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Single cell transcriptional and chromatin accessibility profiling identify the cellular heterogeneity of human medulloblastoma

ABSTRACT: We report the application of single-molecule-based sequencing technology for high-throughput profiling of chromosome occupation and single cell gene expression or single nuclei chromatin accessibility in medulloblastoma tissues or cells. To further define the cellular identity at the single cell level, we characterized 26 MB tissues including 18 newly diagnosed MB tumor tissues by single cell scRNA-seq and scATAC-seq (the assay for transposase-accessible chromatin-sequencing) together with 8 previously reported MB single cell transcriptomics. The newly diagnosed MB tumors included four SHH-MBs (45,731 cells), nine G3-MBs (82,834 cells), thirteen G4 MBs (77,521 cells) with a median of 7,926 cells for scRNA-seq and 9,342 cells for scATAC-seq per MB tumor, respectively. To decipher how dynamic accessibility at the cis-regulatory elements (CREs) relates to the gene regulatory program in TCP-like cells from G3- and G4- MBs, we performed single nuclei ATAC-seq (snATAC-seq) to determine the single-cell chromatin accessibility landscape using high-quality nuclei from a set of fresh G3 and G4 MB tumors that matched with scRNA-seq. Finally, we show that gene expression with chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. Our studies identified a human-specific intermediate progenitor population important for cerebellar neural lineage development and the growth and metastasis of aggressive MBs, highlighting potential therapeutic targets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE198565 | GEO | 2022/10/08

REPOSITORIES: GEO

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Project description:Medulloblastoma (MB) is the most common malignant brain tumor. MB is a cerebellar tumor that occurs mostly in children between the ages of 3-7 years but also in adults. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and G4, each of which with distinct molecular signatures. SHH MBs with MYCN amplification and TP53 mutations and G3 MBs characterized by C-MYC (MYC) overexpression in ~17% of cases from gene amplification with stem like properties, are the most aggressive and least curable with current therapeutic regimen. Using an orthotopic transplant approach, we found that enforced expression of MYCN in cerebellar granule neural progenitors (CGNPs) from 5-7 days old Trp53-null mice induced SHH MBs after transfer in the cortices or cerebella of naive recipient mice. In contrast, overexpression of MYC induced G3 MBs. Because MYCN and MYC bind to the same E-box DNA sequences, we hypothesized that the difference between MYC and MYCN-induced gene expression and tumor identity might be due to their interaction with different partners in CGNPs. In this study, we investigated the role of the Myc interacting zinc finger protein 1 (MIZ1). We found that MIZ1 binds with higher affinity to MYC than to MYCN and that MIZ1 and MYC co-occupy thousands of promoters in G3 MB. Remarkably, enforced expression of a MYC mutant (MYCV394D) that no longer binds to MIZ1 in Trp53-null CGNPs or expression of wild type MYC in CGNPs that lack functional Miz1 resulted in massive changes of the resulting tumorâ??s transcriptional program. Tumors differed from both SHH and G3 medulloblastoma and had a later time of onset compared to MYC-driven G3 medulloblastoma. Our data demonstrate that interaction of MYC with MIZ1 is required for the development of G3 medulloblastoma. ChIP-Seq experiments for Miz1, c-Myc and NMyc from murine medulloblastoma material. Either sorted tumor cells (Miz1 and c-Myc) or spheres from tumor cells (Miz1 and NMyc) were used. Input-samples were sequenced as controls. RNA-Seq from G3 medulloblastomas after restoration of Atoh1 expression.

Project description:Medulloblastomas (MBs) are cerebellar tumors that can be classified into molecularly distinct subgroups that differ in pathology and prognosis. The mechanisms that underlie subgroup specification are largely unknown. While human SHH MBs express MYCN, Group3 (G3) MBs are associated with c-MYC (MYC) overexpression and often show metastasis that confers a poor prognosis. Although MYC proteins are thought to be functionally exchangeable, ectopic expression of Myc or N-myc in Trp53-/-;Cdkn2c-/- cerebellar granule neuron progenitors (GNPs) induces G3 and SHH MBs, respectively, demonstrating that each Myc protein has distinct biological properties. We now show that Myc and N-myc differ in their affinity to Miz1 and that Myc, but not N-myc, effectively recruits Miz1 to its target sites on chromatin. The interaction of Myc with Miz1 is required for the genesis of G3 MB. Myc suppresses ciliogenesis and âreprogramsâ the transcriptome of SHH-dependent GNPs to stem-like cells by repressing genes highly expressed in SHH MB via Miz1. Consistently, target genes of Myc/Miz1 are repressed in human G3 MBs but not in other MB subgroups. Collectively, the data show that the interaction of Myc with Miz1 is a defining hallmark of G3 MB development. In this study, we show that Myc and N-myc differ in their affinity for Miz1, and Myc/Miz1 interaction is required for Group3 medulloblastoma (MB). The gene expression profiles of these tumors were compared to our previously published Group3 MB model as well as SHH model of MB (Kawachi et al., 2012, Cancer Cell). Cerebellar granule neuron progenitors (GNPs) [dka220-222] from postnatal (P) 6 Math1-GFP;Trp53-/-;Cdkn2c-/-. For SHH medulloblastomas, [dka204-206] and [blm015-017 or dka050-dka051], spontaneous medulloblastomas from Cdkn2c-/-;Trp53Fl/Fl;Nestin-Cre (Kawachi et al., 2012, Cancer Cell) and Ptch1+/-;Cdkn2c-/- (Uziel et al., 2005, Genes Dev) were used, respectively. Myc- [dka201-203] and MycV394D (termed MycVD thereafter)- [bvo017-bvo023] tumors were from Trp53-/-;Cdkn2c-/- cerebella of P6-P7 pups. Myc/ÎPOZ tumors [bvo002-bvo006] were obtained from Trp5Fl/Fl;Miz1ÎPOZ/POZ;Nestin-Cre cerebella of P6-P7 pups.

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Single cell transcriptional and chromatin accessibility profiling identify the cellular heterogeneity of human medulloblastoma

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