Abstract
The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.
Highlights
Kruppel-type C2H2 zinc finger (ZNF) proteins with a Nterminal Kruppel-associated box (KRAB) form the largest family of potential transcription factors encoded in the human genome with about 400 members [1,2]
When we controlled the KRAB-AB domain of XFIN which we obtained after RT-PCR from Xenopus laevis larval stage 59 RNA by sequencing, we noticed an additional deoxycytidine insertion in all clones
Functional assays in frog and human cell lines demonstrated that XFIN KRAB-AB behaves like a bona fide KRAB domain, i.e. XFIN KRAB is sufficient to confer transcriptional repression when targeted to the promoter of a reporter cassette and is able to interact with the TRIM28 co-repressor protein
Summary
Kruppel-type C2H2 zinc finger (ZNF) proteins with a Nterminal Kruppel-associated box (KRAB) form the largest family of potential transcription factors encoded in the human genome with about 400 members [1,2]. The KRAB domain may have evolved from an ancestor of the histone H3 methyltransferase PRDM9/Meisetz which is a ZNF protein. This protein appears to be the oldest gene with a KRAB-like domain since an ortholog was discovered in the sea urchin genome based on sequence similarities [8]. PRDM9 plays a prominent role in meiotic recombination and in speciation (reviewed in [9]) Based on the latter and taking into consideration data on KRABZNF evolution, it has been postulated that this transcription factor family might in general be important for speciation [10]
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