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Transcriptional regulation of tracheal fusion cell gene expression

Transcriptional regulation of tracheal fusion cell gene expression Isolate enhancer of three fusion cell expressed genes: dys, CG15252, CG13196 Identify CRM required for fusion cell gene expression Transcriptional pathways in terminally differentiating neurons

mahogony-fernandez
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Transcriptional regulation of tracheal fusion cell gene expression

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  1. Transcriptional regulation of tracheal fusion cell gene expression • Isolate enhancer of three fusion cell expressed genes: • dys, CG15252, CG13196 • Identify CRM required for fusion cell gene expression • Transcriptional pathways in terminally differentiating neurons • Glutamatergic (H-cell sib )and GABAergic (iVUM) neurons • Transcriptional regulation of vglut expression in H-cell sib • Transcriptional regulation gene expression in iVUM neurons

  2. Identify dys Fusion cell Enhancer B2.7 P155 C1.7 T523 dys early enhancer (S12-S17) dys late enhancer (S14-S17)

  3. Sequence alignment of dys155

  4. Motifs Required for dys Activation 1 2 345 6 7 dys155 GATCGG 1 2 TTAAATCAAA 1 2 3 3 GCGCG 4 TGCCTGAC 5 Ets 6 GACCCAAAAC 4 5 6 7 POU 7

  5. Ets protein Pointed is required for dys expression in fusion cells Dys WT pnt

  6. Sequence of alignment of dys523 late enhancer

  7. dys late enhancer is auto-regulated by Dys protein P155 C1.7 dys dys

  8. Identify CG13196 Fusion cell Enhancer CG13196 D92

  9. Sequence alignment of CG16196 enhancer

  10. Motifs Required for CG13196 Expression Motif 8 Motif 8 TCGTG-C AAG TCGTG-D GFP Dys Merge Unmutated D92muC D92muD D92muAAG D92muMotif8

  11. Identify CG15252 Fusion cell Enhancer CG15252 C100

  12. Sequence alignment of CG15252 enhancer

  13. Motifs Required for CG15252 Activation CG15252 Motif 7 AAG AAG C100 GFP Dys Merge TCGTG-C TCGTG-D Unmutated C100muC C100muD C100muAAG C100muMotif9

  14. Co-activator sequences required for CG13196 expression x x C100muCD CG15252 A350 CG13196 A350muBC CG13196 C100 CG15252

  15. Transcriptional pathways in terminally differentiating neurons Sim Sim + En

  16. en * Gene expression porfile in Glutamatergic and GABAergic interneurons TF gene expression iVUM sim per fkh nvy Lim1 H-cell sib sim per fkh nvy Lim1 NF gene expression H-cell sib Vglut GluR1 5-HT 1A CG13565 iVUM Gad1 VIAAT(CG8394) CG15236

  17. Transcriptional regulation of vglut expression in H-cell sib vglut expression in sim, fkh, Lim1, nvy, per mutants? vglut H-cell sib enhancer, identify potential transcription factor binding sites?

  18. Lim1 does not regulate glutamatergic phenotype of H-cell sib H-cell sib sim per fkh nvy Lim1 Lim1/FM7 VGlut Lim1 VGlut/CG13565/En Lim1 VGlut Lim1 VGlut/CG13565/En

  19. vglut expression in Forkhead mutant Sim vglut Merge H-cell sib sim per fkh nvy Lim1 WT fkh

  20. VGlut Genomic Locus 14.5 kb CG3557 4.8 kb 2.8 kb H-cell sib, iVUM, mVUM 2.5 kb mVUM 0.47 kb 3.7 kb 2.7 kb 0.8 kb 0.45 kb 0.6 kb 2.4 kb 0.3 kb 0.3 kb 4.4 kb

  21. VGlut intron 2 contains H-cell sib, iVUM mVUM CG3557 0.6 kb H-cell sib, iVUM, mVUM Sim GFP En Merge iVUMH H-cell sib mVUM

  22. VGlut intron 2-b fragment contains H-cell sib enhancer CG3557 Intron 2-b 0.3kb Sim GFP En Merge

  23. Future Direction on vglut expression in H-cell sib • Role of Fkh in H-cell sib • Cell death in H-cell sib? • caspase-3 staining in sim-tau GFP embryos • Morphology of H-cell sib in sim-tau GFP embryos • H-cell sib gene expression in H99 mutant embryos • vglut expression in sim, nvy and per mutants • Further identify vglut H-cell sib enhancer • mutate potential Fkh binding site in intron2b

  24. en * Transcriptional regulation of gene expression in iVUM TF gene expression iVUM sim per fkh nvy Lim1 H-cell sib sim per fkh nvy Lim1 NF gene expression H-cell sib Vglut GluR1 5-HT 1A CG13565 iVUM Gad1 VIAAT(CG8394) CG15236

  25. Gad1 expression is not regulated by Fkh Gad1 en Sim Merge WT Fkh

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