Kibra Functions as a Tumor Suppressor Protein that Regulates Hippo Signaling in Conjunction with Merlin and Expanded

SummaryThe Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra as another upstream component of the Hippo signaling pathway. We show that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dcr-1 also rescued the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we showed that genetic inhibition of Bel function led to the de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited. Our findings have demonstrated a novel involvement of Bel in regulating transgene expression and its loss triggers a transgene silencing mechanism mediated by protein regulators implicated in RNA processing, piRNA biogenesis, chromatin remodeling and the microRNA pathway

Interrogating the Function of Metazoan Histones using Engineered Gene Clusters

Histones and their post-translational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have non-histone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a facile platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike conclusions drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity during development. These findings highlight the power of engineering histones to interrogate genome structure and function in animals

Principles of Biology I & II (ATLM)

This Grants Collection for Principles of Biology I & II was created under a Round Twelve ALG Textbook Transformation Grant. Affordable Learning Georgia Grants Collections are intended to provide faculty with the frameworks to quickly implement or revise the same materials as a Textbook Transformation Grants team, along with the aims and lessons learned from project teams during the implementation process. Documents are in .pdf format, with a separate .docx (Word) version available for download. Each collection contains the following materials: Linked Syllabus Initial Proposal Final Reporthttps://oer.galileo.usg.edu/biology-collections/1027/thumbnail.jp

Prp22 and Spliceosome Components Regulate Chromatin Dynamics in Germ-Line Polyploid Cells

<div><p>During <i>Drosophila</i> oogenesis, the endopolyploid nuclei of germ-line nurse cells undergo a dramatic shift in morphology as oogenesis progresses; the easily-visible chromosomes are initially polytenic during the early stages of oogenesis before they transiently condense into a distinct ‘5-blob’ configuration, with subsequent dispersal into a diffuse state. Mutations in many genes, with diverse cellular functions, can affect the ability of nurse cells to fully decondense their chromatin, resulting in a ‘5-blob arrest’ phenotype that is maintained throughout the later stages of oogenesis. However, the mechanisms and significance of nurse-cell (NC) chromatin dispersal remain poorly understood. Here, we report that a screen for modifiers of the 5-blob phenotype in the germ line isolated the spliceosomal gene <i>peanuts</i>, the <i>Drosophila</i> Prp22. We demonstrate that reduction of spliceosomal activity through loss of <i>peanuts</i> promotes decondensation defects in NC nuclei during mid-oogenesis. We also show that the Prp38 spliceosomal protein accumulates in the nucleoplasm of nurse cells with impaired <i>peanuts</i> function, suggesting that spliceosomal recycling is impaired. Finally, we reveal that loss of additional spliceosomal proteins impairs the full decondensation of NC chromatin during later stages of oogenesis, suggesting that individual spliceosomal subcomplexes modulate expression of the distinct subset of genes that are required for correct morphology in endopolyploid nurse cells.</p></div

Transgenic overexpression of Pea is silenced in <i>pea</i>-null NC nuclei.

<p>(A-A″) Ovariole that shows the expression of GFP-tagged Pea in the nuclei and the cytoplasm of the nurse cells. (B-B″) Stage-4 control NC nuclei (hRFP, arrow) expresses PeaGFP strongly. Adjacent <i>pea^c89</i> whole germ-line clone (no hRFP) arrests at an earlier stages and does not express PeaGFP.</p

Whole <i>pea^c89</i> germ-line clones arrest at early stages of <i>Drosophila</i> oogenesis.

<p>(A-A″) In wild-type ovarioles, NC nuclei (DAPI; blue) and the nucleolus (Prp38; green) both undergo a transformation from a polytenic chromatin state (compact nucleolus) into a transient condensed phase (partial dispersal of the nucleolus), before a final dispersed state of both NC chromatin and nucleoli. (B-B″) <i>pea^c89</i> mutant germ-line clones (detected by loss of the histone-RFP marker) invariably arrest at approximately stage 2 of oogenesis, as seen by a severely compact nucleolus (Fibrillarin; green). (C-C″) Closeup of the arrested egg chamber reveals a nucleolus (Fibrillarin; green) surrounded by visible polytenic NC nuclei (DAPI; blue). (D-D″) Knockdown of Pea expression outside of the germarium causes frequent arrest during stages 4-5. (E-E″) Stage-8 <i>pea</i>-RNAi egg-chamber with NCCD failure.</p

Schematic of NC chromatin states during <i>Drosophila</i> oogenesis.

<p>(A) Early oogenesis (up to stage 1) marks the formation and budding of the egg chamber from the germarium. The 16-cell syncytium arises from 4 mitotic divisions with incomplete cytokinesis; one cell becomes an oocyte which arrests during meiotic prophase I (with the nucleus condensing to a transcriptionally-quiescent state), while the other fifteen cells abort the meiotic cell cycle and initiate the first endocycle (E1) as polytenic nurse cells. (B) During middle oogenesis (stage 2–10b), NC nuclei facilitate chromosome territory formation by transiently condensing into a ‘5-blob’ configuration during endocycle 5 (E5) and dispersing into a polytene-polyploid state by endocycle 6 (E6) for the remainder of oogenesis. Transient condensation and dispersal occur during stages 4–6 of oogenesis. (C) During late oogenesis (stages 11–14), nurse cells undergo programmed cell death after achieving up to 12 endocycles (E12), dumping their cytoplasmic contents into the oocyte; the oocyte transiently progresses to metaphase I during stage 13, before arrest in preparation for fertilization and egg activation.</p

NC nuclei null for <i>peanuts</i> function display altered spliceosome localization.

<p>(A-A″) Wild-type NC nuclei (marked with histone-RFP, [+])undergoing dispersal display homogenous Prp38 staining, while <i>pea^c89</i> mutant NC nuclei (no histone-RFP, [-]) retain Prp38 localization (green) in interchromatin spaces and a subset of NC chromatin (compare arrow and arrowhead, respectively). Earlier adjacent egg chamber displays wild-type polytene NC nuclei with homogenous Prp38 staining (marked with histone-RFP, [+]). (B-B″) In a stage-7 egg chamber, <i>pea</i>-null mutant NC nuclei (arrowhead) remain undispersed and continue to exhibit enhanced Prp38 localization on chromatin in contrast to wild-type dispersed NC nuclei (arrow).</p

Identification of <i>pea</i> as a putative interactor of <i>otu</i>.

<p>(A-A″) Wild-type stage-9 egg chamber with dispersed NC chromatin and a fully-reticulated nucleoli as marked by fibrillarin (red). (B-B″) <i>otu¹³</i>/+ stage-9 egg chambers with NCCD defects and moderately-reticulated nucleoli. (C-C″) In stage-9 <i>otu¹³</i>/+; <i>Df7130</i>/+ egg chambers (deficiency uncovering <i>pea</i>), moderate enhancement of the 5-blob phenotype is seen along with Fibrillarin accumulation at distal edges of NC nuclei. (D-D″) In <i>otu¹³</i>/+; <i>pea¹</i>/+ egg chambers, moderate enhancement of the 5-blob configuration plus significant retainment of a globular Fibrillarin pattern is frequently seen.</p