53BP1 regulates heterochromatin through liquid phase separation.

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin protein HP1α in a mutually dependent manner. Deletion of 53BP1 results in a reduction in heterochromatin centers and the de-repression of heterochromatic tandem repetitive DNA. We identify domains and residues of 53BP1 required for its LLPS, which overlap with, but are distinct from, those involved in DSB repair. Further, 53BP1 mutants deficient in DSB repair, but proficient in LLPS, rescue heterochromatin de-repression and protect cells from stress-induced DNA damage and senescence. Our study suggests that in addition to DSB repair modulation, 53BP1 contributes to the maintenance of heterochromatin integrity and genome stability through LLPS.

Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation.

The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates disease pathogenesis. Here, we show that upon influenza virus infection, the H4K20me3 methyltransferase Suv4-20h2 binds the viral protein NP, which results in the inactivation of Suv4-20h2 and the dissociation of cohesin from Suv4-20h2. Inactivation of Suv4-20h2 by viral infection or genetic deletion allows the formation of an active chromatin loop at the HoxC8-HoxC6 loci coincident with cohesin loading. HoxC8 and HoxC6 proteins in turn enhance viral replication by inhibiting the Wnt-ß-catenin mediated interferon response. Importantly, loss of Suv4-20h2 augments the pathology of influenza infection in vivo. Thus, Suv4-20h2 acts as a safeguard against influenza virus infection by suppressing cohesin-mediated loop formation.

Pulmonary phagocyte-derived NPY controls the pathology of severe influenza virus infection.

Crosstalk between the autonomic nervous system and the immune system by means of the sympathetic and parasympathetic pathways is a critical process in host defence. Activation of the sympathetic nervous system results in the release of catecholamines as well as neuropeptide Y (NPY). Here, we investigated whether phagocytes are capable of the de novo production of NPY, as has been described for catecholamines. We show that the synthesis of NPY and its Y1 receptor (Y1R) is increased in phagocytes in lungs following severe influenza virus infection. The genetic deletion of Npy or Y1r specifically in phagocytes greatly improves the pathology of severe influenza virus infection, which is characterized by excessive virus replication and pulmonary inflammation. Mechanistically, it is the induction of suppressor of cytokine signalling 3 (SOCS3) via NPY-Y1R activation that is responsible for impaired antiviral response and promoting pro-inflammatory cytokine production, thereby enhancing the pathology of influenza virus infection. Thus, direct regulation of the NPY-Y1R-SOCS3 pathway on phagocytes may act as a fine-tuner of an innate immune response to virus infection, which could be a therapeutic target for lethal influenza virus infection.

Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization.

Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers.

Long time-course monitoring of ZFP809-mediated gene silencing in transgene expression driven by promoters containing MLV-derived PBS.

Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.

The third to fifth zinc fingers play an essential role in the binding of ZFP809 to the MLV-derived PBS.

Members of the kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family mediate a number of cellular processes through binding to target DNA sequences via zinc fingers. Generally, zinc fingers recognize three-nucleotide sequences; however, this rule is not universally applicable. Zinc finger protein 809 (ZFP809) belongs to the KRAB-ZFP family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV) via sequence-specific binding to the primer-binding site (PBS) located downstream of the MoMLV-long terminal repeat (LTR) and the induction of epigenetic modifications at LTR, such as repressive histone modifications and de novo DNA methylation. Previously, we demonstrated the role of the first to fifth zinc fingers of ZFP809 in binding to MLV PBS, indicating these zinc fingers do not recognize MLV PBS as a three-nucleotide sequence. Therefore, in the present study, we constructed truncated and mutated zinc fingers and examined their ability to bind to MLV PBS. The third to fifth zinc fingers of ZFP809 were found to be essential for binding to MLV PBS. Furthermore, the results of the present study indicate that other zinc fingers, which were not directly involved in binding to MLV PBS, may function in potentiating binding and stable protein expression. Further characterization of the amino acid sequences of zinc fingers will help further elucidate the functions and features of KRAB-ZFP and other zinc finger proteins.

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

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.