McLeskey Kiefer, K.K. Ohlemiller, J. Yang, B.W. McDill, J. Kohlhase, M. Rauchman, Expression of a truncated Sall1 transcriptional repressor is responsible for Townes–Brocks syndrome birth defects, Hum. Mol. Genet. 12 (2003) 2221–2227. [23] M. Sakaki-Yumoto, C. Kobayashi, A. Sato, S. Fujimura, Y. Matsumoto, M. Takasato, T. Kodama, H. Aburatani, M. Asashima, N. Yoshida, R. Nishinakamura, The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development, Development 133 (2006) 3005–3013. [24] D. Sweetman, T. Smith, E.R. Farrell, A. Chantry, A. Mu¨nsterberg, The conserved glutamine rich region of chick csal1 and csal3
[25]
[26]
[27]
[28]
779
mediates protein interactions with other spalt family members. Implications for Townes–Brocks syndrome, J. Biol. Chem. 278 (2003) 6560–6566. M. Fu, C. Wang, Z. Li, T. Sakamaki, R.G. Pestell, Minireview: Cyclin D1: normal and abnormal functions, Endocrinology 145 (2004) 5439–5447. C. Netzer, S. Bohlander, M. Hinzke, Y. Chen, J. Kohlhase, Defining the heterochromatin localisation and repression domains of SALL1, Biochim. Biophys. Acta 1762 (2006) 386–391. M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D’Amico, R. Pestell, A. Ben-Ze’ev, The cyclin D1 gene is a target of the betacatenin/LEF-1 pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 5522– 5527. J.A. Diehl, Cycling to cancer with cyclin D1, Cancer Biol. Ther. 1 (2002) 226–231.