October 22^nd, 2021

Dissecting chromatin remodeler-modified oligonucleosome patterns in vitro and in vivo with single molecule sequencing

Nucleosome organization dictates gene regulation

Nucleosome Repeat Length (NRL)

heterochromatin

euchromatin

Nucleosome Free region

(NFR)

�ATP-dependent chromatin remodelers customize nucleosome patterns

Eviction of nucleosomes

Sliding to fixed Regularly spaced arrays (NRLs)

Loading of nucleosomes

How do we currently study various nucleosome patterns? �

�Lai and Pugh, 2017

Via deep sequencing of nuclease-digested chromatin

MNase Digest

Library Prep/Sequencing

Limitations of existing nucleosome mapping techniques

Limitations of existing nucleosome mapping techniques

DNA Mtase

Bisulfite Conversion

Library Prep/Sequencing

Bulk average sequencing fails to capture discrete nucleosome patterns

Can we capture single molecule fiber structures?

Capturing discrete nucleosome patterns with long-read PacBio Single Molecule Real Time sequencing

Sequence (bps)

Polymerase Pulse (time)

SAMOSA:

Single-molecule adenine methylated oligonucleosome sequencing assay

SMRTbell

Abdulhay, McNally et al., 2020

Predicting adenine methylation and nucleosome accessibility

Analysis Pipeline developed by Dr. Colin McNally

Measured Pulse Times

Use a neural network regression model to predict the expected Pulse Times

Deviation of measured Pulse Times from expectation

Apply a cutoff to predict methylation

Predicted methylation status of adenines

Predicted DNA accessibility

Use a hidden markov model to find the most likely path of accessibility

SAMOSA specifically detects nucleosome patterns on in vitro assembled chromatin

Abdulhay, McNally et al., 2020

Can we construct and footprint chromatin fibres made from a native mammalian template?

Inspired by previous work from Korber and Pugh Labs

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SAMOSA detects nucleosome footprints on in vitro assembled mammalian chromatin

SAMOSA detects SNF2h-remodeled nucleosome footprints on in vitro assembled mammalian chromatin

How does SNF2h restructure stretches of nucleosomes?

d1 (bp)

d2 (bp)

SNF2h-mediated nucleosome spacing is heterogenous

SAMOSA adapted in vivo detects genome-wide nucleosome patterns

In vivo SAMOSA: Abdulhay, McNally et al., eLife 2020

SNF2h KO and re-expression mESCs provided by Dirk Schübeler Laboratory

How does SNF2h influence genome-wide nucleosome patterns?

SNF2h KO mESCs

SNF2h WT Add-back mESCs

SNF2h remodeling maintains genome-wide nucleosome regularity

Loss of SNF2h leads to enrichment of irregular fiber and long NRLs

Summary

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• SAMOSA allows us to observe single-molecule nucleosome patterns in vitro and in vivo

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• We can examine the consequences of SNF2h remodeling both in vitro and in vivo with SAMOSA

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• SAMOSA detects discrete nucleosome patterns on in vitro and in vivo mammalian chromatin, and suggest heterogenous ISWI-remodeling outcomes

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Acknowledgements

Dr. Vijay Ramani

(PI, UCSF)

Dr. Colin McNally

(Postdoctoral Fellow, Ramani Lab)

Dr. Laura Hsieh

(Postdoctoral Fellow, Narlikar Lab)

Thank you to everyone in the Ramani, Goodarzi, and Narlikar labs!

Mythili Ketavarapu

(Undergraduate Fellow, UCSB)

Dr. Siva Kasinathan

(Physician-scientist Fellow, Stanford)

Questions?