*The Department of Cell and Molecular Biology, Uppsala University;
The Biomedical Center, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences;
Medical Products Agency, Uppsala
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Abstract |
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Introduction |
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Materials and Methods |
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Results and Discussion |
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Notably, a few KRAB zinc finger genes, which appear in the KRAB A + b and the KRAB A + B families in the distance tree, seem to lack a KRAB b (ZNF222, Zfp93, Zfp94, rKr2) or B (KRAZ1, KZF-1, RITA, ZNF189, ZNF304) exon (all genes marked with an asterisk in fig. 2 ). This might reflect the loss of the exon encoding the b, or the B, box from these genes, or it might be the result of an alternative splicing event. There is strong evidence indicating that these events occurred after the formation of the different families shown in figure 2 (i.e., the KRAB A + b and A zinc finger gene families). The gene encoding ZNF222one of the genes which appear in the A + b family but which seems to lack the exon encoding KRAB bis located within the cluster of KRAB A + b zinc finger genes on human chromosome 19. This indicates that ZNF222 is a true member of the KRAB A + b zinc finger gene family. In addition, upon careful examination of the genomic sequence located between the exons encoding KRAB A and the zinc finger region of ZNF222, a putative KRAB b exon was detected (data not shown). Although the splicing sites bordering this putative exon seem to be intact it remains to be established if they are used. We were unable to perform a similar analysis for the mouse genes Zfp93 and Zfp94 and the rat gene rKr2. However, the human orthologs of both Zfp93 and Zfp94 (HZF6 and ZNF45, respectively) carry a KRAB b box, which shows that these genes do belong to the A + b subfamily. Based on these results, we propose a model in which all the members of the KRAB A + b subfamily originate from one ancestral gene in which the B box diverged into b. Subsequent duplication events resulted in the formation of the KRAB A + b zinc finger gene subfamily. Similarly, the KRAB A zinc finger gene subfamily originates from an ancestral gene in which the exon encoding B was lost completely. However, as mentioned earlier, some of the genes, which in the distance tree appear in the KRAB A + B family, also seem to lack a KRAB B exon. To determine if this is the result of an alternative splicing event or is due to the loss of the exon encoding the B box, we have analyzed the genomic sequences located between the exons encoding KRAB A and the zinc finger region of the human KRAB zinc finger genes KRAZ1, RITA, ZNF189, and ZNF304 for putative KRAB B exons (unfortunately, there is no genomic sequence available for the rat KRAB zinc finger gene KZF-1). The genomic sequences for KRAZ1 and RITA were both found to contain putative KRAB B exons. However, the splicing sites are not canonical, indicating that they may be less efficient. ZNF189 and ZNF304 were both found to contain nucleotide sequences homologous to the KRAB B box, but no open reading frame corresponding to the KRAB B domain could be identified (data not shown). The KRAB B exon has thus been lost in other genes, separate from those belonging to the KRAB A zinc finger gene family.
The KRAB B domain cannot act as a transcriptional repressor by itself. However, it has been shown to increase the repression activity of the A box (Vissing et al. 1995
). On the other hand, the highly divergent b box does not seem to contribute to the repression activity of the KRAB domain (Abrink et al. 2001
). The repression activity of the KRAB domain is thus only modulated by, and not dependent on, the presence of an intact B box. This enables the loss of the KRAB B box or the divergence of B into b without any significant change of function.
Mechanisms Involved in the Increase in the Total Number of Zinc Finger Genes During Evolution
The total number of zinc finger genes appears to have increased dramatically during metazoan evolution. For example, the genome of the plant A. thaliana contains 21 zinc finger genes, as compared with 34 in baker's yeast (S. cervisiae), 68 in a nematode (C. elegans), 234 in an insect (D. melanogaster) and 564 in human beings (Goffeau 1997
; The C. elegans Sequencing Consortium 1998
; Adams et al. 2000
; The Arabidopsis Genome Initiative 2000
; McPherson et al. 2001
; Venter et al. 2001
). In addition, zinc finger genes are often clustered. The clusters consist of evolutionarily related zinc finger genes and are probably a result of a number of consecutive gene duplication events. The KRAB A + b zinc finger genes and the members of the ZNF91 family of KRAB A + B zinc finger genes are both good illustrations of such an event. Duplications of zinc finger genes are most often duplications of single genes (Shannon et al. 1998
and references therein). However, duplications of all or part of a cluster have also been reported (e.g., human chromosome 10; Tunnacliffe et al. 1993
; Jackson et al. 1996
). Evolutionary analysis of zinc finger genes from mouse and human zinc finger clusters shows that different founder genes have been duplicated, lost, and selected independently in each conserved cluster since the divergence of primate and rodent lineages (Dehal et al. 2001
). This indicates that duplication of zinc finger genes is an ongoing process. Following duplication of a gene there are three possible outcomes. The copy can become a nonfunctional pseudogene, retain its function (resulting in increased production of RNA or protein or both), or accumulate molecular changes that may, in time, affect a new function. Duplication-derived paralogous genes, therefore, allow nucleotide substitutions that cause changes in the amino acid sequence. Speciation-derived orthologous genes, on the other hand, strive to preserve their original function. For these genes, nucleotide substitutions which do not affect the amino acid sequence and the function of the gene are predominant. This is also true for KRAB zinc finger genes. Four orthologous pairs and seven paralogous pairs of KRAB zinc finger genes were selected for analysis of nucleotide substitution in their zinc finger regions (table 1
). The analysis shows that the fraction of synonymous substitutions (%S) in the zinc finger region of orthologous genes is significantly higher than the fraction of nonsynonymous substitutions (%N; P < 0.0001; t-test; table 1
). In the zinc finger region of paralogous genes the situation is reversed, and nonsynonymous nucleotide substitutions are more common than synonymous substitutions (P < 0.0001; t-test; table 1
).
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In conclusion, we show that the zinc finger regions of paralogous KRAB zinc finger genes accumulate more changes in their amino acid sequence than do orthologous genes. It should be noted that the amino acids of the KRAB domain, which are involved in the interaction with TIF1-ß and the transcriptional repression exerted by the KRAB domain, are very well conserved in all KRAB zinc finger proteins (Mark, Abrink, and Hellman 1999
; Abrink et al. 2001
). Duplication of KRAB zinc finger genes, therefore, is most likely a first step in the evolution of new transcriptional repressors. The new transcription factor will contain a well-conserved KRAB domain, responsible for transcriptional repression. However, changes in the amino acid sequence of the zinc finger region will slowly lead to altered binding specificity so that new transcription factors will appear which will regulate genes other than those regulated by the ancestral gene. The fact that the residues responsible for the specificity in sequence recognition are separated from those responsible for structural integrity of the domain (Wolfe, Nekludova, and Pabo 2000
) probably allows considerable flexibility in the evolution of novel binding specificities and increases the potential for these proteins to evolve relatively freely.
Expansion of the Zinc Finger Region
Not only has the number of zinc finger genes increased throughout evolution, so has the number of zinc finger motifs within each individual gene. On average, a zinc finger gene of A. thaliana contains one zinc finger motif, whereas the corresponding numbers for S. cervisiae, C. elegans, D. melanogaster, and H. sapiens are 1.5, 2.5, 3.5, and 8, respectively (Venter et al. 2001
). We have analyzed the phylogenetic relationship among the individual zinc finger motifs of 40 KRAB zinc finger genes. Each gene was analyzed separately, and the results clearly indicate the occurrence of internal duplications (fig. 3
). The sizes of these duplications range from a single zinc finger motif to as many as six consecutive zinc finger motifs (e.g., DNABPZ in fig. 3A
). In addition, these duplicated regions can be repeated at least up to four times within the protein (KZF2 in fig. 3A
). Internal duplications, therefore, seem to be a common mechanism involved in the expansion of the number of zinc finger motifs carried in each zinc finger gene and thereby contribute to the evolution of new transcriptional regulators.
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Conclusions |
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The duplication of KRAB zinc finger genes is probably the first step in the evolution of new transcriptional repressors. The new gene, through its well-conserved KRAB domain, is able to interact with TIF1-ß and repress transcription. However, due to accumulating changes in the amino acid sequence of the zinc finger region, the binding specificity of the zinc finger region will slowly change. The binding specificity of the KRAB zinc finger proteins is further modulated by the addition and inactivation of entire zinc finger motifs that take place within the zinc finger region. Together, these mechanisms generate a framework for the evolution of transcription factors with new binding specificities, which might have been essential for the expansion of the KRAB zinc finger gene family during metazoan evolution.
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Acknowledgements |
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Footnotes |
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Keywords: KRAB
Krüppel zinc finger
evolution
Address for correspondence and reprints: Lars Hellman, Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24 Uppsala, Sweden. E-mail: Lars.Hellman{at}icm.uu.se
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