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

Ichida Y, Utsunomiya Y, Yasuda T, Nakabayashi K, Sato T, Onodera M - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, 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.

No MeSH data available.


Related in: MedlinePlus

Domain structures and subcellular localization of intact, truncated, and mutated ZFP809 proteins.(A) Schematic representation of the domain structures of the intact ZFP809 protein (1), a series of truncated proteins (2–10), and a mutated ZFP809 protein with three amino acid substitutions within the KRAB domain (11), assessed for their functionalities in this study. ZF and SD denote zinc finger and spacer domain, respectively. All constructs were attached with the FLAG tag at the N-terminus and cloned into the pLVSIN/IRES/mCherry vector (S1A Fig), and referred to as pLVSIN/CMV/flag-X/ IRES/mCherry vectors (X denotes any of: intact ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB). ΔSD lacks a spacer domain of six amino acids at the C-terminus of ZFP809. (B) Confirmation of the size of the proteins expressed from the eleven vector constructs shown in Fig 1A. 293FT cells transduced with one of the eleven vector constructs were sorted based on mCherry expression. The sorted cells were subjected to Western blot analysis using anti-FLAG antibody (lane C, empty vector (pLVSIN/CMV/flag/IRES/mCherry); lanes 1 to 11, eleven vector constructs). The asterisk indicates non-specific bands. (C) Sub-nuclear localization of intact and truncated/mutated ZFP809 proteins exogenously expressed from lentiviral vectors in 293FT cells detected by immunostaining using the anit-FLAG antibody followed by confocal microscopic analysis. 293FT cells transduced with the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Anti-fibrillarin antibody was also used as a nucleolus marker. Two single-stained, merged, and DAPI-stained images are shown.
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pone.0139274.g001: Domain structures and subcellular localization of intact, truncated, and mutated ZFP809 proteins.(A) Schematic representation of the domain structures of the intact ZFP809 protein (1), a series of truncated proteins (2–10), and a mutated ZFP809 protein with three amino acid substitutions within the KRAB domain (11), assessed for their functionalities in this study. ZF and SD denote zinc finger and spacer domain, respectively. All constructs were attached with the FLAG tag at the N-terminus and cloned into the pLVSIN/IRES/mCherry vector (S1A Fig), and referred to as pLVSIN/CMV/flag-X/ IRES/mCherry vectors (X denotes any of: intact ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB). ΔSD lacks a spacer domain of six amino acids at the C-terminus of ZFP809. (B) Confirmation of the size of the proteins expressed from the eleven vector constructs shown in Fig 1A. 293FT cells transduced with one of the eleven vector constructs were sorted based on mCherry expression. The sorted cells were subjected to Western blot analysis using anti-FLAG antibody (lane C, empty vector (pLVSIN/CMV/flag/IRES/mCherry); lanes 1 to 11, eleven vector constructs). The asterisk indicates non-specific bands. (C) Sub-nuclear localization of intact and truncated/mutated ZFP809 proteins exogenously expressed from lentiviral vectors in 293FT cells detected by immunostaining using the anit-FLAG antibody followed by confocal microscopic analysis. 293FT cells transduced with the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Anti-fibrillarin antibody was also used as a nucleolus marker. Two single-stained, merged, and DAPI-stained images are shown.

Mentions: ZFP809 contains two major domains, one KRAB domain and a domain of seven zinc fingers. As a tool to identify functionally important sub-domains in ZFP809, we generated vector constructs for intact ZFP809 proteins, together with nine types of truncated forms and a mutated form using the lentiviral vector pLVSIN/CMV/IRES/mCherry (S1A Fig). All forms of proteins are tagged with FLAG at the N-terminus. Proteins are forcedly expressed under human cytomegalovirus (CMV) immediate early enhancer-promoter control in mammalian cells. A fluorescent protein, mCherry, is also expressed bicistronically under the control of the CMV promoter. The sub-domain structures of the nine types of truncated forms (referred to as ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, and ΔKRAB_A) are depicted in Fig 1A. The mutated form of ZFP809 protein, referred to as mtKRAB, has amino acid substitutions at the 13th to 15th amino acid residues of the KRAB domain (E13/14A-W15A corresponding to E16/17A-W18A of ZNF268) (Fig 1A and S2 Fig). These amino acid substitutions have been shown to abolish the interaction between KRAB-ZFPs and KAP1 [18]. We confirmed protein expression from the 11 lentiviral vector constructs for ZFP809 proteins shown in Fig 1A by transducing 293FT cells with each of the vectors, followed by sorting mCherry expressing cells and Western blot analysis (Fig 1B). We used these vector constructs to assess the functionality of truncated/mutated forms of ZFP809 proteins in terms of nuclear localization, gene silencing efficiency, and binding ability to PBS.


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

Ichida Y, Utsunomiya Y, Yasuda T, Nakabayashi K, Sato T, Onodera M - PLoS ONE (2015)

Domain structures and subcellular localization of intact, truncated, and mutated ZFP809 proteins.(A) Schematic representation of the domain structures of the intact ZFP809 protein (1), a series of truncated proteins (2–10), and a mutated ZFP809 protein with three amino acid substitutions within the KRAB domain (11), assessed for their functionalities in this study. ZF and SD denote zinc finger and spacer domain, respectively. All constructs were attached with the FLAG tag at the N-terminus and cloned into the pLVSIN/IRES/mCherry vector (S1A Fig), and referred to as pLVSIN/CMV/flag-X/ IRES/mCherry vectors (X denotes any of: intact ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB). ΔSD lacks a spacer domain of six amino acids at the C-terminus of ZFP809. (B) Confirmation of the size of the proteins expressed from the eleven vector constructs shown in Fig 1A. 293FT cells transduced with one of the eleven vector constructs were sorted based on mCherry expression. The sorted cells were subjected to Western blot analysis using anti-FLAG antibody (lane C, empty vector (pLVSIN/CMV/flag/IRES/mCherry); lanes 1 to 11, eleven vector constructs). The asterisk indicates non-specific bands. (C) Sub-nuclear localization of intact and truncated/mutated ZFP809 proteins exogenously expressed from lentiviral vectors in 293FT cells detected by immunostaining using the anit-FLAG antibody followed by confocal microscopic analysis. 293FT cells transduced with the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Anti-fibrillarin antibody was also used as a nucleolus marker. Two single-stained, merged, and DAPI-stained images are shown.
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Related In: Results  -  Collection

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pone.0139274.g001: Domain structures and subcellular localization of intact, truncated, and mutated ZFP809 proteins.(A) Schematic representation of the domain structures of the intact ZFP809 protein (1), a series of truncated proteins (2–10), and a mutated ZFP809 protein with three amino acid substitutions within the KRAB domain (11), assessed for their functionalities in this study. ZF and SD denote zinc finger and spacer domain, respectively. All constructs were attached with the FLAG tag at the N-terminus and cloned into the pLVSIN/IRES/mCherry vector (S1A Fig), and referred to as pLVSIN/CMV/flag-X/ IRES/mCherry vectors (X denotes any of: intact ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB). ΔSD lacks a spacer domain of six amino acids at the C-terminus of ZFP809. (B) Confirmation of the size of the proteins expressed from the eleven vector constructs shown in Fig 1A. 293FT cells transduced with one of the eleven vector constructs were sorted based on mCherry expression. The sorted cells were subjected to Western blot analysis using anti-FLAG antibody (lane C, empty vector (pLVSIN/CMV/flag/IRES/mCherry); lanes 1 to 11, eleven vector constructs). The asterisk indicates non-specific bands. (C) Sub-nuclear localization of intact and truncated/mutated ZFP809 proteins exogenously expressed from lentiviral vectors in 293FT cells detected by immunostaining using the anit-FLAG antibody followed by confocal microscopic analysis. 293FT cells transduced with the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Anti-fibrillarin antibody was also used as a nucleolus marker. Two single-stained, merged, and DAPI-stained images are shown.
Mentions: ZFP809 contains two major domains, one KRAB domain and a domain of seven zinc fingers. As a tool to identify functionally important sub-domains in ZFP809, we generated vector constructs for intact ZFP809 proteins, together with nine types of truncated forms and a mutated form using the lentiviral vector pLVSIN/CMV/IRES/mCherry (S1A Fig). All forms of proteins are tagged with FLAG at the N-terminus. Proteins are forcedly expressed under human cytomegalovirus (CMV) immediate early enhancer-promoter control in mammalian cells. A fluorescent protein, mCherry, is also expressed bicistronically under the control of the CMV promoter. The sub-domain structures of the nine types of truncated forms (referred to as ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, and ΔKRAB_A) are depicted in Fig 1A. The mutated form of ZFP809 protein, referred to as mtKRAB, has amino acid substitutions at the 13th to 15th amino acid residues of the KRAB domain (E13/14A-W15A corresponding to E16/17A-W18A of ZNF268) (Fig 1A and S2 Fig). These amino acid substitutions have been shown to abolish the interaction between KRAB-ZFPs and KAP1 [18]. We confirmed protein expression from the 11 lentiviral vector constructs for ZFP809 proteins shown in Fig 1A by transducing 293FT cells with each of the vectors, followed by sorting mCherry expressing cells and Western blot analysis (Fig 1B). We used these vector constructs to assess the functionality of truncated/mutated forms of ZFP809 proteins in terms of nuclear localization, gene silencing efficiency, and binding ability to PBS.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, 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.

No MeSH data available.


Related in: MedlinePlus