Dev Genes Evol (2014) 224:261–268 DOI 10.1007/s00427-014-0476-x
Characterization of Bbx, a member of a novel subfamily of the HMG-box superfamily together with Cic TieLin Chen & Li Zhou & Yue Yuan & Yin Fang & Yue Guo & HuiZhe Huang & Qin Zhou & XiaoYan Lv
Received: 28 December 2013 / Accepted: 17 July 2014 / Published online: 1 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract High mobility group (HMG)-box proteins, a large and functionally diverse superfamily of architectural protein, are involved in the regulation of DNAdependent processes such as transcription, replication and DNA repair via the HMG-box domain. Bobby sox homolog (BBX), a newly identified HMG-Box protein, may function as a sequence-specific transcription factor. However, its expression pattern and biological functions are largely unknown. In this work, phylogenetic analysis showed that BBX is highly conserved and belongs to a novel subfamily of HMG-Box superfamily together with CIC (capicua homolog). Real time RT-PCR and whole-
mount in situ hybridization in zebrafish embryo revealed that bbx, cica, and cicb were maternally highly expressed from 4 cell to 1K cell stage, and the zygote expression was primarily distributed in the central nervous system (CNS) from 24 to 60 h post-fertilization (hpf). Immunohistochemistry analysis in mouse brain revealed that BBX was weakly expressed in the cerebellum and highly expressed in the cortex and hippocampus. These findings indicate that as a novel HMGbox protein, BBX maybe associated with CNS development and provides useful clues to further study of its biological functions.
Communicated by: Matthias Hammerschmidt TieLin Chen, Li Zhou, and Yue Yuan contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00427-014-0476-x) contains supplementary material, which is available to authorized users. T. Chen : Y. Fang : Q. Zhou Core Facility of Genetically Engineered Mice, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China T. Chen e-mail: [email protected]
Y. Fang e-mail: [email protected]
Q. Zhou e-mail: [email protected]
L. Zhou e-mail: [email protected]
Y. Yuan e-mail: [email protected]
Y. Guo e-mail: [email protected]
H. Huang Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, People’s Republic of China e-mail: [email protected]
X. Lv (*) Department of Dermatology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People’s Republic of China e-mail: [email protected]
L. Zhou : Y. Yuan : Y. Guo : Q. Zhou College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, People’s Republic of China
Present Address: X. Lv 1# Keyuan the Fourth Road, The district of Hi&Tech, Chengdu 610041, People’s Republic of China
Keywords HMG-box superfamily . Central nervous system . Bbx . cica . cicb
Dev Genes Evol (2014) 224:261–268
Material and methods Animals
Introduction High mobility group (HMG)-box proteins are a large and diverse superfamily of HMG proteins. The HMG-box domain is usually found in various DNA-binding proteins such as transcription factors and subunits of chromatin remodeling complexes, playing roles in the regulation of DNAdependent processes such as transcription, replication, and DNA repair (Stros et al. 2007). Based on their DNA binding preference, mammalian HMG-box containing proteins are usually classified into three major groups: the first group contains the non-histone chromosomal proteins, which do not bind to particular duplex DNA sequence, but bind to bent or distorted structures, such as HMG1 and HMG2 (Payet and Travers 1997). The second group includes transcription factors in the nucleus and mitochondria, which produce a discrete footprint on their target promoters, but have no observable specific target DNA sequence, such as UBF and mtTF1 (Kuhn et al. 1994). The last group is highly diverse and consists of sequence-specific transcription factors, which mostly have a single HMG-box and non-acidic C-tails with the ability to bind duplex DNA targets with high sequence specificity, such as SRY, Sox family, TCF1, and LEF1 (Sarkar and Hochedlinger 2013). Accumulating evidences show that the HMG-box superfamily plays an important role in development and various disorders. HMGA1-deficient mice result in diabetes, exhibiting a cardiac hypertrophy and expressing low level of insulin receptor (Semple 2009). Mutations in SRY give rise to XY females with gonad dysgenesis and translocation of SRY to the X chromosome would cause XX male syndrome (Maciel-Guerra et al. 2008). Moreover, testis development, central nervous system (CNS) neurogenesis, oligodendrocyte development, chondrogenesis, and neural crest development are all regulated by the Sox family transcription factors (Kiefer 2007). Bbx, a newly identified HMG-Box protein, contains one HMG-Box domain and a non-acidic C tail. It might function as a sequence-specific transcription factor. A previous study in fission yeast indicated that bobby sox homolog (BBX) activated Cdc10-dependent transcription at G1/S (Sanchez-Diaz et al. 2001). However, its biological function is still largely unknown in higher-ordered vertebrates. In the present study, we characterized a novel subfamily of HMG-box superfamily containing BBX and capicua homolog (CIC). Subsequent expression pattern studies in zebrafish embryo and mouse brain indicated that BBX is associated with central nervous system (CNS) development.
AB wild-type zebrafish were crossed and embryos were collected and kept in Holtfreter’s solution at 28.5 °C until appropriate stage. For in situ hybridization, embryos were treated with 0.003 % PTU (Sigma, Catalog # P7629) in Holtfreter’s solution between 12 and 24 h post-fertilization (hpf) to prevent melanization. C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free facility. Use of zebrafish and mouse in this research was carried out under protocols approved by the Animal Experimental Ethics Committee of Sichuan University.
Bioinformatics analysis and 3-D modeling The accession numbers of proteins used in Fig. 1 are NP_001136040.1 (Homo sapiens, BBX), XP_001150832.1 (Pan troglodytes, BBX), XP_002808361.1 (Macaca mulatta, LOC704762), XP_535728.3 (Canis lupus, BBX), NP_001179688.1 (Bos taurus, BBX), P_081720.2 (Mus musculus, Bbx), NP_001073407.1 (Rattus norvegicus, Bbx), XP_416622.3 (Gallus gallus, BBX), XP_001344562.5 (Danio rerio, LOC100005540), and XM_002932287.2 (Xenopus (Silurana) tropicalis, BBX). The above sequences and human HMG-box domains in Table 1 were aligned for generating a phylogenetic tree using MEGA5 software (http:// www.megasoftware.net/). Alignment was done by ClustalW using the PAM matrix. Phylogenetic tree was constructed using Neighbor-Joining method. 3-D Homology modeling of BBX HMG-box domain (aa80-153) was done by using SWISS-MODEL (http://swissmodel.expasy.org/SWISSMODEL.html) based on template 1wz6 in Protein Data Bank (http://www.rcsb.org/pdb/explore.do?structureId= 1wz6).
Plasmid construction For in vitro synthesis of digoxigenin-labeled RNA probe, the corresponding zebrafish bbx, cica, and cicb probe fragments were cloned by PCR with following primers: bbx (F: 5′-ATAG GATCCAAAGACCCAGAGGTGAAGC-3′; R: 5′-GGCC TCGAGACTTCCCACAGG TATGTCC-3′), cica (F: 5′ATAGGATCCACAGCAACAGTAGCACGACC-3′; R: 5′GGCCTCGA GAATGGCTTGAGCACTTATGG-3′), and cicb (F: 5′-ATAGGATCCCAACTACAGGCTCCTCG TCTC-3′; R: 5′-GGCCTCGAGTTGTCAACCGAATCAAA TGG-3′). Amplified probe fragments were inserted into pBST-II at the site of BamHI and XhoI.
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Fig. 1 Analysis of BBX protein sequences. a. Protein sequence comparisons. Protein sequences of BBX from human, mouse, Xenopus tropicalis and zebrafish, were employed for alignment. Identical amino acids were shaded in black and similar amino acids were shaded in gray. b. Phylogenetic tree construction using MEGA5 software. The identity of amino
acid sequences between human BBX and its orthologs in other species was indicated as percentage. c. Schematic drawing of the protein structure of BBX. NLS nuclear localization sequence; DUF domain of unknown function
Whole-mount in situ hybridization
Wild-type zebrafish embryos were collected and fixed at appropriate stages with 4 % PFA (paraformaldehyde). Embryos were dechorionated when needed and transferred to 100 % methanol and stored at −20 °C for at least 12 h. With BamHI digested plasmid as template, RNA probe was synthesized with T7 RNA polymerase (Fermentas USA, Catalog no. EP0111). Whole-mount in situ hybridization was performed according to the method previously described (Thisse and Thisse 2008). The digoxigenin-labeled probes were recognized by goat anti-digoxigenin antibody (1/3,000) (Roche, Catalog no. 11093274910). Chrome was developed in BM purple AP substrate (Roche, Catalog no. 11442074001) for proper time. Embryos were photographed with a LEICA Z6 APO stereoscope coupled to a LEICA DFC490 camera.
Immunohistochemistry was performed using standard protocols. Briefly, paraffin sections were dewaxed using xylene for 30 min and antigen retrieval was performed by boiling in 10mM sodium citrate with 0.05 % Tween-20 for 15 min. Rabbit anti-BBX antibody (1:200) from Proteintech (Wuhan, China, Catalog no. 17254-1-AP) was incubated overnight at 4 °C and secondary antibody for 2 h at room temperature. Signals were detected using the ABC kit for immunoperoxidase staining form Vector Laboratories (Burlingame, CA, Catalog # SA-5004). RNA extraction and quantitative RT-PCR (qPCR) Total RNA was extracted from cryopreserved tissue using TRIzol from Invitrogen (Shenzhen, China, Catalog no.
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Table 1 Human BBX HMG-box likely proteins Protein names Swiss port Length (aa) HMG-box Identity (%) Score Function BBX CIC HBPI SRY SOX1 SOX2 SOX3 SOX14 SOX21 SOX7 SOX17 SOX18 SOX5 SOX6 SOX13 SOX8 SOX9
Q8WY36 Q96RK0 O60381 Q05066 O00570 P48431 P41225 O95416 Q9Y651 Q9BT81 Q9H612 P35713 P35711 P35712 Q9UN79 P57073 P48436
941 1,608 514 204 391 317 446 240 276 388 414 384 763 828 622 446 509
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
100.00 61.00 39.00 31.00 37.00 37.00 35.00 38.00 36.00 35.00 35.00 34.00% 32.00 32.00 32.00 30.00 30.00
431 256 122 97 132 131 119 130 127 112 120 118 101 103 102 99 101
Cell cycle progression from G1 to S phase in yeast Transcriptional repressor in CNS development Transcriptional repressor binds 5′TTCATTCATTCA3′,Wnt pathway Sex-determining factor, necessary for testes development Transcriptional activator, neuronal development Transcription factor. early embryogenesis, neuronal development Transcription factor, neuronal development, male sex determination Negative regulator of transcription, neuronal development Stem cell differentiation, hair follicle development Transcriptional repressor of Wnt signaling, Binds to 5′AACAAT3′ Binds to 5′AACAAT or AACAAAG3′, inhibits Wnt signaling Trans-activate transcription via binding to 5′AACAAAG3′ Bind to 5′AACAAT3′, oligodendrocyte development Bind to 5′AACAAT3′, neurogenesis and skeleton formation Bind to 5′AACAAT3′, chondrogenesis, neurogenesis, limb development Bind to 5′[AT][AT]CAA[AT]G3′, CNS, limb and facial development Downstream effector of SRY, testis and skeletal development
SOX10 SOX15 TCF1/TCF7 HMG20A HMG20B HMGB1 HMGB2 SP100
P56693 O60248 P36402-4 Q9NP66 Q9P0W2 P09429 P26583 P23497
466 233 380 347 317 215 209 879
1 1 1 1 1 2 2 2
29.00 31.00 30.00 31.00 35.00 32.00 35.00 34.00
95 114 89 94 104 95 113 91
Neural crest development, specification of derivative cell fates Transcription factor binds 5′AACAAT3′ Bind to 5′WWCAAAG3′, cell differentiation and survival Neuronal differentiation, Chromatin remodeling factor Progression through G2 phase, RCOR1/CoREST mediated repression Architectural protein, genome stability, signal transduction Architectural protein, similar to HMGB1, male fertility Repressor/(or activator) of viral and cellular promoters
15596026), following the manufacturer’s protocol. Briefly, 200-mg mouse adult tissues or 20–40 zebrafish embryos were added into 1-ml TRIzol, homogenized with a glass homogenizer and incubated at room temperature for 5 min. After chloroform extraction and precipitation with isopropanol, RNA was washed twice with 75 % ethanol, and finally, the RNA pellet was dissolved in RNase-free water. The RNA concentration was determined using a NanoDrop 2000 spectrophotometer and reverse transcription reactions were performed from 5 ug of total RNA with random primers using RevertAid First Strand cDNA Synthesis Kit from Fermentas (Shenzhen, China, Catalog no. K1622). Real time PCR reactions were performed using UltraSYBR Mixture from CW Bio (Beijing, China, Catalog no. CW0957). Samples were held at 95 °C for 10 min, followed by 40 amplification cycles consisting of a denaturation step at 95 °C for 15 s, an annealing step at 60 °C for 15 s, and an extension step at 72 °C for 15 s. The primer sequences were the following: Reference gene in zebrafish, eef1a1 (F: 5′-CTGGAGGCCAGCTCAAACAT-3′; R: 5′-ATCAAGAAGAGTAGTACCGCTAGCATTAC-3′), bbx (F: 5′-GCATCCACGATTAGACAAC C-3′; R: 5′-GAAGGA CTCTTTACTGGCTTG-3′), cica (F: 5′-AGAGGGTGATGG
TAGGAGGG-3′; R: 5′-GGATGTAACAAACTCGGCACT3′), and cicb (F: 5′-TGCAGCTATCGCTCATCCG-3′; R: 5′GCAAGGGTCACATTTGTTGG-3′). Reference gene in mouse, 18s (F: 5′-GTAACCCGTT GAACCCCATT-3′; R: 5′CCATCCAATCGGTAGTAGCG-3′), Bbx (F: 5′-TTCAACTG GCAGAG ATGTGTC-3′; R: 5′-CTGCTTCCTTCTGGAACG AAGT-3′).
Results and discussion Phylogenetic study of vertebrate BBXs Comparison of human BBX amino acid sequence with corresponding orthologs of other vertebrate species using ClustalW multiple sequence alignment software revealed high similarity between each other (Fig. 1a), with sequence identity ranged from 62.6 to 99.7 % in relative to human (Fig. 1b). Functional domain prediction indicated that BBXs contains a nuclear localization sequence (NLS) and a HMG-Box domain at N terminal, a domain of unknown function (DUF2028) in the
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middle and a non-acidic C-tail (Fig. 1c). Given the NLS and HMG-Box domain, BBX might function as a sequencespecific transcription factor. BBX, a member of a novel subfamily of the HMG-box superfamily together with CIC
BBX HMG-box domain is similar to that found in the Sox family, the level of similarity is insufficient to include this gene as a genuine member of the Sox subfamily (Table. 1). Therefore, these findings indicate that BBX and CIC genes define a new Sox-related HMG subfamily. Spatio-temporal expression pattern of the novel subfamily genes during zebrafish embryogenesis
Alignment indicated that the HMG-Box domain of BBX is evolutionarily conserved among different species (Fig. 2b). 3D modeling revealed a typical L-shaped fold consisting of three a-helices, which might be responsible for binding of HMG-boxes to the DNA minor groove in a sequencespecific fashion like SRY and LEF-1 (Fig. 2c). We used the amino acid sequence of the human BBX HMG-box domain (residues 80–159) as a query to identify BBX HMG-box-like proteins in the human protein database (http://www.uniprot. org/). The search retrieved 25 proteins with significant sequence similarity to the HMG-box domain of human BBX protein half of which are related to neuronal development (Table. 1). The retrieved sequences were aligned and used to construct an unrooted phylogenetic tree (Fig. S1). BBX and CIC (capicua homolog) can be grouped together, which was further confirmed by phylogenetic tree construction just based on the HMG-box domains (Fig. 2a). Although the sequence of
To uncover potential functions of bbx, real time PCR and whole-mount in situ hybridization assay of the novel subfamily of HMG-box superfamily were carried out to evaluate their expression patterns during zebrafish embryogenesis. There are two copies of Cic (cica and cicb) in zebrafish. The data showed that bbx, cica, and cicb were maternally provided and highly expressed from 4 cell to 1K cell stage (Fig. 3a–c and Fig. 3d, a1–c3). At shield stage, all the three genes were weakly and ubiquitously expressed (Fig. 3d, d1–d3). bbx transcripts could be detected at the head from bud stage to 60 hpf (Fig. 3d, e1–j1). Although cicb and cica were both weekly expressed from shield stage to 5–6 somite stage (Fig. 3d, d2–f3), cicb was highly expressed in hindbrain at 5–6 somite stage (Fig. 3d, f3). Specific expression of cica and cicb in the head were apparent from 24 to 60 hpf (Fig. 3d, g2–
Fig. 2 HMG-Box domain analysis of BBX. a. Unrooted phylogenetic construction based on HMG-boxes. Each HMG-box (even the boxes from the same protein) was treated as a single taxonomic unit. b. Alignment of BBX HMG-Box domain from human, mouse, Xenopus
tropicalis and Zebrafish, respectively. Identical amino acids were shaded in black and similar amino acids were shaded in gray. Three α-helices of the HMG-box domain were highlighted by red lines. c. 3D Modeling of human BBX HMG-box domain
Fig. 3 Spatio-temporal expression pattern of bbx, cica, and cicb during zebrafish development. a–c. Real-time PCR of bbx, cica, and cicb in developmental embryos and adult fish. d. In situ hybridization results of bbx, cica, and cicb in embryos at different developmental stages. All the three genes were maternally provided and highly expressed from 4 cell to 1K cell stage (a1–c3). At shield stage, they were weakly and ubiquitously expressed (d1–d3). bbx transcripts could be detected at the head from bud stage to 60 hpf (e1–j1). Although cica and cicb both were weekly
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expressed from shield stage to 5–6 somite stage (d2–f3), cicb transcripts can be specifically detected in hindbrain (f3). Specific expression of cica and cicb in head were apparent from 24 to 60 hpf (g2–j3). 4-cell: animal pole view; 1 K cell and Sphere: Lateral view with animal pole on the top; Shield and Bud: Lateral view with dorsal side to the right; 5–6 Somites, 24, 36, 48, 60, and 72 hpf: Lateral view with anterior to the left; hb: hindbrain
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Real time PCR revealed that Bbx was highly expressed in brain, spleen, testis, and kidney (Fig. 4a). Then, we analyzed the expression of Bbx in those adult organs at protein level by using immunohistochemistry. In mouse brain, BBX was weakly expressed in cerebellum and highly expressed in the cortex and hippocampus (Fig. 4b, 1–5). In line with the previous study which observed mouse BBX expression within ventricular zone progenitor cells within the developing
neocortex and hippocampus (Dixon et al. 2013), our data from zebrafish and mouse brain indicated that BBX is associated with CNS development. At subcellular level, BBX was mainly localized in the nucleus and weakly expressed in the cytoplasm (Fig. 4b, 4) indicating that BBX might function as cytoplasm protein besides transcription factor. Interestingly, Bbx knockout mice were generated by the European Conditional Mouse Mutagenesis Program (Friedel et al. 2007). Although BBx knockout mice generated by the European Conditional Mouse Mutagenesis Program exhibited some phenotypic abnormalities, such as increased plasma IgA and decreased lean body, mass, and cardiac weight (Bassett et al. 2012), they did not exhibit any gross morphological abnormalities of the nervous system using cresyl violet staining (http://www.sanger.ac.uk/mouseportal/phenotyping/ MAEZ/mp-report/nervous-system/). These findings do not rule out a role for BBX during CNS development. Future studies aimed at analysing neural progenitor cell selfrenewal and differentiation may reveal defect and uncover the roles of BBX in mouse CNS development. Besides the brain, BBX might function in other organs. In the spleen, BBX was expressed in the nucleus and cytoplasm,
Fig. 4 Spatio-temporal expression pattern of Bbx in mouse. a. Real-time PCR of bbx in different organs of adult mouse. bbx was highly expressed in brain, spleen, testis, and kidney. b–e. IHC of BBX in mouse adult brain, spleen, testis, and kidney. BBX was weakly expressed in cerebellum (B5) and highly expressed in cortex (B3) and hippocampus (B2). In hippocampus, BBX mainly localized in the nucleus and was weakly expressed in the cytoplasm (B4). In spleen, BBX appeared to be
expressed in the nucleus and cytoplasm (C1–2). In testis, BBX was mainly localized in the nucleus of spermatogonia and spermatocyte, and the cytoplasm of spermatid and mature spermatid cells, no expression was detected in sertoli cells (D1–2). In kidney, BBX was expressed in the nucleus and cytoplasm of renal tubes and collecting tubes, no expression was found in glomeruli (E1–2). DG Dentate gyrus; CA Cornu Ammonis; G Glomeruli
j3). Similar expression patterns also support the above conclusion that these genes are members of the same subfamily and might play a role in CNS development. Cic, a known regulator of embryonic body patterning, is involved in epidermal growth factor receptor (EGFR) signaling, developmental patterning, and cell fate determination (Jimenez et al. 2012). In humans, mutations of Cic are implicated in granule cell lineage and medulloblastoma tumorigenesis (Sahm et al. 2012; Lee et al. 2005). Whether Bbx share a similar function to those of Cic in the CNS system is unknown. Expression pattern of Bbx in mouse
but the cell types need to be confirmed with specific markers (Fig. 4c, 1–2). In testis, BBX was mainly localized in the nucleus of spermatogonia and spermatocyte, and the cytoplasm of spermatid and mature spermatid cells, no expression was found in sertoli cells (Fig. 4d,1–2). In kidney, BBX was expressed in the nucleus and cytoplasm of renal tubes and collecting tubes (Fig. 4, e1–2). The detailed functions of BBX in these organs are required to be further explored.
Acknowledgments We thank Rui Gao for technical assistance. This work was supported by the National Basic Research Program of China Grant 2011CB944002 (to QZ) and the National Natural Science Foundation of China Grant 31271563 (to QZ) Conflict of interest The authors declare that they have no conflict of interest.
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