Characterization of the mouse CP27 promoter and NF-Y mediated gene regulation

Abstract

The cp27 gene is a highly conserved and unique gene with important roles related to craniofacial organogenesis. The present study is a first analysis of the CP27 promoter and its regulation. Here, we have cloned the promoter of the mouse cp27 gene, examined its transcriptional activity, and identified transcription factor binding sites in the proximal promoter region. Two major transcription start sites were mapped adjacent to exon 1. Promoter function analysis of the 5' flanking region by progressive 5' deletion mutations localized transcription repression elements between -1993bp and -969bp and several positive elements between -968bp and the preferred transcription start site. EMSA and functional studies indicated two function-cooperative CCAAT boxes and identified the NF-Y transcription factor as the CCAAT activator controlling transactivation of the CP27 promoter. In addition, this study demonstrated that for its effective binding and function, NF-Y required not only the minimal DNA segment length identified by deletion studies, but also a defined nucleotide sequence in the distal 3' flanking region of the CP27 proximal promoter CCAAT box. These results provide a basis for our understanding of the specific regulation of the cp27 gene in the NF-Y-mediated gene transcription network.

Fig. 1

Nucleotide sequence and putative regulatory elements of the 5′ flanking region of the mouse cp27 gene. The numbering of the nucleotides starts at the first nucleotide of the cDNA (+1). The first nucleotide 5′ of the preferred transcription start site is labeled –1. Selected potential transcription factor binding sequences have been labeled. Abbreviations: consensus binding site for the transcription factor oct-1 (OCT-1), consensus binding site for the gata transcription factor (GATA), consensus binding site for the transcription factor nxk-25 (NXK25), consensus binding site for the transcription factor c-myb (C-MYB), consensus binding site for transcription factor ap1 (AP1), CCAAT box (CAT BOX), E-box (E-BOX), unknown binding site (UNKOWN).

Fig. 2

Determination of the transcription start sites of the mouse cp27 gene. A, primer extension. The oligonucleotide CP 82/61 was end-labeled with (r-³²P) ATP and hybridized with 10 μg of total RNA from NIH 3T3 cells or tRNA. The products of the primer extension reaction were size fractionated and autoradiographed. Lane 1, φX174 Hinf I DNA markers; lane 2, primer extension with tRNA; lane 3, primer extension reaction with RNA from NIH 3T3 cells. B, ribonuclease protection assay. The (r-³²P) ATP-labeled RNA probe was generated using the TOPO-207/+82 plasmid containing a PCR fragment amplified by the sense oligonucleotide CP –207/-186 and the antisense oligonucleotide CP82/61. The labeled probe was hybridized with 10 μg of total RNA from NIH 3T3 cells or yeast RNA. The RNase-resistant products were sized-fractioned and autoradiographed. Lane 1, φX174 Hinf I DNA markers; lane 2, no RNase-resistant product from yeast RNA; lane 3, RNase-resistant products from NIH 3T3 cells. C, mapping transcription start sites by 5′ RACE assay. Rapid amplification of cDNA 5′ ends using total RNAs extracted from NIH 3T3 cells mapped two transcription start sites of the cp27 gene indicated with stars. The first start site corresponded to an A purine residue 140 nucleotides upstream of the ATG initiation codon, and the second one to an A purine residue located 102 nucleotides upstream of the ATG codon.

Fig. 3

Promoter activity in the 5′ flanking region of mouse cp27 gene. A, schematic representation of promoter-reporter plasmids. 1993bp DNA and 5′-truncated fragments of the CP27 promoter upstream of the preferred transcription start site were inserted into the luciferase reporter vector pGL3-Basic in sense orientation. The arrow indicates the preferred transcription start site. The name of each reporter construct was assigned according to the 5′-end nucleotide number and the inserted promoter sequence. All constructs contain a partial exon 1. B, Luciferase activity resulting from the expression of the CP27 promoter-reporter gene constructs. The promoter-reporter gene constructs were transfected into NIH 3T3 cells, and specific luciferase activity was measured 48 hours post transfection. The results were normalized by co-transfection with a pRL-TK reporter plasmid. Error bars represent the standard error for three samples in five independent experiments. The activity of the pGL3-Basic vector transfected in the same experiment is indicated.

Fig. 4

Identification of NF-Y as the CCAAT box binding protein of the mouse CP27 proximal promoter. Nuclear extracts were prepared from NIH 3T3 cells. The extracts were incubated with the ³²P-labeled double-stranded oligoncleotides CP-93/-56 containing the CCAAT box of the mouse CP27 proximal promoter. A, Electrophoretic mobility shift analysis. Nuclear extracts from NIH 3T3 cells were incubated with ³²P-labeled oligonucleotide CP-93/-56 probe in the presence of the various competitors. Lane 1, ³²P-labeled probe only; lane 2, probe plus nuclear extracts; lane 3, twentyfive-fold molar excess of unlabeled wild –type(WT) double-stranded oligonucleotides; lane 4, fifty-fold WT; lane 5, twentyfive-fold molar excess of unlabeled mutated (MUT) double-stranded oligonucleotides; lane 6, fifty-fold molar excess of unlabeled mutated (MUT) double-stranded oligonucleotides. The labels above individual lanes read Pr = probe, NE = nuclear extract, Cp = competition, Mt = mutation. B, effect of heat-treated nuclear extract on the DNA-protein complex. Lane 1, probe only; lane 2, nuclear extracts were incubated with the ³²P-labeled probe; lane 3, the extracts were heated to 85°C for 5 min, centrifuged and then added to the reaction mixture. C, Immunoblot analysis. Nuclear extracts were analyzed by EMSA in the presence of the double-stranded oligoncleotides CP-93/-56. Lanes 1 and 3, probe only; lanes 2 and 4, probe plus nuclear extracts. The EMSA gels were dried and subjected to autoradiography (lane 1 and 2) or blotted onto a nitrocellulose membrane, which was then immunobloted with anti-NF-YA antibody (lane 3 and 4). D, supershift analysis. Lane 1, probe only; lane 2, nuclear extracts were incubated with a ³²P-labeled probe; lane 3–7, the extracts were pre-incubated with anti-NF-YA, NF-YC, NF1, Est1/2 or SOX5 antibodies (respectively) for 15 min and then added to the reaction mixture. The labels above individual lanes read Pr = probe, NE = nuclear extract, Ht = heat treatment, AB = antibody. E, transient transfection experiments. The plasmid pGL-93/-56 CCAATmut is a construct in which the CCAAT box has been mutated. Equal amounts of the mutated or wild-type constructs were transiently transfected into NIH 3T3 cells. pRL-TK was used as internal control for transfection efficiency. The values for relative luciferase activity including standard deviation were derived from at least five independent experiments with triplicate wells. There was a significant difference in luciferase activity between the wild-type and the mutated construct. F, ChIP assay. Chromatin fragments immunoprecipitated with anti-NF-YA antibody were amplified by PCR using primers spanning the CP27 proximal promoter region. Immunoprecipitation with anti-Flag antibody was used as a negative control. Lane 1, positive control; Lane 2, Input DNA fragment amplified by mouse CP27 primers; Lane 3, CP27 target immunoprecipitated by anti-NF-YA antibody; Lane 4, anti-Flag antibody control.

Fig. 5

Effect of site-specific mutation on the NF-Y binding in the 3′ flanking region of the CCAAT box of the mouse CP27 proximal promoter. A, Binding efficiency analysis of the NF-Y on the binding site. Wild-type oligoncleotides or mutated oligonucleotides were labeled with ³²P. These probes were incubated with nuclear extracts and the formation of NF-Y-DNA complexes was analyzed using EMSA. Lane 1, wild-type oligonucleotide probe CP-93/-56; lane 2, deletion-mutated probe CP-93/-56D9; lane 3, deletion-mutated probe CP-93/-56D13; lane 4, site-specific mutated probe CP27-93/-56M10-12. B, Antibody supershift assay. Lanes 1–4, wild-type probe CP-93/-56; lanes 5–8, deletion-mutated probe CP-93/-56D13; lanes 9–12, site-specific mutated probe CP-93/-56M10-12; lanes 1,5, and 9, probe only; lanes 2,6, and 10, probe plus nuclear extract; lanes 3,7, and 11, probe plus nuclear extract and anti-NF-YA antibody; lanes 4,8, and 12, probe plus nuclear extract and anti-NF-YB antibody. The labels above individual lanes in Figs. 6A–C read Pr = probe, NE = nuclear extract, Cp = competition, AB = antibody. C. Transient transfection assays. The oligonucleotide containing the mutated CGGA motif in the 3′ flanking region was substituted for the plasmid pGL-93/-56 M10-12. Equal amounts of the wild-type or mutated constructs were transiently transfected into NIH 3T3 cells and luciferase activity was measured. The data represent the means and standard deviation from five separate experiments. There was a significant difference in luciferase activity between the wild-type and the mutated construct.

Fig. 6

Multiple NF-Y binding elements in the mouse CP27 promoter. A, EMSA. Nuclear proteins were extracted from NIH 3T3 cells. The extracts were incubated with the ³²P-labeled CCAAT box-containing oligonucleotide CP-1255/-1207. lanes 1, probe only; lanes 2, probe plus nuclear extracts; lanes 3, twenty five-fold and lanes 4, fifty-fold molar excess of unlabeled WT double-stranded oligonucleotides CP-1255/-1213; lanes 5, fifty-fold molar excess of unlabeled WT double-stranded oligonucleotides CP-1255/-1234; lanes 6, fifty-fold molar excess of unlabeled WT double-stranded oligonucleotides CP-1233/-1213 (Table 2); lanes 7-9, supershift with anti-NF1, SOX5 or NF-YA antibodies. The labels above individual lanes read Pr = probe, NE = nuclear extract, Cp = competition, AB = antibody. B, Mutation analysis of CAT5 box. The wild type promoter-reporter construct pGL-1255/+48 or its 5′ deletion mutation pGL-1180/+48 or its mutated homologues pGL-1255/+48CAT5-mut was introduced into NIH3T3 cells, and luciferase activity was measured. The values for relative luciferase activity including standard deviation were presented in triplicates in five independent experiments as described before. C, ChIP assay. Chromatin fragments immunoprecipitated with anti-NF-YA antibody were amplified by PCR using primers spanning the CP27 promoter regions from −1254 to −1133bp. Immunoprecipitation with anti-Flag antibody was used as a negative control. Lane1, input DNA fragment amplified by mouse CP27 primers; Lanes 2, CP27 target immunoprecipitated by Anti-NF-YB antibody; Lanes 3, anti-Flag antibody control.

Fig. 7

Effect of NF-Y on the cp27 gene expression. A, function study on the cooperation of multiple CCAAT-boxes to regulate the CP27 promoter. The wild type promoter-reporter construct pGL-1255/+48 or its mutated homologues pGL-1255/+48CAT1-mut, pGL-1255/+48CAT5-mut or pGL-1255/+48CAT1, 5-mut was transfected into NIH3T3 cells, and luciferase activity was measured in triplicates in five independent experiments as described before. B, RT-PCR analysis of NF-YA or NF-YAm29 overexpression efficiency. NIH3T3 cells were transfected with expression vectors pIRES-NF-YA or pIRES-NF-YAm29. After twenty-four hours, total RNA was isolated from transfected cells and reverse transcribed. NF-YA expression level was detected by PCR analysis. RT-PCR products of four separate experiments were loaded onto 1% agarose gels and stained with ethidium bromide. β-actin served as an internal control. C, Luciferase activity of CP27 promoter-reporter gene constructs regulated by overexpression of NF- YA or NF-YAm29. The construct pGL-1255/+48 was co-transfected with either the expression vectors pIRES-NF-YA or pIRES-NF-YAm29 into NIH3T3 cells. Transfection with pIRES-GFP was used as control. Forty eight hours after transfection, cells were harvested and luciferase activity was measured as described above. Results are presented as relative luciferase activity. D, qRT-PCR analysis of CP27 mRNA expression modulated by gene manipulation of NF-YA. NIH3T3 cells were transfected with the expression vectors pIRES-NF-YA or pIRES-NF-YAm29. After 48 hours, total RNA was isolated from the transfected cells and reverse transcribed. CP27 expression levels were measured using real time RT-PCR analysis. Changes in expression levels were measured as n-fold increases of stimulation over the control group using the 2^−ΔΔCt method. CP27 expression was 1.93-fold higher when cells were transfected with NF-YA.