Abstract
The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.
Highlights
The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2zinc finger clusters of four and nine zinc fingers at the COOH terminus
Distribution of the Full-length Kid-1 and KRIP-1 Proteins in the Nucleus—In order to determine the subcellular site of Kid-1 expression, COS-7 cells were transiently transfected with a plasmid encoding a full-length Kid-1 protein with an HA epitope tag at the NH2 terminus. 2–3 days after the transfection, cells were stained with the anti-HA epitope antibody 12CA5 and a fluorescein isothiocyanate- or Cy3-coupled secondary antibody
Using a human autoantibody against the nucleolar transcription factor upstream binding factor (UBF), a clear colocalization of Kid-1 and UBF in patches was detected, demonstrating that the full-length Kid-1 protein is sorted to the nucleolus
Summary
The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. The Kid-1 protein (kidney, ischemia, development) contains 13 Cys2His2zinc fingers at its COOH terminus and KRAB-A and -B domains at its NH2 terminus; it was the first KRAB-zinc finger protein for which a transcriptional repressor activity could be demonstrated [15]. We present a careful analysis of the distribution of the Kid-1 protein demonstrating that expression of Kid-1 leads to the disruption of the nucleolus
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