Species-specific expansion of C2H2 zinc-finger genes and their expression profiles in silkworm, Bombyx mori

Insect Biochemistry and Molecular Biology 38 (2008) 1121–1129

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Species-specific expansion of C2H2 zinc-finger genes and their expression profiles in silkworm, Bombyx mori Jun Duan a, b, Qingyou Xia a, b, *, Daojun Cheng a, Xingfu Zha a, Ping Zhao a, Zhonghuai Xiang a a b

The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Chongqing 400716, China The Institute of Agricultural and Life Sciences, Chongqing University, Chongqing 400030, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 November 2007 Received in revised form 10 August 2008 Accepted 25 August 2008

Most C2H2 zinc-finger proteins (ZFPs) function as sequence-specific DNA-binding transcription factors, and play important roles in a variety of biology processes, such as development, differentiation, and tumor suppression. By searching the silkworm genome with a HMM model of C2H2 zinc-fingers, we have identified a total of 338 C2H2 ZFPs. Most of the ZFP genes were clustered on chromosomes and showed uneven distribution in the genome. Over one third of genes were concentrated on chromosome 11, 15 and 24. Phylogenetic analysis classified all silkworm C2H2 ZFPs into 75 families; 63 of which belong to evolutionarily conserved families. In addition, 188 C2H2 ZFP genes (55.6%) are species-specific to the silkworm. A species-specific expansion of a family with 39 members in a tandem array on chromosome 24 may explain the higher number of species-specific ZFPs in silkworm compared to other organisms. The expression patterns of C2H2 ZFP genes were also examined by microarray analysis. Most of these genes were actively expressed among different tissues on day 3 of the fifth instar. The results provide insight into the biological functions of the silkworm C2H2 ZFP genes in metamorphism and development. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: C2H2 zinc-finger Transcription factor Metamorphism Development Expression pattern Bombyx mori

1. Introduction Zinc-finger proteins (ZFPs) are families of proteins that contain one or more zinc-finger domain(s). Among zinc-fingers, one of the most prevailing types is the C2H2 zinc-finger. Since the first such domain was identified in the transcription factor TFIIIA of Xenopus laevis (Miller et al., 1985), numerous C2H2 type ZFPs have been found in organisms ranging from yeast to human (Bohm et al., 1997; Clarke and Berg, 1998; Englbrecht et al., 2004; Knight and Shimeld, 2001). The C2H2 zinc-finger domain has been shown that could be implicated in DNA-binding, RNA-binding, as well as involved in protein–protein interactions. However, the major function of this domain is DNA-binding. Most of the C2H2 ZFPs are transcription factors, which are involved in a variety of biological processes such as development, differentiation and tumor suppression (Ladomery and Dellaire, 2002; Munoz-Descalzo et al., 2005). For example,

Abbreviations: ZFP, zinc-finger protein; C2H2, Cys(2)/His(2); EST, expressed sequence tag; HMM, hidden Markov model; fru, fruitless; BR-C, Broad-Complex gene; WGS, Whole Genome Shotgun; MCL, Markov cluster algorithm; A/MSG, anterior/median silk gland; PSG, posterior silk gland; ZAD, zinc-finger associated domain; HSP, high-scoring segment pair. * Corresponding author at: The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Tiansheng Road, Beibei, Chongqing 400716, China. Tel.: þ86 23 6825 0099; fax: þ86 23 6825 1128. E-mail address: [email protected] (Q. Xia). 0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.08.005

pattern formation along the anterior–posterior axis of the Drosophila embryo is controlled by a hierarchy of genes. Two key regulatory factors, Hunchback and Kru¨ppel, are C2H2 ZFPs that bind specific DNA sequences to regulate target genes (Stanojevic et al., 1989). Functional studies have demonstrated that Hunchback provides positional information for critical regulation of both segmentation and homeotic genes along the anterior–posterior axis (Wu et al., 2001). Drosophila Kru¨ppel has been suggested to function as a repressor, acting on adjacently expressed gap genes and subordinate pair-rule genes within the segmentation gene cascade (La Rosee-Borggreve et al., 1999). The C2H2 zinc-finger domain typically has a short stretch of 30 amino acids, with two conserved cysteines and two conserved histidines that coordinate a zinc ion that allows the finger to fold into a stable structure. This structure forms two stranded anti-parallel b-sheet and an a-helix to bind specifically on 3 w 4 base pairs along the major groove of DNA (Pavletich and Pabo, 1991; Wolfe et al., 1999). Generally, most of ZFPs have tandem repeats of C2H2 zinc-fingers. Although one zinc-finger can only specifically bind 3 w 4 base pairs, tandem repeats of C2H2 zinc-fingers can recognize a longer DNA sequence. In addition, by substituting critical amino acid residues in the zinc-fingers or linking zinc-fingers with variant DNA-binding specificities, ZFPs can theoretically bind to any given DNA sites. Therefore, zinc-fingers provide an attractive framework for the design of artificial transcription factors. Recent work has demonstrated that artificial C2H2 zinc-finger transcription factors

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could alter expression of specific endogenous genes in cell types ranging from yeast to human (Papworth et al., 2006; Urnov et al., 2005). Due to the importance of natural ZFPs in biological processes and the potential applications used for creating new artificial transcription factors, these genes have attracted considerable interest. However, most of ZFPs have not been well studied in the silkworm, Bombyx mori, which is an economically important insect and a Lepidoptera model for the study of pest control in agriculture (Goldsmith et al., 2005). Recently, an international collaboration has been carried out to assemble a fine genome map of the silkworm, which is based on the 6 and 3 draft sequences that were completed by two independent Whole Genome Shotgun (WGS) sequence projects in 2004 (Mita et al., 2004; Xia et al., 2004). The high quality genome data provide a good opportunity to systematically study C2H2 ZFP genes in the silkworm. Here we present the identification and analysis of these genes using the silkworm genome and microarray data. 2. Materials and methods 2.1. Data sets The updated assembled silkworm genome was used in this study (The International Silkworm Genome Sequencing Consortium, submitted for publication). A high quality reference gene data set, which contains a total of 14,623 genes, was used to identify silkworm C2H2 ZFP genes. Gene datasets for Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster were also downloaded from Ensembl (http://www.ensembl.org) for comparative analysis. Since Ensembl gene datasets contain all transcripts of the same gene, we kept only the longest transcript and discarded shorter isoforms for any given genes to obtain a non-redundant gene dataset. 2.2. Identification of C2H2 ZFP genes The hidden Markov model (HMM) of PF00096 was used to search the deduced proteins of the silkworm genes by HMMER (Eddy, 1998; Finn et al., 2006). The threshold was set up as score greater than 0.0 and E-value less than 0.1. Proteins that contain at least one C2H2 zinc-finger were extracted. Subsequently, the extracted proteins were used to search against the whole Pfam A database by Hmmpfam (Eddy, 1998). If a domain overlaps with other kind of domains, the questionable domains usually have low scores and high E-value. We wrote a PERL script to inspect the overlaps and manually eliminated the questionable domains. This process will not only reduce false-positives for C2H2 zinc-finger detection, but also will identify C2H2 zinc-finger associated domains. The same process was applied to H. sapiens, C. elegans and D. melanogaster C2H2 ZFP identifications. 2.3. C2H2 ZFPs families classification Since tandem repeats of C2H2 zinc-fingers and consensus linker sequence between zinc-fingers often confuse the proteins grouping and determination of evolutionary history, the TRIBE-MCL clustering algorithm was adopted to classify ZFP families (Knight and Shimeld, 2001; Enright et al., 2002). This algorithm, which is based on the Markov cluster (MCL) algorithm, has been shown to be an efficient and reliable method for sequence clustering and grouping of multi-domain structure of proteins (Enright et al., 2002). In order to find the evolutionary conserved family members, the identified C2H2 ZFPs of H. sapiens, C. elegans and D. melanogaster were also analyzed by this method. All-against-all BLAST search was performed by using BLASTP (Altschul et al., 1997). The sequence

similarity threshold E-value was set to 1E-30, and the blast result was parsed and stored in a square matrix. Then the TRIBE-MCL clustering algorithm was used to detect family members. The clustering operations were implemented at inflation values of 2.0. 2.4. Phylogenetic analyses Identified C2H2 ZFPs families were used for phylogenetic analyses. Multiple alignments of protein sequences were made by ClustalW (Thompson et al., 1994). Then neighbor-joining phylogenetic trees were reconstructed by MEGA 4.0 (Tamura et al., 2007). The parameters were chosen as follows: the evolutionary distance was poisson-corrected, gaps were completely deleted, and 1000 iterations were used for calculating bootstrap values. 2.5. Microarray experiment and data analysis A genome-wide 70-mer oligonucleotides microarray with 22,987 probes has previously been customized for the silkworm (Xia et al., 2007). Two hundred ninety nine of the 338 C2H2 zincfinger genes identified in this study were found to have probes on the microarray. The expression patterns of these genes have been surveyed for the 10 representative sample types of Chinese silkworm strains (Dazao) on day 3 of the fifth instar. The representative samples included nine samples, such as the anterior/median silk gland (A/MSG), posterior silk gland (PSG), testis, ovary, fat body, midgut, integument, hemocyte and malpighian tubule, and one specific sample (i.e. head). The detailed experimental process, quality control, consistency in replication and data analysis for these experiments have been described in our previous report (Xia et al., 2007). 3. Results 3.1. Characterization of C2H2 zinc-finger genes in the silkworm genome By searching the new release of the reference gene dataset with the PF00096 of Pfam HMM model, we identified a total of 2337 C2H2 zinc-fingers in silkworm. These zinc-fingers were distributed in 338 proteins, which constitute 2.3% of the annotated genes (Table 1). To date, only four genes have been deposited in GenBank, namely hunchback (O18326), BR-C (BAD24049), JAZ (ABD36224), and a partial sequence of the cubitus interruptus gene Bm-Ci (AF327655). For the other C2H2 ZFPs, we named them as BmZFP1 w BmZFP334 (see Data S1). These genes constitute the first catalogue of C2H2 ZFPs in the silkworm. Approximately 102 C2H2 ZFP genes were found to be expressed when validated by EST evidence using following stringent threshold: BLAST alignments with E-value <1E-30, identities >80% and length coverage >70%. C2H2 ZFPs are critical to gene regulatory networks and thus may influence most biological processes in an organism. We compared the numbers of C2H2 ZFP genes among organisms. As shown in Table 1, there are 646 C2H2 zinc-finger domains distributed in 155 C2H2 ZFPs in C. elegans. Both of the number of C2H2 ZFP genes and the number of C2H2 zinc-fingers increased in the silkworm. In vertebrates, a remarkable increase of the number of ZFP genes and zinc-fingers were observed. For example, in H. sapiens about 7064 C2H2 zinc-fingers were distributed in 794 genes. It appears that there is a positive correlation between an organism’s complexity and the number of C2H2 ZFP genes, as well as their corresponding C2H2 zinc-fingers. The average number of zinc-fingers per ZFP increases from C. elegans to silkworm to H. sapiens, which has the average number of four, seven, 10 zinc-fingers per ZFP, respectively. Although both the silkworm and D. melanogaster are insects, the silkworm has

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Table 1 Comparatively analysis of C2H2 ZFPs in the B. mori, D. melanogaster, C. elegans and H. sapiens. Number of C2H2 ZFP genes

Percent

14,623 14,039 20,060 22,983

338 285 155 743

2.3 2.0 0.8 3.2

Number of C2H2 zinc-finger domains

Species-specific genes Singletons

Genes in speciesspecific families

Total

Percent of Speciesspecific ZFP genes (%)

2337 1785 646 7064

125 96 108 73

63 29 10 87

188 125 118 160

55.6 43.9 76.8 21.5

more zinc-fingers per ZFP than D. melanogaster, an average number seven vs. six zinc-fingers per ZFP, respectively. In addition, the total number of C2H2 zinc-fingers is also much higher in the silkworm. We found 1785 C2H2 zinc-fingers distributed in 285 genes in D. melanogaster vs. 2337 zinc-fingers distributed in 338 genes in the silkworm (Table 1). We also investigated the distribution of the total number of ZFPs per species ranked by the number of zinc-fingers per protein. As shown in Fig. 1A, the silkworm and D. melanogaster have similar numbers of ZFPs with less than 10 C2H2 zinc-fingers per protein. However, D. melanogaster has 31 genes with more than 10 zincfingers, while the silkworm has 54 such genes (Fig. 1A). The corresponding zinc-fingers in this rank are remarkably higher for silkworm, with 886 vs. 455 zinc-fingers for D. melanogaster (Fig. 1B). These results indicate that the higher number of zincfingers of the silkworm in comparison with D. melanogaster is mainly due to the increase of ZFPs that contain greater than 10 zincfingers per gene.

3.2. Associated domain of C2H2 zinc-finger C2H2 ZFPs frequently recruit domains other than the C2H2 zincfingers. These associated domains may assist ZFPs in activating or repressing expression of target genes. In the silkworm, we found that 90 C2H2 ZFPs contained associated domains, and the remaining 248 ZFPs contained only C2H2 zinc-fingers. The most prevalent domain was ZAD, which is implicated in protein–protein interaction. There are 50 ZAD domains distributed in 50 C2H2 ZFPs in the silkworm genome (Table S1). Most ZAD domains are located at the N-terminus of ZFPs. Compared to other organisms, D. melanogaster also has a high number of ZADs (87 genes). However, many other non-insect organisms, such as C. elegans had no ZADs, while human have only

3.3. Gene clustering on chromosomes In the updated data, about 87.4% of genome sequence could be mapped to 28 chromosomes in the silkworm. Based on the high quality map, we could locate 324 ZFP genes on chromosomes. Fig. 2A shows that most of the ZFPs are tandemly clustered on chromosomes, with about 241 genes concentrated into 59 clusters (threshold set as 500 kb for neighboring genes). These ZFP gene

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one ZAD-containing ZFP (Table S1). These results are consistent with the finding that ZADs have lineage-specifically expanded in higher holometabolous insects (Chung et al., 2002, 2007). Another prevalent domain was the BTB domain, which has been demonstrated to function as a protein–protein interaction module (Perez-Torrado et al., 2006). There are 13 BTB-containing C2H2 ZFPs in the silkworm (Table S1). Some of them are evolutionarily conserved with D. melanogaster, such as the sex-determination gene of fruitless and the key regulator of BR-C gene for the ecdysone cascade (Demir and Dickson, 2005; Nishita and Takiya, 2004). As shown in Table S1, the number of BTB-containing C2H2 ZFPs increases from low eukaryotes to high eukaryotes. For example, C. elegans has only one BTB-containing ZFP, while, the silkworm, D. melanogaster, and H. sapiens have 13, 14, and 50 genes, respectively. In contrast to species-specific expansion of ZAD-containing family members, some associated domains have species-specifically been acquired in the silkworm but have not expanded, such as HTH_psq, M, zf-BED, Tubulin, UBA, UBX, AAA_5, ADH_N, ADH_zinc_N, FragX_IP, Gal-bind_lectin (Table S1). Most of these domains can interact with protein or DNAs. For example, UBA, UBX, ADH_N, ADH_zinc_N, FragX_IP can play a role in protein–protein interactions, and zf-BED, HTH_psq can play a role in DNA-binding (Finn et al., 2006).

.e

B. mori D. melanogaster C. elegans H. sapiens

Number of total genes

C

Species

Fig. 1. Number of C2H2 ZFP genes (A) and C2H2 zinc-fingers (B) distribution in B. mori, C. elegans, D. melanogaster and H. sapiens. The different patterns indicate total number of ZFP genes or C2H2 zinc-fingers, which are ranked by zinc-fingers per protein, i.e., whether it contains 1 w 5 zinc-fingers in individual protein, or 6 w 10 zinc-fingers, etc.

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clusters were named as Clu1 w Clu59. The clustered organization results in uneven distribution of C2H2 ZFP genes in the genome. For example, more than one third of C2H2 ZFP genes were located on chromosome 11, 15, and 24, which have 50, 22, and 59 genes, respectively. However, some chromosomes harbor few ZFP genes; there are no ZFP genes on chromosome 28, and chromosomes 7, 14, and 19 contain only four, two, and one ZFP genes, respectively (Fig. 2A).

The largest ZFP gene cluster of Clu51 is on chromosome 24, which consists of 43 C2H2 ZFPs genes organized tandemly over a distance of 650 kb (Fig. 2B). Over half of these genes (24 genes) have more than 10 C2H2 zinc-fingers. As a result, the average number of zinc-fingers per gene for this cluster is 12. In order to investigate the underlying mechanisms of gene expansion of this cluster, the protein sequence similarities of pairwise alignments among all the members were examined by using the HSP score of BLAST alignment. As seen in Fig. 2C, neighboring genes share higher

A

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3

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12

22

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

C

Fig. 2. The distribution of C2H2 ZFP genes on chromosomes in the silkworm. (A) Based on the high quality map, a total of 324 C2H2 ZFP genes could be located on chromosomes. Most of the ZFP genes are tandem clustered on chromosomes, with about 241 genes concentrated into 59 clusters (threshold set as 500 kb for neighboring genes). These clusters were named as Clu1 w Clu59. (B) A diagram of the largest cluster of Clu51 on chromosome 24. This cluster consists of 43 C2H2 ZFP genes organized in a tandem gene cluster. The map shows only the physical order of the C2H2 ZFPs loci in the cluster. The arrowhead indicates the transcriptional orientation. The stars indicate the ZAD-containing ZFP genes, and the number indicates the order. (C) The protein sequence similarities of pairwise alignment between all the members of the largest cluster on chromosome 24 were examined by using HSP score of BLAST alignment. The alignment scores among these ZFPs were displayed in a matrix with increasing color intensity for higher scores, and the numbers on both axes indicate the indicated the order of the genes.

J. Duan et al. / Insect Biochemistry and Molecular Biology 38 (2008) 1121–1129

similarities. Significant examples are the pairs of BmZFP216 and BmZFP217, BmZFP228 and BmZFP229, BmZFP209 and BmZFP210, BmZFP237 and BmZFP238, and the cluster of BmZFP215, BmZFP214, BmZFP213, BmZFP212 and BmZFP227. One possible explanation is that gene expansion of this cluster may be a result of tandem duplication. There are also nine ZAD-containing C2H2 ZFP genes in this cluster. In the case of ZAD-containing ZFP genes on other chromosomes, they also frequently reside in a clustering pattern, such as BmZFP94 and BmZFP96 on chromosome 2, BmZFP189, BmZFP191 and BmZFP192 on chromosome 18, etc. (Table S3). Chromosome 11 also appears to be particularly enriched for C2H2 ZFP genes. Most of these genes are derived from the three largest clusters, e.g. Clu22, Clu23, Clu24. These clusters were located adjacent to each other (<800 kb), and contain 18, 14, and 10 ZFP genes, respectively, indicating that thus clustered organization also played an important role in expanding the number of C2H2 ZFP genes on chromosome 11 (Fig. 2A and Table S3). The average number of zincfingers per gene for these three clusters (Clu22, Clu23 and Clu24) was investigated. The results showed that they have average number nine, five, and seven zinc-fingers per gene, respectively. Therefore, among the largest clusters in silkworm, it seems that only the cluster of Clu51 is related to the higher number of zinc-fingers per ZFP. 3.4. C2H2 ZFP gene families analysis The TRIBE-MCL clustering algorithm was adopted to group the silkworm C2H2 ZFPs into families. The C2H2 ZFPs of C. elegans, D. melanogaster and H. sapiens were also included to find the members of evolutionarily conserved families (see Section 2). The analysis indicated that the silkworm C2H2 ZFPs were classified into 75 gene families, 63 of which belong to evolutionarily conserved families that have members from D. melanogaster, C. elegans or H. sapiens (Fig. S1 and Table S2). In the evolutionarily conserved families, there are 24 families comprised of members from all of the investigated species. This suggests that these families have common ancestors before the radiation of the silkworm, D. melanogaster, C. elegans, and H. sapiens. Some families follow a strict 1:1 orthologous relationship between all the members of investigated species, such as BLIMP-1, MBD2, ZNF622, ZNF277 and the ZNF598 family (Table S2). There are also some families in which members have been expanded. One extreme example is the ZNF91 family, which is the largest C2H2 ZFP gene family. Members of this family have expanded remarkably from C. elegans to H. sapiens. As shown in Table S2, it consists of nine, 72, 79, and 495 members for the C. elegans, silkworm, D. melanogaster and H. sapiens, respectively. Most members shared high similarities with each other. Previous studies have shown that the ZNF91 family has specifically expanded in primates to comprise more than 110 loci in the human genome (Hamilton et al., 2006). Another large family is the Sp/KLF family, which consists of 41 members in the investigated species (Fig. 3). It has been suggested that members of the Sp/KLF family function as transcription factors that may bind GC-boxes and CACCC-boxes through three C-terminal C2H2 zinc-fingers (Suske et al., 2005). In the silkworm genome, there are at least five genes that belong to this family. After inspecting the C2H2 zinc-finger structure, it was found that all five ZFPs in the silkworm contain an array of three C2H2 zinc-fingers at the C-terminus, which is a characteristic of the Sp/KLF family. The linker sequence between zinc-fingers is five amino acids long and highly conserved, which is subject to the consensus sequence of TGE(R/K)(P/R). Phylogenetic analysis showed that the Sp subfamily is clearly separated from the KLF subfamily. BmZFP70, BmZFP144 and BmZFP285 belong to the KLF subfamily (Fig. 3). BmZFP70 is in a clade with the D. melanogaster Luna gene, KLF6 and KLF7 of H. sapiens, suggesting that it might participate in embryonic development and cell differentiation (De Graeve et al., 2003).

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Moreover, BmZFP144 is in a clade with D. melanogaster cbt that might be involved in dorsal closure (Munoz-Descalzo et al., 2005). Another two ZFPs, BmZFP156 and BmZFP157, belong to the SP subfamily and grouped with D. melanogaster Sp1, human Sp8 and Sp5 as a clade (Fig. 3). These two genes are tandem repeated on chromosome 10. Approximately 32 families exclusively belong to the silkworm and Drosophila, such as the families of L(3)neo38, Tiptop, BR-C, Fru, Hkb, Ab, Ken, Sens, etc. (Table S2). These families might be selected to accommodate insect-specific functions of biological processes during evolution. For example, the BR-C family is an insect-specific family. Previous genetic studies have shown that BR-C is ecdysoneinduced transcription factor and a key factor in the insect-specific process of metamorphosis in both D. melanogaster and silkworm (Uhlirova et al., 2003). Another example is the Tio family. The D. melanogaster tiptop gene is required for the segmentation of the distal leg and to switch appendage development from antenna to leg, which is also a specific process in insects (Herke et al., 2005). In contrast to evolutionarily conserved families, there are 12 families that exclusively belong to the silkworm. These speciesspecific families include a total number of 63 members in the silkworm (Table S2). Considering the singleton genes, there are 188,125, 120, 160 species-specific genes in the silkworm, D. melanogaster, C. elegans, and H. sapiens, respectively (Table 1). This suggests that the silkworm has more species-specific ZFPs than other organisms. The largest species-specific family of ZF_FAM24, containing 39 members, has expanded remarkably in the silkworm (Table S2). All the members of this family are located in the largest tandem cluster on chromosome 24 (Fig. 2B). The expansion of this family may explain the higher number of species-specific genes in the silkworm relative to those found in D. melanogaster, C. elegans, and H. sapiens. 3.5. Gene expression profile of C2H2 ZFP genes The gene expressions of C2H2 ZFP genes for 10 representative samples of the Chinese silkworm strains (Dazao) on day 3 of the fifth instar were surveyed by microarray analysis. The expressions of 132 C2H2 ZFP genes could be detected in at least one of the samples. Among these samples, 33 genes were expressed in every investigated sample (Fig. 4C, Table S4). The widely expressed profiles suggest that these genes may play essential roles in the silkworm. For example, BmZFP286 belong to the DNJA5 family (Table S2). It contains a DnaJ domain at the N-terminal followed by two C2H2 zinc-finger domains. The conserved DnaJ domain suggests that BmZFP286 may be involved in protein folding at this stage (Finn et al., 2006). Another example is the BmZFP104 of the Ab family (Table S2). This gene has a BTB domain at the N-terminal and six C2H2 zinc-finger domains at the C-terminal. It is known that the abrupt gene can mediate neuromuscular connectivity in Drosophila (Hu et al., 1995). The wide expression of BmZFP104 in investigated samples suggests that it may be involved in coordinating the movement of these tissues and organs. Eighty-five C2H2 ZFP genes show differential expressions among different samples (Fig. 4B). On the basis of expression profiles, we can divide these genes into three groups. Group A consists of miscellaneous genes, whose gene expression patterns were restricted to specific tissues. For example, the BR-C gene acts as an ecdysoneinduced early gene to regulate the transcription of target genes in metamorphosis. Expression analysis showed that the BR-C gene was expressed in most of the investigated samples with the exception of the hemocyte and silk gland (A/MSG and PSG) (Fig. 4B). BR-C has been induced to prepare these tissues and organs to transform from larva to pupa. Genes in groups B and C were expressed in testis and ovary, and group C had weak expression in A/MSG and PSG. BmZFP150, which belongs to the Fru family, was expressed in both testis and ovary. The fru gene of Drosophila is known as a crucial

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Dm-LUNA Hm-KLF7 83

Hm-KLF6 BmZFP70 Dm-CG12029 Hm-KLF5 Hm-KLF8 Hm-KLF12

78

Hm-KLF3

72

BmZFP285 Dm-CG9895 Cel-KLF1 54

Hm-KLF1

51 88

96

KLF subfamily

Hm-KLF2 98

Hm-KLF4 Cel-F53F8.1

70

Cel-MUA1 Hm-KLF15 BmZFP144

81

Dm-CBT Hm-KLF10

54

Hm-KLF11

84

Hm-KLF16

91

Hm-KLF14 Hm-KLF13

82

Hm-KLF9

57

Cel-SPTF3 Cel-SPTF2 73

Hm-SP2 Hm-SP6 90 BmZFP157 90

Dm-SP1

Sp subfamily

Hm-SP8

67

BmZFP156 Hm-SP5

63

Dm-BTD Hm-SP7 Dm-CG5669 Hm-SP4 Hm-SP1

54 51

Hm-SP3

0.05 Fig. 3. Phylogenetic tree of the Sp/KLF family. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent). Only bootstrap values above 50% are shown. The scale bar indicates an evolutionary distance of amino acid substitutions per position. The prefix indicates the species as follows: Cel, C. elegans; Dm, D. melanogaster; Hm, H. sapiens.

courtship gene of the sex-determination hierarchy (Demir and Dickson, 2005). Previous studies have shown the sex-specific mRNA isoforms were not detected in the silkworm (Ohbayash et al., 2002). Although the role of the silkworm fru gene may not have the role of sex-determination, the expression in only testis and ovary suggests that its function may be related to the sex development. Interestingly, 14 genes were exclusively expressed in only one investigated sample, nine of which belong to the testis-specific

genes, such as BmZFP27, a member of MBD2 family (Fig. 4A). Previous studies have shown that members of MBD2 family play an important role in the DNA methylation-mediated transcriptional repressor, and that an alternative splicing isoform of MBD2 is testis-specific in both humans and mice (Berger and Bird, 2005; Hendrich and Bird, 1998). The testis-specific expression suggested that MBD2 might function in spermatogenesis. Some genes were specifically expressed in other samples. For example, the expression of BmZFP318, which has only

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G

PS

BmZFP302 BmZFP146 BmZFP198 BmZFP258 BmZFP27 BmZFP246 BmZFP79 BmZFP115 BmZFP257 BmZFP318 BmZFP23 BmZFP139 BmZFP22 BmZFP129 BmZFP123 BmZFP305 BmZFP73 BmZFP179 BmZFP265 BmZFP271 BmZFP180 BmZFP289 BmZFP317 BmZFP55 BmZFP7 BmZFP226 BmZFP172 BmZFP127 BmZFP314 BmZFP53 BmZFP83 BmZFP4 BmZFP100 BmZFP136 BmZFP106 BmZFP235 BmZFP242 BmZFP25 BmZFP101 BmZFP293 BmZFP290 hunchback BmZFP321 BmZFP12 BmZFP287 BmZFP169 BmZFP49 BmZFP156 BmZFP181 BmZFP102 BmZFP147 BmZFP243 BmZFP184 BR-C BmZFP182 BmZFP175 BmZFP88 BmZFP89 BmZFP142 BmZFP275 BmZFP197 BmZFP325 BmZFP164 BmZFP262 BmZFP84 BmZFP34 BmZFP82 BmZFP272 BmZFP163 BmZFP200 BmZFP159 BmZFP238 BmZFP195 BmZFP109 BmZFP173 BmZFP108 BmZFP150 BmZFP291 BmZFP11 BmZFP105 BmZFP112 BmZFP33 BmZFP35 BmZFP229 BmZFP284 BmZFP212 BmZFP225 BmZFP40 BmZFP207 BmZFP94 BmZFP249 BmZFP186 BmZFP51 BmZFP76 BmZFP211 BmZFP191 BmZFP283 BmZFP192 BmZFP309

Group A

Group B

Group C

BmZFP306 BmZFP20 BmZFP32 BmZFP201 BmZFP26 BmZFP286 BmZFP85 BmZFP155 BmZFP69 BmZFP104 BmZFP323 BmZFP72 BmZFP144 BmZFP86 BmZFP328 BmZFP56 BmZFP273 BmZFP113 BmZFP166 BmZFP174 BmZFP268 BmZFP205 BmZFP2 BmZFP177 BmZFP311 BmZFP160 BmZFP217 BmZFP215 BmZFP99 BmZFP292 BmZFP228 BmZFP118 BmZFP209

Fig. 4. Analysis of expression patterns for C2H2 ZFP genes of silkworm. Hierarchical clustering with the average linkage method was performed using Cluster software (http:// genome-www.stanford.edu/clustering/), and the data was visualized by matrix2png (http://bioinformatics.ubc.ca/matrix2png/). Gene expression levels are represented by red and green boxes (denoting higher and lower expression levels, respectively). Vertical columns of boxes represent different genes, and horizontal rows represent different samples. (A) The C2H2 ZFP genes that showed tissue-specific expression. First, One-way ANOVA tests were performed to identify the differentially expressed genes across all investigated tissues (P < 0.001). Then, the expression of genes that were significantly different than other tissues with significance level as P < 0.01 and with change ratio >two-fold were considered as tissue-specific genes. (B) The C2H2 ZFP genes that showed differential expression, i.e., the expression was restricted to some samples. Based on the expression, we could class these genes into three groups: A, B and C. Group A is a miscellaneous, and genes’ expressions were restricted to some tissues. Genes in groups B and C were detected to express in testis and ovary, whereas group C has weak expression in A/MSG and PSG. (C) The C2H2 ZFP genes that showed expression in all our investigated tissues.

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six C2H2 zinc-finger domains, was exclusively detected in the ovary. For other genes, BmZFP139 was specific in midgut, BmZFP22 in hemocyte, BmZFP129 in malpighian tubule (Fig. 4A). Similarly, these genes contain only C2H2 zinc-finger domains. The biological roles of these C2H2 ZFPs are unclear, but the tissue-specificity may provide clues about the function of these genes. 4. Discussion The C2H2 ZFPs constitute a large superfamily and many members play important regulatory roles in various organisms. In this study, we have identified C2H2 ZFPs by searching the updated assembled silkworm genome. Compared with D. melanogaster, the silkworm had more zinc-fingers per ZFP gene. Specifically, the silkworm had more ZFPs with more than 10 zinc-fingers per gene. We considered that ZFPs with more C2H2 zinc-fingers per ZFP gene could accommodate more complex regulation networks. First, it has been demonstrated that one C2H2 zinc-finger domain could recognize a set of 3 w 4 DNA bases (Pavletich and Pabo, 1991), so proteins with more repeats of C2H2 zinc-finger domains could recognize longer and more specific DNA sequences of target genes. In addition to the DNA-binding properties, ZFPs can also be implicated in protein–protein interactions. It is plausible that higher organisms may also benefit from the increase of C2H2 zincfinger repeats. As examples, proteins with more domain repeats may interact with more partners in protein–protein interaction networks, and highly connected proteins in interaction networks tend to contain more domain repeats (Ekman et al., 2006). During evolution, several kinds of associated domains of C2H2 zinc-finger have also expanded in a species-specific or lineagespecific manner. One of the most dramatic examples is the KRAB domain, which functions as a transcriptional repressor. ZFPs with a KRAB domain have been shown emerging around the time of tetrapod divergence and they quickly expanded to a group with more than 400 members in humans (Huntley et al., 2006). Our study showed that the KRAB domain is not found in the silkworm. However, another domain, e.g. the ZAD domain, has been remarkably expanded in the silkworm. Most ZAD domains (37 of 50) were encoded by two exons. Checking the exon boundaries of these ZAD domains shows that domain boundaries of 21 ZADs ended adjacent to that of the second exon. This result indicates that there is a strong correlation between the exon and the domain that is encoded by it. It is suspected that the ZAD domain might be derived from exon shuffling. So far, the ZAD domain has only been implicated in protein–protein interaction. Further study of the function of ZAD containing ZFPs may shed light on why the ZAD domains have species-specifically expanded in silkworm and other insects. Most of C2H2 ZFP genes are tandemly clustered on the silkworm genome, which results in an uneven distribution on chromosomes, such as chromosome 11, 15 and 24. Similar clustered organizations have been observed with other organisms. For example, most of the H. sapiens C2H2 ZFP genes are concentrated on certain chromosomes. C2H2 ZFP genes are particularly enriched on chromosome 19, including more than 250 genes clustered at 11 major sites (Dehal et al., 2001). In Drosophila, ZFP genes are also non-randomly distributed throughout the genome, as nearly 100 C2H2 ZFP genes reside on the right arm of the third chromosome (Chung et al., 2002). Therefore it is a common phenomenon that ZFP genes are prone to reside in clustered organization by tandem duplication. Although clustered organization is a common characteristic for C2H2 ZFP genes, the underlying expansion pattern for clustered organization of different organisms may be distinct. A good illustration is the comparison of C2H2 ZFP genes on human chromosome 19 and related mouse clusters. Previous study has shown that these two have strikingly different number of ZFP genes. Evolutionary analysis of a subset of 160 human and 101 mouse KRAB-

contained C2H2 ZFPs suggests that these genes have been speciesspecifically duplicated, lost, and selected in each conserved cluster since the divergence of primate and rodent lineages (Dehal et al., 2001). This indicates that species-specific expansion may also be a characteristic of ZFP genes. Consistently, we observed that many silkworm ZFPs have species-specifically expanded. In particular, a species-specific family of ZF_FAM24 with 39 members has been tandem located at a portion of chromosome 24. These genes may take part in some species-specific biological processes of silkworm, such as spinning silk and metamorphism. The genes resulting from gene duplications may provide the raw materials for generating novel functions. The diversification in sequence and expression pattern of duplicated C2H2 ZFP genes was examined to find whether these genes have acquired novel functions. We observed that most of duplicated C2H2 ZFP genes have experienced considerable sequence diversification after duplication, which may yield proteins with distinct gene targets and DNA recognition specificities (Krebs et al., 2005). The expression patterns of some duplicated C2H2 ZFP genes were also distinct. For example, SP/KLF family members of BmZFP156 and BmZFP157 clustered on chromosome 10 have diverged in sequence (64% sequence identity). BmZFP156 was mainly expressed in ovary on day 3 of the fifth instar, but BmZFP157 was not expressed at this stage. For the species-specific family of ZF_FAM24, BmZFP228 was 29% identical with BmZFP229, and 22% identical with BmZFP115. The microarray data show that BmZFP115 was specifically expressed in testis, BmZFP228 was expressed in every investigated sample and BmZFP229 was expressed in testis and ovary. Expansion of C2H2 ZFP genes and its diversification in expression pattern and sequence may play important roles in the evolution of silkworm and other species (Looman et al., 2002; Shannon et al., 2003). In this study, we have used the microarray to examine the expression patterns of the silkworm C2H2 ZFP genes on day 3 of the fifth instar, which is an important stage for the silk protein synthesis and preparation for metamorphism. One hundred thirty two of the 338 predicted C2H2 ZFP genes were expressed in the investigated tissues. The active expressions of these transcription factors may correspond to the physiological processes of this stage. For example, the sequence and domain structure of BmZFP160 is highly similar with D. melanogaster crol. BmZFP160 has 20 C2H2 zinc-fingers, whereas D. melanogaster crol gene consists of 18 C2H2 zinc-fingers. It has been demonstrated that the crol gene is induced by ecdysone during metamorphosis, and mutation of the crol gene will lead D. melanogaster to die during pupal development with defects in adult head eversion and leg development (D’Avino and Thummel, 1998). Microarray experiments indicated that BmZFP160 was expressed in all the investigated tissues on day 3 of the fifth instar. This suggests that the gene expression might be triggered by ecdysone at this stage and that its encoding protein may be an early regulator responding to ecdysone and plays an important role in preparing these organs for metamorphosis. The results in this study provide an overview of C2H2 ZFPs in the silkworm genome and useful information for further functional studies on these genes. Revealing the roles of these important transcription factors will help us understand the metamorphosis mechanisms and other biological processes in the silkworm. Acknowledgments This work was supported by research grants from the National Basic Research Program of China (No. 2005CB121000), the National Hi-Tech Research and Development Program of China (2006AA10A117), and the National Natural Science Foundation of China (No. 30471313 and No. 30571407).

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Appendix A. Supplemental material Supplementary information for this manuscript can be downloaded at doi: 10.1016/j.ibmb.2008.08.005.

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