Li D et al. (2009), Regulation of ALF promoter activity in Xenopus ...

XB-ART-40207

PLoS One 2009 Aug 17;48:e6664. doi: 10.1371/journal.pone.0006664.

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Regulation of ALF promoter activity in Xenopus oocytes.

Li D , Raza A , DeJong J .

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BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B) were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

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Species referenced: Xenopus laevis
Genes referenced: acta4 cyp26a1 drd1 gpm6b gtf2a1l krt62 tff3.7

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	Figure 1. Isolation and transcription start site mapping of the Xenopus ALF promoter.(A) PCR reactions were performed with X. laevis genomic DNA that had been digested with EcoRV, HincII, PvuII, and StuI. The gene specific primer (GSP) was located 70 base pairs downstream of the 5′ end of ALF mRNA. AP is the adaptor primer. (B) The 1.7 kb HincII ALF promoter fragment contains a DNA transposon and two other repeats, examples of which are aligned in the figure. (C) To map the start site, RT-PCR reactions were performed with oocyte RNA using primers located at various locations throughout the ALF promoter region (S0-S6). The results show strong bands with primers S5 and S6 (lanes 6, 7), weaker bands with primers S3 and S4 (lanes 4, 5), and no bands with primers S1 and S2 (lanes 2, 3). (D) Sequence analysis of nearly 40 RACE clones shows the distribution of start sites throughout the promoter region. The number of hits observed at each position is indicated. Locations of the ATG and primers S3-S6 are indicated. (E) Sodium bisulfite methylation analysis of the ALF promoter shows a high degree of methylation (filled circles) in liver tissue where the gene is normally off, and little to no methylation (open circles) in oocytes where the ALF gene is normally on. Filled cirlces represent methylation while open circles represent no methylation.
	Figure 2. Alignment of ALF promoter sequences from different species.ALF promoters from twelve different species (mouse, rat, chimp, human, macaque, horse, pig, cow, dog, chicken, X. tropicalis, and X. laevis) were aligned. Conserved regions (shaded) could be identified among the first ten of these organisms. The two frog-derived sequences were only weakly similar and were shaded separately. The table shows pairwise identity scores.
	Figure 3. Deletion analysis of ALF promoter constructs reveal a very short active region.(A) Three promoter constructs, ALF1.7, ALF1.0, and ALF0.25 were linked to a luciferase reporter, microinjected into oocytes, and assayed for activity relative to controls pGL3TK (thymidine kinase) and an empty vector (pGL3BASIC). (B) Microinjection experiments with shorter deletions constructs prepared from the ALF0.25 parent (ALF250, ALF205, ALF165, ALF125, ALF85, and ALF45) showed that an 85 base pair construct retained full activity. (C) The relative activity of the ALF0.25 construct was compared in oocytes and Xenopus XLK-WG kidney epithelial cells in comparison to a pGL3-TK reference. (D) The relative activities of differently sized ALF constructs differ in oocytes and XLK-WG epithelial cells. The ALF0.25 construct served as the normalization control.
	Figure 4. Identification of two core promoter elements.(A) A series of mutations and deletions were introduced into the ALF promoter and tested for their effect on activity. Constructs contained nucleotide substitutions (shown in black) or 3′-end deletions (D1A, D2A, and D3A) compared to the wild type controls ALF85 and ALF85+. (B) A set of ALF85 derived constructs injected and tested for activity showed diminished activity with M5A and all the 3′-deletions. (C) A set of ALF85+ derived constructs showed two regions with diminished activity, defined by constructs M5B/M6B and M8B/M9B. (D) Northern blot analysis of wild type and mutant constructs. The top panel shows luciferase RNA levels, the middle panel shows a control 5S rRNA gene hybridization, and the bottom panel shows ethidium bromide-stained 28S and 18S RNA.
	Figure 5. Fine structure mapping of the core promoter elements A and B.(A) A series of three nucleotide substitutions were made in and around the functional elements defined in the previous figure. (B) and (C) Constructs were injected, assayed for luciferase activity, and normalized to a WT (ALF63) control. The results define an upstream A element of about 12 nucleotides (CGTTACGTCAGA) and a downstream B element of about 9 nucleotides (AACTTCCGG).
	Figure 6. Identification of oocyte proteins that interact with the A and B elements.(A) A 96 bp fragment from the ALF85+ promoter was labeled and used as the probe in EMSA assays with oocyte extracts. A summary of the position of the A and B elements and the factor binding sites are shown in the two top lines. Beneath this is shown the relative locations of a series of overlapping oligonocleotide competitors. (B) An additional set of oligonucleotide competitors that contained specific mutations were also used as competitors in the binding assays. (C) Bandshift analysis shows the ALF promoter forms several protein-DNA complexes using oocyte-derived cell-free extracts. The main complexes are indicated by the labels 1, 2, and 3. The P2-05 competitor selectively abolishes complex 1 (lane 2), while the P2-89 competitor selectively abolishes complex 3 and to a lesser extent complex 2 (lane 6). (D) Additional competition assays show that P2-05-M1 but not P2-05-M2 is able to compete for complex 3 (compare lanes 3 and 4). Similarly, the P2-89-M2 competitor but not P2-89-M1 is able to compete for binding of complexes 2 and 3 (compare lanes 7 and 8). (E) Competition with mutant oligos P2-05-M3, P2-89-M3, and P2-89-M4 further refines the binding site to the positions noted in the ‘Complex Formation’ line in (A).
	Figure 7. Effect of maturation and functional analysis of the A and B elements.(A) Deletion of sequences between the A and B elements and the introduction of sequences between the two elements resulted in increased activity for the −5 construct and lowered activity for the −10 construct. The remaining constructs (−15, +5, +10, and +15) showed activity similar to the control. An exchange of elements (EXCAB) led to loss of activity. (B) Separation of the A and B elements using a 256 bp insert resulted in an activity equivalent to the wild type control. Mutation of the repositioned A element (MA) and a combined AB mutant (MAB) resulted in the loss of promoter activity. (C) The effect of oocyte maturation on transcription activity of the ALF85 promoter. Progesterone (P) was added at 2 hour intervals relative to the time of microinjection.
	Figure 8. Effects of activating and repressing regulatory factors on endogenous and introduced promoter DNA in somatic cells and germ cells.Silencing factors are red/black while activating factors are green. Solid lines show paths of function while dashed lines indicate either absence or failure to function. (A) Silencing factors do not repress an introduced germ cell gene promoter in somatic cells, allowing activating factors to (inappropriately) drive transcription. (B) Silencing factors are absent or do not function on endogenous and introduced promoters in germ cells, while activating factors result in transcription from both types of DNA.

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