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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 - PowerPoint PPT Presentation
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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
C1.7P155
T523
dys early enhancer(S12-S17)
dys late enhancer(S14-S17)
Sequence alignment of dys155
GATCGG
TTAAATCAAA
TGCCTGAC
Ets
GACCCAAAAC
GCGCG
POU
1
2
3
4
5
6
7
1 2 3 4 5 6 7
Motifs Required for dys Activation
1 2 3
4 5 6
7
dys155
WT pnt
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 dys
P155 C1.7
D92
CG13196
Identify CG13196 Fusion cell Enhancer
Sequence alignment of CG16196 enhancer
GFP Dys Merge
Unmutated
D92muC
D92muD
D92muAAG
D92muMotif8
TCGTG-DTCGTG-C AAG
Motif 8 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 7 AAG
GFP Dys Merge
Unmutated
C100muC
C100muD
C100muAAG
C100muMotif9
Motifs Required for CG15252 Activation
x xA350muBCCG13196
C100CG15252
C100muCDCG15252
A350CG13196
Co-activator sequences required for CG13196 expression
Transcriptional pathways in terminally differentiating neurons
SimSim + En
Gene expression porfile in Glutamatergic and GABAergic interneurons
TF gene expressionH-cell sib
simperfkhnvy
Lim1
iVUMsimperfkhnvy
Lim1
* en
NF gene expressionH-cell sib
VglutGluR1
5-HT 1ACG13565
iVUMGad1
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
VGlut
Lim1
Lim1
VGlut/CG13565/En
VGlut/CG13565/En
H-cell sibsimperfkhnvy
Lim1
vglut expression in Forkhead mutant
WT
fkh
Sim vglut Merge
H-cell sibsimperfkhnvy
Lim1
VGlut Genomic Locus
14.5 kb
2.4 kb
2.5 kb
2.8 kb
3.7 kb2.7 kb
4.8 kb
0.8 kb 0.6 kb 0.45 kb
0.47 kb
4.4 kb
0.3 kb
0.3 kb
CG3557
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 expressionH-cell sib
simperfkhnvy
Lim1
iVUMsimperfkhnvy
Lim1
* en
NF gene expressionH-cell sib
VglutGluR1
5-HT 1ACG13565
iVUMGad1
VIAAT(CG8394)CG15236
Gad1 expression is not regulated by Fkh
Gad1 en Sim Merge
WT
Fkh