SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells. © 2013 Elsevier B.V.</p

Overall body and bone parameters in NM1 knock-out and wild type mice.

<p>Overall body and bone parameters in NM1 knock-out and wild type mice.</p

PCR primers for genotyping of ES cells and mice mutants.

<p>PCR primers for genotyping of ES cells and mice mutants.</p

Preparation of NM1 knock-out cassette.

<p>(<b>A</b>) WT genomic locus of <i>Myo1c</i> gene. Short homology arm (SA), floxed part (FP), and long homology arm (LA) of appropriate length (0.9; 0.3; 1.7 kb respectively), were cloned to pEasyFlox vector carrying neomycin (NeoR) and thymidine kinase (ThK) selection genes (<b>B</b>). Black lines represent genomic sequences; red line represents sequences derived from pEasyFlox vector. (<b>B</b>). (<b>C</b>) Genomic loci of <i>Myo1c</i> gene with excision of exon -1; P1 – P6 represent different primers needed for genotyping of ES cells and knock-out mice, yellow triangles represent loxP recombination sites. (<b>D</b>) Genotyping of NM1 knock-out mice by PCR. P5 and P6 primers were used to distinguish between wild type (+/+), heterozygous (+/−) and knock-out (−/−) animals. (<b>E</b>) Western blot analysis of NM1 levels in NM1 wild type (+/+) and knock-out (−/−) mice. Fifteen micrograms of protein per lane was loaded, and probed for NM1. Equal loading was monitored by Coomassie Brilliant Blue staining of the band corresponding to actin.</p

Myo1C is able to function in Pol I and Pol II transcription without changes in its expression level.

<p>(<b>A</b>) The level of nascent rRNA was decreased to 80% of WT levels in NM1/Myo1c knock-down cells (U2OS+C8). An overexpression of mouse NM1 (U2OS+C8+NM1) or Myo1c (U2OS+C8+M1c) resistant to shRNA causes restoration of Pol I transcription to almost endogenous levels. As a negative control were used U2OS cells with transduced empty pLKO1.1 vector (U2OS+NC). (<b>B</b>) Both NM1-Flag and Myo1c-Flag interact with Pol II. Extracts from cells overexpressing NM1-Flag and Myo1c-Flag were co-immunoprecipitated with Flag antibody and control IgG. Immunoprecipitates were analyzed by western blotting with antibodies against Flag, Pol II CTD subunit and Myo1c (tail domain recognizing both NM1 and Myo1c). (<b>C</b>) NM1 knock-out does not cause compensatory changes in expression of Myo1c. Expression of Myo1c was measured by RT-qPCR and compared relative to GAPDH expression. Data are presented as mean +/− SD. *** p<0.001.</p

Ratio between NM1 and Myo1c is nearby equal.

<p>(<b>A</b>) HeLa cells were fractionated into cytosolic and nuclear fractions. NM1 and Myo1c amounts were quantified using double fluorescent labeling of western blot membranes after normalization to NM1-GFP band. (<b>B</b>) Total amounts of NM1+Myo1c were compared in mouse skin fibroblasts derived from NM1 knock-out and NM1 wild type mouse. Beta actin signal was used as loading normalizer. (<b>C</b>) Total amounts of NM1+Myo1c were compared in lungs and stomach from NM1 knock out and NM1 wild type mouse. GAPDH signal was used as loading normalizer. (<b>D</b>) The graph shows the amount of NM1 and Myo1c after densitometric quantification of bands from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061406#pone-0061406-g004" target="_blank">figures 4B and 4C</a> showing the ratio between NM1 and Myo1c as compared to actin/GAPDH expression.</p

NM1 knock-out has no effect on cell proliferation and Pol I transcription.

<p>(<b>A</b>) Proliferation of NM1 KO cells (NM1 −/−) is not altered in comparison to WT cells (NM1 +/+). 1×10⁵ cells were seeded on plates (20% confluence; day 0) and let to grow for six days when number of cells was counted again (day 6). (<b>B</b>) Pol I transcription rate in NM1 KO (NM1 −/−) and WT (NM1 +/+) cells is equal. RNA from exponentially growing cells was isolated and expression of 45S pre-rRNA was measured by RT-qPCR. Expression of 45S pre-rRNA is compared relatively to GAPDH expression. Data are presented as mean +/− SD.</p

Red blood cells related phenotype in NM1 knock-out mice.

<p>Red blood cells related phenotype in NM1 knock-out mice.</p

Prdm5 regulates <i>Decorin</i> through a distal enhancer.

<p>A) ChIP-qPCR validation of Prdm5 peaks on selected ECM genes or negative regions from an independent sample immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest “noise” value obtained by ChIP-qPCR on a set of negative regions. B) qRT-PCR analysis of <i>Decorin</i> transcript (<i>Dcn</i>) levels upon Prdm5 knockdown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen-1002711-g004" target="_blank">Figure 4E</a>. Results are presented as average of four independent experiments +/− SD; * = p<0.05. (C) qPCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvarial osteoblasts for <i>Decorin</i>. Expression values were normalized to the control WT samples. *  = p<0.05; T-test, (+/+ n = 14, LacZ/LacZ n = 19 clones). D) Upper panels. Western blot analysis of Decorin levels upon Prdm5 knockdown in cell layers; Tubulin is used as loading control. Lower panels. Western blot analyses of purified proteoglycans from cell culture media from knockdown cells. Tubulin is used as purity control and Fibronectin for equal protein loading. E) ChIP-qPCR with indicated antibodies for Prdm5 binding site upstream of Dcn gene. Meg3 TSS region is used as negative control.</p

Analyses of Prdm5 chromatin interactions.

<p>A) Diagram illustrating the overall distribution of Prdm5 binding sites categorized according to the distance from the nearest TSS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). B) The mean distribution of tags across gene bodies for Prdm5 ChIP-seq (Prdm5-ab1 in blue, Prdm5-ab2 in red and IgG in black). Vertical dashed line at x = 0 represents the TSS. Positions after the TSS are represented as % of the length of the gene. C) Upper panel. Slogos plot produced using the motifs detected by the Weeder program from Prdm5 “shrunk” peaks (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). Lower panel. DNA pulldown assay from nuclear extract of 293 cells overexpressing HA-PRDM5 using biotinylated oligos representing the <i>Col1a1</i> exon 33 (WT) and a mutated control sequence (G-A/T Mut). D) Histogram showing the percentage of H3K9me3 (left panel), H3K4me3 (middle panel) and RNA Polymerase II (right panel) positivity for “Random sampling” (mean value of 100 iterations for 1446 random genes sets) or for Prdm5 target genes. (E) Q-Q plot comparing the quantile distribution of Prdm5 target genes' expression (on Y axis) and all genes (on X axis). Red line is reference line representing equal quantile distribution.</p