(2) we observe intrinsic asymmetry in single-molecule fiber structures assembled on lagging and leading strands. This is neither resolved by CAF-1/ASF1, nor addition of active ISWI remodeler (Isw1a). Will be exciting to identify the factors that restore symmetry in reconstituted setting!(6/n)

I'll leave details for interested readers to dive into, but here are two (of many) highlights: (1) 1st views of nucleosome wrapping states generated by the replisome alone (we see underwound nucleosomes!); we suspect this relates to our observations in mammalian cells in PMID39549698.(5/n)

Next where our group came in. We applied our SAMOSA-ChAAT method (PMID37696956) to these reconstituted replication reactions. The method is spiced up even more, allowing us to footprint single chromatin fibers AND identify replicated DNA through strand-specific BrdUTP detection via PacBio.(4/n)

Building on pioneering work of Kurat, Diffley & colleagues, Bruna, Inge, & Francesca have implemented the pure reconstitution of replication initiation, elongation and termination on fully chromatinized yeast origins. >70 proteins. Extremely challenging, extremely cool, extremely powerful.(3/n)

What about *in vivo*? W/ a mouse model from Zaret group, assessed how perturbed FOXA2 (single mutant allele defective for histone binding) influences nuc structure. Replacing just one allele w/ defective copy led to reproducible shifts in nuc structure in murine hepatocytes in vivo! (9/n)

We found that SOX2 directly influences patterns @ composite SOX2-OCT4 motifs — paradoxically, SOX2 loss leads to (1) more breathable (sub)nucs, & (2) *loss* of least acc nucs; a deeper dive in paper, but we think this may underlie motif dependent vs independent binding modes on chromatin (8/n)

This is a...dense preprint; we look at single-mol co-accessibility, genome-wide distribution, & more. Also looked into TF-nuc interactions in a couple contexts. 1st: to understand if SOX2 directly influences distortion at binding sites, worked w/ @dewitlab.bsky.social to degrade SOX2 in mES. (7/n)

Marty worked out how to classify nucs based on similar intranuc acc, resulting in clusters of different nuc ‘types'; we interpret as unique structurally 'distorted' nuc states in cells! Relative abundance of types shifts depending on genomic region, but we see all types everywhere we look. (6/n)

The transition prob (t) is user-determined. By tuning t, we can force model to break nucs into "subfootprints." We tested if subfootprints were randomly patterned, and were delighted to discover that they weren’t just random subdivisions. They capture what we interpret as subunit nuc structure!(5/n)

IDLI builds on (published) idea 1st postdoc Colin McNally (now sr. ML engineer @ GSK), Hani & I came up with ~4 y ago: combine NNs & HMMs to infer footprints from PacBio seq m6dA-meth DNA. The gist: EcoGII & PB have NN-learnable biases using +/- ctrls; we use as emission probs in 2-state HMM. (4/n)

We’ve hoped (suspected?) that nondestructive chromatin fiber mapping (pioneered by us & others) captures structural aspects missed by nuclease-based techniques. Here, we keep the tiffin cart rolling w/ IDLI: an approach that infers subnuc-res structure from m6dA long-read footprinting data. (3/n)