Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing

Abstract

Zinc finger protein 809 (ZFP809) is a member of the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family, and is highly expressed in mouse immature cells. ZFP809 is known to inhibit the expression of transduced genes driven by Moloney murine leukemia virus (MoMLV)-typed retroviral vectors by binding to the primer binding site (PBS) located downstream of the MLV-long terminal repeat (LTR) of the vectors and recruiting protein complexes that introduce epigenetic silencing marks such as histone modifications and DNA methylation at the MLV-LTR. However, it remains undetermined what domains of ZFP809 among the KRAB domain at N-terminus and the seven zinc fingers are critical for gene silencing. In this study, we assessed subcellular localization, gene silencing ability, and binding ability to the PBS of a series of truncated and mutated ZFP809 proteins. We revealed the essential role of the KRAB A box for all functions assessed, together with the accessory roles of a subset of zinc fingers. Our data also suggest that interaction between KAP1 and the KRAB A box of ZFP809 is critical in KAP1-dependent control of gene silencing for ZFP809 targets.

Functional and Structural Features of Zinc Finger Protein 809

“…It has been shown to inhibit the transcriptional expression of MoMLV and is required for the silencing of endogenous retroviruses (ERVs) during mouse embryonic development [1,11]. Furthermore, domains with diverse functions have been shown within ZFP809 [12,13]. Here we discuss the functions and features of functional domains within ZFP809 in light of the findings of recent studies.…”

“…binding to the PBS. We investigated subcellular localization, gene silencing ability, and binding ability to the MLV-PBS using a series of truncated and mutated ZFP809 proteins [12,13]. We revealed that the KRAB domain is required for nuclear localization and silencing of transgene expression driven by the MLV-LTR and bind to the MLV-PBS in conjunction with the zinc fingers (Figure 1B) [12,13].…”

“…We investigated subcellular localization, gene silencing ability, and binding ability to the MLV-PBS using a series of truncated and mutated ZFP809 proteins [12,13]. We revealed that the KRAB domain is required for nuclear localization and silencing of transgene expression driven by the MLV-LTR and bind to the MLV-PBS in conjunction with the zinc fingers (Figure 1B) [12,13]. Although previous studies have indicated that zinc fingers within KRAB-ZFPs bind to specific DNA sequences and directly interact with three-nucleotide sequences [3,10], our results demonstrated that individual zinc fingers, particularly the third, fourth, and fifth zinc fingers, are required for the binding of ZFP809 to the MLV-PBS and that other zinc fingers contribute to stable protein expression [12,13] (Figure 1B).…”

“…We revealed that the KRAB domain is required for nuclear localization and silencing of transgene expression driven by the MLV-LTR and bind to the MLV-PBS in conjunction with the zinc fingers (Figure 1B) [12,13]. Although previous studies have indicated that zinc fingers within KRAB-ZFPs bind to specific DNA sequences and directly interact with three-nucleotide sequences [3,10], our results demonstrated that individual zinc fingers, particularly the third, fourth, and fifth zinc fingers, are required for the binding of ZFP809 to the MLV-PBS and that other zinc fingers contribute to stable protein expression [12,13] (Figure 1B). According to previous studies, ZFP809 interacts with regions within the MLV-PBS (underlined sequence) and does not recognize threenucleotide sequences, indicating that not all zinc fingers are necessarily involved in target DNA binding [21][22][23][24].…”

ZNF224 Protein: Multifaceted Functions Based on Its Molecular Partners

“…Interestingly, a novel function of the KRAB domain has been recently highlighted. Indeed, published data showed that the zinc finger domains, while possessing NLSs [ 39 , 40 , 41 ], cooperate with the KRAB domain for the nuclear localization of KRAB-ZFPs [ 42 , 43 , 44 ]. In detail, in their elegant papers, Wang and colleagues used mutagenesis studies and confocal microscopy analysis to show that the KRAB domain and zinc fingers collaborate for nuclear localization, but they also display different subnuclear targeting activities.…”