📋 Browse Articles

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments. Show less

We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression. Show less

The formation of the sex vesicle, or XY body, during male meiosis and pairing of the sex chromosomes are thought to be essential for successful spermatogenesis. Despite its cytological discovery a century ago, the mechanism of XY body formation, particularly heterochromatinization of the sex chromosomes, has remained unclear. The HP1 class of chromobox genes are thought to encode proteins involved in the packaging of chromosomal DNA into repressive heterochromatin domains, as seen, for example, in position-effect variegation. Study of the distribution of a murine HP1-like chromodomain protein, M31, during spermatogenesis revealed spreading from the tip of the XY body in mid-stage pachytene spermatocytes to include the whole of the XY body in late-pachytene spermatocytes. We also demonstrate that the formation of the XY body during spermatogenic progression in neonatal mice coincides with the expression of a novel nuclear isoform of M31, M31(p21). These results support the view that a common mechanistic basis exists for heterochromatin-induced repression, homeotic gene silencing, and sex-chromosome inactivation during mammalian spermatogenesis. Show less

Locus control regions (LCRs) are gene regulatory elements in mammals that can overcome the highly repressive effects normally associated with heterochromatic transgene locations (for example the centromere) in mice. Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced-a phenomenon known as position effect variegation (PEV). We show here that PEV can also occur when the transgene is non-centromeric and that the extent of variegation can be developmentally regulated. Furthermore, by overexpressing a mammalian homologue (M31) of Drosophila melanogaster heterochromatin protein 1 (HP1; refs 7,8) in transgenic mouse lines that exhibit PEV, it is possible to modify the proportion of cells that silence the transgene in a dose-dependent manner. Thus, we show M31 overexpression to have two contrasting effects which are dependent on chromosomal context: (i) it enhanced PEV in those lines with centromeric or pericentromeric transgene locations; and (ii) it suppressed PEV when the transgene was non-centromeric. Our results indicate that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals. Show less

Mammalian heterochromatin proteins 1 (HP1alpha, HP1beta, and HP1gamma) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin remodeling. Using antibodies specific for each HP1 isoform, we showed that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1alpha and HP1beta are strictly colocalized. In mitotic HeLa cells, all of HP1alpha and a fraction of HP1beta and HP1gamma remain associated with chromosomes. Immunostaining of spread HeLa chromosomes showed that HP1alpha is mainly localized on centromeres as shown previously for HP1beta, while HP1gamma is distributed on discrete sites on the arms of chromosomes. Biochemical analysis showed that HP1alpha and HP1gamma are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1beta apparently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 isoforms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their targeting at different chromatin sites, generating pleiotropic effects. Show less

The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin. Show less

HP1-like chromobox genes comprise an evolutionarily conserved family of genes that encode components of centromeric heterochromatin. In order to investigate the role of the murine HP1-like gene, M31, in heterochromatin formation we have isolated its gene and characterised its transcripts and protein products. PCR products that represent M31 transcripts were detected at the one-cell stage and were maternal in origin. Maternal provision of M31 transcripts may reflect a need for M31 in the formation of a functional centromere in order that there is proper segregation of chromosomes during the early cleavage divisions; studies in fission yeast and Drosophila have suggested a crucial role for HP1-like genes in centromere function. There are three protein products encoded by the M31 gene. Surprisingly, the two smaller products are found almost exclusively in the cytoplasm. Show less

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one. Show less

Contrabithorax (Cbx) alleles are dominant mutations in the Ultrabithorax (Ubx) gene of Drosophila that cause its ectopic expression in the mesothorax. We have studied the role of the homologous chromosome in the mesothoracic phenotype in several Cbx heterozygotes. None of the Cbx alleles studied shows variations in phenotype with extra doses of the Ubx gene. Only Cbx2 and CbxIRM (a revertant of Cbx1) show synapsis-dependent gene expression ("transvection"). The mesothoracic phenotypes of CbxIRM and Cbx2 heterozygotes are strongly modified when the homologous chromosome carries breakpoints proximal to or at the Ubx locus or null alleles of this gene. These lesions in the homologous chromosome enhance the Cbx2 mutant phenotype and reduce that of CbxIRM one. The genetic analysis of these transvection effects suggests that the transcription of the CbxIRM and Cbx2 alleles depends on RNAs of short radius of action from the homologous Ubx gene. Show less

Cbx1 is a dominant mutation of the bithorax complex (BX-C) of Drosophila partially transforming the second thoracic (T2) segment towards the third one (T3). Molecular analysis has shown that Cbx1 arose from a transposition within the BX-C of a DNA fragment of 17 kb containing pbx+ inserted into the Ubx area. In addition to the dominant phenotype, the Cbx1 mutation produces a set of recessive homeotic transformations that we show are characteristic of the Ubx mutations. We present evidence that the dominant and the recessive transformations arise from different mechanisms and suggest the dominant transformation is caused by an alteration of the normal regulatory role of pbx+ resulting in an adventitious expression of some Ubx+ products in T2, while the Ubx phenotype is caused by the breakpoint of the insertion. Show less