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Regulation of promoter occupancy during activation of cryptobiotic embryos from the crustacean Artemia franciscana

Ana Martinez-Lamparero, Marie-Carmen Casero, Javier Ortiz-Caro and Leandro Sastre^*

Instituto de Investigaciones Biomédicas CSIC/UAM, C/ Arturo Duperier No. 4, 28029 Madrid, Spain

View larger version (28K): [in a new window]   Fig. 1. Functional analysis of the Actin302 gene promoter in mammalian cells. (A) Several fragments (1 to 8) of the Actin302 promoter region were generated and cloned into the pXP2 luciferase reporter vector. The fragments differed in their 5' ends, which are indicated by a solidus on the nucleotide sequence of the promoter with the corresponding name shown above. The 3' end of all the fragments was coincident with the end of exon 1, also indicated by a solidus. Nucleotides are numbered from the ATG initiation codon in exon 2 (the intron is excluded for the numeration). (B) The different fragments indicated in A were cloned into the pXP2 reporter vector and subsequently transfected into Bsc40 cells. Cells were collected 48 h after transfection and luciferase activity was determined. Abbreviations: pXP2, the activity of the empty reporter vector; 302, the activity of the complete promoter shown in A; 1 to 8, the activity of fragments 1 to 8.

View larger version (59K): [in a new window]   Fig. 2. Analysis of proteins binding to the activator region of the Actin302 promoter. An oligonucleotide corresponding to nucleotides –409 to –388 of the Actin302 promoter was used as a probe in an electrophoretic mobility shift assay. Either cyst nuclear extracts (10 µg; lanes 2–4), nauplius nuclear extracts (10 µg; lanes 5–7) or both (lanes 8,9) were incubated with the probe before electrophoretic analysis. Fifty times excess unlabeled oligonucleotide was added as a competitor. Lanes marked `S' correspond to samples in which the same oligonucleotide was used as competitor, while lanes marked `N' contain samples in which a non-related oligonucleotide, corresponding to nucleotides –1397 to –1349 of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) promoter 2, was used as competitor. Arrows indicate the migration of nauplius-specific retardation complexes.

View larger version (16K): [in a new window]   Fig. 3. Functional analysis of promoter 2 of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-encoding gene in mammalian cells. (A) A 1376 nt fragment of SERCA promoter 2, from nucleotides –1477 to the end of exon 1 (nucleotide –102), named 2.0, and smaller fragments generated from the internal restriction sites BstEII, BalI and ClaI to nucleotide –102, were cloned into the pXP2 luciferase reporter vector. DNA from these plasmids was transfected into Bsc40 cells, and luciferase activity was determined 48 h after transfection. Columns 2.0, BstEII, BalI and ClaI indicate the activity obtained after transfection with these vectors, while column (–) indicates the result of transfecting the reporter vector without the promoter region. (B) The region –1477 to –1311 of SERCA promoter 2 was isolated and divided into smaller fragments through PCR reactions. The complete fragment, either in its natural orientation (C) or inverted (F5) with respect to the transcription initiation site, or the smaller fragments (F1–F4 and F6–F8) were cloned into the pT109 reporter vector, which contains the minimal thymidine kinase promoter. DNA from the different plasmids was transfected into Bsc40 cells, and the associated luciferase activity was determined 48 h later. The relative activities obtained after transfection of the reporter vector without promoter (pT109) or containing the complete fragment (C, F5) or smaller fragments (F1–F4, F6–F8) are indicated.

View larger version (100K): [in a new window]   Fig. 4. Analysis of protein binding to the activator region of promoter 2 of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-encoding gene. An oligonucleotide corresponding to nucleotides –1397 to –1349 of SERCA promoter 2 was used as probe in an electrophoretic mobility shift assay. The probe was incubated with either 10 µg of nauplius or cyst nuclear extracts. A fifty times excess of the same (S) unlabeled oligonucleotide (lanes 3, 7) or two different non-related (N) oligonucleotides (lanes 4, 8 and 5, 9, respectively) were used as competitors. The first non-related oligonucleotide corresponds to nucleotides –1347 to –1316 of the same SERCA promoter 2 (lanes 4, 8) and the second to the consensus binding site for the transcription factor Oct-1 (lanes 5, 9). Arrows indicate the migration of specific retardation complexes.

View larger version (54K): [in a new window]   Fig. 5. In vitro analysis of protein-binding regions of the promoter of the Na+/K+-ATPase 1 subunit-encoding gene. (A) The indicated amounts of cyst or nauplius nuclear extracts were incubated with an asymmetrically labeled probe from the proximal region of the Na+/K+-ATPase 1-subunit-encoding gene promoter. After incubation, the samples were subjected to partial DNase I digestion and analyzed in 6% polyacrylamide–7 mol l–1 urea gels. Nucleotide sequencing reactions of the same fragment were used to locate the digestion products. The open box on the right indicates the protected region that was analyzed in B. (B) An oligonucleotide probe of the region indicated in A, corresponding to nucleotides –207 to –188, was incubated with nauplius (lanes 2–4; 15 µg), cyst (lanes 6–8; 20 µg) or both (lane 5) nuclear extracts and analyzed by polyacrylamide gel electrophoresis (PAGE). The same unlabeled oligonucleotide was used as a specific competitor (S; lanes 3, 7), while an oligonucleotide from nucleotides –240 to –221 of the Actin302 promoter was used as a non-related competitor (N; lanes 4, 8). Arrows indicate the migration of nauplius-specific retardation complexes.

View larger version (50K): [in a new window]   Fig. 6. In vitro analysis of protein-binding regions of the Actin302 proximal promoter region. (A) Nauplius or cyst nuclear extracts, or both, were incubated with an asymmetrically labeled probe from the proximal region of the Actin302 gene promoter. After incubation, samples were partially digested with DNase I and analyzed in 6% polyacrylamide–7 mol l–1 urea gels. Sequencing reactions of the same promoter region were analyzed in parallel to determine the nature of the digestion products. The open box to the right indicates the migration corresponding to the probe used in B. (B) The region corresponding to the footprint shown in A (nucleotides –240 to –221) was used to synthesize an oligonucleotide probe, which was incubated with nauplius (15 µg; lanes 2–4), cyst (20 µg; lanes 6–8) or both nuclear extracts (lane 5). Complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). 50 times excess of the same unlabeled oligonucleotide was used as a specific competitor (S; lanes 3, 7). An oligonucleotide corresponding to nucleotides –207 to –188 of the Na+/K+-ATPase 1-subunit-encoding gene promoter was used as a non-specific competitor (N; lanes 4, 8). The arrow indicates the specific retardation complex obtained after incubation with nauplius nuclear extracts.

View larger version (65K): [in a new window]   Fig. 7. In vitro analysis of protein-binding regions of promoter 2 of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-encoding gene. (A) The indicated amounts of cyst or nauplius nuclear extracts were incubated with an asymmetrically labeled probe derived from the proximal region of promoter 2 of the SERCA gene. Samples were subjected to limited digestion with DNase I before analysis in 6% polyacrylamide–7 mol l–1 urea gels. Sequencing reactions of the same promoter region were analyzed in parallel to identify DNase I digestion bands. The open box on the right indicates the protected region that was further analyzed in B. (B) An oligonucleotide probe corresponding to nucleotides –262 to –238 of promoter 2 of the SERCA gene was incubated with nauplius (lanes 2–6) or cyst (lanes 7–11) nuclear extracts and analyzed by polyacrylamide gel electrophoresis (PAGE). Fifty times excess unlabeled oligonucleotides were used as competitors. The same oligonucleotide used as probe was the specific competitor (S; lanes 5, 10), and an oligonucleotide corresponding to nucleotides –207 to –188 of the Na+/K+-ATPase 1 subunit gene promoter was the non-specific competitor (N; lanes 6, 11). Arrows indicate the migration of specific retardation complexes.

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