Mutations in the insulator protein Suppressor of Hairy wing induce genome instability.

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.

Condensin II subunit NCAPH2 associates with shelterin protein TRF1 and is required for telomere stability.

Condensin II subunits are known to be expressed and localized to interphase nuclei of eukaryotic cells. Although some studies have shown that condensin II likely exerts axial compaction forces, organizes chromosome territories, and has possible transcriptional modulatory functions, the full range of condensin II interphase activities are not known. In particular, it is not known if condensin II interphase activities are generally genome-wide or if they have additional local activities unique to specific chromosomal structures such as telomeres. Here, we find that NCAPH2 interacts with TRF1 and these two proteins co-localize at telomeres. Depletion of NCAPH2 leads to ATR-dependent accumulation of 53BP1 and γH2AX DNA damage foci, including damage specific to telomeres. Furthermore, depletion of NCAPH2 results in a fragile telomere phenotype and apparent sister-telomere fusions only days after NCAPH2 depletion. Taken together these observations suggest that NCAPH2 promotes telomere stability, possibly through a direct interaction with the TRF1 shelterin component, and prevents telomere dysfunction resulting from impaired DNA replication. Because proper telomere function is essential for chromosome integrity these observations reveal a previously unappreciated function for NCAPH2 in ensuring genome and telomere stability.

Characterization of a cdc14 null allele in Drosophila melanogaster.

Cdc14 is an evolutionarily conserved serine/threonine phosphatase. Originally identified in Saccharomyces cerevisiae as a cell cycle regulator, its role in other eukaryotic organisms remains unclear. In Drosophila melanogaster, Cdc14 is encoded by a single gene, thus facilitating its study. We found that Cdc14 expression is highest in the testis of adult flies and that cdc14 null flies are viable. cdc14 null female and male flies do not display altered fertility. cdc14 null males, however, exhibit decreased sperm competitiveness. Previous studies have shown that Cdc14 plays a role in ciliogenesis during zebrafish development. In Drosophila, sensory neurons are ciliated. We found that the Drosophila cdc14 null mutants have defects in chemosensation and mechanosensation as indicated by decreased avoidance of repellant substances and decreased response to touch. In addition, we show that cdc14 null mutants have defects in lipid metabolism and resistance to starvation. These studies highlight the diversity of Cdc14 function in eukaryotes despite its structural conservation.

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

Condensin II Regulates Interphase Chromatin Organization Through the Mrg-Binding Motif of Cap-H2.

The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization.

TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase η.

We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.

The way Wnt works: components and mechanism.

The canonical Wnt/ß-catenin pathway is an ancient and evolutionarily conserved signaling pathway that is required for the proper development of all metazoans, from the basal demosponge Amphimedon queenslandica to humans. Misregulation of Wnt signaling is implicated in many human diseases, making this pathway an intense area of research in industry as well as academia. In this review, we explore our current understanding of the molecular steps involved in the transduction of a Wnt signal. We will focus on how the critical Wnt pathway component, ß-catenin, is in a "futile cycle" of constant synthesis and degradation and how this cycle is disrupted upon pathway activation. We describe the role of the Wnt pathway in major human cancers and in the control of stem cell self-renewal in the developing organism and in adults. Finally, we describe well-accepted criteria that have been proposed as evidence for the involvement of a molecule in regulating the canonical Wnt pathway.

Condensins and 3D Organization of the Interphase Nucleus.

Condensins are conserved multi-subunit protein complexes that participate in eukaryotic genome organization. Well known for their role in mitotic chromosome condensation, condensins have recently emerged as integral components of diverse interphase processes. Recent evidence shows that condensins are involved in chromatin organization, gene expression, and DNA repair and indicates similarities between the interphase and mitotic functions of condensin. Recent work has enhanced our knowledge of how chromatin architecture is dynamically regulated by condensin to impact essential cellular processes.

XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling.

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing ß-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.

Chromatin insulators specifically associate with different levels of higher-order chromatin organization in Drosophila.

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F-58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.