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. 2020 Aug 11;133(15):jcs243303. doi: 10.1242/jcs.243303

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© 2020. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Screening of proteins for CENP-B-dependent assembly on ectopic alphoid^tetO DNA using a fluorescence microscopy-based interaction-trap (FMIT) assay. (A) Experimental design of the FMIT assay. Expression plasmids of a bait or prey are co-transfected into the CENP-B KO HeLa-Int-03 cell line, which has a synthetic alphoid repeat array containing tetO and CENP-B box sequences (alphoid^tetO DNA) at an ectopic integration site. Colocalization of EYFP signal and the Halo tag-TMR ligand signal can be detected when an interaction exists between bait and prey and/or when the prey recognizes an alphoid^tetO chromatin change induced by the bait. Negative and positive example images are shown in the upper right and lower right panels, respectively. In the positive case, tetR–EYFP–CENP-B^Full recruits the Halo-tag fused to the CENP-B dimer domain (Halo–CENP-B 541–599). The yellow squares indicate the location of tetR–EYFP protein spots on the alphoid^tetO DNA site (shown magnified in inset images). Scale bars: 5 µm. (B) Schematic structure of the bait (tetR–EYFP–CENP-B) and negative control (tetR–EYFP alone) used in this figure. DBD, DNA-binding domain; DDE, DDE-superfamily endonuclease domain; Acidic:1, acidic domain 1; Acidic:2, acidic domain 2; Dimer, dimerization domain. (C) Exploration of protein assembly on the ectopic alphoid^tetO DNA integration site by CENP-B tethering. The bait and prey expression plasmids were co-transfected into the HeLa-Int-03 CENP-B KO cell line. The cells were fixed 1 d after transfection. More than 50 cells were analyzed to calculate the frequency of Halo positive EYFP-spots for each prey. The tetR–EYFP–CENP-B binds to not only tetO, but also to the CENP-B box. (D) CENP-B-independent (tetR–EYFP alone bait, blue bars) and CENP-B-dependent (tetR–EYFP–CENP-B bait, orange bars) assembly of Halo-tag fused histone modifiers, chaperones, CENPs and heterochromatin proteins was evaluated as the percentage of cells exhibiting colocalization of bait and prey on the ectopic alphoid^tetO integration site. K4me, K9me, K27me and K36me indicate H3 lysine methyl transferases. K9de, K27de and K36de indicate H3 lysine demethylases. CBX, chromobox proteins; PcG, polycomb-group proteins; HDAC, histone deacetylases; HAT, histone acetyltransferases.