Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.


Statistics
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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 Sequencing data generated for this study are available at the Sequence Read Archive under accession PRJNA622577. Copy number data for high-risk neuroblastoma were downloaded from https://github.com/padpuydt/copynumber_HR_NB/27. Public data supporting the findings of this manuscript were downloaded from the Gene Expression Omnibus under accessions GSE90683, GSE80152, GSE24447, GSE37385, GSE18927 and GSE28874 and from ArrayExpress under accession E-MTAB-6570. Medulloblastoma ChIP-seq data were downloaded from https://pecan.stjude.cloud/dataset/northcott. Corresponding BigWig und narrowPeak files can be downloaded from https://data.cyverse.org/dav-anon/iplant/home/konstantin/helmsaueretal/. An accompanying UCSC genome browser track hub is provided for

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Sample size
The sample size for copy number data was given by the sample size of the public data. We chose four cell lines as in-depth case studies. We did not test specific hypotheses with these data for which sample size calculation would have been applicable but rather describe two cases of enhancer hijacking in class II amplicons.
Data exclusions No data was excluded.

Replication
For large parts of our analysis, we used published sequencing data that had not been acquired in replicates. When we acquired ChIP-seq/ ATAC-seq data ourselves, this was not done with replicates. Hi-C data was acquired in technical duplicates and merged after individual inspection of the data. FISH experiments were done once per cell line.
Randomization Not applicable. The study did not have different experimental groups.

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Not applicable. The study did not have experimental groups such that investigators could not be blinded to group allocation.

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Cell line identity was verified by STR genotyping (Genetica DNA Laboratories, Burlington, NC and IDEXX BioResearch, Westbrook, ME).

Mycoplasma contamination
Absence of Mycoplasma sp. contamination was determined with a Lonza MycoAlert system (Lonza Group Ltd., Basel, CH).
Commonly misidentified lines (See ICLAC register) No commonly misidentified lines were used in this study.

ChIP-seq Data deposition
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Replicates
This study is mainly based on published ChIP-seq data which had not been acquired in replicates. For additional data acquired, this was not performed with replicates. Peak statistics were initially derived from comparison to the read distribution in matched input samples, and then compared between samples, as described in detail in the Methods. Hi-C was acquired in technical duplicates and merged after inspection.