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