Fig. 1.

System to investigate the interplay between CTCFL and CTCF in somatic cells. a Schematic representation of the similarities and differences between CTCF and CTCFL. Figure adapted from Marshall et al. [34]. The DNA binding domain of both proteins is composed of 11 zinc fingers. ZFs 1–10 and ZF11 belong to the C₂H₂ and C₂HC class of ZFs, respectively. Shared and different amino acids in CTCF and CTCFL are shown in green and yellow, respectively. Blue circles indicate zinc ions. Histidines and cysteines that form coordinate bonds with zinc are marked. b Scheme of genetic modifications in the Ctcf locus and the doxycycline-inducible transgenic Ctcfl or Ctcf knocked-in at the Tigre locus. The endogenous Ctcf contains the auxin-inducible degron (AID) and the eGFP tag on both alleles. Both Ctcf and Ctcfl transgenes harbor an N terminal 3× FLAG tag and C terminal mRUBY2 as well as TetO-3G element and rtTA3G for doxycycline-induced expression. c Experimental strategy for expression of dox-inducible CTCF/CTCFL transgenes in the presence and absence of CTCF using the auxin-inducible degron system. Addition of indole-3-acetic acid (IAA), a chemical analog of auxin, leads to transient and reversible degradation of CTCF, while addition of doxycycline (Dox) leads to induction and expression of the respective transgene. The four conditions used in our analysis are as follows: U, untreated cells; I, IAA treated for CTCF depletion; D, Dox induced expression of transgenic CTCF/CTCFL; ID, IAA plus Dox treated for depletion of endogenous CTCF and induction of transgene expression. d Western blot using FLAG antibody shows that the level of expression of transgenes are comparable across the cell types (CTCF and CTCFL in D and ID conditions). CTCF has a predicted molecular weight of 84 kDa and CTCFL, 74 kDa. However, CTCF is known to migrate as a 130-kDa protein [39]. Since the transgenes are expressed as fusion proteins with FLAG tag and mRuby2, which together adds another 29 kDa, the resulting proteins migrate at 159 and 103 kDa, respectively. e Western blot with CTCF antibody shows the presence of endogenous and transgenic CTCF. Histone H3 serves as a loading control in d and e. “M” is the molecular weight ladder and the molecular weights are marked. f, g Flow cytometry and microscopy confirmed that the level of mRuby2 expression of transgenic CTCF and CTCFL are comparable