GSEA analyses for NtcA on repressed genes

John Aach

May 15, 2006

Methods

• The Su et.al. (2005) supplementary material contains the sequence downstream of the NtcA box and also the 10 box itself (where found). From this it was possible to find the distance into this downstream sequence of the start of the 10 box. There were also genes listed that had NtcA sites without -10 boxes. Note that due to construction of the Su et.al. data sets, 10 boxes upstream of NtcA sites are not considered.

• GSEA tests were performed on lists of genes, some of which are described below, considering only the 6 and 48 hour time points in MED4 and MIT9313, using the Direction parameter of gsea.m to look for enrichment among repressed genes.

– Negative control: For the 6 hour time point, a GSEA test was run with the top 15 ranked NtcA sites for genes with both orthologs and non-orthologs ranked separately (see Methods in manuscript). These are parameters equivalent to those run for 6 hour enrichment of induced genes, except that the direction of the test was reversed so as to look for enrichment among repressed genes. This test is a negative control because the fact that these genes were enriched among activated genes means that they should not also have been enriched among repressed genes. Remaining tests considered sets of genes with NtcA sites without 10 boxes or with NtcA sites close to 10 boxes which might be occluded by NtcA binding.

– A list was prepared of genes with that were identified in Su et.al. (2005) as having NtcA sites without downstream 10 boxes. These could be sites that block transcription initiated by upstream binding sites for the RNA polymerase and associated factors. The situation is diagrammed below. GSEA tests looking for enrichment among repressed genes were conducted using this list and considering the top 15 sites for genes listed as having orthologs and as not having orthologs, ranked separately.

Methods (continued)

– A list was prepared of genes with that were identified in Su et.al. (2005) as having NtcA sites that were within 15bp of a downstream 10 box. These could be sites that are so close to the 10 box that NtcA binding occludes it, thereby blocking transcription. The situation is diagrammed below. There are only a very small number of these sites, and all were used in the GSEA regardless of NtcA site rank: MED4 has 16 (9 for genes with and 7 for genes without orthologs) and MIT9313 has 8 (6 for genes with and 2 for genes without orthologs).

gene

NtcARNAP

XX

geneNtcA

RNAPXX-10 box

Results

• All tests were negative for enrichment (P>>.05). The smallest P value was .6544 (MIT9313 time point 48 hr for NtcA sites within 15bp of a 10 box).

MED4 GSEA tests for NtcA site enrichment among repressed genes

0 200 400 600 800 1000 1200 1400 1600 1800-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05ES=0.0225274

P=0.9562P

c=0.481466

le=50

MED4 T6 repression: top 15 NtcA sites for both genes with + without orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

Negative control

0 0.05 0.1 0.15 0.2 0.25 0.3 0.350

200

400

600

800

1000

1200ES=0.0225274

P=0.9562P

c=0.481466

MED4 T6 repression: top 15 NtcA sites for both genes with + without orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

Negative control

0 200 400 600 800 1000 1200 1400 1600 1800-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05ES=0.0201592

P=0.9618P

c=0.456128

le=1328

MED4 T6 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 6hr

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.40

200

400

600

800

1000

1200

1400ES=0.0201592

P=0.9618P

c=0.456128

MED4 T6 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 6hr

0 200 400 600 800 1000 1200 1400 1600 1800-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08ES=0.0782916

P=0.7426P

c=1.84667

le=895

MED4 T48 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 48hr

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.40

200

400

600

800

1000

1200ES=0.0782916

P=0.7426P

c=1.84667

MED4 T48 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 48hr

0 200 400 600 800 1000 1200 1400 1600 1800-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1ES=0.0466228

P=0.8666P

c=0.795495

le=1621

MED4 T6 repression: all genes with NtcA sites < 15bp from -10 box: score plot

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 6h

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.50

200

400

600

800

1000

1200

1400ES=0.0466228

P=0.8666P

c=0.795495

MED4 T6 repression: all genes with NtcA sites < 15bp from -10 box: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 6h

0 200 400 600 800 1000 1200 1400 1600 1800-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06ES=0.0486287

P=0.9008P

c=0.5994

le=71

MED4 T48 repression: all genes with NtcA sites < 15bp from -10 box: score plot

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 48h

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

200

400

600

800

1000

1200ES=0.0486287

P=0.9008P

c=0.5994

MED4 T48 repression: all genes with NtcA sites < 15bp from -10 box: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 48h

MIT9313 GSEA tests for NtcA site enrichment among repressed genes

0 500 1000 1500 2000 2500-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05ES=0.00408905

P=0.997P

c=0.301528

le=2241

MIT9313 T6 repression: top 15 NtcA sites for both genes with + without orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

Negative control

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.40

200

400

600

800

1000

1200

1400ES=0.00408905

P=0.997P

c=0.301528

MIT9313 T6 repression: top 15 NtcA sites for both genes with + without orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

Negative control

0 500 1000 1500 2000 2500-0.25

-0.2

-0.15

-0.1

-0.05

0ES=0

P=1P

c=0.293152

le=2250

MIT9313 T6 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 6hr

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.40

200

400

600

800

1000

1200ES=0

P=1P

c=0.293152

MIT9313 T6 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 6hr

0 500 1000 1500 2000 2500-0.15

-0.1

-0.05

0

0.05

0.1ES=0.0573243

P=0.8262P

c=0.999778

le=847

MIT9313 T48 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: score plot

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 48hr

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.40

200

400

600

800

1000

1200ES=0.0573243

P=0.8262P

c=0.999778

MIT9313 T48 repression: top 15 NtcA sites for genes w/o -10box both with + w/o orthologs: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

gene

NtcARNAP

XX No -10 boxTime = 48hr

0 500 1000 1500 2000 2500-0.45

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05ES=0.00625559

P=0.9924P

c=0.345553

le=2236

MIT9313 T6 repression: all genes with NtcA sites < 15bp from -10 box: score plot

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 6h

0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

200

400

600

800

1000

1200ES=0.00625559

P=0.9924P

c=0.345553

MIT9313 T6 repression: all genes with NtcA sites < 15bp from -10 box: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 6h

0 500 1000 1500 2000 2500-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15ES=0.134464

P=0.6544P

c=3.84253

le=1134

MIT9313 T48 repression: all genes with NtcA sites < 15bp from -10 box: score plot

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 48h

0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

200

400

600

800

1000

1200ES=0.134464

P=0.6544P

c=3.84253

MIT9313 T48 repression: all genes with NtcA sites < 15bp from -10 box: null distribution

gene number

runn

ing

enric

hmen

t sc

ore

geneNtcA

RNAPXX-10 box

-10 occludedTime = 48h