Transcription factor cCP2 controls gene expression in chicken embryonic stem cells

Abstract

cENS-1/cERNI genes have been shown to be expressed very early during chicken embryonic development and as well as in pluripotent chicken embryonic stem (CES) cells. We have previously identified a promoter region, which is specifically active in CES cells compared to differentiated cells. In order to understand the molecular mechanisms which regulate the cENS-1/cERNI promoter, we analyzed the cis-acting elements of this promoter in CES and differentiated cells. We identified a short sequence, named the B region, 5'-CAAG TCCAGG CAAG-3', that exhibits a strong enhancer activity in CES and differentiated cells. Mutation of the B region in the whole cENS-1 promoter strongly decreases the promoter activity in CES cells, suggesting that this region is essential for activating the promoter. The B region is similar to the previously described response element for the transcription factor CP2 and we show by supershift experiments that a protein complex containing CP2 is bound to this B response element. All these results identify a nuclear factor belonging to the CP2 transcription factor family that is crucial for the activation of the cENS-1/cERNI promoter. The pattern of expression of cCP2 in early chicken embryo before gastrulation is very similar to that of cENS-1/cERNI which strongly suggests that cCP2 also plays an essential role in gene expression early in embryonic development.

Figure 1

Structure of cENS-1 promoter. The sequence from –738 to +83 relative to the transcription start site (TSS) is presented. The TSS is marked with right-angled arrows and the putative TATA box in bold. This sequence was analyzed by MatInspector Professional release 6.1. Transcription factors with 1.00 core similarity and more than 0.95 matrix similarities are highlighted in gray and labeled above the sequence. (+) and (–) indicate, respectively, the sense and antisense strands. The A and B response elements identified in this study are underlined.

Figure 2

Functional analysis of deletion mutants of the cENS-1 promoter. Deletions were generated as described in Materials and Methods; a schematic representation of each reporter construct is shown on the left. These constructs were transfected into CES cells, CES cells induced to differentiate with retinoic acid for 48 h and into QT6 cells. Firefly luciferase activity was normalized for transfection efficiency using the Renilla luciferase activity. Values are relative to the activity obtained with the largest promoter fragment (p738-luc) in CES cells for three independent experiments, each performed in triplicate.

Figure 3

Functional analysis of the –308/–180 region. (A) One or three copies of the –308/–180 region fused to the pGL2 promoter construct were assayed by transfection experiments in CES cells and in QT6 cells. Values are relative to the activity obtained in each cell line with the empty pGL2-promoter (SV40) construct. (B) One or three copies of the –308/–180 region fused to the p179-luc construct were assayed by transfection into CES and QT6 cells. Values are relative to the activity obtained with p179-luc construct. Data represent ratios of firefly luciferase versus Renilla luciferase activities and values are means of at least three independent transfection experiments. The orientation of the –308/–180 is shown by the sense of the arrows.

Figure 4

The –308/–180 region contains two binding sites for CES cells nuclear factors. (A) Map of the seven probes used to cover the –308/–180 region. Each probe is designated with a roman numeral. (B) EMSAs were performed with each double-stranded radiolabeled oligonucleotide probe and nuclear extracts prepared from CES cells as described in Materials and Methods. The positions of DNA–protein complexes throughout are indicated by arrows. Lanes 1, 4, 7, 10, 13, 16, 19: labeled probes alone; lanes 2, 5, 8, 11, 14, 17, 20: labeled probes with nuclear extracts from CES cells; lanes 3, 6, 9, 12, 15, 18, 21: competition experiments with 100-fold molar excess of wild-type unlabeled probes.

Figure 5

Fine mapping of A and B binding sites by methylation interference. (A) Methylation interference assays were performed with methylated –251/–223 (A region) or –297/–277 (B region) probes radiolabeled at the 5′-end of the sense or antisense strands. The probes were incubated with CES cell nuclear extracts. The free (F) and retarded (R) probes were then sequenced and compared with the G+A sequence obtained with the non-methylated probes. The G residues implicated were identified by a disappearance or a decrease in the signal. (B) Summary of the interactions. The guanine nucleotides identified by methylation interference assays are presented in bold on a double-stranded DNA fragment.

Figure 6

Binding of chicken nuclear proteins to the B region. (A) Sequences of the oligonucleotides used. DNA binding sequence is underlined and mutations are marked with an asterisk and bold characters. Mutated nucleotides on the B region are numbered. (B) EMSAs were performed with the B wild-type labeled probe alone (lane 1) or with nuclear extracts prepared from CES cells (lane 2). The position of the DNA–protein complex is indicated by an arrow. Competition experiments were performed with a 10-fold molar excess (lane 3) or 100-fold molar excess (lane 4) of the unlabeled wild-type B oligonucleotide or with a 100-fold molar excess of the B mutated C₁, G₄, C₁₁ and G₁₄ unlabeled oligonucleotide (lanes 5, 6, 7 and 8, respectively). (C) B mutated C₁, A₂, A₃, G₄, T₅, C₆, C₇ oligonucleotides were labeled and used for EMSAs with CES cell nuclear extracts (lanes 1, 2, 3, 4, 5, 6 and 7, respectively). (D) EMSAs were performed with each double-stranded radiolabeled probe alone (lanes 1, 5, 9, 13) or with CES cell nuclear extracts (lanes 2, 6, 10, 14). The position of the DNA–protein complex is indicated by an arrow. Competition experiments were performed with a 100-fold molar excess of either the mutated B unlabeled oligonucleotide (lanes 3, 7, 11, 15) or the B wild-type unlabeled oligonucleotide (lanes 4, 8, 12, 16).

Figure 7

Functional analysis of the B region. (A) Multimerized wild-type or mutated B region fused to the pGL2-promoter construct were assayed by transfection into QT6 and CES cells. For each cell line, values are relative to the activity obtained with the pGL2-promoter construct. Data represent ratios of firefly luciferase versus Renilla luciferase activities, and values are the means of three independent experiments. (B) The B region was disrupted by targeted mutagenesis on the p456-luc construct to give p456 Bmut-luc. The cytosine residue of the first CNRG box in the B response element was substituted by an adenine residue (see Materials and Methods). These two constructs were transfected into CES cells and promoter activity assayed. Values are relative to the activity obtained with the p456-luc. (C) Labeled wild-type B oligonucleotide was used as a probe with CES cell nuclear extracts (lane 1). Complex is indicated by a black arrow. Competition with 100-fold molar excess of unlabeled wild-type or mutated B oligonucleotides is shown, respectively, on lanes 2 and 3; 250, 25 and 2.5 ng of monoclonal mouse anti-CP2 or 250 ng of IgG control antibody were incubated with CES nuclear extracts after the addition of B labeled probe (lanes 4 to 7, respectively). Antibody-dependent supershift is indicated by the top black arrow. (D) Immunoblots realized with nuclear extracts from undifferentiated and differentiated CES cells (lanes 1 and 2, respectively) and HeLa cells (lane 3) incubated with a monoclonal mouse anti-CP2.

Figure 8

Expression of CP2 in chicken embryo. Whole-mount in situ hybridization of CP2 mRNA. Embryos were hybridized to an RNA antisense probe located in the coding sequence of CP2. (A) Stage X embryo (EG) dorsal side; a.o., area opaca; m.z., marginal zone. (B) Stage XIII embryo (EG) dorsal side; k.s., koller’s sickle. (C) Stage 3 embryo dorsal side; p.s., primitive streak; p.n.p., prospective neural plate. (D) Stage 3+ embryo dorsal side. (E) Stage 4+ embryo; n.n.e., non-neural ectoderm; n.p., neural plate. (F) Stage 5 embryo; ch., chord. (G) Stage 7+ embryo; n.f., neural fold; s., somite. (H) Stage 8 embryo. (I) Stage 9 embryo. (J) Stage 12 embryo.