Loss of zinc-finger protein 212 leads to Purkinje cell death and locomotive abnormalities with phospholipase D3 downregulation

Rin Khang,Jee-Yin Ahn,Eunsang Kwag,Ji-Yeong Lee,Areum Jo,Jinsu Park,Jeong-Yun Choi,Joo-Ho Shin,Yun-Song Lee,Hanna Kim,Dong-Gyu Jo,Okjae Koo,Inwoo Hwang,Hark Kyun Kim,Yunjong Lee,Hojin Kang

doi:10.1038/s41598-021-02218-x

Rin Khang, Jee-Yin Ahn + Show 14 more

Open Access

https://doi.org/10.1038/s41598-021-02218-x

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Abstract

Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212–PLD3 relationship is important for Purkinje cell survival.

Highlights

Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown
We conducted immunofluorescence staining of ZNF212 in HEK293 cells and found that ZNF212 was predominantly located in the nucleus (Fig. S2a)
We validated the specificity of ZNF212 antibody in HEK293 cells transfected with Small interfering RNA (siRNA)-ZNF212 (Fig. S2b) and investigated the protein levels of Zfp[212] in various mouse organs; Zfp[212] was robustly expressed in the brain (Fig. 1b, c)

Summary

Introduction

Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp[212] and Pld[3] in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212–PLD3 relationship is important for Purkinje cell survival. The Krüppel-associated box (KRAB) domain-containing zinc-finger protein (K-ZNF) group is the largest transcription regulator family and is expressed in tetrapods[1]. Zfp[212] (a mouse homolog of human ZNF212) is highly expressed in the cerebellum (Cb). Deletion of Zfp[212] resulted in cerebellar Purkinje cell death, followed by ataxia-like movement disorders

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