Supplementary Figure Legends; Sup. Figure 1: (A-B) ChIP was performed in U251 and T98G cells with -146C>T mutant

Tert promoter and HCT116 with -146C wild type Tert promoter against active histone marks

(H3K4Me3 and H3K9Ac) followed by qPCR with primers specific for Tert promoter region

proximal to TSS. Graph shows qPCR results with % input method as indicated. (C) Gene

expression analysis performed by RT-qPCR for Tert gene and data normalized to actin levels.

n=2 for all the experiments, error bars indicate mean±SD of 2 independent experiments.

Sup. Figure 2: (A) Graph shows qPCR analysis of Tert expression normalized to actin levels

in BLM cells. (B) Graph shows telomerase activity (TRAP) in BLM6 and BLM14 cells. (C)

Telomere length analysis using southern hybridization method in BLM6 and BL14 in passage

6 (P6). (D) Telomere length analysis using southern hybridization method in BLM6 and

BLM14 in passage 10 (P10), P18 and P28. (E) Representative confocal microscopy images

of ALT associated PML bodies. BLM6 and BLM14 cells were stained with TRF2 and PML

antibodies and ALT associated PML bodies were shown as yellow color. ALT+ U2OS cells

were used as a positive control that TRF2 and PML co-localization in nucleus was shown by

arrows in the merged image.

Sup. Figure 3: (A) Jaspar transcription factor prediction tool detected 11 GABPA motifs in

chr5:1,556,087-1,558,758 region (T-INT1). Score and relative score of GABPA sites along

with start and end positions are shown. GABPA sites are present in sense (1) or anti-sense

strand (-1). Predicted GABPA site sequence in T-INT1 region is also shown. (B) ChIP was

performed against GABPA followed by qPCR with primers specific to 5’ end of

chr5:1,556,087-1,558,758 (T-INT1) in BLM6 (-146C>T) and BLM14 (-146C) cells. ChIP q-

PCR primers were targeting ~290 to 450 nucleotides of chr5:1,556,087-1,558,758 (T-INT1).

Error bars indicate the mean ± SD of the two independent experiments. P values were

calculated by two tailed Student’s t test method.

Sup. Figure 4: (A) BLM cells were transfected with siControl (siCont), siGabpa or siBRD4.

48h post transfection TRAP assay was performed. Graph shows telomerase activity (TRAP)

of BLM cells upon siRNA transfection. (B) GABPA and BRD4 depletion in BLM6 cells with

mutant Tert promoter decreases colony formation and this is not as significant as the effect of

these depletions in BLM14 cells with WT Tert promoter. (C) Quantification of the colonies

in B. (D) Tert and (E) BRD4 expression is shown upon siCont and siBRD4 transfection of

Fadu cells that harbor wild type Tert promoter. (F) Graph shows expression of Tert isofoms

as indicated on x-axis. Actin was used for normalizing the data. Error bars indicate mean±SD

of 3 independent experiments. P values were calculated by student’s t-test method.

Sup. Figure 5: ChIP was performed against GABPA followed by qPCR with primers

specific to 5’ end of chr5:1,556,087-1,558,758 in HCT116 -146C and HCT116 -146C>T

cells. ChIP q-PCR primers were targeting ~290 to 450 nucleotides of chr5:1,556,087-

1,558,758. Error bars indicate the mean ± SD of the two independent experiments. P values

were calculated by two tailed Student’s t test method.

Sup. Figure 6: (A) Genotyping PCR was performed from genomic DNAs of T-INT1 WT

and KO cells. Agarose gel electrophoresis for the PCR products from indicated cell types is

shown. WT PCR product was of 4530bp and is indicated in red arrow while KO PCR product

was of 550bp and is indicated in orange arrow. DNA ladder is shown in lane 1 and 8. (B)

Graph shows qPCR analysis of GABPA expression normalized to actin levels in BLM T-

INT1 WT and BLM T-INT1 KO cells. (C) ChIP was performed against BRD4 in BLM T-

INT1 WT and BLM T-INT1 KO cells with primers specific to TSS region of Tert promoter.

Error bars indicate the mean ± SD of the two independent experiments. P values were

calculated by two tailed Student’s t test method.

 

A B

Sup. Fig1

α-H3K4Me3

α-H3K9Ac

α-H3K4Me3

α-H3K9Ac

CTert Expression

Mutant Tert Promoter Wild Type Tert Promoter

ATert Expression

BTRAP Assay

E MergedTRF2PML

U2O

SB

LM

6B

LM

14

Sup. Fig2

C D

1 2 3 4 5 6

BL

M14

BL

M6

1 2

BL

M14

BL

M6

BL

M14

BL

M6

BL

M14

BL

M6

P10 P18 P28

M (kb)

P6

3

4

5

6

8

9

10

12

7

3

4

5

6

8910

12

7

2

1

M (kb)

Chr5:1,556,087-1,558,758

A

B

Fo

ld r

ecru

itm

ent

Anti-GABPAof chr5:1,556,087-1,558,758

Sup. Fig3

BLM Cells TRAP Assay

A

FFadu CellsTert Expression

D Fadu CellsBRD4 Expression E

Sup. Fig4

BLM6

BLM14

si-Cont si-GABPA si-BRD4

BLM6 BLM14

B

C

Anti-GABPAof chr5:1,556,087-1,558,758

HCT116-146C>T

HCT116-146C

Sup. Fig5

GABPA Expression

BLMT-INT1

KO

BLMT-INT1

WT

Sup. Fig6

B CAnti-BRD4

BLMT-INT1

KO

BLMT-INT1

WT

2 31 4 5 6 7 8

BL

M T

-IN

T1

WT

BL

M T

-IN

T1

KO

A37

5 T

-IN

T1

WT

A37

5 T

-IN

T1

KO

U25

1 T

-IN

T1

WT

U25

1 T

-IN

T1

KO

DN

A L

AD

DE

R

DN

A L

AD

DE

R

4530bp 

550bp 500bp  

1000bp  

3000bp   

A