1A

IP/

Inp

ut

Figure 1: E47 is present on tRNA genes

1B

IgG

TF

IIB

E47

Brf

1

WB: Brf1

Inpu

t

IP

A) IP/Input enrichment of E47 at 6 active tRNA genes and one inactive tRNA gene (tRNA81Leu-TAA) measured via Chromatin IP. An IP with SL1 antibody is used as control. ETC19 (Extra TFIIIC binding site) and ApoE gene regions are used as no enrichment control.

B) Protein immunoprecipitation performed with the shown antibodies or IgG as a control. Brf1 could be precipitated by an E47 antibody more than a TFIIB or IgG pull-downs. 10% input is used in the first lane.

2A

Fig 2 E47 negatively affects pol III transcript levels

2B

2C

0

1

2

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4

Hdac1 Hdac2 Gcn5 cMyc p300

5S rRNAtRNA-LeutRNA-ThretRNA-iMet

Fol

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er C

ontr

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iRN

A

2D

Fol

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er C

ontr

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iRN

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Fol

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ALegend in next page

A) E47 siRNA knockdown induces 5sRNA and tRNA levels monitored via qPCR. Levels of 3 active tRNAs measured and 5sRNA are depictedrelative to a control siRNA treatment. A value of 1 would mean no change

B) E47 siRNA knockdown enhances recruitment of Brf1 and RPC155 at tRNA genes. Chromatin IP to determine recruitment of Brf1 and RPC155 at 3 tRNA genes upon knockdown of E47 protein levels. Antibodies are used for proteins depicted on the x-axis. Presence of Brf1 and RPC155 is shown relative to a control siRNA treatment. A value of 1 would mean no change.

C) E47 siRNA knockdown affects recruitment of chromatin remodelers at tRNA genes. Chromatin IP to determine recruitment of HDAC and HAT complexes at 3 tRNA genes and 5sRNA upon knockdown of E47 protein levels. Antibodies are used for proteins depicted on the x-axis. Presence of HDAC and HAT complexes are shown relative to a control siRNA treatment. A value of 1 would mean no change.

D) E47 siRNA knockdown enhances presence of permissive chromatin marks and reduces presence of repressive marks at tRNA genes. Chromatin IP to determine recruitment of chromatin marks at tRNA genes upon knockdown of E47 protein levels. Antibodies are used for the appropriate histone modifications are depicted on the x-axis. Presence of acetylated or methylated histone forms is shown relative to a control siRNA treatment. A value of 1 would mean no change.

IP/ I

nput

Fig 3: Id2 protein is detected on tRNA genes

3A

3C

Inp

ut

IgG

TF

IIB

Id2

WB: Brf1

Brf

1

IP

3B

Fol

d ov

er C

ontr

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iRN

A

ChIP after E47siRNA

A) IP/Input enrichment of Id2 at 6 active tRNA genes measured via Chromatin IP. An IP with SL1 antibody is used as control. ETC19 (Extra TFIIIC binding site),ApoE gene and an inactive tRNA, tRNA81-Leu gene regions are used as no enrichment control.

B) E47 knockdown affects recruitment of Id2 at tRNA genes. Chromatin IP to determine recruitment of Id2 at 5S rRNA and RNA genes upon knockdown of E47 protein levels. H3 antibody was used a control. Id2 absence at tRNA genes is shown relative to a control siRNA treatment. A value of 1 would mean no change.

C) Protein immunoprecipitation performed with the shown antibodies or protein A/G beads. Brf1 could be precipitated by an Id2 antibody more than a TFIIB or just beads pull-downs. 10% input is used in the first lane.

Fig 4: Id2 positively regulates pol III gene transcription

4A 4B

5S rRNA

tRNA

Actin

Id2

Ad

-Id

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Id2 mRNA

ARPP P0

Vec

tor

Western Blot

RT-PCR

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4C

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Fol

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A A) Id2 siRNA knockdown reduces 5sRNA and tRNA expression levels as monitored via qPCR. Levels of 3 active tRNAs measured and 5sRNA are depicted relative to a control siRNA treatment. A value of 1 would mean no change.

B) Overexpression of Id2 using an adenoviral vector upregulates 5S rRNA and tRNA expression. Western blot on the left show increased levels of Id2 protein. Semi-quantitative Reverse Transcriptase PCR on the right shows increased Id2 mRNA levels as well as induced 5S rRNA and tRNA levels. ARPP0 gene was used as a control.

C) Id2 siRNA knockdown diminishes recruitment of Brf1 and RPC155 at tRNA genes. Chromatin IP to determine recruitment of Brf1 and RPC155 at 3 tRNA genes upon knockdown of Id2 protein levels. Presence of Brf1 and RPC155 is shown relative to a control siRNA treatment. A value of 1 would mean no change.

35S-Brf1

35S-Luciferase

Supplemental Fig 1: Id2 and E47 form in vitro and in vivo complexes with pol III transcription machinery

s1A

s1B

A) GST tagged and purified E47 and Id2 proteins immunoprecipitate 35S labelled in vitro transcribed Brf1. A purified protein extract from GST-Vector expressing cells, and 35S labelled in vitro transcribed Luciferase were used as a negative control.

B) Protein immunoprecipitation performed with the antibodies or protein A/G beads as shown above each lane. RPF155, TFIIIC 220 and TBP could be precipitated by an Id2 and E47 antibody more than just beads pull-down. 10% input is used in the first lane.

Sup Fig 2: Knock down of E2A transcripts induces Pol III transcribed genes

5S rRNA

tRNA

Actin

E47E12

ARPP P0

WesternBlot

RT-PCR

E47 mRNA E2A

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Knockdown of E47 using siRNA upregulates 5S rRNA and tRNA expression. Western blot on the left shows decreased levels of E2A proteins (upper band E47, lower band E12).

Semi-quantitative Reverse Transcriptase PCR on the right shows reduced E2A transcript levels as well as induced 5S rRNA and tRNA levels. ARPP0 gene was used as a control.

Actin

Id2

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Western Blot

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ol s

iRN

A Id

2 si

RN

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RT-PCR

ARPP P0 mRNA

Id mRNA

5S rRNA

tRNA

Sup Fig 3 :Id2 depletion negatively affects pol III transcribed genes

Knockdown of Id2 using siRNA downregulates 5S rRNA and tRNA expression. Western blot on the left shows decreased levels of Id2 proteins.

Semi-quantitative Reverse Transcriptase PCR on the right shows reduced Id2 transcript levels as well as decreased 5S rRNA and tRNA levels. ARPP0 gene was used as a control.