The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith-Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.

In human and mouse most imprinted genes are arranged in chromosomal clusters. This linked organization suggests coordinated mechanisms controlling imprinted expression. We have sequenced 250 kb in the centre of the mouse imprinting cluster on distal chromosome 7 and compared it with the orthologous Beckwith-Wiedemann gene cluster on human chromosome 11p15.5. This first comparative imprinting cluster analysis revealed a high structural and functional conservation of the six orthologous genes identified. However, several striking differences were also discovered. First, compared with the mouse the human sequence is approximately 40% longer, mostly due to insertions of two large repetitive clusters. One of these clusters encompasses an additional gene coding for a homologue of the ribosomal protein L26. Second, pronounced blocks of unique direct repeats characteristic of imprinted genes were only found in the human sequence. Third, two of the orthologous gene pairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showed apparent differences in imprinting between human and mouse, whereas others like Tssc6/TSSC6 were not imprinted in either organism. Together these results suggest a significant functional and structural variability in the centre of the imprinting cluster. Some genes escape imprinting in both organisms whereas others exhibit tissue- and species-specific imprinting. Hence the control of imprinting in the cluster appears to be a highly dynamic process under fast evolutionary adaptation. Intriguingly, whereas imprinted genes within the cluster contain CpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6 do not. This and additional comparisons with other imprinted and non-imprinted regions suggest that CpG islands are key features of imprinted domains.

Domina (Dom) is a novel member of the FKH/WH transcription factor gene family of Drosophila. Two alternatively polyadenylated Dom transcripts of 2.9 and 3.9 kb encode a 719-amino-acid protein with a FKH/WH domain and a putative acidic transactivation domain. Dom is mainly expressed in the central and peripheral nervous system. Homozygous mutants show rough eyes, irregular arrangement of bristles, extended wings, defective posterior wing margins, and a severely diminished vitality and fertility. Heterozygous Dom flies are morphologically wild type but show suppression of position-effect variegation. Consistently with this chromatin effect DOM protein is accumulated in the chromocenter and, as expected from a transcription factor, is found at specific euchromatic loci. Sequence comparison suggests that DOM of Drosophila is homologous to the chordate WHN proteins. The chromatin modifying capability of DOM is probably based on the FKH/WH domain, which shows a remarkable structural similarity to the winged-helix structures of H1 and the central globular domain of H5.

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles. Molecular & Cellular Proteomics 5:234 -244, 2006.As protein-protein interactions (PPIs) 1 are central to most biological processes, their systematic identification is considered a key strategy for uncovering the complex organization principles of functional cellular networks (1-3). A number of experimental and computational techniques have been developed to determine all the potential and actual PPIs in selected model organisms (4 -8) and humans (9 -11).Because protein arrays allow the parallel detection of addressable elements in a single experiment (12-18), they have been recognized as a promising technology for the identification of PPIs (19,20) or other specific biochemical activities (20 -23), and much effort has been devoted to their development in recent years. Zhu et al. (20) e.g. printed 5,800 purified yeast proteins onto coated glass slides and used them successfully in proof-of-principle experiments for the detection of novel calmodulin-and phospholipid-interacting proteins. Protein arrays were also used efficiently for identifying novel targets of protein kinases (24 -26). In principle, the production of protein arrays and their utilization for systematic large scale interaction and activity screens has proven viable. One major drawback, however, has been the necessity to use purified proteins. Protein purification is usually difficult, time-consuming, and expensive. For array-based studies, especially high throughput systematic screening (19), technologies are needed that permit...

DNA methyltransferases (Dnmts) mediate the epigenetic modification of eukaryotic genomes. Mammalian DNA methylation patterns are established and maintained by co-operative interactions among the Dnmt proteins Dnmt1, Dnmt3a and Dnmt3b. Owing to their simultaneous presence in mammalian cells, the activities of individual Dnmt have not yet been determined. This includes a fourth putative Dnmt, namely Dnmt2, which has failed to reveal any activity in previous assays. We have now established transgenic Drosophila strains that allow for individual overexpression of all known mouse Dnmts. Quantitative analysis of genomic cytosine methylation levels demonstrated a robust Dnmt activity for the de novo methyltransferases Dnmt3a and Dnmt3b. In addition, we also detected a weak but significant activity for Dnmt2. Subsequent methylation tract analysis by genomic bisulphite sequencing revealed that Dnmt3 enzymes preferentially methylated CpG dinucleotides in a processive manner, whereas Dnmt2 methylated isolated cytosine residues in a non-CpG dinucleotide context. Our results allow a direct comparison of the activities of mammalian Dnmts and suggest a significant functional specialization of these enzymes.

The PEV-modifying winged-helix/forkhead domain transcription factor JUMU of Drosophila is an essential protein of pleiotropic function. The correct gene dose of jumu is required for nucleolar integrity and correct nucleolus function. Overexpression of jumu results in bloating of euchromatic chromosome arms, displacement of the JUMU protein from the chromocenter and the nucleolus, fragile weak points, and disrupted chromocenter of polytene chromosomes. Overexpression of the acidic C terminus of JUMU alone causes nucleolus disorganization. In addition, euchromatic genes are overexpressed and HP1, which normally accumulates in the pericentric heterochromatin and spreads into euchromatic chromosome arms, although H3-K9 di-methylation remains restricted to the pericentric heterochromatin. The human winged-helix nude gene shows similarities to jumu and its overexpression in Drosophila causes bristle mutations.

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