SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-Cre$^{ERT2}$::Smarcb1$^{flox/flox}$ model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway

Fuzzy Tandem Repeats Containing p53 Response Elements May Define Species-Specific p53 Target Genes

Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species

Of mice and men: fuzzy tandem repeats and divergent p53 transcriptional repertoires

International audienceThe clinical importance of tumor suppressor p53 makes it one of the most studied transcription factors. A comparison of mammalian p53 transcriptional repertoires may help identify fundamental principles in genome evolution and better understand cancer processes. Here we summarize mechanisms underlying the divergence of mammalian p53 transcriptional repertoires, with an emphasis on the rapid evolution of fuzzy tandem repeats containing p53 response elements

Transactivation of murine <i>Ncoa1</i> by p53 relies on a cluster of p53 half-sites.

<p>(A) Sequence of the clustered p53 REs upstream of the <i>Ncoa1</i> gene, represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1D</a>. Numbers are relative to the TSS. The parentheses indicate a putative half-site with a rare variant of the core CWWG motif: evidence that p53 may bind a half-site with a CWWA core was reported <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen.1002731-Jaiswal1" target="_blank">[42]</a>, but only 2% of the sequences bound by p53 contain a p53RE with a CWWA core in its second half-site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen.1002731-Smeenk1" target="_blank">[19]</a>. (B) MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results, from 7 experiments, were plotted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1A</a>. (C) A 2.5 kb-long fragment containing sequences upstream of the Ncoa1 TSS (from −3.5 kb to −1 kb) was cloned before a luciferase reporter gene, and transfected in p53^−/− MEFs alone (<i>IV</i>), or with an expression vector for WT p53 (<i>IV</i>+p53WT), or mutant p53 (<i>IV</i>+p53R270H), then luciferase activities were measured. Luciferase activities were determined with the same sequences after deletion of the clustered putative p53 REs (sequences from −3.1 kb to −1 kb; plasmid <i>V</i>). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1E</a>. (D) ChIP assay was performed in doxorubicin-treated p53^−/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as above. (E) Fuzzy tandem repeats within the <i>Ncoa1</i> cluster. Integrated <i>mreps</i> results are represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1G</a>. (F) The cluster of p53 REs at the <i>Ncoa1</i> locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human <i>Ncoa1</i>loci and plotted as before. (G) Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. After stress, p21 mRNAs levels increased (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1H</a>), but not Ncoa1 mRNA levels. Results, from 3 independent experiments, were normalized and plotted as before.</p

Murine <i>Rbl2/</i>p130 is a p53 target gene.

<p>(A) WT and p53^−/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) before RNA extraction and real-time PCR quantification, in 8 independent experiments. Data were normalized to control mRNA levels, then a value of 1 was assigned to mRNA amounts in unstressed WT cells. (B) MEFs were treated as in (A), then protein extracts were immunoblotted with antibodies to p130, p53 and Gapdh. (C) MEFs were left untreated (Unt) or treated with Nutlin (Nut) before RNA quantification, in 4 independent experiments. (D) Putative p53 REs, identified using Consite and a positional frequency matrix (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#s4" target="_blank">Methods</a>), were plotted along the <i>Rbl2</i>/p130 locus as lollipops, with greytones according to scores (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen.1002731.s010" target="_blank">Table S1</a>). Numbers are relative to the transcription start site (TSS). Black box: exon 1. Below, the cluster sequence is shown, with p53 putative binding half-sites in bold (putative binding sites have 0–3 mismatches with the consensus; matches in capital letters and mismatches in lowercase; perfect half-sites are boxed). (E) The 6 kb upstream the TSS were cloned before a luciferase reporter gene, and the plasmid was transfected in p53^−/− MEFs alone (<i>I</i>), or with an expression vector for WT p53 (<i>I</i>+p53WT) or mutant p53 (<i>I</i>+p53R270H) to measure luciferase. Likewise, luciferase was measured with plasmids containing 2.5 kb of sequences downstream the TSS with (<i>II</i>) or without (<i>III</i>) the clustered p53 REs. Results, from 3 independent experiments, were normalized to control renilla luciferase, then a value of 1 was assigned to luciferase in cells transfected with reporter plasmids alone. (F) ChIP assay was performed in doxorubicin-treated MEFs, with an antibody against p53, or rabbit IgG as a control. Immunoprecipitates were quantified using real-time PCR and normalized to input DNA on an irrelevant region, in 3 independent experiments. (G) Integrated results of the cluster sequence analysis with <i>mreps</i>. Numbers are relative to the TSS. (H) Human lung fibroblasts were treated and mRNAs were quantified as in (A), in 3 independent experiments. Similar results were obtained with foreskin fibroblasts. (I) Putative p53 response elements were searched for and plotted along the human <i>Rbl2</i>/p130 locus as in (D). The region homologous to the murine clustered p53 REs is below, with putative half-sites as before (those with a single mismatch are underlined). The parentheses indicate a nonamer that might be a putative half-site with a deletion within the core CWWG motif (the minus sign indicates the deletion), a situation observed in about 5% of p53 binding half-sites <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen.1002731-Riley2" target="_blank">[41]</a>.</p

A partial divergence in the regulation of <i>Rbl2</i>/p130, <i>Ncoa1</i>, and <i>Klhl26</i> among rodents.

<p>(A) Homologous regions of the <i>Rbl2</i>, <i>Ncoa1</i> and <i>Klhl26</i> loci from mouse and rat were analyzed with Consite, and results were plotted along the map as before. At each rat locus, 0–2 putative p53 REs were found to map at the same position as a cluster of p53 REs at the homologous murine locus region. Their sequence is indicated below the map. (B) Primary REFs were left untreated (Unt) or treated for 24 h with doxorubicin (Dox) at 0.5 µg/ml, or Nutlin at 10 µM, before RNA extraction and real-time PCR quantification. Results were normalized to control mRNA levels, then the mean amount of mRNAs in unstressed cells was assigned a value of 1. Data are from 3 independent experiments.</p

Comparative Consite analysis of <i>Rbl2</i> sequences from 11 mammalian species.

<p>The sequences for the first 2.5 kb downstream of the <i>Rbl2</i> transcription start site (mouse, rat, dog, cattle, rhesus monkey, gibbon, chimp, human) or translation start site (rabbit, horse, elephant) were analyzed using Consite as before. Results are represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1D</a>.</p

p53 transactivates murine <i>Klhl26</i> via a cluster of p53 half-sites.

<p>(A) Sequence of the cluster of p53 half-sites in <i>Klhl26</i> intron 1, represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1D</a>. Numbers are relative to the TSS. (B) WT and p53^−/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results from 3 independent experiments. (C) A 1 kb-long fragment from <i>Klhl26</i> intron 1 (from +0.1 kb to +1.1 kb) was cloned before a luciferase reporter gene, to test for p53-dependent reporter activity. The reporter plasmid was transfected in p53^−/− MEFs alone (<i>VI</i>), together with an expression vector for p53 WT (<i>VI</i>+p53WT) or for mutant p53 (<i>VI</i>+p53R270H) and luciferase activities were measured. Similarly, luciferase activities were determined with the same sequences after deletion of clustered putative p53 half-sites (sequences from +0.39 to +0.64 kb were deleted; plasmid <i>VII</i>). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1E</a>. (D) ChIP assay was performed in doxorubicin-treated p53^−/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as before. (E) Integrated results of the <i>mreps</i> DNA sequence analysis, represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1G</a>. (F) The cluster of p53 REs at the <i>Klhl26</i> locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human <i>Klhl26</i> loci and plotted as before. (G) <i>Klhl26</i> is not induced in response to stress in human fibroblasts. Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and quantification. After stress, p21 mRNAs levels increased (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1H</a>), but not Klhl26 mRNA levels. Results, from 4 independent experiments, were normalized and plotted as before.</p

Comparative Consite analyses of <i>Ncoa1 and Klhl26</i> sequences from 7 mammalian species.

<p>The 4 kb of DNA sequences upstream of the <i>Ncoa1</i> transcription start site (left), or the 2 kb surrounding the <i>Klhl26</i> transcription start site (right), were analyzed in 7 mammalian species using Consite as before. Results are represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002731#pgen-1002731-g001" target="_blank">Figure 1D</a>. Shorter lines, or dashed lines within brackets, indicate incomplete sequences.</p