Mutations in promotors

Mutations in promotors

Outlook JOURNAL CLUB Genome-wide screen for protein–protein interaction in yeast Adele Rowley ar14034@ glaxowellcome.co.uk Using the yeast 2 hybri...

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Outlook

JOURNAL CLUB

Genome-wide screen for protein–protein interaction in yeast

Adele Rowley ar14034@ glaxowellcome.co.uk

Using the yeast 2 hybrid system, Uetz et al.1 report the first genome-wide study in Saccharomyces cerevisiae to investigate protein–protein interactions. The yeast two-hybrid system exploits the observation that the DNAbinding and transcriptional-activation domains of a transcription factor such as Gal4 are separable, and that they can activate transcription, even when expressed on different fusion proteins, provided that they are brought into close proximity at the promotor of a suitable reporter gene. This is achieved by fusing each domain to one or other of a pair of proteins that physically interact and then combining these constructs in the same yeast cell. Furthermore, because yeast cells exist in two complementary mating types, it is relatively easy to introduce DNA-

binding and activation-domain fusions to one another via a simple transformant-mixing strategy. The authors have used two approaches to assay the ability of DNA-binding domain fusions to interact with all possible activation-domain fusions. In the first, 192 individual DNA-binding-domain fusions were tested against almost all potential activation-domain constructs in an arrayed library format; in the second, DNA-binding-domain fusions were mixed with pooled activation-domain constructs. The first approach, while of lower throughput, resulted in a higher number of interactions/bait. Overall, 957 putative interactions (109 of which had been previously discovered), involving 1004 proteins, were identified. To manipulate the large data set, Uetz et al. developed bioinformatics tools to view

Heterochromatin revealed

Jonathan Hodgkin [email protected]

Large stretches of the genomes of plants and animals are taken up with heterochromatin, which has distinctive molecular and cytological properties, when compared with the gene-rich euchromatin. Heterochromatin is singularly hard to clone and sequence, and is therefore largely ignored in large-scale genome-sequencing efforts. However, it is well known to have important biological properties, such as supporting centromere function and causing longrange position effects on gene activity. These papers on a segment of the Arabidopsis genome offer a welcome look inside the ‘black box’ of a heterochromatic knob on the 4S chromosome arm, which is the first piece of cytologically recognizable heterochromatin to be fully sequenced. The region is ~500 kb across and, at its core, contains a tandem array of a 2-kb transposon-related repeat. This is surrounded by complex arrangements of retro-

transposons and DNA transposons from several different families, some of which have not previously been described in Arabidopsis. Almost no predicted or expressed genes are found in the sequence, and none at all at its core. Examination of genetraps1 revealed that 33 had landed in flanking euchromatin, but none in the heterochromatin. This could be because of a silencing effect such that integrations in the heterochromatin are not detected, but might also be caused by a tendency for genetraps to integrate only around expressed genes. Recombination in the region is also strikingly correlated with the presence of expressed genes, being reduced tenfold or more in the heterochromatin. In the accompanying paper, Fransz et al.2 examine the cytogenetics of this chromosome arm by fluorescence in situ hybridization. They find that the knob has probably arisen from a small inversion event, exchanging the position of

Mutations in promotors Diethard Tautz [email protected] 204

The assumption that regulatory sequences should be prime targets for the introduction of evolutionary novelties is almost commonplace today. But so far the studies demonstrating this experimentally are, in fact, very rare.

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The b gene in Maize codes for a helix–loop–helix-domain protein that is required for the activation of the anthocyanin pigmentation pathway. Variance in anthocyanin pigmentation is of direct evolutionary relevance in Maize as it

protein connections and to integrate data with other pertinent information, for example homologues in other organisms. The results of this study clearly tether many previously unassigned genes to networks of interacting proteins and reveal new connections within and between pathways, even alluding to the existence of previously unidentified pathways. Combined with other genome-wide efforts and an existing wealth of genetic information, this study marks another milestone in progress towards a complete understanding of regulatory mechanisms in this organism. 1 Uetz, P. et al. (2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627

part of the pericentromeric chromatin and adjacent euchromatin, because some ecotypes have the presumed ancestral arrangement of sequences and lack the knob. These authors have also carefully measured the condensation and decondensation of different chromosomal segments during meiotic stages, demonstrating dynamic changes and differences in the behaviour of euchromatin and the different types of heterochromatin. The results open the way to detailed understanding of what controls chromosomal condensation in different parts of the genome.

1 The CSHL/WUGSC/PEB Arabidopsis Sequencing Consortium (2000) The complete sequence of a heterochromatic island from a higher eukaryote. Cell 100, 377–386 2 Fransz, P.F. et al. (2000) Integrated cytogenetic map of chromosome 4S of A. thaliana. Structural organization of heterochromatic knob and centromere region. Cell 100, 367–376

might be involved in such things as resistance to UV, cold tolerance or parasite defense. The promotor region of the b gene has previously been studied in detail and it was shown that an allele that led to a switch from anthocyanin synthesis in vegetative tissues to seed tissues was caused by a large insertion in the promotor region. Selinger and Chandler1 have now sequenced the

JOURNAL CLUB

promotor regions of 18 different wildtype alleles, for which they have carefully determined the phenotypic effects in appropriate test crossings. This yields fascinating insights into the evolutionary dynamics of this promotor region and allows some general conclusions. There are three distinct clades of promotor sequences, which differ by large numbers of nucleotide replacements between the clades, but show only relatively few differences within them. Nevertheless, even promotors from

different clades can yield similar coloration phenotypes. On the other hand, alleles with strong and distinctive patterns of expression can be very closely related to alleles with weak expression, which suggests that distinctive patterns have arisen repeatedly and independently. In fact, most of the changes in expression pattern might have been caused by insertions and deletions of transposable elements, of which a large number of different ones were found in the promotor regions of the different alleles. This

Outlook

suggests that the potential for new mutations in promotor regions by far exceeds the rate of point mutations, which would allow the fast evolution of differences in expression patterns. 1 David A. Selinger and Vicki L. Chandler (1999) Major recent and independent changes in levels and patterns of expression have occurred at the b gene, a regulatory locus in maize. Proc. Natl. Acad. Sci. U. S. A. 96, 15007–15012

Wnt–TGFb signalling interaction? Two recent papers suggest that Wnt and TGFβ signalling can interact either synergistically or antagonistically in early Xenopus development. Baker et al.1 show that, contrary to some earlier reports, Wnt and Wnt signalling components can act as neural inducers in Xenopus embryos. sia and Xnr3 are two genes that are induced by Wnt signalling and have neural-inducing properties. However, when expression of sia and Xnr3 was blocked, using dominant-negative frizzled (a Wnt receptor), neural induction by Wnt was unaffected. By contrast, a dominant-negative version of Tcf (a transcription factor that is activated by Wnt signalling) blocks both responses. This suggests that Wnt can act via alternative pathways, each requiring Tcf. The expression of TGFβ-family member bone morphogenic protein 4 (BMP4) is reduced dorsally during gastrulation, and this was hypothesized to be caused by signals from the dorsal

organizer, such as noggin, that block BMP signalling and, hence, self-activation of Bmp expression. However, Baker et al. show that although Wnt and noggin both induce neural tissue, only Wnt represses Bmp4 expression at the gastrula stage. They suggest that βcatenin is increased dorsally, both in the animal hemisphere and in the organizer region, by Wnt signalling that arises from the post-fertilization cortical rotation. This initially induces neural tissue via a decrease in Bmp4 expression, but independently of signals from the organizer, which act later. Nishita et al.2 demonstrate a direct interaction between components of the Wnt and TGFβ signalling pathways to control the expression of the Xenopus organizer-specific gene twin, a close relative of sia. The twin promoter contains binding sites for the transcription factors lymphoid enhancer binding factor 1 (Lef1) and its relative Tcf, and also for Smads (downstream effectors

of TGFβ signalling). Nishita et al. show that Smad4 co-immunoprecipitates with β-catenin and with Lef1. Wnt signalling causes β-catenin to translocate to the nucleus, where it interacts with Lef1 and Tcf. The authors present evidence that Smad4 enters the nucleus along with β-catenin, and that Smad4 and Lef1 and Tcf can co-activate some, but not all Wnt-responsive genes (including twin and sia). They suggest that Wnt and TGFβ could act not only synergistically in this way, but also antagonistically if there is competition for a limited pool of Smad4. 1 Baker, J.C. et al. (1999) Wnt signaling in Xenopus embryos inhibits Bmp4 expression and activates neural development. Genes Dev. 31, 3149–3159 2 Nishita, M. et al. (2000) Interaction between Wnt and TGFβ signalling pathways during formation of Spemann’s organizer. Nature 403, 781–785

Alison Snape [email protected]

Author’s correction Elaine Ostrander, Francis Galibert and Donald F. Patterson (2000) Canine genetics comes of age. Trends Genet. 16, 117–124 On page 122 of the March issue, the authors would like to correct the statement ‘The first example of candidate cloning of a canine disease gene was X-linked severe combined immunodeficiency (XSCID) in the Basset Hound’ to read the following: ‘The first example of cloning a canine disease gene using linkage mapping of the canine locus and selection of a candidate gene based on the orthologous region of a human chromosome was X-linked severe combined immunodeficiency (XSCID) in the Basset Hound.’ This is in recognition of the important contributions made to the field of canine disease genes by Evans et al., summarized in Ref. 43 of the article. S0168-9525(00)01977-6

Erratum Complex evolution of the inositol-1-phosphate synthase gene among archea and eubacteria by Nandita Bachhawat and Shekhar C. Mande Trends Genet. 16, 111–113 We apologize to the authors for mistakes in the spelling of Shekhar C. Mande’s name and email address (which should be [email protected]) S0168-9525(00)02016-3

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