PLoS One. 2015 Sep 29;10(9):e0139274. doi: 10.1371/journal.pone.0139274.

Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing.

Ichida Y1, Utsunomiya Y1, Yasuda T1, Nakabayashi K2, Sato T3, Onodera M1.
  • 1Department of Human Genetics, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan.
  • 2Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan.
  • 3Department of Biosciences and Informatics, Keio University, Yokohama, Kanagawa, 223-8522, Japan.

 

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.

PMID: 26417948

 

Supplements

Zinc finger protein 809 (ZFP809) is a member of the kruppel-associated box-containing ZFPs (KRAB–ZFPs), which represent the largest single family of transcription factors in mammals[1]. ZFP809 has been shown to inhibit the transcriptional expression of Moloney murine leukemia virus (MoMLV) through binding to the MLV-derived primer binding site (PBS) located downstream of the long terminal repeat (LTR) and interacting with KRAB-associated protein 1 (KAP1)[1, 2]. Furthermore, previous studies have demonstrated that the ZFP809/KAP1 complex recruits heterochromatin protein 1 (HP1), an ERG-associated protein with a SET domain (ESET, a H3K9 methyltransferase), the nucleosome remodeling and deacetylation complex including histone deacetylation 1 (HDAC1), and DNA methyltransferase 3A (DNMT3A). This protein complex represses the activity of the LTR by inducing epigenetic modifications at the LTR, such as repressive histone modifications and de novo DNA methylation [3–6]. ZFP809 also is required for the silencing of endogenous retroviruses (ERVs) during mouse embryonic development [7, 8].

Furthermore, ZFP809 contains a KRAB domain at the N-terminus and seven zinc fingers at the C-terminus. It is highly expressed in immature murine cells, including embryonic stem cells (ESCs) and embryonic carcinoma cells [2, 7]. Previous studies on KRAB–ZFPs have indicated that the KRAB domain interacts with KAP1, and zinc fingers bind to target sequences and interact with proteins other than KAP1 [1]. Therefore, we hypothesized that domains within ZFP809 have different functions, including subcellular localization, ability to silence transgene expression driven by the MLV–LTR, and 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[9, 10]. We revealed that the (1) KRAB A box domain and the first and second zinc fingers are required for the proper nuclear localization of ZFP809, (2) KRAB domain and the first to fifth zinc fingers are required for silencing of transgene expression driven by the MLV–LTR, (3) ZFP809 binds to MLV PBS through the third to fifth zinc fingers and cooperative roles of other zinc fingers may contribute to stable protein expression (Figure 1). 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.

Moreover, we found that ZFP809 has six linkers between zinc fingers, three of which contain the T(S)GEKP sequence, which are conserved among C2H2 zinc fingers [11–13]. Previous studies have demonstrated that the phosphorylation of T(S)GEKP sequences leads to the inactivation of zinc finger binding to target DNA [11–13]. Therefore, we focused on the linker between zinc fingers and revealed that these linkers affect the ability of ZFP809 to prime gene silencing and the precise nuclear localization of ZFP809 [14].

 

 

fig 1

Figure 1. Functional domains within ZFP809. ZFP809 has a KRAB domain at the N-terminus and seven zinc fingers at the C-terminus. The KRAB domain is required for nuclear localization (except the nucleolus), gene silencing, and binding to the MLV–PBS. The first and second zinc fingers are required for the proper nuclear localization and the third, fourth, and fifth zinc fingers bind the MLV–PBS. In this figure, “A” indicates KRAB_A box, “B” indicates KRAB_B box, and “ZF” indicates zinc finger.

 

References

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  14. Y. Ichida, Y. Utsunomiya, M. Onodera (2016) Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization.    Biochem. Biophy. Res. Commun, Available online 12 February 2016.

 

 

 

 

 

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