Collected Supplemental Figures...

Supplemental Figure 4Supplemental Figure 2 Supplemental Figure 5 Supplemental Figure 7B

Supplemental Figure 7: Biniou targets a large number of transcription factors

and components of signaling cascades

Supplemental Figure 7ASupplemental Figure 6

Supplemental Figure 4: Endogenous expression of the CG2330 gene

Supplemental Figure 3

Supplemental Figure 2:

Biniou ChIP binding to the three characterised Biniou-regulated enhancers


Expression profiling of biniou mutant

embryos

The 1,164 genes with differential expres-sion were clustered by K-medians clustering, using Pearson uncentered correlations to calculate the distance metric (TIGR-Mev, (Saeed et al. 2003)). Each row corresponds to one gene, each column corresponds to one developmental condition, in the following order from left to right: biniou loss-of-function 5-6 hrs, 6-7hrs, 7-8hr, 8-9hrs, 9-10hrs and 13-14hrs, ectopic biniou 4-5hrs, 5-6hrs, 6-7hrs. All measurements are ratios of biniou mutant embryos over stage matched wild type embryos. Yellow represents increased expression in mutant compared to wild-type, blue represents decreased expression, and black is no change. The degree of change is indicated by the scale on the bottom (log2). The data falls into four major clusters, two of which have significantly reduced levels of expression (clusters 1 & 2), indicating a genetic dependency on biniou for their expression. Genes known to be expressed in visceral muscle are indicated. Cluster 2 contains a number of genes that only change expression late in development, suggesting a role in later aspects of VM morphogen-esis or function. Some of these genes are also likely to be secondary responses to the lack of VM at these late stages. For example, HLH54F is specifically expressed in the longitudinal muscle and does not change expression in biniou mutants at early stages of development when the trunk VM cells are still present. Rather this gene is only differentially expressed at later stages when the longitudinal VM migration is blocked due to a lack of trunk VM. Surprisingly few genes had increased expression with ectopic expression of biniou. We believe that this result is biological as bagpipe is ectopically expressed. The fact that Biniou is not sufficient to ectopically express many genes likely reflects the need for extensive combinatorial regulation, which is strongly supported by our ChIP-on-chip experi-ments.

(A-C) Schematic overview of genomic regions containing characterised Biniou-enhancers: The gene is depicted at the bottom of the panel, with exons in black and introns in grey. Genomic fragments on the tiling arrays are indicated as stacks of four horizontal grey bars in their corresponding genomic position. Each single bar represents the results from one ChIP-on-chip time-period, with the earliest (6-8 hrs, stages 10-11) positioned at the top of the stack and the latest (12-14 hrs, stage 15) at the bottom of the stack. Blue arrow represents the direction of developmental time. The red bars indicate Biniou binding, light red bars indicate binding just below our stringent cut-off. The green double-headed arrows indicate the known enhancers. Note, Biniou binds to all three characterised Bagpipe enhancers, indicating a potential regulatory connection in other VM subtypes, in addition to the trunk VM.

Motif clover_result_bin_early_regions clover_result_bin_contin_regions clover_result_bin_late_regions

Score Pval Score Pval Score Pval

OVER - REPRESENTED RESULTS

FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0

FLYREG_ey 15.7 0.002

FLYREG_hkb 7.99 0.007

FLYREG_kni -2.04 0

FLYREG_toy 8.19 0.001

JASPAR_CORE_MA0011 Broad-complex_2 ZN-FINGER, C2H2

-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003

JASPAR_FAMILY_MF0002 bZIPCREB/G-box-like subclass bZIP

-4.3 0.005

JASPAR_FAMILY_MF0003 REL classREL

15.2 0.006

JASPAR_FAMILY_MF0005 Forkheadclass Forkhead

25.8 0.001 98.4 0 44.2 0

JASPAR_FAMILY_MF0011 HMG classHMG

0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001

Michal_Mad_01 33.6 0

Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006

Michal_pho_01 12.4 0

Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0

TRANSFAC_CF2II_01;CF2-II;CF2-II;CF2-II; Species: fruit fly, Drosophilamelanogaster.

11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0

TRANSFAC_CROC_01;Croc;Crocodileregulator of head development;Croc;Species: fruit fly, Drosophilamelanogaster.

27.6 0

TRANSFAC_DL_01;dl;dorsal;Dl;Species: fruit fly, Drosophilamelanogaster.

13 0.003 12 0.001

TRANSFAC_HAIRY_01;Hairy;Hairy;H;Species: fruit fly, Drosophilamelanogaster.

5.83 0.01

TRANSFAC_HSF_01;HSF;heat shockfactor (Drosophila);HSTF; Species:fruit fly, Drosophila melanogaster.

-2.2 0.004


6.36 0

TRANSFAC_TATA_01; TATA; cellularand viral TATA box elements;TBP;Species: human, Homo sapiens.;TBP;Species: mouse, Mus musculus.;TBP;Species: fruit fly, Drosophilamelanogaster.

-2.37 0.005

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

ttk-V

ME

1 kb

ttk

ttk-VME

fd64a-V

ME

1 kb

LATE

EARLY

fd64a CG1134

fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

5' 3'bap

1 2

3

C

B

A

4)2)1)

3)

F

E

D

C

B

A

CG

2330

Stage 11 Stage 13 Stage 15/16

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

Continuously-bound Late-boundEarly-bound

In situ hybridisation using a DIG-labelled probe directed against the coding region of CG2330. There is no detectable expression at stage 11, with weak expression initiating at stage 12. From stage 13-16 CG2330 is expressed in the trunk VM, somatic muscle and pharyngeal muscle. It is not possible from these in situs to distinguish between trunk circular or longitudinal muscle. However, the Biniou-bound enhancer drives specific expression in the longitudinal VM (manuscript Fig. 1D’).

The 143 newly identified Biniou direct target genes are highly enriched in a number of functional classes of genes. Most notable is the enrichment of ‘transcriptional regulators’: A comparison to a curated database (FlyTF (Adryan and Teichmann 2006)) identified ~20% of Biniou target genes as transcription factors (Supple. Fig. 7A). This group provides a useful resource to further explore the transcriptional network governing VM development. Biniou also targets components of many of the major developmental signalling pathways, which likely impart spatial inputs to Biniou-bound CRMs (supple. Fig. 7B): FGF (bnl, stumps), EGF (star), Hh (pxb), Wg (pan (dTCF)), Notch-Delta (neur). Biniou also binds to CRMs of two components of the semaphorin signalling pathway: off-track (Fig. 2E: otk) and mical. Both genes are expressed in the nervous system, where they play a role in receiving and integrating axonal guidance cues (Winberg et al. 2001; Terman et al. 2002). As these genes were unexpected targets for a muscle specific transcription factor, we re-examined their expression to determine if they are also expressed in the developing visceral muscle. In situ hybridization of off-track revealed that it is expressed in the circular VM during the stages of VM migra-tion, and that this expression depends on biniou activity (Fig. 5E). Moreover, the Binou-bound enhancer recapitulates the endogenous genes’ expression (Fig. 2E).

Biniou direct target genes implicated in cell signalling

Flybase ID Gene Pathway

FBgn0004364 18 wheeler (18w) Toll-likeFBgn0022131 atypical protein kinase C (aPKC) GPCR-signaling

transductionFBgn0014135 branchless (bnl) FGFFBgn0000490 Decapentaplegic (dpp) DppFBgn0000463 Delta (Dl) Notch/DeltaFBgn0000546 Ecdysone receptor (EcR) EcdysoneFBgn0024222 immune response deficient 5 (ird5) TollFBgn0034590 Magi Maguk proteinFBgn0002932 Neuralized (neur) Notch/DeltaFBgn0019664 pangolin (pan) WgFBgn0003177 polychaetoid (pyd) Maguk proteinFBgn0053207 Pxb HhFBgn0053529 Rapgap1 RTK/RASFBgn0026181 Rho-kinase (rok) Rho signalingFBgn0003310 Star (S) EGFFBgn0020299 stumps FGFFBgn0041184 Suppressor of cytokine signaling at 36E (Socs36E) JNKFBgn0033134 Tetraspanin 42El (Tsp42El) membrane signaling

complex

Biniou direct target genes annotated as TFs (www.FlyTF.org)

Flybase ID Gene

FBgn0015903 apontic (apt)FBgn0004862 bagpipe (bap)FBgn0010460 bunched (bun)FBgn0000251 caudal (cad)FBgn0008649 delilah (dei)FBgn0011763 DP transcription factor (Dp)FBgn0000492 Drop (Dr)FBgn0002633 E(spl) region transcript m7 (HLHm7)FBgn0000546 Ecdysone receptor (EcR)FBgn0001981 escargot (esg)FBgn0004895 forkhead domain 64A (fd64A)FBgn0004914 Hepatocyte nuclear factor 4 (Hnf4)FBgn0022740 HLH54F (HLH54F)FBgn0001235 homothorax (hth)FBgn0000448 Hormone receptor-like in 46 (Hr46)FBgn0001269 invected (inv)FBgn0011236 ken and barbie (ken)FBgn0008651 ladybird late (lbl)FBgn0039039 lame duck (lmd)FBgn0005630 longitudinals lacking (lola)FBgn0040765 luna (luna)FBgn0030505 NFAT (NFAT)FBgn0003002 odd paired (opa)FBgn0019664 pangolin (pan)FBgn0003118 pointed (pnt)FBgn0003460 sine oculis (so)FBgn0004579 spalt major (salm)FBgn0003866 teashirt (tsh)FBgn0003870 tramtrak (ttk)FBgn0003862 trithorax (trx)FBgn0016076 vrille (vri)FBgn0004606 Zn finger homeodomain 1 (zfh1)

Biniou & Mef2 co-bound enhancers

jakobsen et al

1











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

ttk-V

ME

1 kb

ttk

ttk-VME

fd64a-V

ME

1 kb

LATE

EARLY

fd64a CG1134

fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

5' 3'bap

1 2

3

C

B

A

4)2)1)

3)

F

E

D

C

B

A

CG

2330


bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12








complex


Flybase ID Gene



jakobsen et al

2











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

ttk-V

ME

1 kb

ttk

ttk-VME

fd64a-V

ME

1 kb

LATE

EARLY

fd64a CG1134

fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

5' 3'bap

1 2

3

C

B

A

4)2)1)

3)

F

E

D

C

B

A

CG

2330


bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12








complex


Flybase ID Gene



jakobsen et al

3

Supplemental Figure 3: Biniou enhancer binding determines the timing

of enhancer activity

(A more detailed description of the genomic location and spatial expression of the

enhancers shown in manuscript Fig. 2)

(A-F) Schematic overview of the genomic regions used in Figure 2 (manuscript): Thegene is depicted at the bottom of the panel, with exons in black and introns in grey.

Genomic fragments on the tiling arrays are indicated as stacks of four horizontal grey

bars in their corresponding genomic position. Each single bar represents the resultsfrom one ChIP-on-chip time-period, with the earliest (10-11) positioned at the top of

the stack and the latest (stage 15) at the bottom of the stack. Blue arrow representsthe direction of developmental time. The red bars indicate Biniou binding. The black

double headed arrow represents the cloned region used to make transgenic reporter

lines. All enhancer regions are named by the gene locus followed by –VME (VisceralMuscle Enhancer)

(A) The VME region 5’ to the ttk locus initiates expression at stage 10 in the trunkand hindgut VM, followed shortly thereafter by expression in the somatic mesoderm.

The visceral mesoderm expression if absent by stage 13, and all traceable enhanceractivity in the somatic muscle is absent by stage 15. The time-span of enhancer

expression in the VM (stages 10-12) match the specific Biniou binding during theearly ChIP time points (note only the upper bars in supple. Fig. 3A are red, lower are

grey)

(B) The fd64a-early-VME initiates expression at stage 10 specifically in the hindgutVM, and switches off activity at late stages of development. (There is also transientexpression detectable in cells at the ventral side of the embryo at stage 13).

In contrast the fd64a-late-VME is expressed from stage 13 to 15 in a subset of cellswithin the anterior and posterior regions of the trunk VM. Note, the time-span of

Biniou binding to both enhancers reflects the timing of enhancer activity (supple. Fig.

3B: fd64a-early-VME ChIP enrichments at stages 10-11 only (red bars), while thelate-VME has ChIP binding only at the last two time-periods (stages 13-15, red bars).

In the case of fd64a, Biniou impinges on two different regulatory modules, which

both have restricted temporal and spatial expression.

(C) The HLH54F-VME drives specific expression in the caudal VM from stages 10-16, recapitulating the full expression of the endogenous gene.

jakobsen et al

4

(D) The otk-VME, located in the last intron of this gene, drives expression in thetrunk VM and hindgut VM from stages 11-16. Interestingly, this enhancer’sexpression coincides with regions of the trunk VM that demarcate the future

compartments of the midgut. This enhancer nicely recapitulates the endogenousgenes expression in the VM (compare manuscript Fig. 2E to 5E)

(E) The intronic VME for the mib2 gene initiates expression at early stage 11 in thetrunk and hindgut VM as well as the somatic muscle. At stage 13 expression is also

detected in the foregut VM. At stage 15-16 the somatic muscle expression ismaintained, however there is no detectable expression in the trunk VM, despite

continued Biniou enhancer binding. Both the VM and somatic muscle expression of

this enhancer closely matches the expression of the mib2 gene.

(F) The ken-VME initiates expression in the salivary glands and in some segmentallyclusters of neuronal cells. At stage 14-15 enhancer expression initiates in the anterior

part of the foregut VM, matching the late binding of Biniou detected by ChIP (red

bars). Fluorescent in situ hybridisation with biniou showed specific co-localisation inthe foregut VM at stages 14 and 15 (see manuscript Fig. 2I).

jakobsen et al

5











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

bin 5

-6

bin 6

-7

bin 7

-8

bin 8

-9

bin 9

-10

bin 1

3-14

over

exp

4-5

over

exp

5-6

over

exp

6-7

ttk-V

ME

1 kb

ttk

ttk-VME

fd64a-V

ME

1 kb

LATE

EARLY

fd64a CG1134

fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

5' 3'bap

1 2

3

C

B

A

4)2)1)

3)

F

E

D

C

B

A

CG

2330


bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12

bap

6-8

bin 6

-8

bin 8

-10

bin 1

0-12

bin 1

2-14

Mef

2 6-

8

Mef

2 8-

10

Mef

2 10

-12








complex


Flybase ID Gene



jakobsen et al

6











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

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EARLY

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fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

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1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

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1 2

3

C

B

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4)2)1)

3)

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CG

2330


bap

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bin 6

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complex


Flybase ID Gene



jakobsen et al

7

Supplemental Figure 5: Over-represented motifs distinguish between

the three temporal groups of Biniou-bound enhancers

Each temporal group of Biniou-bound enhancers (manuscript Fig. 2A) wassearched for overrepresented known transcription factor binding motifs (TFBSs)

using the Clover program (Frith et al. 2004). The ChIP bound regions were repeatmasked. A set of 104 matrices for transcription factors from Drosophila

melanogaster was obtained from FlyReg (Bergman et al. 2005), Transfac (Matys et

al. 2006), Jaspar (Vlieghe et al. 2006) and the literature. Clover was used to searcheach temporal group (early, continuously and late bound) using default parameters

with all matrices. This analysis reported 33 different transcription factor signatures tobe over-represented in at least one of the three temporal groups, compared to

background (unenriched regions of the genome covered on the tiling array). The

results shown in the table contain the clover score, and also the Pval. We use the Pvalas the determining factor for significant enrichment.

The majority of motifs are enriched in all three groups, however the following arespecific to smaller sub-groups:

Early-bound enhancers: knirps (kni), Broad complex Zn finger

Early and Continuous: Beaf-32, HMG class, Mef2Continuous-bound: Aef1, toy, Rel, Mad, Pho, croc, pan, ey, hairy, HSF,

TBP

Continuous and late: BEAF-32, Broad complex Zn finger, Dl(All: Biniou, CF2II + others)

jakobsen et al

8











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

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EARLY

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fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

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1 2

3

C

B

A

4)2)1)

3)

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2330


bap

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10

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2 10

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complex


Flybase ID Gene



jakobsen et al

9

Supplemental Figure 6: Combinatorial binding of Biniou & Mef2

We performed ChIP-on-chip experiments with Mef2 at the same stages of

development as Biniou, on the same microarray platform as reported previously(Sandmann et al. 2006). The Mef2 binding data was integrated with the Biniou

binding data for all 292 Biniou-bound genomic regions. To assess the relative

occupation of Mef2 on each of the three temporal groups of Biniou-enhancers, weclustered each group to distinguish regions that are bound by co-bound by Mef2 from

those that or not. For each cluster, the Biniou-ChIP experiments are shown on the left

with a white line separating the Mef2 ChIP time-series on the right (Both Bin andMef2 contain ChIPs at 6-8 hrs, 8-10hrs and 10-12 hrs). The intensity of yellow

reflects the degree of enrichment, shown in the scale (log2).

There is substantial combinatorial binding of Biniou and Mef2 on the early-

bound and continuous-bound enhancers, matching the enrichment of Mef2 motifs in

these groups (indicated by the red lines to the left of the upper clusters). 65.1% of theBiniou early-bound enhancers are cobound by Mef2 and 50.4% of the continuous-

bound enhancers are co-bound by Biniou and Mef2, at the same stages ofdevelopment. In contrast, only 20.1% of the Biniou late-bound enhancers are co-

bound by Mef2. This was surprising as Mef2 plays a prominent role in muscle

differentiation. Interestingly, we do not find significant Biniou binding to a largeclass of enhancers for muscle contractile proteins that are regulated by Mef2 (e.g.

Mlc1, Mlc2). We suggest that there may be two independent differentiation programsduring VM development, one governed by Mef2 regulating more general muscle

proteins, and a second, more VM specific, program driven by Biniou.

jakobsen et al

10











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

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EARLY

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fd64a-e-VME

fd64a-l-VME

HL

H5

4F-V

ME

1 kb

HLH54F CG5009

HLH54F-VME

otk-V

ME

1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

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1 kb

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1 2

3

C

B

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4)2)1)

3)

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CG

2330


bap

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bap

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8

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2 8-

10

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2 10

-12








complex


Flybase ID Gene



jakobsen et al

11











embryos






FLYREG_Aef1 16.5 0

FLYREG_BEAF-32 -3.49 0 -4.03 0 -3.77 0.001

FLYREG_BEAF-32B -4.25 0.007 -3.86 0.01

FLYREG_Top2 32.3 0.001 104 0 58.7 0

FLYREG_Trl 6.7 0.001 42.8 0 11.3 0

FLYREG_bin -2.07 0 -1.97 0 -2.12 0



FLYREG_kni -2.04 0



-2.96 0.01


-0.349 0.009 4.77 0 2.13 0


6.28 0 2.28 0.003


-4.3 0.005


15.2 0.006


25.8 0.001 98.4 0 44.2 0


0.591 0.001 3.43 0.007

Michal_Adf1_01 98.7 0.002

Michal_Dref_01 -3.82 0.002 -4.55 0.001 -4.07 0.001


Michal_Mef2_01 12.2 0 30.3 0

Michal_pan_01 36.5 0.006


Michal_srp_01 -3.38 0.001 -3.93 0 -3.64 0.001

Michal_z_01 0.779 0


11.3 0.001 21.9 0 17.4 0


13 0.002 19.6 0.005 20.8 0


27.6 0


13 0.003 12 0.001


5.83 0.01


-2.2 0.004


6.36 0


-2.37 0.005

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H5

4F-V

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1 kb

HLH54F CG5009

HLH54F-VME

otk-V

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1 kbotk

otk-VME

mib

2-V

ME

1 kb

mib2 CG10449

mib2-VME

ken-V

ME

1 kb

kenCG4581

ken-VME

1 kb

5' 3'ßTub60D

1 kbdpp

5' 3'

1 kb

5' 3'bap

1 2

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C

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4)2)1)

3)

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2330


bap

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8

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10

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2 10

-12








complex


Flybase ID Gene



jakobsen et al

12


Supplemental Figure 8: Two independently raised polyclonal antibodies

against Biniou give similar ChIP results

To examine the similarity between the Biniou antibodies we examined the

enrichments of a known Biniou-bound region compared to a negative region by real-time

PCR. In these assays, variability in enrichment levels was just as frequently caused by

differences in chromatin preparations, as by the antibodies. The similarity of the ChIP

results obtained with the two antibodies was also assessed more globally by plotting the

mean of the enrichment values for the two repeats performed with antibody-1 (RDI)

against the mean of the enrichment obtained with antibody-2 (REP). All features with a

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

-0.5 0 0.5 1 1.5 2 2.5 3 3.5

AB IP mean >0.7 log2-enrich

Regression Statistics

Multiple R 0.604345036

R Square 0.365232923

Adjusted R Square 0.364292527

Standard Error 0.369906419

Observations 677

AB IP mean >1.0 log2-enrich

Regression Statistics

Multiple R 0.725939354

R Square 0.526987945

Adjusted R Square 0.524992114

Standard Error 0.361571851

Observations 239

B

A

-1

jakobsen et al

13

single repeat log2-enrichment above 0.7 in the 6-8 hrs time point are displayed, mean for

the RDI-antibody on the Y-axis, and the mean for REP-antibody on the X-axis (Supple.

Fig. 8A). The enrichment ratios for both antibodies are very similar for ratios >0.7 log2.

To quantify the degree of similarity we performed a regression analysis on all features

with a mean for both antibodies >0.7 or >1.0 log2 (cut-offs shown as green and red bars,

respectively, in Supple. Fig. 8A). This analysis indicates that the ChIP results from both

antibodies are highly correlated. As observed for expression profiling analysis, the

correlation co-efficient is higher for features with higher enrichment values (Supple. Fig.

8B).

We also analysed the data using both a paired and unpaired SAM test. These

showed only very minor differences in the number of regions found to be significantly

bound, again indicating that the ChIP is very reproducible between the two antibodies. As

there were no clear differences between the antibodies, all four hybridisations (two for

each antibody) were used as biological replicates for the detection of significantly bound

regions. Thus, by pooling all sources of variance, a genomic region needs to be robustly

enriched in multiple experiments to be called significant.

jakobsen et al

14

Supplemental Methods:

Experimental methods for expression profiling biniou mutants:

Homozygous biniou loss of function mutant embryos (biniou1l allele) were isolated

from their balancer siblings using an automated embryo sorter (Furlong et al. 2001a;

Furlong et al. 2001b). Wild-type embryos (Canton S) were collected and processed in

parallel. One-hour collections were sorted at stage 9-10 and aged to the appropriate stage

after sorting. For each one-hour developmental time-period five individual embryo

collections and sorts were conducted. A small aliquot of all samples was isolated and

formaldehyde fixed to estimate the staging of wild type and mutant embryos. The four

samples with the tightest staging were used to extract RNA, which was hybridised to

arrays containing one 60-oilgo for every predicted Drosophila gene (INDAC oligos). For

ectopic expression of Biniou, embryos were collected from transheterozygous parents of

the following genotype: twi-24B-Gal4 X UAS-Biniou-HA. The HA-tag had no apparent

affect on Biniou activity, determined by the ability of Biniou-HA to ectopically express

FasIII. All samples were hybridised in pairs of wild type and mutants, with appropriate

dye swaps to reduce the effect of dye biases.

The microarray data was normalised using print-tip LOWESS normalisation. A

one-class SAM analysis was performed on four independent biological repeats for each

time point. Genes with a q < 0.01 and a fold enrichment of log2 > 0.7 or < −0.7 at one or

more time-point were considered to be differentially expressed. For the ectopic

expression, we only considered genes with elevated expression (q<0.01 and fold change

log2 > 0.7).

Target gene assignment

All genes within 60kb of a Biniou-bound region (30kb 5’ and 3’) were assigned a

score, which was the product of four variables: distance between the gene and the bound

region (D = distance), a change in expression in biniou mutant embryos (L=loss of

function), a change of expression in embryos with ectopic biniou expression (E=ectopic),

and additional information on the gene’s expression pattern (S, for supporting data, based

on the BDGP in situ database (Tomancak et al. 2002), Flybase, or the literature). 60kb

was selected as the distance to sample around a ChIP-bound region as the gene density in

jakobsen et al

15

Drosophila is relatively high. This distance will cover on average 10 genes in vicinity of

the enhancer and therefore should include the correct target gene.

All sampled genes received a score between 0 and 1 for each of the variables

described above. Distance scores are linearly related to the physical distance between an

enriched region and each neighboring gene. This transformation method was found to

perform at least as well as a more complex sigmoidal. A perfect distance score of 1 was

obtained for genes with loci overlapping the position of enriched regions. Neutral scores

of 0.6 (e.g. for genes with no change in the expression profiling experiments or where no

in-situ information is available) were found to be optimal through iterative optimization of

the algorithm (Sandmann et al., 2006, 2007).

A total score of 0.217 was chosen as a cut-off for target gene assignment. This

ensured that a ChIP-bound region was not assigned to a gene based on proximity alone.

At least one piece of supporting evidence is necessary to overcome this threshold, since a

perfect distance score of 1 and neutral scores of 0.6 in all other categories produces a total

score of 0.216 (1 * 0.6 * 0.6 * 0.6 = 0.216).

An example of an assignment of a ChIP-bound region to a gene is shown below. If

the assignment was based solely on proximity, CG3407 would be assigned as the target

gene of this Bagpipe-bound region (indicated in red). With our automated assignment,

slp1 receives the highest score, although this gene is located ~9.5 kb from the ChIP-bound

region. This Bagpipe-bound region is sufficient to drive reporter gene expression in a

pattern that recapitulates the endogenous slp1 genes expression in the visceral muscle,

validating the automated gene assignment (manuscript Fig. 4C). This example clearly

demonstrates the importance of using an assignment approach not based on proximity

alone. Using this method, the three known Biniou-bound enhancers were correctly

assigned to the appropriate target gene. In addition, all enhancers shown in Figure 1, 2

and 4 (main manuscript) recapitulate all or some of the assigned target genes expression.

jakobsen et al

16

Target gene assignment: Visualization of the genomic environment of an enriched region with both ChIP-on-chip and expression profiling data displayed

A) The genes are depicted above or below the ChIP-on-chip data, reflecting if they are on the plus or minus strand, respectively. Expression profiling data of biniou mutant embryos is shown for each time-point above or below the gene (Genomic fragments on the tiling arrays are indicated as stacks of five horizontal grey bars in their corresponding genomic position. Each single bar represents the results from one ChIP-on-chip time-period, with Bagpipe (10-11) positioned at the top of the stack, then Biniou (10-11) and the latest time-period (stage 15) at the bottom of the stack. The red bar indicates Bagpipe binding. Note, there is no Biniou binding to this genomic region. The black double headed arrow represents the cloned region used to make transgenic reporter lines. Scores are shown for each gene (D: score, gene distance to enriched fragment, L: loss-of-function, E: ectopic expression, S: supplemental score, Total score in red). The Bagpipe-bound region was assigned to slp1, as this gene has the highest total score.

B) Flow-scheme for automated gene assignment.

Estimating overrepresentation of binding sites in Biniou-bound regions: We used all known Biniou, Mef2, Tinman and Bagpipe sites to generate a position

weight matrix using the MEME algorithm (Bailey and Elkan 1994).

MEME was run with the following parameters:

meme footprints_fasta_file -dna -bfile DrosophilaBackground.freq -nostatus -

nmotifs 1 -maxw 14 -revcomp -minw 6 -mod anr

DrosophilaBackground.freq:

jakobsen et al

17

A 0.3 C 0.2 G 0.2 T 0.3

The following position weight matrices (PWMs) were generated and used to search for

enrichment into different groups of Biniou-bound CRMS using MAST (Bailey and

Gribskov 1998b; Bailey and Gribskov 1998a).

We performed a filtering step, where only Biniou sites matching the RYAAAYA, Mef2 sites containing an invariable TANNNNTA motif, and Tinman sites with an invariable CACTT motif were considered. The optimal p-value cut-off was determined by benchmarking MAST on a set of enhancer sequences known to contain functional binding sites. The p-values used were: 2e-04 for Biniou and Bagpipe, 1.5e-04 for Mef2 and 3.4e-04 for Tinman.

MAST was used to scan the genomic sequence of all the fragments on the tiling

array. Predicted motifs were counted within the Biniou pulled down regions and the non-

pulled down (background) regions. The significance of enrichment of predicted sites was

estimated by a binomial exact test using the R statistical programming environment.

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