Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Elisa Oberbeckmann; Vanessa Niebauer; Shinya Watanabe; Lucas Farnung; Manuela Moldt; Andrea Schmid; Patrick Cramer; Craig L. Peterson; Sebastian Eustermann; Karl-Peter Hopfner; Philipp Korber

Nuclear DNA is packaged into chromatin based on a repeating building block, the nucleosome core particle (NCP; (Kornberg, 1974; Olins and Olins, 1974) ), where 147 base pairs (bp) of DNA are wound around a histone protein octamer (Kornberg and Lorch, 1999; Luger et al., 1997; Olins and Olins, 2003) . Packaging by nucleosomes orchestrates all genomic processes (Lai and Pugh, 2017) . Nucleosomes mainly occur in regular arrays where they are aligned to each other such that the lengths of linker DNA between NCPs are about constant within an array. Linker lengths may vary among arrays in the same cell (Baldi et al., 2018b; Chereji et al., 2018; Ocampo et al., 2016; Valouev et al., 2011) and differ on average between cell types and species (van Holde, 1989) . Arrays are often phased, i.e., aligned relative to a genomic reference point. A combination of both in vivo studies (Ganapathi et al., 2011; Hartley and Madhani, 2009; Kubik et al., 2018; Tsankov et al., 2011; van Bakel et al., 2013; Yan et al., 2018; Yarragudi et al., 2004) and in vitro reconstitutions indicated that these 6 Senior author 7 These authors contributed equally. 8 Lead contact * Correspondence: [e-mail], [e-mail], [e-mail] genomic alignment points or "barriers" often reflect the binding of abundant, sequence-specific DNA binding proteins, like the general regulatory factor (GRFs) Reb1, Abf1, or Rap1 in budding yeast or other architectural factors like CTCF in mammals (Wiechens et al., 2016) or Phaser in flies (Baldi et al., 2018a) . Throughout eukaryotes, phased arrays are prominent at active promoters. Nucleosomedepleted regions (NDRs) at the core promoter are flanked by arrays that begin with the so called +1 nucleosome close to the transcription start site (TSS) and cover the gene body (Baldi et al., 2020; Lai and Pugh, 2017) . This organization is important for transcription fidelity as mutants with impaired array phasing show aberrant transcription initiation (Challal et al., 2018; Hennig et al., 2012; Kubik et al., 2019; Pointner et al., 2012; Smolle et al., 2012) . While nucleosome arrays are likely the most pervasive and longest known chromatin organization, their generation is still not explained. Specifically, regular spacing requires fixed distances between nucleosomes, and phasing requires a fixed distance between array and reference point. What sets these distances? In vivo and in vitro data suggest that ATP dependent chromatin remodeling enzymes (remodelers) are key to the answer. Remodelers are conserved in eukaryotes and mobilize, reconfigure, or disassemble/reassemble nucleosomes upon ATP hydrolysis (Clapier and Cairns, 2009; Clapier et al., 2017) . They are subdivided into the SWI/SNF, ISWI, CHD, and INO80 families, according to their main ATPase sequence features. Besides the core ATPase, remodelers often contain additional domains and subunits that bind the nucleosome, regulate activity and targeting, and convert their DNA tracking activity into the remodeler-specific chemo-mechanical reaction. For example, nucleosome disassembly is accomplished only by SWI/SNF family members and histone exchange only by INO80 family members, while nucleosome sliding is catalyzed by most remodelers. Particularly relevant for array generation is an ATPdependent nucleosome spacing activity, by which some remodelers convert irregular arrays into arrays of regularly spaced nucleosomes. Remodelers of the ISWI, CHD, and INO80 (Ito et