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ANIMAL GENETICS |
Department of Dairy Science, University of Wisconsin-Madison, 1675 Observatory Dr., Madison, WI 53706
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cattle • comparative analysis • gene expression • imprinting
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, there is accumulating evidence that conservation of imprinted genes should not be assumed. From 1 study, of 63 examined protein-coding imprinted genes, 35 in human and 54 in mouse, only 26 genes were reported to be conserved in both species (Morison et al., 2005). Recently, we reported that the coatomer protein complex, subunit gamma 2; decorin; succinate dehydrogenase complex, subunit D; and solute carrier family 38, member 4 genes are not imprinted in cattle, whereas their orthologues are imprinted in mouse or human (Khatib, 2005a; Zaitoun and Khatib, 2006). Identification of species-specific imprinted genes is necessary to understand the evolution of genomic imprinting and to elucidate mechanisms leading to allele-specific expression. Given that most imprinted genes have been identified in mouse and human, and the small number of reported imprinted genes in cattle, the objectives of this study were to investigate the conservation of imprinting and pattern of expression of 6 cattle genes and to compare their allelic expression with that of human and mouse orthologues. For this purpose, we chose to investigate the imprinting status of 6 genes known to be imprinted in mouse: CD81, also known as target of antiproliferation antibody 1, tumor-suppressing subtransferable candidate 4 (TSSC4), and oxysterol-binding protein homologue 1 (OBPH1) genes clustered on bovine chromosome 29; the paternally expressed gene 10 (PEG10) and ankyrin repeat and suppressor of cytokine signaling box-containing protein 4 (ASB4) genes clustered on bovine chromosome 4; and the 5-hydroxytryptamine (serotonin) 2A receptor (HTR2A) microdomain gene on bovine chromosome 12. In addition, we report the comparative expression analysis of these genes among cattle, mouse, and human.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DNA Extraction and PCR Amplification
Samples from various organs of fetuses and their dams were obtained from a local slaughterhouse. All specimens were preserved in RNALater RNA Stabilization Reagent (Qiagen, Valencia, CA) to avoid RNA degradation. Tissues were ground with a mortar and pestle in liquid N into a fine powder, which then was used for either RNA or DNA extraction. Genomic DNA was extracted from tissues using the AquaPure Genomic DNA kit (BioRad, Hercules, CA). The PCR amplifications were conducted under the following conditions: reaction mixtures contained 50 ng of genomic DNA, 50 ng of each primer, 200 µM of each deoxynucleoside triphosphate, 2.5 µL of 10 x PCR buffer (Promega, Madison, WI), 1.5 units of Taq DNA polymerase (Promega), and distilled water to a 25-µL final volume. Cycling conditions were as follows: denaturation for 5 min at 95°C and then 26 cycles of 94°C for 45 s, touchdown annealing from 63°C to 50°C for 45 s (with a temperature drop rate of 2°C/2 cycles), 72°C for 45 s, and final elongation cycle at 72°C for 7 min. Table 1 shows the primer sequences used to amplify the genomic DNA and the PCR product sizes.
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Total RNA was isolated from various tissue types using RNeasy kit (Qiagen). RNase-free DNase I was applied directly to the RNA extraction column. To control for genomic DNA contamination, another round of RNase-free DNase I (Sigma, St. Louis, MO) was applied to the already eluted RNA. Both DNase I treatments were done according to the instructions of the manufacturers, except for the incubation time, which lasted 1 h. The reverse transcription PCR (RT-PCR) was performed using the OneStep RT-PCR Kit (Qiagen). The RT-PCR cycling conditions included incubation at 50°C for 30 min, 95°C for 15 min, and then touchdown PCR conditions, as described for genomic DNA PCR amplifications. The primer sequences used in the RT-PCR reactions are shown in Table 1
. For genes OBPH1, CD81, TSSC4, and ASB4, primers were designed across exons to eliminate the possibility of mistyping due to genomic DNA contamination in the RT-PCR reactions. Because of exon size limitations in PEG10 and HTR2A genes, primers were designed in 1 exon. However, to exclude the possibility of DNA contaminations in samples amplified with these genes, RNA samples were used as templates with more than 1 pair of primers for PCR amplifications in the absence of the enzyme reverse transcriptase.
Polymorphism Detection and Allele-Specific Gene Expression
To search for polymorphisms in the 6 cattle genes, in silico analysis was performed to identify mismatches between coding sequences of these genes and bovine expressed sequence tags deposited in the GenBank database using the basic local alignment search tool. Candidate SNP were further examined by direct sequencing of either pooled RNA samples or individual genomic DNA samples. The RNA pools were constructed from 4 to 10 tissues obtained from 4 to 10 animals and amplified in RT-PCR reactions. The PCR and RT-PCR products were purified from agarose gel using the GFX PCR DNA Purification Kit (Amersham Biosciences, Piscataway, NJ) and sequenced using BigDye terminator (Applied Biosystems, Foster City, CA). Data were analyzed using Applied Biosystems’ Sequencing Analysis (version 5.0). The SNP were identified by visually inspecting each base in all sequencing traces from the pooled RNA samples. Confirmation of SNP was carried out by individually amplifying and sequencing genomic DNA samples from individuals that composed the pools. The SNP identified in heterozygous individuals were used to assess genomic imprinting. The principle is that an imprinted gene would exhibit hemizygosity (monoallelic expression), whereas a biallelically expressed gene (not imprinted) would exhibit heterozygosity at the SNP. Dams of heterozygous individuals were genotyped to identify parental origin of expressed alleles in cases of monoallelic expression.
Sequence homology among cattle, human, and mouse genes was analyzed using the ClustalW program (Thompson et al., 1994; http://www.ebi.ac.uk/clustalw/).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The RT-PCR analysis revealed that CD81 was expressed in all 26 fetal tissues examined. Tissue types were ovary, skeletal muscle, liver, pituitary, mammary gland, kidney, brain, adrenal gland, spleen, heart, pancreas, cartilage, eye, and cotyledon. Also, CD81 was expressed in adult tissues including caruncle, ovary, lung, spleen, and liver. A search for polymorphisms in CD81 revealed 2 different transition substitutions, A/G and C/T at positions 1098 and 1138, respectively (Genbank accession no. NM_001035099). Out of 36 animals examined, 4 individuals were heterozygous: fetus 3, fetus 23, and dam 11 were heterozygous for the A/G SNP, and fetus 9 was heterozygous for the C/T SNP. Utilizing both SNP, examination of CD81 revealed biallelic expression in all 31 different bovine fetal and adult tissues, including placental tissues (Figure 1A, 1B, and 1C). Thus, it was clear that CD81 is not imprinted in cattle.
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). Both SNP were employed to analyze the imprinting status of TSSC4 in 6 placental and another 6 nonplacental tissues obtained from the 5 heterozygous fetuses. The TSSC4 gene was monoallelically expressed in cotyledon tissues obtained from fetuses 14, 23, and 24 and in amnion tissues obtained from fetuses 22, 23, and 24 (Table 2). In contrast, TSSC4 was biallelically expressed in all examined nonplacental tissues, such as skeletal muscle, spleen, intestine, kidney, and liver of fetus 15 and in the heart of fetus 23 (Table 2). Examples of monoallelic and biallelic expression of TSSC4 are shown in Figure 1a. Sequencing of genomic DNA of the dams of fetuses 22 and 23 revealed that those dams were heterozygous for SNP 293, so the parental origin of the expressed allele in placental tissues could not be determined. In contrast, the dams of fetuses 14 and 24 were homozygous for alleles C and T, respectively, so for these fetuses, TSCC4 expression was clearly maternal.
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along with the genomic DNA sequence (Figure 1G). Expression Patterns and Imprinting of PEG10 and ASB4 Genes
The RT-PCR analysis of the tissue distribution of PEG10 transcripts revealed that PEG10 was expressed in all tissue types obtained from 3 fetuses (Table 3). Sequencing of pooled RNA samples revealed 1 transition polymorphism (A/G) at position 1317 (GenBank accession no. XM_870465). Table 3 shows the expression status of PEG10 in tissues obtained from 3 fetuses found to be heterozygous for SNP 1317. Tissues of fetuses 1 and 2 expressed the G allele (Figure 1K), whereas tissues of fetus 3 expressed the A allele. Genotyping of dams of fetuses 1 and 2 showed that those dams were homozygous for the A allele. So, the origin of the expressed alleles in these 2 fetuses was paternal.
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, fetal brain, cartilage, and skeletal muscle tissues expressed ASB4 in both fetuses examined, whereas lung, eye, cotyledon, and mammary gland did not express ASB4 in either fetus. Expression of ASB4 in other examined tissues was not consistent between the 2 fetuses (Table 4). In contrast to fetal tissues, ASB4 transcripts were not observed for any dam tissue examined, except the heart (Table 4). Sequencing of PCR products revealed 2 heterozygous fetuses for a transition SNP (A/G) at position 11120. This SNP was employed to assess the imprinting status in fetal tissues expressing ASB4. Sequencing of RT-PCR products obtained from these tissues revealed biallelic expression of ASB4. Examples of biallelic expression of ASB4 using genomic DNA as a control are shown in Figures 1L and 1M.
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The expression pattern of HTR2A was examined in 31 cattle tissues obtained from 7 individuals. The HTR2A gene was expressed in all tissue types examined including heart, brain, spleen, liver, thyroid, intestine, lung, kidney, mammary gland, cotyledon, cartilage, eye, and ovary. Sequencing of PCR products, amplified from a total of 36 fetuses and dams, revealed 1 SNP (A/C) at position 3344 (GenBank accession no. NM_001001157), apparent in 7 fetuses and 1 dam. Sequencing of RT-PCR products obtained from tissues of heterozygous individuals showed that HTR2A was biallelically expressed in all examined tissues. Examples of biallelic expression of HTR2A along with genomic DNA as a control are shown in Figures 1N and 1O.
Table 5 summarizes the imprinting status of the examined genes and their coding sequence similarity among cattle, human, and mouse. All 6 genes were imprinted in mouse, whereas only 2 genes were found to be imprinted in either human or cattle. Furthermore, a greater sequence similarity was observed between cattle and human genes than between cattle and mouse or between human and mouse genes.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Expression Analysis of the CD81, TSSC4, and OBPH1 Genes
The CD81 gene was expressed in all fetal and adult cattle tissues tested. Similarly, transcripts of the gene have been observed in all human and mouse whole embryos and in the different fetal and placental tissues examined (Caspary et al., 1998; Lewis et al., 2004; Umlauf et al., 2004; Monk et al., 2006). In this study, CD81 was biallelically expressed in placental tissues and in all other fetal and adult tissues examined. Thus, we conclude that CD81 is not imprinted in cattle. Likewise, the human CD81 gene was reported to be not imprinted in somatic cell panels (Gabriel et al., 1998) and in different fetal and placental tissues (Monk et al., 2006). In contrast to cattle and human, mouse Cd81 has been reported to be imprinted, with predominant expression from the maternal allele in placental tissues in embryos aged 9.5 to 17.5 d postcoitum (Paulsen et al., 2000; Lewis et al., 2004; Umlauf et al., 2004). It is worth noting that in a different study, Cd81 showed predominant maternal expression only early in mouse development, until embryonic day 8.5, and it showed biallelic expression in both embryonic and placental tissues at later stages of development (Caspary et al., 1998). Based on the aforementioned studies in cattle, human, and mouse, it is likely that CD81 is a tissue- and species-specific imprinted gene.
The RT-PCR analysis revealed that transcripts of TSSC4 were expressed in all tested fetal and adult tissue types. Likewise, human TSSC4 has been found to be expressed in a wide range of fetal and adult tissues (Lee et al., 1999). Thus, the expression pattern of this gene seems to be ubiquitous. Imprinting analysis showed bovine TSSC4 to be maternally expressed in placental tissues, like the mouse gene (Paulsen et al., 2000; Umlauf et al., 2004), but in contrast to the human orthologue, which was reported to be not imprinted (Lee et al., 1999; Monk et al., 2006).
Tissue distribution of OBPH1 transcripts revealed that OBPH1 was expressed in all cattle tissue types obtained from fetuses and dams. Similar ubiquitous expression has also been reported in mouse and human (Engemann et al., 2000; Higashimoto et al., 2002). However, allele expression analysis in fetal and adult tissues showed that OBPH1 is biallelically expressed in both placental and nonplacental tissues in cattle in contrast to mouse (Engemann et al., 2000; Lewis et al., 2004; Umlauf et al., 2004) and human (Higashimoto et al., 2002).
Thus, in our comparative analysis study, cattle CD81, TSSC4, and OBPH1 showed no consistency with the mouse or human orthologues regarding imprinting status. In our study, CD81 was reported to be imprinted only in mouse, TSSC4 was shown to be imprinted in mouse and cattle but not in human, and OBPH1 was reported to be imprinted in mouse and human but not in cattle. These results are not too surprising given that CD81, TSSC4, and OBPH1 were reported to be placenta-specific imprinted genes in mouse. Recently, the imprinting status of CD81 and TSSC4 was studied in human placental tissues and found to be not imprinted (Monk et al., 2006). The authors suggested that placenta-specific expression of these genes in mouse is regulated by histone modifications, whereas the human orthologues are probably regulated by different mechanisms. Indeed, the imprinting results of our study together with results from mouse and human studies show that not 1 of CD81, TSSC4, and OBPH1 was found to be consistently imprinted in all 3 species. In addition, differences in placenta-specific imprinting could be due to different morphogenesis and endocrine function in human and mouse (Malassine et al., 2003).
Expression Analysis of the PEG10 and ASB4 Genes
In mouse, Peg10 and Asb4 are located on proximal chromosome 6 within a 1-Mb cluster of imprinted genes (Ono et al., 2003). The PEG10 gene is known to be imprinted in mouse and human, but its imprinting status in cattle is not known. We found that PEG10 is imprinted in cattle—paternally expressed in all tissues examined, like the mouse (Ono et al., 2003), human (Ono et al., 2001), and pig (Zhang et al., 2006) orthologues. Thus, paternal expression of PEG10 is conserved among mammalian species examined, although tissue distribution of its transcripts differs.
The expression pattern of ASB4 in cattle fetal tissues, although not consistent in the 2 fetuses examined, was similar to that found in mouse in d-15.5 fetuses (Mizuno et al., 2002). In contrast, ASB4 transcripts were not observed in any adult tissues examined, except the heart. This result was in agreement with the expression pattern of Asb4 in adult mouse tissues reported by Kile et al. (2000). The observed downregulation of ASB4 in adults suggests that this gene could play an important role in embryo growth and development in addition to its known functions in inhibition of cytokine signaling reported by Kile et al. (2000).
The Asb4 gene has been reported to be imprinted in mouse, but its imprinting status in human and cattle has not been reported. Contrary to our results in cattle, Mizuno et al. (2002) reported that Asb4 was maternally expressed in all fetal mouse tissues examined. Recently, the expression of Asb4 has been reported in both androgenetic and parthenogenetic embryos (Ogawa et al., 2006). The authors suggested that disruption in imprinting, in some specific cases, might be due to missing mechanisms that coordinate in trans the interaction between the paternal and maternal alleles (Ogawa et al., 2006). The conflicting imprinting data for the mouse Asb4 gene could be due to polymorphic imprinting (Monk et al., 2006), strain-specific imprinting differences, or partial imprinting of Asb4. Indeed, in the allelic expression analysis reported by Mizuno et al. (2002), traces of the paternal allele could be observed for some tissues.
Expression Analysis of the Microdomain Gene HTR2A
The HTR2A gene was expressed in all cattle tissue types examined. Although human and mouse genes have not been tested specifically for their expression in a wide range of tissues, HTR2A/Htr2a expression has been observed in all tissues examined (Kato et al., 1996, 1998; Bunzel et al., 1998; Pastinen et al., 2003), as in cattle. In contrast to the biallelic expression of HTR2A in cattle, Kato et al. (1998) reported that Htr2a showed maternal expression in mouse brain, ovary, and eye. In our study, these 3 tissues showed biallelic expression.
The data on imprinting status of HTR2A from human studies are inconsistent. Kato et al. (1996) reported that HTR2A showed exclusive maternal expression in human fibroblasts. On the other hand, Bunzel et al. (1998) reported that HTR2A showed polymorphic imprinting in adult human brains. Pastinen et al. (2003) found that HTR2A was randomly monoallelically expressed in human lymphoblasts. Random monoallelic expression of autosomal nonimprinted genes has recently been reported for different genes (Sano et al., 2001; Khatib, 2005b). Thus, based on the aforementioned human studies and on our results, HTR2A is imprinted in neither human nor cattle, but it is maternally expressed in mouse. This type of species-specific imprinting has been reported for many genes (http://www.otago.ac.nz/IGC).
In summary, in our comparative expression analysis study, we report the expression pattern and genomic imprinting of 6 cattle genes. Imprinting of CD81, TSSC4, OBPH1, ASB4, and HTR2A was not conserved among mouse, human, and cattle species, and only PEG10 was imprinted in all 3 species. Thus, it is most likely that the genes examined in this study are species-specific imprinted genes. Of the 6 genes examined in this study, all known to be imprinted in mouse, only 2 have been found to be imprinted in either human or cattle. The lack of conservation of imprinted genes between mouse and human and cattle could be attributed to evolutionary relationships among these species. The estimated divergence time between the ancestor of rodents and the common ancestor of artiodactyls and primates is about 81 million years (Li et al., 1990). Our comparative sequence alignment analysis showed greater sequence similarity between cattle and human than between cattle and mouse genes. These results support the hypothesis that rodents have a greater rate of nucleotide substitution than primates and artiodactyls (Li et al., 1990; Kumar and Subramanian, 2002). Consequently, as Monk and colleagues (2006) suggested, human or cattle genes might be under transition from monoallelic to biallelic expression.
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Footnotes |
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2 Corresponding author: hkhatib{at}wisc.edu
Received for publication March 8, 2007. Accepted for publication August 31, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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