Experimental Strategies to Manipulate the Cellular Levels of the Multifunctional Factor CTCF

Abstract

Cellular homeostasis is the result of an intricate and coordinated combinatorial of biochemical and molecular processes. Among them is the control of gene expression in the context of the chromatin structure which is central for cell survival. Interdependent action of transcription factors, cofactors, chromatin remodeling activities, and three-dimensional organization of the genome are responsible to reach exquisite levels of gene expression. Among such transcription factors there is a subset of highly specialized nuclear factors with features resembling master regulators with a large variety of functions. This is turning to be the case of the multifunctional nuclear factor CCCTC-binding protein (CTCF) which is involved in gene regulation, chromatin organization, and three-dimensional conformation of the genome inside the cell nucleus. Technically its study has turned to be challenging, in particular its posttranscriptional interference by small interference RNAs. Here we describe three main strategies to downregulate the overall abundance of CTCF in culture cell lines.

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization

“…Yet, genome-wide assays after RNAi revealed only very limited consequences, with CTCF depletion leading to slightly reduced intra-TAD chromosomal contacts, slightly increased inter-TAD contacts and modest transcriptional changes with no clear link to folding defects (Zuin et al, 2014). Genetic manipulation of CTCF has proven difficult as it is essential for development (Moore et al, 2012; Sleutels et al, 2012; Soshnikova et al, 2010; Wan et al, 2008) and proliferation of cultured cells (González-Buendía et al, 2014), hampering the understanding of the exact role of CTCF in mammalian chromosome folding and genome functions. It is currently unclear to what extent CTCF is actually required for chromatin architecture and which levels of genome organization this factor controls.…”

A negatively-charged region within carboxy-terminal domain maintains proper CTCF DNA binding

“…44 In addition, the CTD region is highly conserved in vertebrates (Figure 1E). Owning to the lethality of CTCF deletion in mice or in cultured cells, 45,46 we tried to delete the CTCF NTD or CTD and found that deletion of NTD, but not CTD, is lethal in cultured cells. Specifically, we screened 254 single-cell clones for deletion of NTD, and could not find a single homozygous cell clone.…”