Biology subjects

Cavalli, G.

Publications and source records attributed to Cavalli, G..

4 recordsLinked to original sources

bioRxiv2018

Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome organization while maintaining spatiotemporal information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. We used Hi-M to study chromosome organization during the awakening of the zygotic genome in intact Drosophila embryos. By visualizing chromosome architecture and gene expression simultaneously in single nuclei, we were able to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture in time and space will be key to understand the mechanisms and consequences of the 4D organization of the genome.

cell biology↗

bioRxiv2018

PRC1-dependent compaction of Hox gene clusters prevents transcriptional derepression during early Drosophila embryogenesis

Summary paragraphPolycomb-group (PcG) proteins are conserved chromatin factors that maintain the silencing of key developmental genes, notably the Hox gene clusters, outside of their expression domains [1-3]. Polycomb repressive complex 2 (PRC2) trimethylates lysine K27 of histone H3 [4], and PRC1 collaborates with PRC2 in gene silencing. Genome-wide studies have revealed large H3K27me3 chromatin domains bound by PcG proteins, and Polycomb domains fold into distinct nuclear structures [5-9]. Although PRC1 is involved in chromatin compaction [10-16], it is unknown whether PRC1-dependent transcriptional silencing is a consequence of its role on higher-order chromatin folding. This is because depletion of PRC1 proteins typically induces both chromatin unfolding and ectopic transcription, and ectopic transcription can open chromatin by itself. To disentangle these two components, we analysed the temporal effects of two PRC1 proteins, Polyhomeotic (Ph) and Polycomb (Pc), on Hox gene clusters during Drosophila embryogenesis. We show that the absence of Ph or Pc affects the higher-order chromatin folding of Hox clusters prior to ectopic Hox gene transcription, demonstrating that PRC1 primary function during early embryogenesis is to compact its target chromatin. During later embryogenesis, we observed further chromatin opening at Hox complexes in both Ph and Pc mutants, which was coupled to strong deregulation of Hox genes at this stage of development. Moreover, the differential effects of Ph and Pc on Hox cluster folding matches the differences in ectopic Hox gene expression observed in these two mutants, suggesting that the degree of Hox derepression in PcG mutants depends on the degree of structural constraints imposed by each PcG component. In summary, our data demonstrate that binding of PRC1 to large genomic domains during early embryogenesis induces the formation of compact chromatin to prevent ectopic gene expression at later time-points. Thus, epigenetic mechanisms such as Polycomb mediated silencing act by folding chromatin domains and impose an architectural layer to gene regulation.

developmental biology↗

bioRxiv2017

Single-cell absolute contact probability detection reveals that chromosomes are organized by modulated stochasticity

At the kilo- to mega-base pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combined high-content super-resolution microscopies with state-of-the-art DNA labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We found that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we were able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs were to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

biophysics↗

bioRxiv2017

The Drosophila Dosage Compensation Complex activates target genes by chromosome looping within the active compartment

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific-lethal dosage compensation complex (DCC) identifies X chromosomal High Affinity Sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

genomics↗