Defining CTCF physiological roles: rules of the "CTCF code"

Monday, September 22, 2014 — Poster Session II
4:00 p.m. – 6:00 p.m.	FAES Academic Center	NIAMS	GEN-16

* FARE Award Winner

Authors

• L. Vian
• H. Nakahashi
• W. Resch
• K.R. Kieffer Kwon
• A. Oksanen
• M. Dose
• N. Pruett
• S. Nelson
• J. Qian
• W. Dubois
• R.D. Phair
• R. Casellas

Abstract

CTCF is an 11-zinc-finger DNA binding protein implicated in nuclear architecture and gene regulation. However, its precise role in development is unknown because CTCF deletion leads to embryonic lethality. In the mammalian genome, there are ~50,000 CTCF binding sites, often displaying sequence variability. By ectopically expressing ZF mutants in activated B cells, we showed that CTCF uses different ZF combinations to recognize diverse motifs. For instance, ZFs9-11 are required for CTCF to binding a particular 20-bp upstream sequence. As this motif is present in a relatively small fraction of CTCF binding sites, the overall genomic profile of CTCF is unaffected. This observation raised the possibility to develop partial CTCF knockouts, defective in CTCF activity at specific loci without compromising cell viability. To this end we have used TALEN and CRISPR-Cas9 genome editing technology to generate ZF mutants in B cell lines. Following our prediction, ZF11 mutants were viable whereas targeting of ZF3, which is required for CTCF binding to most sites, led to lethality. Thus, we begun a ZF11 mutants comprehensive deep-sequencing (including polymeraseII, cohesin recruitment) and transcriptome analysis. Preliminary data shows localized transcriptional defects in ZF11 mutants. Results validation and its implications vis-à-vis CTCF nuclear architecture will be discussed.

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