Functional Study On A Novel KRAB Zinc Finger Protein ZNF333

Transcription repressors of the KRAB-zinc finger protein family are important in development and differentiation. We identified a new KRAB zinc finger protein, ZNF333, in the critical region of a polysyndactyly locus. Highly homologous duplicated KRAB domains have been identified on the N-terminal in ZNF333 and typical zinc finger domain on the C-terminal. ZNF333 has several phosphorylation sites. There are two isoforms of ZNF333, ZNF333-I and ZNF333-II through alternative splicing. ZNF333-I has highly homologous duplicated KRAB domains and zinc finger domain, while ZNF333-II only has one KRAB domain without zinc finger domain.Indirect immunofluorescence displayed ZNF333-I and ZNF333-II localizing in nucleus and the localization is independent from DNA binding domain. ZNF333-I localized primarily within 30-50 clustered subnuclear structure, producing a speckled appearance with a faint diffuse background in the entire nucleus, with the exception of the nucleoli. ZNF333-II, the short splicing variant, shows 3-6 patches that may be nucleoli. When coexpressed ZNF333-I and ZNF333-II, ZNF333-I altered the localization and subnuclear clusters, colocalized with short splicing variant.We examined the effect of a series of amino-terminal deletions constructs on the transcriptional activity of ZNF333 using mammalian monohybrid system in HEK293 cell line. KRAB-2 domain showed strong transcriptional repression activity, while KRAB-1 domain and two spacers didn't show this activity. Furthermore, substitution mutations at the highly conserved amino acids in the KRAB-2A domain ( DV to AA ) disrupted its repression activity, indicating that KRAB-2A is responsible for the repression activity.To determine whether the transcriptional activity by ZNF333 was limited to HEK293, we compared the transrepressing activity of ZNF333 in several different cell lines using monohybrid analysis. The KRAB 2A domain is required and sufficient for transcription repression in HEK293, Hela and K562 cell lines. However, in Jurkat cells, both the KRAB 1A and KRAB 2A domains are strongly active repressors, whereas in the HepG2 cells neither domain show significant activities. Thus, the two KRAB domains of the same ZNF protein, regulated by alternative pre-mRNA splicing, have cell-specific activities in transcriptional repression. The differences in the degree of transrepression among cell lines may be related to different levels of expression of co-repressors which interact with ZNF333.