KRAB and zinc fingers function cooperatively for the precise nucleoplasmic, but not nucleolar, localization of KRAB-ZFPs.

<p>A, Analysis of subcellular localization of various ZNF268 mutants. B, Analysis of the endogenous subcellular localization of 116 human KRAB-ZFP genes. The subcellular localization data of 116 human KRAB-ZFP genes were retrieved from the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>), and the percentages of their subcellular localization were calculated based on the following rules. First of all, the localization pattern of these genes was analyzed based on their distribution in cytoplasm and nucleus. Then the genes that showed presence in the nucleus were further analyzed based on their distribution in cytoplasm and nucleolus. Specifically, they were classified into the following four groups: exclusively whole nucleus; whole nucleus and cytoplasm; nucleus but not nucleolus; nucleus but not nucleolus and cytoplasm. C, Subcellular localization of ZNF300 and KOX1 and their KRAB domains. KOX1 and ZNF300 genes were FLAG-tagged and transfected into HeLa cells. Control is the cells transfected with empty vector. The FLAG-tagged proteins were labeled with FITC. The white arrows indicate the nucleolus region with negative DAPI staining in the nucleus.</p

Both zinc fingers and KRAB domains are necessary for nuclear localization of ZNF268a.

<p>A and C, Schematic representation of ZNF268a and the truncation constructs. Numbers in parentheses indicate the number of zinc fingers the mutants contain. B and D, All the constructs were fused to GFP and transfected into HeLa cells and subjected to confocal analysis 24; N/C, nucleus and cytoplasm.</p

KRAB and zinc fingers of ZNF268 contain nuclear localization domains with different subnuclear localization.

<p>A, Schematic representation of ZNF268 structure. ZNF268 was divided into 9 regions. UD, unknown domain; SD, spacer domain; figures indicate each of the zinc fingers. The subcellular distribution of each region is indicated. N, nucleus; N/C, nucleus and cytoplasm; ZF, zinc fingers; numbers in parentheses near ZF indicate the numbers of zinc fingers the mutants contain. B, Subcellular localization of the nine regions of ZNF268. Each region of ZNF268 was fused to GFP and transfected into HeLa cells. Twenty-four hours later, confocal fluorescence analysis was performed; C, Different subnuclear distribution of zinc fingers and KRAB. Each region of ZNF268 fused to GFP was transfected into HeLa cells, together with FLAG-tagged ZNF268 KRAB. Twenty-four hours later, cells were subjected to confocal analysis. KRAB was labeled with TRITC. The white arrows indicate the nucleolus region with negative DAPI staining in the nucleus.</p

The effect of the KRAB mutation (E16/17A-W18A and D8A/V9A) on nuclear localization of ZNF268a and the truncated mutants.

<p>A, Schematic representation of a(1–4), a(1–8), a(1–16), and a-GFP mutations at the indicated site of the KRAB domain (E16/17A-W18A). B, The above constructs were transfected into HeLa cells for confocal analysis. The subcellular distribution of each construct is indicated in panel A. N, nucleus; N/C, nucleus and cytoplasm; C, Schematic representation of a(1–4), a(1–8), a(1–16), and a-GFP mutation at the indicated site of the KRAB domain (D8A/V9A); D, The above constructs were transfected into HeLa cells for confocal analysis.</p

Zinc Fingers Function Cooperatively with KRAB Domain for Nuclear Localization of KRAB-Containing Zinc Finger Proteins

<div><p>Multiple nuclear localization domains have been identified in nuclear proteins, and they finely control nuclear import and functions of those proteins. ZNF268 is a typical KRAB-containing zinc finger protein (KRAB-ZFP), and previous studies have shown that the KRAB domain reinforces nuclear localization of KRAB-ZFPs by interacting with KAP1. In this study, we find that some of 24 zinc fingers of ZNF268 also possess nuclear localization activity. Results of mutagenesis studies suggest that KRAB and zinc fingers are both necessary, and they function both independently and cooperatively for the nuclear localization of ZNF268. However, the subnuclear targeting activities of KRAB and zinc fingers are different. KRAB targets proteins in nucleoplasm, but not in the nucleolus, which is mediated by interaction with KAP1, while zinc fingers target proteins in the whole nucleus uniformly. The cooperative activities of KAP1-KRAB-zinc fingers result in the precise nucleoplasmic, but not nucleolar localization of KRAB-ZFPs. Our studies reveal a novel mechanism for the subcellular localization of KRAB-ZFPs and may help us to further explore their biological functions.</p></div

KRAB-KAP1 interaction mediates the nucleoplasmic localization of KRAB-ZFPs.

<p>A, Colocalization of KRAB and KAP1 in the nucleoplasm, but not nucleolus. HeLa cells were transfected with the indicated GFP expression plasmids. Twenty-four hours later, cells were incubated with anti-KAP1 antibody and subjected to labeling with TRITC conjugated secondary antibody for confocal analysis. B, The subcellular localization of ZNF268 KRAB mutants. Plasmids expressing wild-type (WT) or mutant ZNF268 KRAB-GFP (E16/17A-W18A) together with control (left panel) or FLAG-KAP overexpressing plasmid (right panel) were transfected into HeLa cells subjected to confocal analysis 24 hours later. The white arrows indicate the nucleolus region with negative DAPI staining in the nucleus. C, Model for subcellular localization of KRAB-ZFPs. The KRAB domain and Zinc finger domain both contain the NLS and their cooperative functions contribute to the precise nucleoplasmic, but not nucleolar localization.</p