Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail

Abstract

Background: Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date.

Results: Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay.

Conclusions: Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Keywords: Cryptic; EGF-CFC; Left-right-axis; Snail; Transcription.

Fig. 1

Genomic organization of Cryptic gene and putative Snail binding sites in the promoter region. Nucleotide sequence of Cryptic promoter region is shown in the boxes. The Cryptic promoter sequence was analysed for the Snail binding elements (SBE). Schematic location of the two predicted Snail binding sites- CACGTG (at −453 to −447 bp relative to TSS, SBE1) and CAGGTG (at −2745 to −2751 relative to the TSS, SBE2) is represented. TSS: Transcription Start Site

Fig. 2

Cryptic promoter activity in cells over expressing Snail. Plasmid construct expressing Snail was transfected in increasing concentrations (as indicated) in HEK 293 cells along with reporter constructs for Cryptic promoter activity by cloning the Cryptic promoter region upstream of the firefly luciferase gene. (a) The full length Cryptic promoter, (b) Promoter region containing a single Snail binding element (SBE1), and (c) deleted Snail binding elements are co-expressed with increasing concentrations of the vector expressing Snail. (d) Luciferase acivity is also measured for the Cryptic promoter construct either containing SBEI or SBEII mutant or full-length and for the vector alone. Empty reporter vector is used as vector control, pCDNA3 is used as control for Snail transfection and Beta-galactosidase construct is utilized to ensure equal transfection. The relative luciferase activity is plotted as a function of increasing Snail expression. Experiments are carried out in triplicates and repeated at least 3 times. Data with p < 0.05 is considered significant

Fig. 3

Endogenous Cryptic levels are attenuated by Snail expression and are restored upon Snail depletion in PANC1 cells. a Cryptic and Snail levels are measured by western blotting after transfecting different amounts of Snail/control/shRNA plasmids (2/4/6 μg of Snail plasmid and 4 μg of shRNA plasmid and total amount of plasmid made up to 8 μg with pCDNA3 empty vector). Equal loading is confirmed by beta-actin. The blot is representative of 3 experiments (n = 3). qPCR is performed on reverse transcribed samples to estimate the mRNA levels of (b) Snail and (c) Cryptic and is normalized to beta-actin expression (n = 4)

Fig. 4

Interaction of Cryptic promoter region with endogenous Snail in nuclear lysates of PANC1 cells. Total nuclear protein extract (NPE) was isolated from PANC1 cells that express Snail endogenously. (a) Schematic of oligonucleotide duplex corresponding to Cryptic promoter region 15 bp upstream and 15 bp downstream of the Snail binding element was used for electrophoteric shift, (b) Schematic of the Snail binding element was mutated (showed in red). (c) the schematic complex of oligonucleotide supershift is depicted. (d) Lane 1 represents the biotinylated probe. Lanes 2 and 3 represent the incubation of increasing amounts of NPE with wild type probe. Lanes 4 and 5 are obtained upon incubating the NPE with the wild-type oligonucleotides with IgG control or Snail specific antibodies. Lanes 6 represents the mutated Snail binding element Lane 7 represents the incubation of the NPE with SBE mutated oligonucleotide. NPE: nuclear protein extract, * represents 10 μg NPE; blue and red arrows represent shift and supershifts, respectively

Fig. 5

Interaction of Snail with the Cryptic-promoter in-vivo. Chromatin Immunoprecipitation (ChIP) was performed in PANC1 cells for the two putative Snail binding sites using a) semi-quantitative or b) qPCR. The cells expressing endogenous Snail were cross linked using formaldehyde followed by shearing and immunoprecipitation using a Snail specific or IgG control antibody. The resulting chromatin was reverse cross linked and amplified using the primers flanking the two putative Snail binding sites. Equal loading was confirmed by the amplification of input chromatin. The resulting blot (4A) and the quantification (4B) is representative of 3 experiments (n = 3)

Fig. 6

Proposed mechanism of Snail mediated L-R axis specification through Cryptic repression. (Up,left) Low endogenous expression of Snail on the left side of the developing embryo permits Cryptic-mediated Nodal signalling, causing left-side specification. (Up, right) Relatively higher levels of Snail on the right side suppress Cryptic-mediated Nodal signalling resulting right-side specification. (Bottom) A Snail mutant background is reported to aberrantly activate Nodal signalling. The de-repression of Cryptic in a mutant Snail background may cause bi-laterally symmetrical activation of Nodal signalling and thereby random organ positioning