The functions currently known for members of the KRAB-containing protein family include transcriptional repression of RNA polymerase I, II, and III promoters, binding and splicing of RNA, and control of nucleolus function. The functions of most of the family have not been well studied, but a few examples are as follows. The human Kid1 protein can bind to heteroduplex DNA structures and is localized to the nucleolus [10
]. Once in the nucleolus, Kid1 induces nucleolar disintegration and greatly reduces the synthesis of ribosomal RNA by RNA polymerase I, which takes place in this sub-nuclear compartment. Moreover, the KRAB domain of Kid1 is necessary for both of these phenomena, suggesting that the protein may repress transcription by RNA polymerase I. Because the number and size of the nucleolus correlates with the activity level of RNA polymerase I, its repression may contribute to the disintegration of the nucleolus. Interestingly, however, the KRAB domain of Kox1, which has the same domain structure as Kid1 and therefore belongs to the same subfamily, cannot repress transcription by RNA polymerase I in Gal4-based assays [11
]. Thus, it is likely that the KRAB domain functions differently in the full-length Kid1 protein than in a chimeric fusion protein (as used in the Gal4 assay) or that the KRAB domains of Kox1 and Kid1 behave differently at RNA polymerase I promoters. More studies are needed to differentiate between these possibilities.
In contrast to Kid-1, human Znf74 is found in discrete granular structures in the nucleus, is tightly associated with the nuclear matrix, binds to RNA, and interacts with RNA polymerase II [12
]. This KRAB-containing protein contains a truncated KRAB A domain and 12 different C2
zinc-finger motifs that are sufficient for targeting the protein to the nuclear matrix as well as for RNA binding. In addition, Znf74 interacts with the hyperphosphorylated form of RNA polymerase II and colocalizes with it in nuclear domains that are enriched in splicing factors. These findings suggest that Znf74 may regulate gene expression through both transcriptional and post-transcriptional mechanisms. KS1, which has ten zinc-finger domains and both KRAB A and B boxes, is a strong repressor of RNA polymerase activity by the Kap1-mediated mechanism described below [5
]. KS1 is also a suppressor of the neoplastic transformation that is mediated by several oncogenes [13
The biochemical functions of KRAB-containing proteins described above are thought to be critical to their cellular roles, which include cell differentiation, cell proliferation, apoptosis, and neoplastic transformation. Krim-1B, a KRAB-containing protein with nine zinc-finger motifs, antagonizes the growth regulatory properties of the oncogene product c-Myc by binding to it via the second zinc finger [14
]. The interaction between Krim-1B and c-Myc decreases the transcriptional transactivation of c-Myc that is dependent on c-Myc binding to the E-box in the promoters of its target genes. Other KRAB-containing proteins are involved in the regulation of cell proliferation. The leucine zipper and sterile-alpha motif protein kinase (ZAK) has been implicated in the regulation of cell-cycle arrest by decreasing cyclin-E expression, and a KRAB-containing protein has been shown to be associated with ZAK, playing a role in this phenomenon [15
]. The expression of the KRAB-containing protein AJ8, for instance, is developmentally regulated in embryonic tibiae and calvariae, suggesting a role in the maturation of bone cells, and the overexpression of AJ8 in osteoblastic cells represses known markers of osteoblast differentiation [16
]. Some KRAB proteins also appear to be involved in the regulation of apoptosis. Myeloid cells transfected with the cDNA of the KRAB-containing protein ZK1 are more sensitive to cell death induced by ionizing radiation than non-transfected cells [17
]. Together, these examples support a role for KRAB-containing proteins in the regulation of morphogenesis. Consequently, several laboratories, including mine, have been investigating the functional association of these proteins with pathophysiological processes. Although there has not been any definitive proof on the causal role of KRAB-containing proteins in human diseases, using gene-mapping techniques, some KRAB-containing proteins have been proposed to be candidate genes for developmental and neoplastic disorders, as well as for schizophrenia [18
]. The lack of functional evidence at this point makes this association tenuous, however. A better understanding of the molecular mechanisms underlying the functions of KRAB-containing proteins will have important biological implications.
Mechanism of function
Studies by three laboratories have identified a 100 kDa core-pressor protein for KRAB domains, known as Kap1, Tif1β, or Krip1 [20
]. Binding to a RING-B-box coiled-coil (RBCC) motif of Kap1 is an absolute requirement for KRAB-containing proteins to mediate transcriptional repression. These elegant studies [20
] demonstrated that Kap1 binds to KRAB domains as an oligomer, functioning as a scaffold to recruit heterochromatin protein 1 isoforms (HP1α, HP1β, and HPlγ), histone deacetylases (HDACs), and Setdb1, a novel SET-domain protein that methylates lysine 9 of histone h3. Interestingly, HP1 proteins bind to Lys9-methylated histone h3 in order to condense chromatin [23
]. Together, these findings have recently led to the proposal of the model shown in Figure [27
]. The model predicts that KRAB-containing proteins bind to their corresponding DNA sequence, triggering the recruitment of Kap1; subsequently, Kap1 forms a scaffold containing HP1, Setdb1, and an HDAC, and silences gene expression by forming a facultative heterochromatin environment on a target promoter. This model would suggest a KRAB-mediated stepwise assembly of a powerful corepressor complex. Further examination is needed, however, of whether the complex is instead preformed and then recruited by a KRAB-domain on particular promoter. Also, as these proteins can all be regulated by post-transla-tional modifications, it is not clear whether the corepressor complexes predicted by the model always contain Kap1, HP1, and SETDB1. Despite these questions, the building of this model is one of the most significant steps forward in this field of research.
Figure 3 A current model for the complex formed by KRAB-containing proteins and other proteins. A KRAB-containing protein binds specifically to a gene promoter through its multiple zinc fingers. A trimeric Kap1 complex binds to the KRAB domain of the KRAB-containing (more ...)