Reconstituting Drosophila Centromere Identity in Human Cells.

The centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, centromere protein (CENP)-A, yet how its self-propagation is achieved remains poorly understood. Here, we develop a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C, and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify CAL1 to be required for dCENP-C loading onto chromatin in cooperation with dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of Drosophila centromere identity.

Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila.

In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.

Polycomb group-mediated gene silencing mechanisms: stability versus flexibility.

Polycomb group (PcG) proteins are highly conserved chromatin factors that repress transcription of particular target genes in animals and plants. PcG proteins form multimeric complexes that act on their target genes through the regulation of post-translational histone modifications, the modulation of chromatin structure and chromosome organization. PcG proteins have long been considered as a cellular memory system that stably locks regulatory chromatin states for the whole lifespan of the organism. However, recent work on the genome-wide distribution of PcG components and their associated chromatin marks in vertebrate cells and Drosophila have challenged this view, revealing that PcG proteins confer dynamic transcriptional control of key developmental genes during cell differentiation and development.

Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex.

In Drosophila, the function of the Polycomb group genes (PcGs) and their target sequences (Polycomb response elements (PREs)) is to convey mitotic heritability of transcription programmes--in particular, gene silencing. As part of the mechanisms involved, PREs are thought to mediate this transcriptional memory function by building up higher-order structures in the nucleus. To address this question, we analysed in vivo the three-dimensional structure of the homeotic locus bithorax complex (BX-C) by combining chromosome conformation capture (3C) with fluorescent in situ hybridization (FISH) and FISH immunostaining (FISH-I) analysis. We found that, in the repressed state, all major elements that have been shown to bind PcG proteins, including PREs and core promoters, interact at a distance, giving rise to a topologically complex structure. We show that this structure is important for epigenetic silencing of the BX-C, as we find that major changes in higher-order structures must occur to stably maintain alternative transcription states, whereas histone modification and reduced levels of PcG proteins determine an epigenetic switch that is only partially heritable.

Role for Brm in cell growth control.

Recently, we have shown implication of Brm, the catalytic subunit of the SWI/SNF chromatin remodeling complex, in repression of cyclin A expression in quiescent cells. Here, we have examined the fate of cells lacking Brm throughout the cycle. We find that despite elevated levels of cyclins A and E, these cells can respond to serum starvation, however, without reaching a canonical G(0) phase as they continue to express high levels of c-Myc and have an abnormally large average size. The response to serum starvation can be correlated with increased levels of Rb proteins p130 and p107 as well as increased association of p27 with the cyclin-dependent kinases, possibly compensating for the higher levels of G(1) cyclins by reducing their associated kinase activity. After serum stimulation, reentry into the cycle occurs normally, but the S phase is delayed and shorter. In addition, the M phase has an increased duration, and we observed frequent faulty chromosome segregation events in anaphase. Altogether, our data suggest that cells can partially overcome the absence of Brm by activating several compensatory mechanisms to control the cell cycle. However, they remain profoundly affected, unable to enter a canonical quiescent state, presenting a shorter S phase, and finally unable to perform correct chromosome segregation.

PU.1/Spi-1 binds to the human TAL-1 silencer to mediate its activity.

The TAL-1/SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for primitive hematopoiesis and for adult erythroid and megakaryocytic development. Activated transcription of TAL-1 as a consequence of chromosomal rearrangements is associated with a high proportion of human T cell acute leukemias, showing that appropriate control of TAL-1 is crucial for the formation and subsequent fate of hematopoietic cells. Hence, the knowledge of the mechanisms, which govern the pattern of TAL-1 expression in hematopoiesis, is of great interest. We previously described a silencer in the 3'-untranslated region of human TAL-1, the activity of which is mediated through binding of a tissue-specific 40 kDa nuclear protein to a new DNA recognition motif, named tal-RE. Here, we show that tal-RE-binding activity, high in immature human hematopoietic progenitors is down regulated upon erythroid and megakaryocytic differentiation. This expression profile helped us to identify that PU.1/Spi-1 binds to the tal-RE sequences in vitro and occupies the TAL-1 silencer in vivo. By expressing a mutant protein containing only the ETS domain of PU.1 in human erythroleukemic HEL cells, we demonstrated that PU.1 mediates the transcriptional repression activity of the silencer. We found that ectopic PU.1 is not able to induce silencing activity in PU.1-negative Jurkat T cells, indicating that PU.1 activity, although necessary, is not sufficient to confer transcriptional repression activity to the TAL-1 silencer. Finally, we showed that the silencer is also active in TAL-1-negative myeloid HL60 cells that express PU.1 at high levels. In summary, our study shows that PU.1, in addition to its positive role in TAL-1 expression in early hematopoietic progenitors, may also act as a mediator of TAL-1 silencing in some hematopoietic lineages.

Cyclin A repression in quiescent cells is associated with chromatin remodeling of its promoter and requires Brahma/SNF2alpha.

Cell cycle-dependent expression of cyclin A is controlled by transcriptional repression in early phase of the cell cycle. In this study, we directly examine the chromatin structure of the mouse cyclin A promoter through in vivo micrococcal nuclease footprinting. We describe here that cyclin A repression is associated with two positioned nucleosomes and that histones progressively lose DNA contact synchronously with gene activation. This particular nucleosomal organization is disrupted by mutations of the cyclin A bipartite repressor sequence. Moreover, the same sequence recruits the chromatin remodeling factor Brahma/SNF2alpha (Brm) onto the cyclin A promoter. Accordingly, cyclin A proximal promoter is not wrapped around nucleosomes and not repressed in quiescent cells lacking Brm. These results provide molecular explanations for the transcriptional repression state of cyclin A, as well as insights into the action of Brm chromatin remodeling factor as cell cycle regulator.