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Isolation and sequence analysis of the cDNA encoding subunit C of human CCAAT-binding transcription factor
Gene 197 (1997) 161–163
Isolation and sequence analysis of the cDNA encoding subunit C of human CCAAT-binding transcription factor Vladimir Dmitrenko, Oleg Garifulin, Vadim Kavsan * Institute of Molecular Biology and Genetics, National Academy of Sciences of the Ukraine, Zabolotnogo st. 150, 252627 Kiev, Ukraine Received 11 February 1997; accepted 16 April 1997
Abstract Sets of cDNA clones corresponding to genes with different expression specificity, ‘brain-specific’, ‘common’, ‘liver-specific’, were identified by differential hybridization of a human fetal brain cDNA library with total cDNA probes of human fetal brain and human fetal liver. Nucleotide sequence analysis revealed that one of the ‘common’ clones contained the cDNA encoding subunit C of human CCAAT-binding transcription factor. The isolated human CBF-C cDNA is 1977 nt long and consists of the fulllength 3∞-untranslated region (781 nt) with a poly(A) tail at the 3∞ end, 185 nucleotides of 5∞-untranslated region and the open reading frame (1011 nt), encoding a 337-aa protein with 91.7% homology with the translated region of rat CBF-C cDNA. © 1997 Elsevier Science B.V. Keywords: Differential hybridization; cDNA library; Nucleotide sequence; Human fetal brain; Protein similarity
1. Introduction Promoters of eukaryotic genes include a complex set of cis-regulatory elements. One such element, required for optimal activity of many promoters (Myers et al., 1986; Jones et al., 1987), contains a conserved CCAAT sequence, which is often found between 80 and 120 nt upstream of the transcription initiation site (Benoist et al., 1980; Efstratiadis et al., 1980; McKnight and Tijan, 1986). Several proteins specifically recognizing CCAAT elements have been described (for a review, see Chodosh et al. (1988)). However, the relationship amongst these proteins has only recently become clear. It has been shown that the CCAAT-binding transcription factor (CBF, earlier also called NF-Y and CP1) is a heteromeric DNA-binding protein consisting of three subunits: CBF-A, CBF-B, and CBF-C (Maity et al., 1992). CBF-A and CBF-C interact with each other to form a complex; CBF-B does not interact with CBF-A or CBF-C individually but associates with the CBF* Corresponding author. Tel. +380 44 2663498; Fax +380 44 2663498; e-mail: [email protected] Abbreviations: aa, amino acid(s); cDNA, DNA complementary to RNA; mRNA, messenger RNA; ORF, open reading frame; CBF, CCAAT-binding transcription factor; CBF-A, -B, -C, subunit A, B, C, respectively, of the CCAAT-binding transcription factor. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 7 8- 1 1 19 ( 97 ) 0 02 5 5 -2
A–CBF-C complex (Sinha et al., 1995). The isolation of cDNA has been reported for CBF-A and CBF-B of rat (Maity et al., 1990; Vuorio et al., 1990) and mouse ( Van Huijsduijnen et al., 1990); cDNA of human CBF-A has also been cloned (Becker et al., 1991), and CBF-C cDNA has recently been isolated from rat liver cDNA library (Sinha et al., 1995). In this paper, we describe the identification and nucleotide sequence analysis of cDNA coding for subunit C of human CBF-C.
2. Experimental and discussion 2.1. Identification of cDNA for human CBF-C Differential hybridization of a gridded human fetal brain cDNA library was carried out using cDNA probes synthesized on total poly (A)+ RNA isolated from human fetal brain and human fetal liver. The synthesis of these total cDNA probes and hybridization analysis were performed according to standard procedures (Sambrook et al., 1989). The ‘grids’, i.e., filters with robotically spotted high density arrays of cDNA clones of an ordered human fetal brain cDNA library (Lehrach et al., 1990) and cDNA clones themselves were kindly provided by H. Lehrach (Reference Library Database,
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Max Planck Institute of Molecular Genetics, Berlin, Germany). The hybridization analysis allowed us to identify the sets of cDNA clones, which corresponded to genes with different expression specificity, ‘brainspecific’, ‘liver-specific’, and ‘common’ or ‘housekeeping’. The nucleotide sequence of cDNA inserts was determined by the dideoxynucleotide chain termination method (Sanger et al., 1977). Nucleotide sequence homology searches in GenBank and other sequence databases, carried out using the BlastN algorithm (Altschul et al., 1990), revealed that one ‘common’ cDNA clone, ICRFp507N0593, contained a cDNA homologous to the cDNA encoding subunit C of rat CCAAT-binding transcription factor (Sinha et al., 1995). 2.2. Sequence analysis of cDNA for human CBF-C The isolated human CBF-C cDNA is 1977 nt long and consists of the full 3∞ untranslated region (781 nt) with a poly(A) tail at the 3∞ end, full translated region, and 185 nucleotides of untranslated 5∞ sequence. The canonical polyadenylation signal sequence AATAAA is localized 13 nucleotides upstream of the poly(A) tail. Human and rat CBF-C cDNAs differ significantly in their 5∞ and 3∞ untranslated regions. The published sequence of the 5∞ untranslated region of rat CBF-C cDNA (Sinha et al., 1995) seems to be incomplete (only 84 nt) and has practically no homology with human CBF-C cDNA. The 3∞ regions have more homology, but again the published rat sequence appears to be incomplete, only 114 nt (Fig. 1). The nucleotide sequence of human CBF-C cDNA contains a 1011 bp ORF encoding a protein (337 aa) with 91.7% homology to the translated region of rat CBF C cDNA. Four nucleotide changes lead to amino acid substitutions in positions 9, 52, 215 and 331. Additionally, three deletions eliminate codons in positions 250–251 and 298, restore the reading frame, making rat CBF-C 3 aa acid shorter than human CBF-C. Like rat CBF-C, the human factor is rich in glutamine residues and shows no homology with known protein–protein interaction motifs, suggesting that CBF may represent a unique heteromeric DNA binding protein. Hybridization of human CBF-C cDNA with both fetal brain and fetal liver cDNA probes shows that
Fig. 1. Comparison of human and rat CBF-C cDNAs and proteins. The deduced amino acid sequence is shown for the human protein. The positions of amino acids (in italics) and nucleotides are indicated on the right. Asterisks indicate the nucleotide differences. Dashes indicate the absence of nucleotides. A putative polyadenylation signal is underlined. The nt sequence reported here has been deposited in the EMBL nucleotide sequence database (accession no. Z74792).
V. Dmitrenko et al. / Gene 197 (1997) 161–163
human CBF-C is ubiquitous, with an mRNA transcribed from a ‘housekeeping’ gene. The specific role of CBF in the regulation of eukaryotic gene expression remains to be established. The availability of human CBF-C cDNA should help studies aimed at a better understanding of these functions. 2.3. Conclusions
(1) Analysis of the cDNA clone ICRFp507N0593 from human fetal brain cDNA library reveals a very high homology with subunit C of rat CCAAT-binding transcription factor. (2) Human CBF-C is a ubiquitous protein encoded by a ‘housekeeping’ gene and may be part of a unique heteromeric DNA binding protein.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Becker, D.M., Fikes, J.D., Guarente, L., 1991. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc. Natl. Acad. Sci. USA 88, 1968–1972. Benoist, C., O’Hare, K., Breathnach, R., Chambon, P., 1980. The ovalbumin gene: sequence and putative control region. Nucleic Acids Res. 9, 127–142. Chodosh, L.S., Baldwin, A.S., Carthew, R.W., Sharp, P.A., 1988. Human CCAAT-binding proteins have heterologous subunits. Cell 53, 11–24. Efstratiadis, A., Posakony, J.W., Maniatis, T., Lawn, R.M., O’Connell, C., Spritz, R.A., DeRiel, J.K., Forget, B.G., Weissman, S.M.,
163
Slightom, J.L., Blechl, A.E., Smithies, O., Baralle, F.E., Shoulders, C.C., Proudfoot, N.J., 1980. The structure and evolution of the human b-globin family. Cell 21, 653–668. Jones, K.A., Kadonaga, G.T., Rosenfelad, P.G., Kelly, T.G., Tjian, R., 1987. A cellular DNA-binding protein that activates eucaryotic transcription and DNA replication. Cell 48, 78–89. Lehrach, H., Drmanac, R., Hoheisel, J., Larin, Z., Lennon, G., Monaco, A., Nizetic, D., Zehetner, G., Poustka, A., 1990. Hybridization fingerprinting in genome mapping and sequencing. In: Davies, K.E., Tilghman, S.M. ( Eds.), Genome Analysis. Volume 1. Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 39–81. Maity, S.N., Sinha, S., Routeshouser, E.C., De Crombrugghe, B., 1992. Three different polypeptides are necessary for DNA binding of the mammaian heteromeric CCAAT-binding factor. J. Biol. Chem. 267, 16574–16580. Maity, S.N., Vuorio, T., De Crombrugghe, B., 1990. The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor. Proc. Natl. Acad. Sci. USA 87, 5378–5382. McKnight, S., Tijan, R., 1986. Transcriptional selectivity of viral genes in mammalian cells. Cell 46, 795–805. Myers, R.M., Tilly, K., Maniatis, T., 1986. Fine structure genetic analysis of a b-globin promoter. Science 232, 613–618. Sambrook, J., Fritch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F.S., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Sinha, S., Maity, S.N., Lu, J., De Crombrugghe, B., 1995. Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein–DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. Proc. Natl. Acad. Sci. USA 92, 1624–1628. Van Huijsduijnen, R.H., Li, X.Y., Black, D., Matthes, H., Benoist, C., Mathis, D., 1990. Co-evolution from yeast to mouse: cDNA cloning of the two NF-Y (CP-1/CBF ) subunits. EMBO J. 9, 3119–3127. Vuorio, T., Maity, S.N., De Crombrugghe, B., 1990. Purification and molecular cloning of the ‘A’ chain of a rat heteromeric CCAATbinding protein. J. Biol. Chem. 265, 22480–22486.
Isolation and sequence analysis of the cDNA encoding subunit C of human CCAAT-binding transcription factor Vladimir Dmitrenko, Oleg Garifulin, Vadim Kavsan * Institute of Molecular Biology and Genetics, National Academy of Sciences of the Ukraine, Zabolotnogo st. 150, 252627 Kiev, Ukraine Received 11 February 1997; accepted 16 April 1997
Abstract Sets of cDNA clones corresponding to genes with different expression specificity, ‘brain-specific’, ‘common’, ‘liver-specific’, were identified by differential hybridization of a human fetal brain cDNA library with total cDNA probes of human fetal brain and human fetal liver. Nucleotide sequence analysis revealed that one of the ‘common’ clones contained the cDNA encoding subunit C of human CCAAT-binding transcription factor. The isolated human CBF-C cDNA is 1977 nt long and consists of the fulllength 3∞-untranslated region (781 nt) with a poly(A) tail at the 3∞ end, 185 nucleotides of 5∞-untranslated region and the open reading frame (1011 nt), encoding a 337-aa protein with 91.7% homology with the translated region of rat CBF-C cDNA. © 1997 Elsevier Science B.V. Keywords: Differential hybridization; cDNA library; Nucleotide sequence; Human fetal brain; Protein similarity
1. Introduction Promoters of eukaryotic genes include a complex set of cis-regulatory elements. One such element, required for optimal activity of many promoters (Myers et al., 1986; Jones et al., 1987), contains a conserved CCAAT sequence, which is often found between 80 and 120 nt upstream of the transcription initiation site (Benoist et al., 1980; Efstratiadis et al., 1980; McKnight and Tijan, 1986). Several proteins specifically recognizing CCAAT elements have been described (for a review, see Chodosh et al. (1988)). However, the relationship amongst these proteins has only recently become clear. It has been shown that the CCAAT-binding transcription factor (CBF, earlier also called NF-Y and CP1) is a heteromeric DNA-binding protein consisting of three subunits: CBF-A, CBF-B, and CBF-C (Maity et al., 1992). CBF-A and CBF-C interact with each other to form a complex; CBF-B does not interact with CBF-A or CBF-C individually but associates with the CBF* Corresponding author. Tel. +380 44 2663498; Fax +380 44 2663498; e-mail: [email protected] Abbreviations: aa, amino acid(s); cDNA, DNA complementary to RNA; mRNA, messenger RNA; ORF, open reading frame; CBF, CCAAT-binding transcription factor; CBF-A, -B, -C, subunit A, B, C, respectively, of the CCAAT-binding transcription factor. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 7 8- 1 1 19 ( 97 ) 0 02 5 5 -2
A–CBF-C complex (Sinha et al., 1995). The isolation of cDNA has been reported for CBF-A and CBF-B of rat (Maity et al., 1990; Vuorio et al., 1990) and mouse ( Van Huijsduijnen et al., 1990); cDNA of human CBF-A has also been cloned (Becker et al., 1991), and CBF-C cDNA has recently been isolated from rat liver cDNA library (Sinha et al., 1995). In this paper, we describe the identification and nucleotide sequence analysis of cDNA coding for subunit C of human CBF-C.
2. Experimental and discussion 2.1. Identification of cDNA for human CBF-C Differential hybridization of a gridded human fetal brain cDNA library was carried out using cDNA probes synthesized on total poly (A)+ RNA isolated from human fetal brain and human fetal liver. The synthesis of these total cDNA probes and hybridization analysis were performed according to standard procedures (Sambrook et al., 1989). The ‘grids’, i.e., filters with robotically spotted high density arrays of cDNA clones of an ordered human fetal brain cDNA library (Lehrach et al., 1990) and cDNA clones themselves were kindly provided by H. Lehrach (Reference Library Database,
162
V. Dmitrenko et al. / Gene 197 (1997) 161–163
Max Planck Institute of Molecular Genetics, Berlin, Germany). The hybridization analysis allowed us to identify the sets of cDNA clones, which corresponded to genes with different expression specificity, ‘brainspecific’, ‘liver-specific’, and ‘common’ or ‘housekeeping’. The nucleotide sequence of cDNA inserts was determined by the dideoxynucleotide chain termination method (Sanger et al., 1977). Nucleotide sequence homology searches in GenBank and other sequence databases, carried out using the BlastN algorithm (Altschul et al., 1990), revealed that one ‘common’ cDNA clone, ICRFp507N0593, contained a cDNA homologous to the cDNA encoding subunit C of rat CCAAT-binding transcription factor (Sinha et al., 1995). 2.2. Sequence analysis of cDNA for human CBF-C The isolated human CBF-C cDNA is 1977 nt long and consists of the full 3∞ untranslated region (781 nt) with a poly(A) tail at the 3∞ end, full translated region, and 185 nucleotides of untranslated 5∞ sequence. The canonical polyadenylation signal sequence AATAAA is localized 13 nucleotides upstream of the poly(A) tail. Human and rat CBF-C cDNAs differ significantly in their 5∞ and 3∞ untranslated regions. The published sequence of the 5∞ untranslated region of rat CBF-C cDNA (Sinha et al., 1995) seems to be incomplete (only 84 nt) and has practically no homology with human CBF-C cDNA. The 3∞ regions have more homology, but again the published rat sequence appears to be incomplete, only 114 nt (Fig. 1). The nucleotide sequence of human CBF-C cDNA contains a 1011 bp ORF encoding a protein (337 aa) with 91.7% homology to the translated region of rat CBF C cDNA. Four nucleotide changes lead to amino acid substitutions in positions 9, 52, 215 and 331. Additionally, three deletions eliminate codons in positions 250–251 and 298, restore the reading frame, making rat CBF-C 3 aa acid shorter than human CBF-C. Like rat CBF-C, the human factor is rich in glutamine residues and shows no homology with known protein–protein interaction motifs, suggesting that CBF may represent a unique heteromeric DNA binding protein. Hybridization of human CBF-C cDNA with both fetal brain and fetal liver cDNA probes shows that
Fig. 1. Comparison of human and rat CBF-C cDNAs and proteins. The deduced amino acid sequence is shown for the human protein. The positions of amino acids (in italics) and nucleotides are indicated on the right. Asterisks indicate the nucleotide differences. Dashes indicate the absence of nucleotides. A putative polyadenylation signal is underlined. The nt sequence reported here has been deposited in the EMBL nucleotide sequence database (accession no. Z74792).
V. Dmitrenko et al. / Gene 197 (1997) 161–163
human CBF-C is ubiquitous, with an mRNA transcribed from a ‘housekeeping’ gene. The specific role of CBF in the regulation of eukaryotic gene expression remains to be established. The availability of human CBF-C cDNA should help studies aimed at a better understanding of these functions. 2.3. Conclusions
(1) Analysis of the cDNA clone ICRFp507N0593 from human fetal brain cDNA library reveals a very high homology with subunit C of rat CCAAT-binding transcription factor. (2) Human CBF-C is a ubiquitous protein encoded by a ‘housekeeping’ gene and may be part of a unique heteromeric DNA binding protein.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Becker, D.M., Fikes, J.D., Guarente, L., 1991. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc. Natl. Acad. Sci. USA 88, 1968–1972. Benoist, C., O’Hare, K., Breathnach, R., Chambon, P., 1980. The ovalbumin gene: sequence and putative control region. Nucleic Acids Res. 9, 127–142. Chodosh, L.S., Baldwin, A.S., Carthew, R.W., Sharp, P.A., 1988. Human CCAAT-binding proteins have heterologous subunits. Cell 53, 11–24. Efstratiadis, A., Posakony, J.W., Maniatis, T., Lawn, R.M., O’Connell, C., Spritz, R.A., DeRiel, J.K., Forget, B.G., Weissman, S.M.,
163
Slightom, J.L., Blechl, A.E., Smithies, O., Baralle, F.E., Shoulders, C.C., Proudfoot, N.J., 1980. The structure and evolution of the human b-globin family. Cell 21, 653–668. Jones, K.A., Kadonaga, G.T., Rosenfelad, P.G., Kelly, T.G., Tjian, R., 1987. A cellular DNA-binding protein that activates eucaryotic transcription and DNA replication. Cell 48, 78–89. Lehrach, H., Drmanac, R., Hoheisel, J., Larin, Z., Lennon, G., Monaco, A., Nizetic, D., Zehetner, G., Poustka, A., 1990. Hybridization fingerprinting in genome mapping and sequencing. In: Davies, K.E., Tilghman, S.M. ( Eds.), Genome Analysis. Volume 1. Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 39–81. Maity, S.N., Sinha, S., Routeshouser, E.C., De Crombrugghe, B., 1992. Three different polypeptides are necessary for DNA binding of the mammaian heteromeric CCAAT-binding factor. J. Biol. Chem. 267, 16574–16580. Maity, S.N., Vuorio, T., De Crombrugghe, B., 1990. The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor. Proc. Natl. Acad. Sci. USA 87, 5378–5382. McKnight, S., Tijan, R., 1986. Transcriptional selectivity of viral genes in mammalian cells. Cell 46, 795–805. Myers, R.M., Tilly, K., Maniatis, T., 1986. Fine structure genetic analysis of a b-globin promoter. Science 232, 613–618. Sambrook, J., Fritch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F.S., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Sinha, S., Maity, S.N., Lu, J., De Crombrugghe, B., 1995. Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein–DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. Proc. Natl. Acad. Sci. USA 92, 1624–1628. Van Huijsduijnen, R.H., Li, X.Y., Black, D., Matthes, H., Benoist, C., Mathis, D., 1990. Co-evolution from yeast to mouse: cDNA cloning of the two NF-Y (CP-1/CBF ) subunits. EMBO J. 9, 3119–3127. Vuorio, T., Maity, S.N., De Crombrugghe, B., 1990. Purification and molecular cloning of the ‘A’ chain of a rat heteromeric CCAATbinding protein. J. Biol. Chem. 265, 22480–22486.