Evidence of pioneer factor activity of an oncogenic fusion transcription factor

Abstract

Summary Recent characterizations of pioneer transcription factors provide insights into their structures and patterns of chromatin recognition associated with their roles in cell fate commitment and transformation. Intersecting with these basic science concepts, identification of pioneer factors (PFs) fused together as driver translocations in childhood cancers raises questions of whether these fusions retain the fundamental ability to invade repressed chromatin, consistent with their monomeric PF constituents. This study defines the cellular and chromatin localization of PAX3-FOXO1, an oncogenic driver of childhood rhabdomyosarcoma (RMS), derived from a fusion of PFs. To quantitatively define its chromatin-targeting functions and capacity to drive epigenetic reprogramming, we developed a ChIP-seq workflow with per-cell normalization (pc-ChIP-seq). Our quantitative localization studies address structural variation in RMS genomes and reveal insights into inactive chromatin localization of PAX3-FOXO1. Taken together, our studies are consistent with pioneer function for a driver oncoprotein in RMS, with repressed chromatin binding and nucleosome-motif targeting.

Pioneer factors in development and cancer

“…Work with MNase-sequencing revealed that gene activation is highly predictive of strongly positioned nucleosomes near the promoter ( Kaplan et al., 2010 ; Schones et al., 2008 ). The nucleosomal binding preferences for PFs can be challenging to measure by chromatin sequencing at steady state, and rapidly inducible control over PFs is important for their experimental characterization ( Sunkel et al., 2021 ). Of note, the induced DNA unwrapping upon binding to Reb1 motifs near the entry/exit sites is supported by the predominance of sub-nucleosomal MNase fragments at its binding sites ( Henikoff et al., 2011 ).…”

“…While the ability of PFs to recognize nucleosomes containing their motifs is a central attribute, a key question is, is this process limited by the local context in highly repressed regions of the genome? One powerful method to address this question relies on sequencing and proteomics analysis of regions of the genome that are refractory to endonuclease cleavage (sonication-resistant heterochromatin; srHC) ( Becker et al., 2017 ; Fernandez Garcia et al., 2019 ; Sunkel et al., 2021 ). The conceptual basis for this is that the effects exerted by the mechanical force of sonication would be influenced by local chromatin repression.…”

“…This addresses a common experimental bias in epigenetics, which leads to over-representation of accessible DNA in ChIP-seq, CUT&Tag, and MNase-seq experimental workflows (see Table 1 ). However, recent studies have enabled meta-analyses of ATAC-seq data with ChIP-seq data, computationally inferring nucleosome positioning from NucleoATAC ( Beati and Chereji, 2020 ; Schep et al., 2015 ; Sunkel et al., 2021 ). Integrations of PF localization and nucleosome positioning will continue to illuminate the genomics of PFs.…”

“…As mutation rates are higher in srHC ( Carone and Lawrence, 2013 ), it will be important to understand functional regulation in heterochromatin regions and whether one of the functions of srHC is for functional repression of mutations or repeat elements. With studies connecting the heterochromatin targeting of H4K20-methylated at H3K9me3-enriched regions, it will be interesting to further investigate unbiased proteomics approaches for srHC to understand how repressive histone marks and PFs may be interrelated ( Sunkel et al., 2021 ).…”

“…The major translocations PAX3/7-FOXO1 comprise fusion partners PAX3 and PAX7 that have individually demonstrated pioneer function ( Hatta and Cirillo, 2007 ; Magli et al., 2019 ; Mayran et al., 2018 ). Recent evidence suggests that PAX3-FOXO1 has intrinsic pioneer activity to bind to repressive chromatin and nucleosomal motifs ( Sunkel et al., 2021 ). PAX3 also ranks highly as a vulnerability in RMS ( Figure 3 ) ( Dharia et al., 2021 ).…”