UpSET recruits HDAC complexes and restricts chromatin accessibility and acetylation at promoter regions.

Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.

Nuclear envelope budding: Getting large macromolecular complexes out of the nucleus.

Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair.

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding.

Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although a general outline of the cellular events occurring during NE budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE bud formation. Using a multidisciplinary approach, we identify Wash, its regulatory complex (SHRC), capping protein and Arp2/3 as new molecular components involved in the physical aspects of NE bud formation in a Drosophila model system. Interestingly, Wash affects NE budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE bud formation, and directly by blocking NE bud formation along with its SHRC, capping protein and Arp2/3. In addition to NE budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms, suggesting new avenues for exploration in both normal and disease biology.

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

Wash exhibits context-dependent phenotypes and, along with the WASH regulatory complex, regulates Drosophila oogenesis.

WASH, a Wiskott-Aldrich syndrome (WAS) family protein, has many cell and developmental roles related to its function as a branched actin nucleation factor. Similar to mammalian WASHC1, which is embryonic lethal, Drosophila Wash was found to be essential for oogenesis and larval development. Recently, however, Drosophila wash was reported to be homozygous viable. Here, we verify that the original wash null allele harbors an unrelated lethal background mutation; however, this unrelated lethal mutation does not contribute to any Wash oogenesis phenotypes. Significantly, we find that: (1) the homozygous wash null allele retains partial lethality, leading to non-Mendelian inheritance; (2) the allele's functions are subject to its specific genetic background; and (3) the homozygous stock rapidly accumulates modifications that allow it to become robust. Together, these results suggest that Wash plays an important role in oogenesis via the WASH regulatory complex. Finally, we show that another WAS family protein, SCAR/WAVE, plays a similar role in oogenesis and that it is upregulated as one of the modifications that allows the wash allele to survive in the homozygous state.

Molecular basis for chromatin binding and regulation of MLL5.

The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His-Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.

Degringolade, a SUMO-targeted ubiquitin ligase, inhibits Hairy/Groucho-mediated repression.

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. In this study, we show that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho (Gro/transducin-like enhancer (TLE)) during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. Our findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes.

Drosophila embryos close epithelial wounds using a combination of cellular protrusions and an actomyosin purse string.

The repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular, which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process, expansion, coalescence, contraction and closure, and describe the molecular dynamics of each phase. Specifically, we find that myosin, E-cadherin, Echinoid, the plasma membrane, microtubules and the Cdc42 small GTPase respond dynamically during wound repair. We demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular protrusions and actomyosin ring contraction along the leading edge of the wound.

The Drosophila STUbL protein Degringolade limits HES functions during embryogenesis.

Degringolade (Dgrn) encodes a Drosophila SUMO-targeted ubiquitin ligase (STUbL) protein similar to that of mammalian RNF4. Dgrn facilitates the ubiquitylation of the HES protein Hairy, which disrupts the repressive activity of Hairy by inhibiting the recruitment of its cofactor Groucho. We show that Hey and all HES family members, except Her, interact with Dgrn and are substrates for its E3 ubiquitin ligase activity. Dgrn displays dynamic subcellular localization, accumulates in the nucleus at times when HES family members are active and limits Hey and HES family activity during sex determination, segmentation and neurogenesis. We show that Dgrn interacts with the Notch signaling pathway by it antagonizing the activity of E(spl)-C proteins. dgrn null mutants are female sterile, producing embryos that arrest development after two or three nuclear divisions. These mutant embryos exhibit fragmented or decondensed nuclei and accumulate higher levels of SUMO-conjugated proteins, suggesting a role for Dgrn in genome stability.

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds.

To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.

Two Septin complexes mediate actin dynamics during cell wound repair.

Cells have robust wound repair systems to prevent further damage or infection and to quickly restore cell cortex integrity when exposed to mechanical and chemical stress. Actomyosin ring formation and contraction at the wound edge are major events during closure of the plasma membrane and underlying cytoskeleton during cell wound repair. Here, we show that all five Drosophila Septins are required for efficient cell wound repair. Based on their different recruitment patterns and knockdown/mutant phenotypes, two distinct Septin complexes, Sep1/Sep2/Pnut and Sep4/Sep5/Pnut, are assembled to regulate actin ring assembly, contraction, and remodeling during the repair process. Intriguingly, we find that these two Septin complexes have different F-actin bending activities. In addition, we find that Anillin regulates the recruitment of only one of two Septin complexes upon wounding. Our results demonstrate that two functionally distinct Septin complexes work side by side to discretely regulate actomyosin ring dynamics during cell wound repair.

Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino.

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.

Cytoskeleton responses in wound repair.

Wound repair on the cellular and multicellular levels is essential to the survival of complex organisms. In order to avoid further damage, prevent infection, and restore normal function, cells and tissues must rapidly seal and remodel the wounded area. The cytoskeleton is an important component of wound repair in that it is needed for actomyosin contraction, recruitment of repair machineries, and cell migration. Recent use of model systems and high-resolution microscopy has provided new insight into molecular aspects of the cytoskeletal response during wound repair. Here we discuss the role of the cytoskeleton in single-cell, embryonic, and adult repair, as well as the striking resemblance of these processes to normal developmental events and many diseases.

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins.

BACKGROUND: Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. RESULTS: We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. CONCLUSIONS: All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

Wound repair: Two distinct Rap1 pathways close the gap.

Groups of cells often coordinate their movements during normal development, cancer invasion, and wound repair. These coordinated migrations require dynamic cytoskeleton and cell-junction remodeling. Two distinct Rap1 pathways are required to regulate this dynamic remodeling for rapid wound closure.

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds.

To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, we find that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, our results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.

Coordinated efforts of different actin filament populations are needed for optimal cell wound repair.

Cells are subjected to a barrage of daily insults that often lead to their cortices being ripped open and requiring immediate repair. An important component of the cell's repair response is the formation of an actomyosin ring at the wound periphery to mediate its closure. Here we show that inhibition of myosin or the linear actin nucleation factors Diaphanous and/or dishevelled associated activator of morphogenesis results in a disrupted contractile apparatus and delayed wound closure. We also show that the branched actin nucleators WASp and SCAR function nonredundantly as scaffolds to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and to slow wound closure. Removing linear and branched actin simultaneously results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear F-actin filaments that undergo a chiral swirling movement to close the wound, uncovering a new mechanism of cell wound closure. Taken together, we demonstrate the roles of different actin substructures that are required for optimal actomyosin ring formation and the extraordinary resilience of the cell to undergo wound repair when it is unable to form different subsets of these substructures.

Scar/WAVE has Rac GTPase-independent functions during cell wound repair.

Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular structures such as lamellipodia and contractile actomyosin rings. Wiskott-Aldrich Syndrome (WAS) family proteins are downstream effectors of Rho family GTPases that usually function in a one-to-one correspondence to regulate branched actin nucleation. In particular, the WAS protein Scar/WAVE has been shown to exhibit one-to-one correspondence with Rac GTPase. Here we show that Rac and SCAR are recruited to cell wounds in the Drosophila repair model and are required for the proper formation and maintenance of the dynamic actomyosin ring formed at the wound periphery. Interestingly, we find that SCAR is recruited to wounds earlier than Rac and is still recruited to the wound periphery in the presence of a potent Rac inhibitor. We also show that while Rac is important for actin recruitment to the actomyosin ring, SCAR serves to organize the actomyosin ring and facilitate its anchoring to the overlying plasma membrane. These differing spatiotemporal recruitment patterns and wound repair phenotypes highlight the Rac-independent functions of SCAR and provide an exciting new context in which to investigate these newly uncovered SCAR functions.