CASZ1 induces skeletal muscle and rhabdomyosarcoma differentiation through a feed-forward loop with MYOD and MYOG

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.

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Software and code
Policy information about availability of computer code Data collection Encode atac_dnase_pipelines was used for ATAC-seq data collection and analysis; trimmomatic 0.39 and Partek Flow was used for RNAseq data collection and analysis. ChIP-seq data collection and analysis is shown in ChIP-seq reporting summary. The software and tools are installed on NIH Biowulf.  (2017) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All the home generated RNA-seq, ChIP-seq and ATAC-seq datasets, the GEO accession number is GSE126147. Link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126147.
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes were chosen based on expected phenotypes and previous experience with assay variability, as well as the minimum number of animals that required to do power calculation.

nature research | reporting summary
October 2018 Data exclusions No data were excluded from these assays.

Replication
Each experiment was done in replicated. The numbers were indicated in figure legend. For example, for cell proliferation assay, cell differentiation assay were repeated three or more times. RNA-seq experiments were performed in three biological replicates. Moreover, majority of the experiments were also confirmed through using different techniques and using multiple cell lines, such as the regulation of ERMS cell proliferation by CASZ1 was confirmed in two ERMS cell lines; transcriptome regulated by CASZ1 was analyzed in three cell lines; differentially expressed genes identified from RNA-seq was confirmed by realtime PCR; gene expression regulation was validated by both realtime PCR and western blot in different cell lines; the regulation of CASZ1 by RAS-MEK pathway was confirmed by both genetic inhibition and pharmaceutic inhibition of RAS-MEK pathway.
Randomization N=20 of 4 to 6 weeks old SCID female Beige mice for each cell line were randomly divided in 2 groups: one group of mice received normal chow diet while the other group received Dox-containing chow diet for over 5 days. All mice were injected with 2 x 10^6 cell suspension into the left hind limb at the gastrocnemius muscle of the mice. Each group of the mice continue to receive the same diet as before the cell injection.

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October 2018
Specificity of antibody against CASZ1 was confirmed by peptide competition (Liu et al., Plos One 2011), transient siRNA against CASZ1 protein, followed by western blotting. To validate CASZ1 antibody for ChIP-seq, we performed immunoprecipitation of formaldehyde cross-linked cells that with low or high CASZ1 expression, followed by western blotting. We also compared the ChIP-seq results of CASZ1b-3xTy1 fusion protein transfected cells using anti-CASZ1 antibody and anti-Ty1 antibody and demonstrated the results are significantly correlated (pearson's correlation=0.89).

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) C2C12 and HEK293T cell lines were obtained from ATCC. Rhabdomyosarcoma RD and SMS-CTR cell lines were originally obtained from ATCC, and RH30 was originally obtained from Peter Houghton's lab, where this cell line was established.

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Laboratory animals
Groups of 4 to 6 weeks old female Fox Chase SCID Beige: CB17.B6-PrkdcscidLyst bg/Crl (SCID Beige) mice were injected with 2 x 10^6 in 100μl cell suspension mixed with matrigel (1:1) into the left hind limb at the gastrocnemius muscle. In all experiments, the leg dimensions were measured twice a week with digital calipers to obtain two diameters of the tumor diameter, from which the tumor volume was determined. All mice were euthanized when any of the tumor diameters were approaching 20 mm in any dimension.

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No wild animals were used in this study.

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All Xenograft studies were approved by the National Cancer Institute's Animal Care and Use Committee (ACUC), and all animal care was in accordance with institutional guidelines.
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Accession key: abahoaowhtcffix Files in database submission