Beat Suter

Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning.

Here we show that the Drosophila homologue of Lissencephaly-1, DLis-1, acts together with Bicaudal-D (Bic-D), Egalitarian (Egl), dynein and microtubules to determine oocyte identity. DLis-1 is further required for nurse-cell-to-oocyte transport during oocyte growth, and for the positioning of the nucleus in the oocyte. Immunostaining of DLis-1 protein reveals a cortical localization that is independent of microtubules. DLis-1 may function in this position as a cortical anchor for the other nuclear-localization factors. DLis-1 and Bic-D are further required for nuclear localization in the developing nervous system, indicating that homologues of Bic-D, dynein and Egl-like proteins may also be involved in vertebrate neural migration and that their absence may cause a Miller–Dieker-like lissencephaly.

Xpd/Ercc2 regulates CAK activity and mitotic progression

General transcription factor IIH (TFIIH) consists of nine subunits: cyclin-dependent kinase 7 (Cdk7), cyclin H and MAT1 (forming the Cdk-activating-kinase or CAK complex), the two helicases Xpb/Hay and Xpd, and p34, p44, p52 and p62 (refs 1–3). As the kinase subunit of TFIIH, Cdk7 participates in basal transcription by phosphorylating the carboxy-terminal domain of the largest subunit of RNA polymerase II. As part of CAK, Cdk7 also phosphorylates other Cdks, an essential step for their activation. Here we show that the Drosophila TFIIH component Xpd negatively regulates the cell cycle function of Cdk7, the CAK activity. Excess Xpd titrates CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality, whereas a decrease in Xpd results in increased CAK activity and cell proliferation. Moreover, Xpd is downregulated at the beginning of mitosis when Cdk1, a cell cycle target of Cdk7, is most active. Downregulation of Xpd thus seems to contribute to the upregulation of mitotic CAK activity and to regulate mitotic progression positively. Simultaneously, the downregulation of Xpd might be a major mechanism of mitotic silencing of basal transcription.

Immune-Deficient Drosophila melanogaster: A Model for the Innate Immune Response to Human Fungal Pathogens

We explored the host-pathogen interactions of the human opportunistic fungus Candida albicans using Drosophila melanogaster. We established that a Drosophila strain devoid of functional Toll receptor is highly susceptible to the human pathogen C. albicans. Using this sensitive strain, we have been able to show that a set of specific C. albicans mutants of different virulence in mammalian infection models are also impaired in virulence in Drosophila and remarkably display the same rank order of virulence. This immunodeficient insect model also revealed virulence properties undetected in an immunocompetent murine model of infection. The genetic systems available in both host and pathogen will enable the identification of host-specific components and C. albicans genes involved in the host-fungal interplay.

T-loop phosphorylation stabilizes the CDK7–cyclin H–MAT1 complex in vivo and regulates its CTD kinase activity

Cyclin‐dependent kinase (CDK)7–cyclin H, the CDK‐activating kinase (CAK) and TFIIH‐associated kinase in metazoans can be activated in vitro through T‐loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T‐loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T‐loop. Mutation of both phosphorylation sites causes temperature‐dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7–cyclin H–MAT1 complex towards the C‐terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate‐specific increase in activity caused by T‐loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH‐associated CDK7, and thereby regulate transcription.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo

ABSTRACT TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

Lissencephaly-1 promotes the interaction of dynein with dynactin and facilitates motor complex association with mRNA cargos.

The Ste20-like kinase misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing drosophila eye

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. Here, we show that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. We propose that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells.

Xpd, a Structural Bridge and a Functional Link

Xpd, one of nine polypeptide subunits of the general transcriptionffactor TFIIH plays an organizing role in TFIIH assembly. This structural function in protein complex assembly appears to be used by cells in a dynamic way to regulate and co-ordinate the diverse cellular functions of the different sub-complexes in transcription, DNA repair and cell cycle progression.

Drosophila Xpd Regulates Cdk7 Localization, Mitotic Kinase Activity, Spindle Dynamics, and Chromosome Segregation

The trimeric CAK complex functions in cell cycle control by phosphorylating and activating Cdks while TFIIH-linked CAK functions in transcription. CAK also associates into a tetramer with Xpd, and our analysis of young Drosophila embryos that do not require transcription now suggests a cell cycle function for this interaction. xpd is essential for the coordination and rapid progression of the mitotic divisions during the late nuclear division cycles. Lack of Xpd also causes defects in the dynamics of the mitotic spindle and chromosomal instability as seen in the failure to segregate chromosomes properly during ana- and telophase. These defects appear to be also nucleotide excision repair (NER)–independent. In the absence of Xpd, misrouted spindle microtubules attach to chromosomes of neighboring mitotic figures, removing them from their normal location and causing multipolar spindles and aneuploidy. Lack of Xpd also causes changes in the dynamics of subcellular and temporal distribution of the CAK component Cdk7 and local mitotic kinase activity. xpd thus functions normally to re-localize Cdk7(CAK) to different subcellular compartments, apparently removing it from its cell cycle substrate, the mitotic Cdk. This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER. Xpd dynamically localizes Cdk7/CAK to and away from subcellular substrates, thereby controlling local mitotic kinase activity. Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system. This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes.