June 15, 2017;
Genes coding for cyclin-dependent kinase inhibitors are fragile in Xenopus.
Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c
and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c
and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals'' health. Also, another gene coding CDKn1b
is lost besides CDKn1c
and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c
.S expression is higher than that of CDKn2c
.L, and a conserved noncoding sequence (CNS) of CDKn2c
genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus.
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Structures of genes involved in cell-cycle regulation in the X. laevis genome. (a) Summaries of gene structures. Most genes are retained as homeologous pairing genes. Black characters, a homeologous pairing gene; magenta characters, a singleton gene. Genes lost (ccd3, CDKn1c (p57), CDKn2a (p16)) and amplified (ccnb1.2, ccnb5, ccndx, CDKnx (p27Xic)) are given in parentheses and bold, respectively. Colored squares and lines represent a positive (blue) or negative (black) role in the cell-cycle. (b) Pie charts summarize the structures of genes categorized into each group. Numbers in each chart indicate genes subdivided into each category, and those out of charts indicate percentages of genes with altered structures.
Comparisons of amino acid sequences of CDKnx (Xic1) among X. tropicalis (Xtr) and chromosomes L (XlL) and S (XlS) of X. laevis. Alignments were calculated from data based on the gene models (X.tropicalis ver. 9, X. laevis J-strain ver. 9.1) using BLAST Xenopus (Xenbase). CDKnx on X. laevis 7L was originally reported as Xic1 by Su et al. Each N terminus, which does not affect the activity as CDKI, is not conserved among them.
Expression profiles of cdkn2c genes from L and S chromosomes. Abscissa indicates developmental stages (a) or adult tissues (b). RNA-seq data generated in duplicate for seventeen developmental stages and fourteen adult tissues were used for calculations of TPM, and construction of each graph ( Session et al., 2016). The ordinate indicates TPM values. RNA-seq experiments were done twice with similar results, and representative data are shown.
Identification of cis-regulatory sequences, which contribute to the cdkn2c.S unique expression. (a) A comparison of non-coding sequences in cdkn2c genes among X. laevis chromosomes L and S and X. tropicalis. Upper panel: a genomic locus surrounded by dotted line indicates the conserved non-coding sequences (CNSs), which are retained in cdkn2c.S of X. laevis and cdkn2c of X. tropicalis, but are different from cdkn2c.L of X. laevis. Lower panel: sequences conserved between cdkn2c.S in X. laevis and cdkn2c in X. tropicalis are surrounded by black squares, and predicted binding motifs for transcription factors are indicated. (b) The CNSs in (a) were used for transgenic reporter analysis with X. laevis.
Comparisons of binding abilities of CDKn2c.L and CDKn2c.S to CDK4. (a) Amino acid alignments among CDKn2c of X. tropicalis and CDKn2c.L and CDKn2c.S of X. laevis. The twenty residues responsible for association and inhibition for CDK4 in CDKn2a (p16) and CDKn2c (p18) of human are also shown. Alterations of amino acids are colored as indicated in (a). (b) Co-immunoprecipitation assay to compare binding abilities to CDK4 between CDKn2c.L and CDKn2c.S. Lysates were prepared from cells expressing the indicated protein, and used for immunoprecipitation with anti-flag antibody. The samples were subjected to immunoblotting with anti-CDK4 (upper) and anti-flag antibodies (lower), respectively. Experiments were done twice with similar results, and representative data are shown.
Syntenies of cdk7 and ccnh loci in X. tropicalis and X. laevis. Abbreviations for species and chromosome numbers: X. tropicalis (XTR1), X. laevis (XLA1L and XLA1S). The p- and q-arms of chromosome 1 are denoted by letters in the figure, respectively.
Comparative analysis of developmental speed between X. laevis and X. tropicalis. (a) Appearances of X. laevis and X. tropicalis. (b) Appearances of developing embryos. The stages of X. laevis and X. tropicalis at each time point are described above the photo. (c) Growth of X. laevis and X. tropicalis is summarized. After fertilization, eggs of X. laevis and X. tropicalis were simultaneously cultured at 26 °C, and times were determined at the indicated stages (b and c).