The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues. Peter Lorenz, Sabine Dietmann, Thomas Wilhelm, Dirk Koczan, Sandra Autran, Sophie Gad, Gaiping Wen, Guohui Ding, Yixue Li, Marie-Françoise Rousseau-Merck, et al. To cite this version: Peter Lorenz, Sabine Dietmann, Thomas Wilhelm, Dirk Koczan, Sandra Autran, et al.. The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles ofC2H2 zinc finger evolution associated with unique expression profiles in human tissues.. BMC Genomics, BioMed Central, 2010, 11 (1), pp.206. 10.1186/1471-2164-11-206. inserm-00651122 HAL Id: inserm-00651122 https://www.hal.inserm.fr/inserm-00651122 Submitted on 12 Dec 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lorenz et al. BMC Genomics 2010, 11:206 http://www.biomedcentral.com/1471-2164/11/206 RESEARCH ARTICLE Open Access TheResearch ancient article mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues Peter Lorenz1, Sabine Dietmann2, Thomas Wilhelm3, Dirk Koczan1, Sandra Autran4, Sophie Gad5, Gaiping Wen6,7, Guohui Ding8, Yixue Li8, Marie-Françoise Rousseau-Merck4 and Hans-Juergen Thiesen*1 Abstract Background: Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation. Results: Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities. Conclusions: The ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny. Background tion [1-7]. Members of the most prominent subfamily con- Evolution of tetrapodes coincides with the expansion of tain the Krüppel-associated box (KRAB) transcriptional Krüppel-type C2H2 zinc finger (ZNF) genes leading to the repressor domain at their N-terminus [8-10]. The Krüppel- largest gene family involved in transcriptional gene regula- type zinc finger domain was originally identified in Xeno- * Correspondence: [email protected] pus laevis TFIIIA [11] and the Krüppel mutant of Droso- 1 Institute of Immunology, University of Rostock, Schillingallee 70, 18055 phila melanogaster [12]. It is known as nucleic acid Rostock, Germany interaction domain but can also contribute to protein-pro- Full list of author information is available at the end of the article © 2010 Lorenz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lorenz et al. BMC Genomics 2010, 11:206 Page 2 of 30 http://www.biomedcentral.com/1471-2164/11/206 tein interactions [13-16]. The classical zinc finger fold con- found in the genome of amphibians (Xenopus laevis/tropi- sists of an approximately 30-amino acid unit of two calis) compared with the human genome for which compre- antiparallel β strands linked to an amphipathic α-helix with hensive studies list 300-400 KRAB-ZNF protein-coding two cysteines and two histidines coordinating a zinc ion to genes [4,5,40,6]. Thus, KRAB-zinc finger genes presum- stabilize the structure [17]. DNA-binding-specificity relies ably coevolved with or occurred shortly after the appear- on amino acids within the α-helix reaching into the major ance of tetrapodes and underwent a huge expansion during groove of DNA [18]. According to the crystal structure of mammalian evolution. It was hypothesized that KRAB-like DNA bound EGR1/Zif268, the residues most crucial for sequences in the histone methyltransferase Meisetz date the DNA binding are localized at positions -1, 3 and 6 with origin of this domain back to the last common ancestor of respect to the start of the α-helix. Position 2 is also involved chordates and echinoderms [41]. However, this KRAB-like in DNA binding, but makes contact to the complementary domain is closely related to the KRAB-like domain of the strand [13]. Zinc finger domains usually occur in arrays of SSX proteins that does not interact with TRIM28 and con- multiple C2H2 zinc finger modules comprising from only a sequently does not initiate transcriptional repression [42]. few up to more than thirty units [19]. The individual units Initial mapping data on KOX zinc finger genes already are separated by a conserved sequence (consensus TGEKP) indicated that most ZNF genes are clustered in the human called HC link. genome [43]. This was confirmed by detection of 23 chro- The KRAB domain was originally described as heptad mosomal KOX gene ZNF loci [44]. Numerous ZNF gene repeat of leucines in KOX1/ZNF10 [8] and shown to be clusters have been defined over the years [45-48,3,49] and evolutionarily conserved [9], for review see [20]. The catalogued to a total of about 60-90 genomic loci [4,5,40], KRAB domain of KOX1 consists of a KRAB-A and a depending on definition. Diversification of ZNF genes dur- KRAB-B subdomain of which the KRAB-A subdomain ing evolution is reflected by duplication and deletion of mediates transcriptional repression [21-23] and the KRAB- zinc finger domains thereby modifying recognition speci- B part enhances the repression in conjunction with KRAB- ficities for RNA/DNA binding [50,2,51]. Individual degen- A [24]. Later on, different KRAB-B subdomains with dif- erate non-functional zinc finger domains do occur within a ferent properties have been discovered [19,25,4]. The sequence as well as after truncation by introduction of an KRAB domain, early postulated as a protein-protein inter- in-frame stop codon (reviewed for KRAB-ZNFs in [52]). action domain [8], has been shown to interact with the Lineage-specific expansions and losses within ZNF clusters RBCC domain of TRIM28 (tripartite motif-containing 28, contribute to evolutionary adaptation [2,3,49,51,5,6]. also known as KAP-1, KRIP-1, TIF1β; reviewed in [20]). In this manuscript we focus on the human ZNF cluster at This protein is considered essential for KRAB-mediated 8q24.3 that had not been investigated in detail. It was cho- transcriptional repression and recruits various chromatin- sen because of the presence of several KOX ZNF genes modifying protein complexes, thus leading to a repressive (ZNF7/KOX4, ZNF16/KOX9 and ZNF34/KOX32) for chromatin state [26-28]. It is most likely that all KRAB which we have a longstanding interest [44]. We sequenced C2H2 zinc finger proteins mediate transcriptional repres- the locus as part of the german HUGO initiative on chromo- sion in a sequence-specific manner. It is currently unre- some 8 and identified seven ZNF genes. These genes form solved how many target genes are regulated by an a paralog group well separated from other ZNF subfamilies.