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Keywords:

  • WD repeats;
  • bromodomain;
  • transcription;
  • mouse development;
  • Down syndrome

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Human WDR9 has been mapped to chromosome 21, within one of the Down syndrome (DS) critical regions. Here, we study the expression pattern of the murine Wdr9 gene and its protein product. We show that Wdr9 is broadly expressed in the mouse embryo by means of in situ hybridization and immunohistochemistry. Wdr9 expression levels are dynamic during embryonic development as revealed by Northern blot analysis. We further show that WDR9 is a nuclear protein associated with BRG1, a SWI/SNF complex component. We also demonstrate that a polyglutamine-containing region of the protein functions as a transcriptional activation domain. We propose that WDR9 is a transcriptional regulator involved in chromatin remodeling through the action of two bromodomains and contacts to the SWI/SNF complex. These results may provide a molecular basis for the association of WDR9 with DS. Developmental Dynamics 227:608–614, 2003. © 2003 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Down syndrome (DS) is the leading cause of congenital heart disease and the most frequent genetic cause of mental retardation. Other clinical features include abnormalities in the craniofacial region, intestine, and immune system. Although a few genes have been implicated in the etiology of DS (Reeves et al.,2001), the molecular basis for its clinical features is far from understood. Genetic studies on partial trisomy patients have designated some regions on chromosome 21 as Down syndrome critical regions (DCRs) that correlate with specific phenotypes (Korenberg et al.,1992; Delabar et al.,1993). Identification of genes and characterization of their function in these regions should lead to an understanding of the molecular nature of DS defects. DCR-2 has been associated with certain facial and dermatoglyphic features, mental retardation, congenital heart disease, as well as duodenal stenosis (Korenberg et al.,1994). Human WDR9 was identified as a transcribed sequence within DCR-2 (Vidal-Taboada et al.,1998; Hattori et al.,2000). The expression of this transcript was further examined recently, and results indicated that human WDR9 is widely expressed in different human adult tissues and several cell lines with tissue-specific transcripts (Ramos et al.,2002).

The mouse Wdr9 gene has been mapped to the distal portion of chromosome 16, which is syntenic with human chromosome 21 (Pletcher et al.,2001). Ts65Dn mice that have segmental trisomy for mouse chromosome 16 show several features mimicking human Down syndrome (Reeves et al.,2001). Alternative splicing gives rise to two predicated Wdr9 open reading frames encoding C-terminal isoforms (GenBank accession no. AJ292467). WDR9 contains eight WD repeats in its N-terminus and two bromodomains in the central portion of the protein (Fig. 1).

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Figure 1. A schematic diagram of murine WDR9 protein. It contains eight WD repeats and two bromodomains (Bromo) as indicated. The coding region corresponding to a was used as a probe for in situ hybridization as well as Northern blotting analysis. Region b was used as the epitope for antibody generation. The coding sequence of region c was cloned into a GAL-DBD vector to generate GAL-WDR9f. Shaded and black boxes indicate a polyglutamine region and putative nuclear localization signals, respectively.

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The WD repeat-containing proteins have been implicated in a wide variety of cellular functions, such as transcriptional regulation, RNA processing, signal transduction, cytoskeleton assembly, and cell cycle (Smith et al.,1999; Li and Roberts,2001, and references therein). The bromodomain has been identified as a protein motif that binds specifically to acetylated lysine residues in histone tails and has been found in several chromatin-associated factors (Dyson et al.,2001, and references therein), many of which are components of transcriptional regulatory complexes. The presence of these functional domains, as well as a potential link with DS, led us to speculate that WDR9 may be involved in transcriptional regulation and/or other important cellular processes. Here, we report on studies of the murine Wdr9 gene and its protein products. We show that Wdr9 is broadly expressed during mouse embryonic development and that its protein products are located in the nucleus in NIH3T3 and HEK293 cells. We also obtained evidence that WDR9 interacts with BRG1, a component of the SWI/SNF chromatin remodeling complex, and that it harbors a transcriptional activation domain. These results strongly suggest that WDR9 acts as a transcriptional regulator in vivo, providing a molecular basis for its possible association with DS.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

A partial Wdr9 cDNA (nucleotides 2027 to 2516) was isolated from a 9.5 days postcoitus (dpc) mouse embryonic library. To examine the spatial and temporal pattern of Wdr9 expression, we performed in situ hybridization and Northern analysis with mouse embryonic samples from different developmental stages. Whole-mount in situ hybridization on 10.5 dpc mouse embryos revealed that Wdr9 transcripts are broadly distributed (Fig. 2A). The signal appears to be more intense in the craniofacial region, first branchial arch, and limb bud. In situ hybridization on 12.5 dpc embryonic sections gave more detailed information on the Wdr9 expression pattern. Wdr9 is strongly expressed in the frontonasal region, prevertebral column, and most of the visceral organs, such as liver, heart, lung, and gastrointestinal tract (Fig. 2C,E–H). The signals in the brain and spinal cord are relatively weak but detectable. This finding is consistent with a recent study on human chromosome 21 orthologous genes expression in mouse (Gitton et al.,2002), which reported absent or low abundance Wdr9 transcripts in neonatal brain. Northern analysis on a mouse embryo full-stage blot detected an 8.3-kb Wdr9 transcript during most developmental stages. The message begins to increase at 7.5 dpc and peaks at 10.5 to 11.5 dpc, an important stage in organogenesis. The expression level starts to decrease from 14.5 dpc and is very weak by late embryonic stages (Fig. 2I).

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Figure 2. Wdr9 transcripts are expressed broadly during embryonic development. A,B: Whole-mount in situ hybridization on 10.5 days postcoitus (dpc) mouse embryos. C,D: In situ hybridization on 12.5 dpc mouse embryonic parasagittal sections. A 490-bp fragment, indicated in Figure 1 was digoxigenin-labeled or [35S]UTP-labeled to generate antisense (A,C) and sense probes (B,D). Wdr9 transcripts are widely distributed in the embryos. Signals are more intense in the craniofacial area and first branchial arch (arrowheads in A). Heart and limb bud are also positive for Wdr9 signal (arrows in A). E–H: In situ hybridization on embryonic sections reveals Wdr9 message in the head and frontonasal region, such as medulla oblongata (m), tongue muscle (t), and nasal septum (n). Wdr9 transcripts are also present in the prevertebral column (v) and most of the internal organs such as heart (h), liver (lv), lung (lu), and intestinal tract (d). I: Northern blotting analysis on mouse embryonic full-stage blot (from 4.5 to 18.5 dpc). Upper panel, a transcript at around 8.3 kb was detected throughout development. Lower panel, loading control for the total RNA used for the full-stage blot.

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A search of the protein database (BLASTp) revealed similarity in the WD repeat region with several proteins in a variety of organisms. These proteins include WDR5 (BIG-3), which has been implicated recently in osteoblast differentiation (Gori et al.,2001), and mouse NDRP, which is expressed in developing and regenerating neurons (Kato et al.,2000). Of interest, hTAF100, a component of TFIID (Dynlacht et al.,1993; Dubrovskaya et al.,1996), shows significant homology to WDR9 at the WD repeat region. The surface of WD repeats have been proposed to regulate interactions with other proteins or ligands. Because similar surfaces are likely to bind a common partner, the homology to hTAF100 implicates WDR9 in a transcriptional regulatory role. In addition, the presence of several potential nuclear localization signals suggests that WDR9 is a nuclear protein (Fig. 1). Two bromodomains within WDR9 also suggest an association with chromatin. These observations prompted us to hypothesize that WDR9 functions as a transcriptional regulator. To examine the expression of WDR9 protein, we generated a polyclonal antibody against C-terminal residues 2135-2177 (Fig. 1, region b). Immunostaining showed that WDR9 is localized to the nucleus of NIH3T3 cells (Fig. 3) and HEK293 cells (data not shown). Moreover, it appears to be excluded from the pericentric heterochromatin (compare Fig. 3C with 4′,6-diamidine-2-phenylidole-dihydrochloride staining of 3D). The absence of WDR9 in transcriptionally silenced heterochromatin further supports a role in transcriptional activation.

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Figure 3. WDR9 is located in the nucleus of NIH3T3 cells. A,C: NIH3T3 cells were seeded on coverslips and were immunolabeled with anti-WDR9 antibody. The secondary antibody was conjugated with fluorescein isothiocyanate. WDR9 showed nuclear localization. B,D: Nuclei were revealed by 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining. Arrowheads in D point to regions of intensely stained pericentric heterochromatin. Note the lack of WDR9 in these areas (arrowheads in C).

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Immunohistologic analysis on 12.5 dpc mouse embryonic sections showed that the expression pattern of WDR9 protein generally correlates with the mRNA (compare Fig. 4A and Fig. 2C). Immunoreactive signals are broadly present in the frontonasal region, central, and posterior trunk (Fig. 4C–E). More detailed analysis showed that WDR9 is expressed in ectodermal derivatives such as surface epithelium, neural tissue of the brain (Fig. 4F,G), and spinal cord (data not shown). Intensive staining was also observed in dorsal root ganglia (Fig. 4H). In the developing circulatory system, both the endocardial lining and myocardial cells of the heart ventricular wall are positive for WDR9 (Fig. 4I). WDR9 is also present in mesenchyme of the cardiac cushion near the site of fusion with neural crest-derived aorticopulmonary septal tissue. Some of the positive cells are relatively large with extensive cytoplasmic processes distinguishable from the surrounding smaller mesenchymal cells (Fig. 4J, arrowhead and asterisk). Atrioventricular septal defects are common in children with DS. The separation of the cardiac tube into atria (A), ventricles (V), and outflow tract during development is achieved by mesenchymal outgrowths known as cardiac cushions. Signals from the AV mesenchyme modulate the transformation of endothelial cells into cushion mesenchyme (Eisenberg and Markwald,1995). The expression of WDR9 in this critical site may indicate a function in regulating this cell-transformation process. However, because neural crest also contributes to cushion formation, we cannot exclude a strictly neural crest origin for such WDR9-expressing cells.

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Figure 4. The expression of WDR9 in the mouse embryo. A: Immunohistochemistry was performed on 12.5 days postcoitus (dpc) mouse embryonic parasagittal sections. B: Absence of signals in control using antibody preabsorbed with antigen. C–E: Higher magnifications showing the expression WDR9 in the head (frontonasal), thoracic, and abdominopelvic regions, respectively. Note wide expression in many tissues and organs, such as tongue muscle (t), nasal septum (n), heart ventricles (hv), and endocardial cushion (e), lung (lu), the prevertebral column (v), liver (lv), intestinal tract (d), metanephros (k), and genital tubercle (g). The box in D is magnified in J. F,G: WDR9 is expressed in the surface ectoderm (arrow in F), neural tissue of the midbrain (MB), hindbrain (HB), and choroid plexus (CP) in the roof of the fourth ventricle. H: Intensive staining is observed in dorsal root ganglia (DG). I: In the heart ventricle, WDR9 is present in cardiocytes but absent from erythrocytes. The inset shows a higher magnification revealing nuclear localization of WDR9 in the endothelial cells that line the ventricular cavity. J: Higher magnification of the atrioventricular site of the heart (see box in D) that is near the fusion site of aorticopulmonary septal tissue and endocardial tissue. Endothelial cells lining the cavity (arrows) are positive for immunoreactive signals. Arrowheads point to strongly stained mesenchymal cells that are distinguishable from the surrounding smaller mesenchymal cells (asterisk) within the cushion. K: WDR9 is also detected in the cartilage primordia of the prevertebral condensations (C), the mesenchyme of the intervertebral discs (IVD), as well as the notochord (N). L,M: Immunoreactive signals in the lung epithelium (LE), duodenal epithelium (DE), and surrounding mesenchymal cells. Inset in L shows cytoplasmic localization of WDR9 in the lung epithelial cells (arrow points to nucleus devoid of immunoreactive signal). N: WDR9 is expressed at high levels in the liver. Multiple cell types contain WDR9, including megakaryocytes (arrow), hepatocytes (arrowhead), and erythroblasts (open triangle). O: The pancreatic primordium (arrowheads) and surrounding coelomic epithelium (arrows) are positive for WDR9 signal. P: WDR9 is detected in the nephrogenic mesenchyme (semicircle) and primitive collecting duct (arrow). Q: Immunoreactive signals are present in the genital tubercle region. Note the cytoplasmic localization of the signals in the lung epithelium, duodenal epithelium, dorsal root ganglia, and epithelium of the choroid plexus.

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WDR9 is also expressed in the prevertebral column, including the mesenchyme that will form the intervertebral disc as well as the cartilage primordium (Fig. 4K). Immunoreactive signals are also present in endodermal derivatives such as lung (Fig. 4L), duodenal epithelium (Fig. 4M), and associated mesenchyme. Positive signals can be detected in multiple cell types such as erythroblasts, hepatocytes, and megakaryocytes in the liver sinusoids (Fig. 4N). WDR9 is also expressed in the pancreatic primordium (Fig. 4O). In the metanephron, WDR9 can be found in the nephrogenic mesenchyme and the primitive collecting duct (Fig. 4P, semicircle and arrow, respectively). The ectoderm and mesenchymal cells in the genital tubercle also express WDR9 (Fig. 4Q).

In some tissues, the levels of Wdr9 transcript and protein product are not well correlated. For example, although transcripts are relatively abundant in tongue musculature, the immunoreactive signal is weak, but present (Figs. 2E vs. 4C). Conversely, the antibody strongly detects WDR9 in the central nervous system (CNS), whereas transcript levels are low. Nonetheless, comparison of Figure 2C and D shows that Wdr9 transcripts are indeed present in the CNS, consistent with their presence in adult brain (Ramos et al.,2002). Tissue-specific mechanisms affecting mRNA and protein stability and translational efficiency could easily account for discrepancies in transcript and protein levels.

Of interest, WDR9 showed differential subcellular localization among different embryonic tissues. For example, it is mainly cytoplasmically localized in the surface ectoderm, dorsal root ganglia, lung, and intestinal tract epithelium. But in most of the CNS, prevertebral cartilage, and myocardial cells, WDR9 is largely nuclear. These observations suggest that WDR9 function is differentially regulated through the control of its subcellular distribution.

Western blotting of cellular extracts from different human and mouse cell lines detected a signal at approximately 240 kDa, consistent with a predicated molecular mass of 248 to 253 kDa. However, there are additional immunoreactive bands in HEK293 cells with slightly different molecular weights (Fig. 5A). We also observe additional bands at much lower molecular weight (80–160 kDa) that could arise from degradation or alternative transcripts noted by others (Ramos et al.,2002). The detection of immunoreactive proteins in cells of neural (HEK293), germ cell/primitive ectoderm (P19), and embryonic fibroblast (NIH3T3) origins confirms the broad tissue distribution of WDR9.

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Figure 5. WDR9 functions as a transcriptional regulator. A: Western blotting analysis detected the expression of WDR9 in HEK293, P19, and NIH3T3 cells. The immunoreactive band(s) is approximately 240 kDa. Note additional bands at much lower molecular weight (80 to 160 kDa). B: Coimmunoprecipitation showed that WDR9 associates with BRG1 but not TBP or TAFII250. inp, whole cell extract input; IP, immunoprecipitated species. C: The polyglutamine region in WDR9 function as a transcriptional activation domain. GAL-WDR9f–induced reporter luciferase gene expression. Experiments were performed twice in duplicate, and error bars represent the standard error of the mean.

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To further investigate the function of WDR9, we studied its association with TBP and TAF250, two major components of the general transcription factor complex TFIID, as well as BRG1, a component of the ATP-dependent SWI/SNF chromatin remodeling complex. By coimmunoprecipitation, we demonstrated that WDR9 is associated with BRG1 but not TBP or TAF250 (Fig. 5B). The BRG1 interaction domain in WDR9 has not been mapped but could involve the WD repeats.

Polyglutamine residues function as transactivation domains in several factors including Sp1 (Gill et al.,1994). To examine the possible activity of the polyglutamine region within WDR9 (Fig. 1), we generated GAL-WDR9f, a fusion of WDR9 residues 632 to 846 (Fig. 1, region c) to the GAL DNA-binding domain (GAL-DBD) and measured its activity on an appropriate reporter construct. GAL-WDR9f induced reporter gene activity up to sevenfold compared with the unfused GAL-DBD vector (Fig. 5C). The above results are consistent with a role for WDR9 as a transcriptional activator.

The expression pattern of WDR9 as well as its potential role in transcriptional regulation makes it an important candidate for future investigations. Further characterization of its role in transcription and development should add to our understanding of its possible association with DS.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Northern Blot

A mouse embryonic full-stage blot (Seegene; MESB1002-1) was probed with a 490-bp fragment of the murine Wdr9 cDNA (nt 2027-2516, coding sequence for region “a” in Fig. 1).

In Situ Hybridization

Digoxigenin-labeled and [35S]UTP-labeled cRNA probes were generated by using the same region as that used for Northern blot. Whole-mount in situ hybridization was carried out as previously described (Folberg et al.,1997). In situ hybridization on sections was performed according to Wilkinson (1992).

Antibodies and Immunostaining

A partial Wdr9 est clone (IMAGE:3369701) was cloned into the vectors pMALP2 and pGEX-2T for the generation of MBP and GST fusion proteins in Escherichia coli. Rabbits were immunized with an MBP-WDR9 fusion protein (resides 2135-2177 of murine WDR9, Fig. 1 region b). Antiserum recognizing both WDR9 isoforms was purified by affinity chromatography over a GST-WDR9 (fusion protein of GST with the same murine WDR9 residues as above) column as previously described (Lamolet et al.,2001). Eluted immunoglobulin G was subsequently dialyzed and concentrated with a Millipore Ultrafree centrifugal filter. Immunofluoresence staining on NIH3T3 and HEK293 cells was performed essentially as described (Saleh et al.,2000). Immunohistologic analysis on embryonic sections was adapted from (Daniels et al.,1996). Samples were analyzed under a Nikon ECLIPSE E800 microscope with a Nikon DXM1200 digital camera.

Immunoprecipitation Assays and Immunoblotting

HEK293 cell were harvested and lysed in EBC lysis buffer (120 mM NaCl, 50 mM Tris-Cl pH 8.0, 0.5% NP40, and protease inhibitors) at 4°C for 30 min followed by brief sonication. Whole cell extracts were precleared with protein A agarose (Upstate) and then incubated with 1 μg of primary antibody, followed by addition of protein A agarose. Precipitates were washed once with high salt NETN (20 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5% NP40, 0.5 M NaCl), twice with NETN (without 0.5 M NaCl), and eluted with 1× sodium dodecyl sulfate (SDS) sample buffer. Cellular extracts or eluted proteins were separated by SDS-polyacrylamide gel electrophoresis. Antibodies used in Western blotting are anti-TAFII250 (Upstate Biotech), anti-TBP, and anti-BRG1 (Santa Cruz). Secondary antibodies were conjugated with horseradish peroxidase and detected by enhanced chemiluminescence (MEN Life Science).

Transfection and Luciferase Activity Assay

GAL-WDR9f was constructed by subcloning cDNA sequences encoding WDR9 residues 632-846 (Fig. 1, region c) 3′ to GAL-DBD sequence. A luciferase reporter construct driven by five GAL4 binding sites was cotransfected with the GAL-DBD or GAL-WDR9f expression vectors into HEK293 cells by calcium phosphate method. A cotransfected lacZ reporter driven by the Rous sarcoma virus LTR was used to normalize transfection efficiency. Luciferase assays were performed as described previously (Phelan et al.,1995).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank Erzsébet Nagy Kovács for technical support. H.H. is the recipient of an Internal Scholarship of the Faculty of Medicine, McGill University. M.F. is a Chercheur-National of the Fonds de la Recherche en Santé du Québec. M.F. received funding from the Canadian Institutes of Health Research.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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