Genome-wide Rules of Nucleosome Phasing in Drosophila

Abstract

Graphical Abstract Highlights d Phased nucleosome arrays are mapped genome-wide in Drosophila d A DNA motif strongly associated with phased arrays in flies and mice is identified d Phaser, a previously uncharacterized protein, binds this motif d Phased arrays are generated by local barrier elements and chromatin remodelers SUMMARY Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genomewide reconstitution of physiological chromatin in Drosophila embryo extracts, we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif. It also revealed how the global activity of the chromatin remodelers CHRAC/ ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.

Biophysical regulation of local chromatin structure

“…Further examination identified an ATACG motif which binds a novel zinc-finger protein, Phaser. The function of this factor, other than its ability to position nucleosomes in vivo and in vitro, remains unknown [61]. Perhaps Phaser reflects an emergent class of proteins which by binding tightly at specific sites in chromatin can alter local chromatin structure for some unknown purpose.…”

A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

“…Preblastoderm embryos contain all components required to support the first 14 rapid nuclei divisions, including chromatin assembly and DNA damage signalling (67,86,87). Preblastoderm embryo extracts allow the efficient assembly of complex chromatin with unique dynamic properties (27,88), which enabled the pioneering discovery of ATP-dependent nucleosome sliding factors (10–12) and fundamental properties of chromatin organization (13).…”

“…Indeed, the ISWI-containing nucleosome sliding factors NURF, CHRAC and ACF have been first identified and isolated from this extract (10–12). We recently reconstituted chromatin genome-wide and discovered faithful nucleosome phasing at prominent sites (13).…”

Basis of Specificity for a Conserved and Promiscuous Chromatin Remodeling Protein

“…There is an abundance of literature suggesting that Drosophila ACF complex, the Isw2 ortholog, is a nonspecific nucleosome spacing and assembly factor that evenly spaces phased nucleosome arrays against defined genomic barriers 10,18,49 . We wondered if the WAC domain of Drosophila Acf1 was different from that of Itc1, so we performed sequence alignment of WAC domains and compared to Acf1 from the Drosophila genus.…”

“…The CHD and ISWI families of ChRPs have been characterized as nonspecific nucleosome sliding and spacing factors in vitro [6][7][8][9][10][11][12] . In yeast, flies, and mammals ChRPs generate evenly spaced nucleosome arrays at transcription start sites (TSSs) and organize genomic chromatin at other defined boundaries [12][13][14][15][16][17][18][19][20] . However, relatively little is known about the in vivo biological regulation of these spacing factors, and it is not understood how they can accurately and reproducibly position nucleosomes throughout the genome in different cellular contexts.…”