1

Probing the mechanism and function of 3D genome

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• TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

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• Exploring the relationship between TADs architecture and SHM susceptibility in human B cells

Yubao Cheng

Siyuan Wang’s Lab

Yale University

2

Higher-order chromosome organization

Ezgi Süheyla Doğan et al., Nature Plants, 2018

Tom Misteli, Cell, 2007

David U.Gorkin et al., Cell Stem Cell, 2014

Michael Levine et al., Cell, 2014

Job Dekker et al., Cell, 2016

Wendy A. Bickmore et al., Cell, 2013

Thomas Cremer et al., Cold Spring Harb Perspect Biol, 2010

Giancarlo Bonora et al., Curr Opin Genet Dev, 2014

3

Topologically associating domains (TADs)

Jesse R. Dixon et al., Nature, 2012

Elphège P. Nora et al., Nature, 2012

Tom Sexton et al., Cell, 2012

Chunhui Hou et al., Molecular Cell, 2012

Suhas S.P. Rao et al., Cell, 2014

Adrian L. Sanborn et al., PNAS, 2015

Geoffrey Fudenberg et al., Cell Reports, 2016

Suhas S.P.Rao., Cell, 2017

Wibke Schwarzer et al., Nature, 2017

4

Bogdan Bintu et al., Science, 2018

Takashi Nagano et al., Nature, 2013

Ilya M. Flyamer et al., Nature, 2017

Tim J Stevens et al., Nature, 2017

Takashi Nagano et al., Nature, 2017

Longzhi Tan et al., Science, 2018

Highly variant TAD-like structures (single-cell domains)

• Loop extrusion by cohesin is dispensable
• Epigenetics or transcription ?
• Xi and Xa show distinct epigenetic states;

Xi is largely devoid of cohesin binding

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Do single-cell domains exist on both Xi and Xa?

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Chromatin tracing on X chromosomes across two TADs

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TADs attenuate on Xi and exist on Xa

Anand Minajigi et al., Science, 2015

Xinxian Deng et al., Genome Biology, 2015

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Single-cell domains are present on both Xi and Xa

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Single-cell domains showed different distributions on Xi and Xa, but with similar strengths

Boundary probabilities

Boundary strengths

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“Fbn2 loop is both rare and dynamic, with a looped fraction of ~3-6.5% and a median loop lifetime of ~10-30 min.”

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“Fbn2 loop is both rare and dynamic, with a looped fraction of ~3-6.5% and a median loop lifetime of ~10-30 min.”

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Do epigenetic modifications or transcription contribute to single-cell domain formation?

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TSA+NaBu

(histone acetylation ↑)

DMOG

(histone methylation ↑)

GSK126

(H3K27me3 ↓)

α-amanitin

(transcription ↓)

Single-cell domains persist under perturbations

ZdCyd

(DNA methylation ↓)

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Boundary frequencies and strengths of the single-cell domains are similar after treatment

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Chromatin traces from different conditions are highly dispersed among the clusters

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Does Xi compaction happen at single TAD level?

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Xi compaction presents at the TAD-to-chromosome scale, but not at the TAD/sub-TAD scale

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Summary

• Both Xi and Xa contain single-cell domains with similar frequency and strength

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• Single-cell domains are resistant to tested epigenetic and transcription perturbations

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• Xi compaction presents at the TAD-to-chromosome scale

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What are the functions of TADs?

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• Transcription regulation
• DNA replication-timing
• Somatic hypermutation (SHM) ?

Benjamin D. Pope et al., Nature, 2014

Malte Spielmann, et al. Nature Review, 2018

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Generation of the diversity of the immunoglobulin repertoire

https://www.anaptysbio.com/technology/

Somatic hypermutation (SHM)

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Genome-wide SHM susceptibility mapping

Filip Senigl, et al. 2019, Cell Reports

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What mechanism is underlying hot vs. cold TADs?

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statistical significance of enrichment

Filip Senigl, et al. 2019, Cell Reports

Genome-wide SHM susceptibility mapping

Super-enhancer

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statistical significance of enrichment

Filip Senigl, et al. 2019, Cell Reports

Genome-wide SHM susceptibility mapping

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How is the genome spatially organized across multiple length scales as germinal center B cells develop and activate SHM?

How does 3D genome architecture influence the targeting of SHM in the normal and oncogenic contexts?

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https://www.nyuoralcancer.org/oral_cancer/oral_cancer.html

Zlatko Dembic, in The Cytokines of the Immune System, 2015

Stebegg … Graca. Front. Immunol. 2018

SHM takes place in germinal center

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Multiplexed Imaging of Nucleome Architectures (MINA)

RNA MERFISH

Chromatin tracing/ DNA MERFISH

Miao Liu, et al. 2020, Nature Communications

Siyuan Wang, et al. 2016, Science

Kok Hao Chen, et al. 2015, Science

IF

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Tonsil MERFISH identified different cell types

1440 um

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CD35 IF

100 um

Tonsil MERFISH identified different cell types

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New MERFISH library design

Data from:�HAMISH W. KING at al., �SCIENCE IMMUNOLOGY, 2021

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Chromatin tracing showed consistent results

ChrX, 30kb scale

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Chromatin tracing library design

50 consecutive 10 kb regions,

200 oligos for each region

BCL6 TAD

FDG4 TAD

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Chromatin tracing row data

FISH Round 1 (z projection)

DAPI

10 um

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Future plans

• Analysis the chromatin folding of hot TAD (BCL6) and cold TAD (FGD4) in different cell types in tonsil (10kb scale parallel chromatin tracing)

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• Analysis the “nuclear organization properties” of TADs in tonsil (chromatin scale tracing or DNA MERFISH)
• Clustering and phase separation
• Association with nuclear lamina, nucleoli, nuclear pores

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• Study the association between SHM susceptibility and chromatin folding in different B cell lymphomas (combine with genetic perturbation)

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Wang Lab:

Dr. Siyuan Wang

Miao Liu

Mengwei Hu

Bing Yang

Yanbo Chen

Jonathan Radda

Shengyan Jin

Tyler Jensen

Morley Willoughby

Ruihuan Yu

David’s lab:

Dr. David Schatz

Jianshu Wang

Filip Senigl

Huseyin Saribasak

Anurupa Devi Yadavalli

Jukka Alinikula

​

Dr. Ann Haberman

Acknowledgement

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Thank you!