Transcriptional regulation of tracheal fusion cell gene expression Isolate enhancer of three fusion cell expressed genes: dys, CG15252, CG13196 Identify.

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Presentation transcript:

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

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

Sequence alignment of dys155

GATCGG TTAAATCAAA TGCCTGAC Ets GACCCAAAAC GCGCG POU Motifs Required for dys Activation dys155

WTpnt Dys Ets protein Pointed is required for dys expression in fusion cells

Sequence of alignment of dys523 late enhancer

dys late enhancer is auto-regulated by Dys protein dys P155C1.7

D92 CG13196 Identify CG13196 Fusion cell Enhancer

Sequence alignment of CG16196 enhancer

GFPDysMerge Unmutated D92muC D92muD D92muAAG D92muMotif8 TCGTG-DTCGTG-CAAG Motif 8 Motifs Required for CG13196 Expression

CG15252 C100 Identify CG15252 Fusion cell Enhancer

Sequence alignment of CG15252 enhancer

CG15252 C100 TCGTG-DTCGTG-C AAGMotif 7AAG GFPDysMerge Unmutated C100muC C100muD C100muAAG C100muMotif9 Motifs Required for CG15252 Activation

xx A350muBC CG13196 C100 CG15252 C100muCD CG15252 A350 CG13196 Co-activator sequences required for CG13196 expression

Transcriptional pathways in terminally differentiating neurons Sim Sim + En

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

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

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

vglut expression in Forkhead mutant WT fkh SimvglutMerge H-cell sib sim per fkh nvy Lim1

VGlut Genomic Locus 14.5 kb 2.4 kb 2.5 kb 2.8 kb 3.7 kb 2.7 kb 4.8 kb 0.8 kb 0.6 kb 0.45 kb 0.47 kb 4.4 kb 0.3 kb CG3557

0.6 kb VGlut intron 2 contains H-cell sib, iVUM mVUM

CG3557 VGlut intron 2-b fragment contains H-cell sib enhancer Intron 2-b 0.3kb

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

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

Gad1 expression is not regulated by Fkh Gad1enSimMerge WT Fkh