The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

Somatic chromosome pairing has a determinant impact on 3D chromatin organization.

In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. Here, we address this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. Here, we identify the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. We also report that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, we show that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, our results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.

The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo.

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too.

WEADE: A workflow for enrichment analysis and data exploration.

Data analysis based on enrichment of Gene Ontology terms has become an important step in exploring large gene or protein expression datasets and several stand-alone or web tools exist for that purpose. However, a comprehensive and consistent analysis downstream of the enrichment calculation is missing so far. With WEADE we present a free web application that offers an integrated workflow for the exploration of genomic data combining enrichment analysis with a versatile set of tools to directly compare and intersect experiments or candidate gene lists of any size or origin including cross-species data. Lastly, WEADE supports the graphical representation of output data in the form of functional interaction networks based on prior knowledge, allowing users to go from plain expression data to functionally relevant candidate sub-lists in an interactive and consistent manner.

Decoding the Regulatory Logic of the Drosophila Male Stem Cell System.

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. We elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. We identified key roles of nucleoporins and V-ATPase proton pumps and demonstrate their importance in controlling germline development from the support side. To make our dataset publicly available, we generated an interactive analysis tool, which uncovered conserved core genes of adult stem cells across species boundaries. We tested the functional relevance of these genes in the Drosophila testis and intestine and found a high frequency of stem cell defects. In summary, our dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types.

Identification and functional analysis of healing regulators in Drosophila.

Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the overall steps in wound healing, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process. However, they do allow the less understood aspects of the healing response to be explored, e.g., which signal(s) are responsible for initiating tissue remodeling? How is sealing of the epithelia achieved? Or, what inhibitory cues cancel the healing machinery upon completion? Answering these and other questions first requires the identification and functional analysis of wound specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method that is healing-permissive and that allows live imaging and biochemical analysis of cultured imaginal discs. We performed comparative genome-wide profiling between Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. Sets of potential wound-specific genes were subsequently identified. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in healing assays. This non-saturated analysis defines a relevant set of genes whose changes in expression level are functionally significant for proper tissue repair. Amongst these we identified the TCP1 chaperonin complex as a key regulator of the actin cytoskeleton essential for the wound healing response. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound healing response.