A Method to Inflict Closed Head Traumatic Brain Injury in <em>Drosophila</em>

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

University of Wisconsin-Madison

Deacetylation of histones by the SIN3 complex is a major mechanism utilized in eukaryotic organisms to repress transcription. Presumably, developmental and cellular phenotypes resulting from mutations in SIN3 are a consequence of altered transcription of SIN3 target genes. Therefore, to understand the molecular mechanisms underlying SIN3 mutant phenotypes in Drosophila, we used full-genome oligonucleotide microarrays to compare gene expression levels in wild type Drosophila tissue culture cells versus SIN3-deficient cells generated by RNA interference. Of the 13,137 genes tested, 364 were induced and 35 were repressed by loss of SIN3. The approximately 10-fold difference between the number of induced and repressed genes suggests that SIN3 plays a direct role in regulating these genes. The identified genes are distributed throughout euchromatic regions but are preferentially excluded from heterochromatic regions of Drosophila chromosomes suggesting that the SIN3 complex can only access particular chromatin structures. A number of cell cycle regulators were repressed by loss of SIN3, and functional studies indicate that repression of string, encoding the Drosophila homologue of the yeast CDC25 phosphatase, contributes to the G2 cell cycle delay of SIN3-deficient cells. Unexpectedly, a substantial fraction of genes induced by loss of SIN3 is involved in cytosolic and mitochondrial energy-generating pathways and other genes encode components of the mitochondrial translation machinery. Increased expression of mitochondrial proteins in SIN3-deficient cells is manifested in an increase in mitochondrial mass. Thus, SIN3 may play an important role in regulating mitochondrial respiratory activity.

The SIN3 deacetylase complex represses genes encoding mitochondrial proteins: implications for the regulation of energy metabolism.

Cambiamenti dinamici nella struttura della cromatina, indotta dalla modificazione post traduzionale di istoni, giocano un ruolo fondamentale nella regolazione della trascrizione eucariotica. Qui riportiamo quello istone H2B &egrave; fosforilata a evolutivamente conservato Ser33 (H2B-S33) dal dominio carbossi-terminale chinasi (CTK) della subunit&agrave; della drosofila TFIID TAF1. Fosforilazione di H2B-S33 presso il promotore della stringa gene regolatore ciclo cellulare e il gigante del gene di segmentazione coincide con l&#39;attivazione di transcriptional. Eliminazione di attivit&agrave; TAF1 CTK in drosofila cellule ed embrioni riduce l&#39;attivazione trascrizionale e fosforilazione di H2B-S33. Questi dati rivelano che H2B-S33 &egrave; un substrato fisiologico per la CTK TAF1 e che la fosforilazione H2B-S33 &egrave; essenziale per gli eventi di attivazione trascrizionale che promuovono lo sviluppo e la progressione del ciclo cellulare.

TAF1 attiva la trascrizione di fosforilazione della serina 33 in istone H2B.

&#x67D3;&#x8272;&#x8D28;&#x7ED3;&#x6784;&#xFF0C;&#x8BF1;&#x5BFC;&#x7684;&#x7EC4;&#x86CB;&#x767D;&#xFF0C;&#x7FFB;&#x8BD1;&#x540E;&#x4FEE;&#x9970;&#x7684;&#x52A8;&#x6001;&#x53D8;&#x5316;&#x5728;&#x8C03;&#x8282;&#x771F;&#x6838;&#x8F6C;&#x5F55;&#x7684;&#x57FA;&#x672C;&#x4F5C;&#x7528;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x7684;&#x7EC4;&#x86CB;&#x767D; H2B &#x78F7;&#x9178;&#x5316;&#x5728;&#x8FDB;&#x5316;&#x4FDD;&#x5B88; Ser33 (H2B S33) &#x7684;&#x679C;&#x8747; TFIID TAF1 &#x4E9A;&#x57FA;&#x7684;&#x7FA7;&#x57FA;&#x672B;&#x7AEF;&#x6FC0;&#x9176;&#x57DF; &#xFF08;&#x6377;&#x901A;&#xFF09;&#x3002;&#x78F7;&#x9178;&#x5316; H2B S33 &#x5728;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x8C03;&#x63A7;&#x57FA;&#x56E0;&#x5B57;&#x7B26;&#x4E32;&#x548C;&#x5206;&#x5272;&#x57FA;&#x56E0;&#x5DE8;&#x5934;&#x542F;&#x52A8;&#x5B50;&#x4E0E;&#x8F6C;&#x5F55;&#x6FC0;&#x6D3B;&#x4E0D;&#x8C0B;&#x800C;&#x5408;&#x3002;TAF1 &#x6377;&#x901A;&#x6D3B;&#x52A8;&#x5728;&#x679C;&#x8747;&#x7EC6;&#x80DE;&#x548C;&#x80DA;&#x80CE;&#x7684;&#x6D88;&#x9664;&#x51CF;&#x5C11;&#x4E86;&#x8F6C;&#x5F55;&#x6FC0;&#x6D3B;&#x548C; H2B S33 &#x7684;&#x78F7;&#x9178;&#x5316;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; H2B S33 &#x662F; TAF1 &#x6377;&#x901A;&#x751F;&#x7406;&#x57FA;&#x677F;&#x548C; H2B S33 &#x78F7;&#x9178;&#x5316;&#x662F;&#x8F6C;&#x5F55;&#x6FC0;&#x6D3B;&#x4E8B;&#x4EF6;&#xFF0C;&#x4EE5;&#x4FC3;&#x8FDB;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x8FDB;&#x7A0B;&#x548C;&#x53D1;&#x5C55;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

TAF1 &#x901A;&#x8FC7;&#x78F7;&#x9178;&#x5316;&#x4E1D;&#x6C28;&#x9178; 33 &#x4E2D;&#x7EC4;&#x86CB;&#x767D; H2B &#x6FC0;&#x6D3B;&#x8F6C;&#x5F55;&#x3002;

Dynamic changes in chromatin structure, induced by posttranslational modification of histones, play a fundamental role in regulating eukaryotic transcription. Here we report that histone H2B is phosphorylated at evolutionarily conserved Ser33 (H2B-S33) by the carboxyl-terminal kinase domain (CTK) of the Drosophila TFIID subunit TAF1. Phosphorylation of H2B-S33 at the promoter of the cell cycle regulatory gene string and the segmentation gene giant coincides with transcriptional activation. Elimination of TAF1 CTK activity in Drosophila cells and embryos reduces transcriptional activation and phosphorylation of H2B-S33. These data reveal that H2B-S33 is a physiological substrate for the TAF1 CTK and that H2B-S33 phosphorylation is essential for transcriptional activation events that promote cell cycle progression and development.

TAF1 activates transcription by phosphorylation of serine 33 in histone H2B.

Les changements dynamiques dans la structure de la chromatine, induite par modification post-traductionnelle des histones, jouent un r&#xF4;le fondamental dans la r&#xE9;gulation de transcription chez les eucaryotes. Nous rapportons ici que histone H2B est phosphoryl&#xE9;e au niveau de l&#x26;#39;&#xE9;volution, conserv&#xE9; Ser33 (H2B-S33) par le domaine carboxy-terminal kinase (CTK) de la sous-unit&#xE9; chez la drosophile TFIID TAF1. Phosphorylation de H2B-S33 au promoteur de la cha&#xEE;ne du cycle cellulaire et g&#xE8;ne r&#xE9;gulateur du g&#xE8;ne de segmentation g&#xE9;ant co&#xEF;ncide avec l&#x26;#39;activation transcriptionnelle. &#xC9;limination de l&#x26;#39;activit&#xE9; TAF1 CTK dans les cellules de drosophile et les embryons r&#xE9;duit l&#x26;#39;activation transcriptionnelle et la phosphorylation de H2B-S33. Ces donn&#xE9;es r&#xE9;v&#xE8;lent que H2B-S33 est un substrat physiologique de la CTK TAF1 et que la phosphorylation H2B-S33 est essentiel pour les &#xE9;v&#xE9;nements d&#x26;#39;activation transcriptionnelle qui favorisent la progression du cycle cellulaire et le d&#xE9;veloppement.

TAF1 active la transcription par phosphorylation de la s&#xE9;rine 33 dans histone H2B

TAF1 aktiviert die Transkription durch die Phosphorylierung von Serin 33 in Histon H2B.

&#x30D2;&#x30B9;&#x30C8;&#x30F3;&#x306E;&#x7FFB;&#x8A33;&#x5F8C;&#x4FEE;&#x98FE;&#x306B;&#x3088;&#x308A;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x308B;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x69CB;&#x9020;&#x306E;&#x52D5;&#x7684;&#x5909;&#x5316;&#x306F;&#x3001;&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x8EE2;&#x5199;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30D2;&#x30B9;&#x30C8;&#x30F3;H2B&#x304C;&#x3001;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;TFIID&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;TAF1&#x306E;&#x30AB;&#x30EB;&#x30DC;&#x30AD;&#x30B7;&#x30EB;&#x672B;&#x7AEF;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#xFF08;CTK&#xFF09;&#x304C;&#x9032;&#x5316;&#x7684;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;Ser33&#xFF08;H2B-S33&#xFF09;&#x304C;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x8ABF;&#x7BC0;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6587;&#x5B57;&#x5217;&#x3068;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C6;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x907A;&#x4F1D;&#x5B50;&#x5DE8;&#x4EBA;&#x306E;&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x3067;H2B-S33&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306F;&#x3001;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x4E00;&#x81F4;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x7D30;&#x80DE;&#x3068;&#x80DA;&#x306B;&#x304A;&#x3051;&#x308B;TAF1 CTK&#x6D3B;&#x6027;&#x306E;&#x9664;&#x53BB;&#x306F;&#x3001;H2B-S33&#x306E;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3092;&#x4F4E;&#x6E1B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;H2B-S33&#x306F;&#x3001;TAF1 CTK&#x306E;&#x751F;&#x7406;&#x7684;&#x57FA;&#x8CEA;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x305D;&#x306E;H2B-S33&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306F;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306E;&#x9032;&#x884C;&#x3068;&#x958B;&#x767A;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x5316;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x3002;

TAF1&#x306F;&#x3001;&#x30D2;&#x30B9;&#x30C8;&#x30F3;H2B&#x306E;33&#x306E;&#x30BB;&#x30EA;&#x30F3;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x8EE2;&#x5199;&#x3092;&#x6D3B;&#x6027;&#x5316;

Chromatin &#xAD6C;&#xC870;, &#xD788;&#xC2A4;&#xD1A4;, posttranslational &#xC218;&#xC815;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB41C; &#xB3D9;&#xC801; &#xBCC0;&#xD654;&#xB294; &#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC758; &#xC804;&#xC0AC; &#xC870;&#xC808;&#xC5D0; &#xADFC;&#xBCF8;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xC5EC;&#xAE30;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xD788;&#xC2A4;&#xD1A4; h2b&#xC5D0; &#xB300; &#xD55C; phosphorylated &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xC874; Ser33 &#xBCF4;&#xACE0;&#xC11C; (H2B S33) &#xCD08;&#xD30C;&#xB9AC; TFIID &#xC18C; &#xB2E8;&#xC704; taf1&#xC758; carboxyl &#xD130;&#xBBF8;&#xB110; &#xD0A4; &#xB3C4;&#xBA54;&#xC778; (CTK)&#xC5D0; &#xC758;&#xD574;. &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xC720;&#xC804;&#xC790;&#xB97C; &#xADDC;&#xC81C; &#xBB38;&#xC790;&#xC5F4; &#xBC0F; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xD654; &#xC9C4; &#xAC70; &#xB300; &#xD55C; &#xBC1C;&#xAE30;&#xC778;&#xC5D0; H2B s33&#xC758; &#xC778; &#xC0B0;&#xD654;&#xB294; transcriptional &#xD65C;&#xC131;&#xD654;&#xC640; &#xC77C;&#xCE58; &#xD55C;&#xB2E4;. TAF1 CTK &#xD65C;&#xB3D9; &#xCD08;&#xD30C;&#xB9AC; &#xC138;&#xD3EC; &#xBC0F; &#xBC30;&#xC544;&#xC758; &#xC81C;&#xAC70; transcriptional &#xD65C;&#xC131;&#xD654; &#xBC0F; H2B s33&#xC758; &#xC778; &#xC0B0;&#xD654;&#xB97C; &#xAC10;&#xC18C; &#xC2DC;&#xD0A8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xACF5;&#xAC1C; H2B S33 TAF1 CTK&#xC5D0; &#xB300; &#xD55C; &#xC0DD;&#xB9AC; &#xC801; &#xAE30;&#xD310; &#xACE0; H2B S33 &#xC778; &#xC0B0;&#xD654; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xC9C4;&#xD589; &#xBC0F; &#xAC1C;&#xBC1C;&#xC744; &#xCD09;&#xC9C4; &#xD558;&#xB294; transcriptional &#xC815;&#xD488; &#xC778;&#xC99D; &#xC774;&#xBCA4;&#xD2B8;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Taf1&#xB294; &#xD788;&#xC2A4;&#xD1A4; h2b&#xC5D0;&#xC5D0;&#xC11C; &#xB5A0;&#xB4E4;&#xACE0; 33&#xC758; &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xC804;&#xC0AC;&#xB97C; &#xD65C;&#xC131;&#xD654;&#xD569;&#xB2C8;&#xB2E4;.

Los cambios din&#xE1;micos en la estructura de la cromatina, inducida por la modificaci&#xF3;n postraduccional de las histonas, juegan un papel fundamental en la regulaci&#xF3;n de la transcripci&#xF3;n eucari&#xF3;tica. Aqu&#xED; mostramos que la histona H2B es fosforilados en Ser33 conservadas evolutivamente (H2B-S33) por el dominio de la quinasa carboxilo-terminal (CTK) de la subunidad de TFIID TAF1 Drosophila. La fosforilaci&#xF3;n de H2B-S33 en el promotor de la cadena del ciclo celular gen regulador y la gigante segmentaci&#xF3;n gen coincide con la activaci&#xF3;n transcripcional. Eliminaci&#xF3;n de la actividad TAF1 CTK en c&#xE9;lulas de Drosophila y de los embriones reduce la activaci&#xF3;n de la transcripci&#xF3;n y la fosforilaci&#xF3;n de H2B-S33. Estos datos revelan que H2B-S33 es un sustrato fisiol&#xF3;gico para el CTK TAF1 y que la fosforilaci&#xF3;n de H2B-S33 es esencial para los eventos de activaci&#xF3;n de la transcripci&#xF3;n que promueven la progresi&#xF3;n del ciclo celular y el desarrollo.

TAF1 activa la transcripci&#xF3;n por la fosforilaci&#xF3;n de la serina 33 en la histona H2B

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, we analyzed the splicing of the Drosophila melanogaster TAF1 pre-mRNA. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. We demonstrate that TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells was mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing was mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing.

ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage.

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, we generated a Drosophila model of A-T. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, we hypothesize that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T.

Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia.

FK-228 is a potent histone deacetylase (HDAC) inhibitor with tremendous therapeutic potential against a wide array of human cancers. We describe the development of analogs that share FK-228's novel mechanism of activation and HDAC inhibition.

Cyclic Disulfides as Functional Mimics of the Histone Deacetylase Inhibitor FK-228.

Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

The effect of ATM knockdown on ionizing radiation-induced neuronal cell cycle reentry in Drosophila.

To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE). The TCE functions in the 5' untranslated region of Mst(3)CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp) genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID) subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and understanding the dual role of the TCE in translational and transcriptional regulation.

The Drosophila Translational Control Element (TCE) is required for high-level transcription of many genes that are specifically expressed in testes.

Neurodegeneration is a hallmark of the human disease ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the Nuclear Factor-κB (NF-κB) transcription factor Relish, a key regulator of the Immune deficiency (Imd) IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-κB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-κB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-κB protein in glial cells.

The innate immune response transcription factor relish is necessary for neurodegeneration in a Drosophila model of ataxia-telangiectasia.

Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

A Drosophila model of closed head traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. Our genome-wide association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood-brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction.

Rebeccah J. Katzenberger

Department of Cell and Regenerative Biology

University of Wisconsin-Madison

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Rebeccah J. Katzenberger

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Regenerative Biology

University of Wisconsin-Madison

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Department of Cell and Regenerative Biology