Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging

Bo Gu; Colin J Comerci; Dannielle G McCarthy; Saumya Saurabh; W E Moerner; Joanna Wysocka

doi:10.1016/j.molcel.2020.10.001

Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging

Mol Cell. 2020 Nov 19;80(4):699-711.e7. doi: 10.1016/j.molcel.2020.10.001. Epub 2020 Oct 21.

Authors

Bo Gu¹, Colin J Comerci², Dannielle G McCarthy³, Saumya Saurabh⁴, W E Moerner⁵, Joanna Wysocka⁶

Affiliations

¹ Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
² Department of Chemistry, Stanford University, Stanford, CA, USA; Biophysics Program, Stanford University, Stanford, CA, USA.
³ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁴ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Department of Chemistry, Stanford University, Stanford, CA, USA; Biophysics Program, Stanford University, Stanford, CA, USA. Electronic address: wmoerner@stanford.edu.
⁶ Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA; Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: wysocka@stanford.edu.

Abstract

CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.

Keywords: CTCF; Pol II; chromatin; cohesin; quantitative analysis; super-resolution imaging; topological associated domain; transcription.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
CCCTC-Binding Factor / genetics
CCCTC-Binding Factor / metabolism*
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
Cells, Cultured
Chromatin / genetics
Chromatin / metabolism*
Chromosomal Proteins, Non-Histone / genetics
Chromosomal Proteins, Non-Histone / metabolism*
Chromosomes, Mammalian
Cohesins
Embryonic Stem Cells / cytology*
Embryonic Stem Cells / metabolism
Genetic Loci
Genome
Image Processing, Computer-Assisted
Mice
Microscopy, Fluorescence / methods*
Proteins / genetics
Proteins / metabolism*
Transcription, Genetic*

Substances

CCCTC-Binding Factor
Cell Cycle Proteins
Chromatin
Chromosomal Proteins, Non-Histone
Ctcf protein, mouse
Proteins
WAPL protein, mouse

Abstract

Publication types

MeSH terms

Substances

Grants and funding