Supplementary Figure 1.

Repression of miRNAs in MycER tumors. Northern blot analysis of miRNAs in MycERtumors. ‘Expression Ratio’ indicates the level of miRNA expression in the MycON state relative to the MycOFF state. Specific hybridization conditions, as shown in Figs. 1c and 4b, were used for miR-30b and let-7a. tRNALys served as a loading control (representative blot shown).

miR-34a

miR-146a

miR-150

miR-195

miR-15a

miR-26a

miR-22

miR-29a

miR-30b

let-7a

miR-16

Myc

ON

Myc

OFF

0.31

0.37

0.34

0.29

0.51

0.57

0.35

0.46

0.10

0.83

0.41

Expressionratio

tRNALys

miR-34a

miR-30a*

miR-30a

miR-22

miR-30e

miR-30e-3p

miR-30d

miR-146

let-7i

miR-30b

let-7e

miR-29b

miR-30c

miR-26b

miR-29a

miR-29c

let-7b

miR-15a

miR-195

let-7c

miR-26a

let-7g

let-7a

let-7d

miR-150

miR-16

let-7f

Nam

alw

a

Man

ca

Raj

i

Nor

mal

B C

ells

-7 7

Fold expression

a

Myc

Actin

Luc

shR

NA

Myc

shR

NAb

miR-15a

miR-26aLu

c sh

RN

A

miR-22

miR-16

miR-195

miR-146a

miR-30b

U6 snRNA

let-7a

1.35

1.52

1.52

1.50

1.31

1.63

1.34

1.19

FoldinductionM

ycsh

RN

A

c

Supplementary Figure 2.

Myc represses miRNAs in Burkitt’s lymphoma cells. (a) Analysis of previously published miRNA expression profiling data (He et al., Nature, 2005) demonstrates that most Myc-repressed miRNAs are expressed at lower levels in Burkitt’s lymphoma cells compared to normal B cells. (b) Western blot showing Myc knockdown by lentivirally-expressed shRNA in EW36 Burkitt’s lymphoma cells. shRNA directed against luciferase (Luc) served as a negative control. (c) Myc knockdown results in upregulation of miRNAs in EW36 cells. miR-29a was not upregulated by Myc shRNA under these conditions and miR-34a and miR-150 were not expressed at detectable levels in this cell line (not shown).

100%

50%100%

50%100%

50%100%

50%

20 kbC1 N

miR-29b-1/29a

Mouse

Rat

Opossum

Chicken

C2

0

20

40

60

80

100

C1 C2 N

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc associates with a conserved region upstream of the miR-29b-1/29a cluster. (a) VISTA analysis of phylogenetic conservation encompassing the miR-29b-1/29a cluster as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 3.

a

b

Myc associates with a conserved region upstream of the miR-30d/30b cluster. (a) VISTA analysis of phylogenetic conservation encompassing the miR-30d/30b cluster as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in

Supplementary Figure 4.

a

b

5 kb

miR-30b

C N

Mouse

Rat

Opossum

Chicken

100%

50%100%

50%100%

50%100%

50%

miR-30d

0

20

40

60

80

100

C N

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc associates with a conserved region upstream of miR-34a. (a) VISTA analysis of phylogenetic conservation encompassing miR-34a as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 5.

a

b

100%GPR157

100%

50%

50%100%

50%100%

50%

miR-34a

10 kbC1 N1

Mouse

Rat

Opossum

Chicken

N3N2C2C3

0

20

40

60

80

100

C1 C2 C3 N1 N2 N3

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc associates with a conserved region upstream of miR-146a. (a) VISTA analysis of phylogenetic conservation encompassing miR-146a as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 6.

a

b

Mouse

Rat

Opossum

Chicken

10 kb

miR-146a

CN

100%

50%100%

50%100%

50%

PTTG1

100%

50%

0

20

40

60

80

100

120

C N

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc associates with a conserved region upstream of the miR-195/497 cluster. (a) VISTA analysis of phylogenetic conservation encompassing the miR-195/497 cluster as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 7.

a

bC2N C4C3C1

1 kb

Mouse

Rat

Opossum

Chicken

100%

50%100%

50%100%

50%100%

50%

MGC49942 BCL6B

miR-195 miR-497

0

20

40

60

80

100

120

C1 C2 C3 C4 N

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc does not associate with conserved regions upstream of miR-150. (a) VISTA analysis of phylogenetic conservation encompassing miR-150 as described in Figure 3a. Amplicons shown in Figure 3b are bolded and underlined. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 8.

a

b

2 kbC1

Mouse

Rat

Opossum

Chicken

NC3C2

miR-150

RPS11 FCGRT100%

50%100%

50%100%

50%100%

50%

C5 C4 C6

0

20

40

60

80

100

C1 C2 C3 C4 C5 C6 N

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Myc does not associate with conserved regions upstream of the miR-30a/30c-2 cluster. (a) VISTA analysis of phylogenetic conservation encompassing the miR-30a/30c-2 cluster as described in Figure 3a. (b) Real-time PCR analysis of Myc chromatin immunoprecipitates as described in Figure 2c.

Supplementary Figure 9.

a

b

5 kb

Mouse

Rat

Opossum

Frog(X. tropicalis)

miR-30c-2

100%

50%100%

50%100%

50%100%

50%

C6orf155

miR-30a

C2 C4C1 C3 C6C5

0

20

40

60

80

100

C1 C2 C3 C4 C5 C6

Amplicon

Fold

enr

ichm

ent

High Myc (-tet) Low Myc (+tet)

Retroviral miRNA expression levels in Myc3 and 38B9 cells. (a) Numbers below blots represent the expression level of each miRNA relative to the non-transformed B cell line YS-PB11. All quantifications were normalized to to loading control (tRNALys, not shown) and to P493 (low Myc) RNA which was loaded on each gel to allow direct comparison of miRNA levels across blots. (b) Retroviral miR-150 expression was compared to MycOFF tumors since this miRNA was not expressed in YS-PB11 cells (not shown).

Supplementary Figure 10.

miR-22

miR-15a

miR-34a

miR-26a

miR-30b

miR-150

miR-195

0.10 0.73

0.36 6.78

0.01 2.68

0.21 5.06

0.08 1.33

0.11 1.0 0.02 5.93

0.16 3.61

0.10 0.74

0.15 32.7

0.19 5.15

0.17 3.58

0.09 4.45

0.01 3.54

0.15 9.35

1.0

1.0

1.0

1.0

1.0

1.0

miR-160.13 0.60 0.12 0.701.0

Myc3 38B9

MS

CV

-em

pty

MS

CV

-miR

P49

3 (L

ow M

yc)

MS

CV

-em

pty

MS

CV

-miR

P49

3 (L

ow M

yc)

YS-P

B11

P49

3 (L

ow M

yc)

b

a

MycERtumor Myc3 38B9

Myc

ON

Myc

OFF

MS

CV

-em

pty

MS

CV

-miR

P49

3 (L

ow M

yc)

MS

CV

-em

pty

MS

CV

-miR

P49

3 (L

ow M

yc)

miR-26a

miR-22

miR-16

miR-150

miR-195

1.0 0.520.730.90 0.320.660.27 0.420.49

miR-34a

2 126 3624 48 60 72+ -Time after removal of Tet (hr)Tet

0.27

1.0 1.622.082.60 1.081.950.30 1.141.46 0.64

1.0 0.871.251.32 0.761.240.27 0.740.85 0.63

1.0 0.690.850.91 0.610.760.50 0.740.74 0.54

1.0 0.841.141.22 0.351.090.09 0.470.64 0.20

1.0 0.891.070.88 0.640.990.23 0.730.86 0.57

tubulin

Myc

2 126 3624 48 60 72+ -Time after removal of Tet (hr)Tet

b

a

Kinetics of miRNA repression following Myc-induction in P493-6 cells. (a) Western blot demonstrating Myc induction following removal of tetracycline (tet). Leftmost tet (+) or tet (-) lanes represent cells grown with or without tet for 72 hours. (b) Northern blots demonstrating miRNA repression following tet release. Numbers below blots represent expression level of each miRNA relative to tet (+) level, normalized to loading control (tRNALys, not shown). Under these conditions, P493-6 cells do not begin proliferating until 48 hours after tet removal and do not reach maximal growth rates until at least 72 hours after tet removal (our unpublished observations and O’Donnell et al., Mol Cell Bio, 2006).

Supplementary Figure 11.

Supplementary Table 3. Specific wash temperatures for northern blotting.

miR-30 family Specific wash temperature (oC)

miR-30a 44

miR-30b 44

miR-30c 48.5

miR-30d 56

miR-30e 45.5

let-7 family Specific wash temperature (oC)

let-7a 58

let-7b 54

let-7d 54

let-7e 44.5

let-7g 47

let-7i 47.5

miR-98 49.5

miR-99/100 family Specific wash temperature (oC)

miR-99a 48

miR-99b 44

miR-100 48

Supplementary Table 4.

Primer sequences for real-time PCR

miRNA transcription unit Amplicon Forward primer sequence(5'-3')

Reverse primer sequence(5'-3')

Genomic coordinates(Human May 2004 assembly)

miR-15a/miR-16-1 U TGGGCACTGTGCTAAATAAATGA TGAGCAATAAACACGATTAATTCGTAA chr13:49,555,189-49,555,239

miR-15a/miR-16-1 S ATACCGCCTCTTAACCCCCC CATGCGTAAAAATGTCGGGAA chr13:49,554,223-49,554,273

miR-15a/miR-16-1 D AATCGTTAGCTCGAAGCCCC GGGAGGAGTGTTCACGGGT chr13:49,553,109-49,553,159

miR-22 U CTTCTCTCGGCCCAAGACG AACTCTAACCCCCGCTCCC chr17:1,565,542-1,565,592

miR-22 S CTGGCTCTGATTGGCAAGGA TCGTGCAATTCCGCCC chr17:1,566,424-1,566,474

miR-22 D ACCTTAGGGTAGAGGGAGGGCT CATGGCCCATCCCCTAATTT chr17:1,567,079-1,567,129

miR-26a-1 D GGAGAGACTGGGAGCGAGTGT CAAACTCACAACCTCCCGGT chr3:37,878,976-37,879,027

miR-26a-2 U CAACCTTCGAATCCCGAAAG GAGTCCTAGGTCCGCCCAC chr12:56,527,775-56,527,825

miR-26a-2 S CTCCATCTGTGAGCGGCC AAAATAGCAAAGCTCCCGACTG chr12:56,526,949-56,526,999

miR-26b U CAAAATAGTAACGACGAGTGAAAAGAA TGGTCTTTTTCCTCGTTTATGAAGTT chr2:219,089,009-219,089,059

miR-26b S GCTCTTGACGTCCTTGCGAG TTCTCTCCTGTCTGGTGGTCG chr2:219,089,633-219,089,683

miR-26b D AGGTGAGGAAACTGAGGCAGG AGGAAACCCCCGAAGAGTTC chr2:219,090,606-219,090,656

miR-29b-1/miR-29a C1 CACCAACTGAAAACCTGCCA GAATGAACGTTGTGAAATCCCTC chr7:130,055,217-130,055,267

miR-29b-1/miR-29a C2 TGCGCGTGACCAGAAAAGTA GCCTCAGATTGGTTCGCTTG chr7:130,055,889-130,055,939

miR-29b-1/miR-29a N CCTTTCACTCCCAGCCCAAT CCACCATGTGGCTATGACACAG chr7:130,058,638-130,058,688

miR-29b-2/miR-29c C AGGGAGCCAACATGGAGACA CGTTGGAAAGTTGTTTACCTTGC chr1:204,384,665-204,384,715

miR-29b-2/miR-29c N ACTCCAAAGACTGTGTTTCTGCC TTATGGAGCAGGCTGCAGTG chr1:204,365,311-204,365,361

miR-30a/miR-30c-2 C1 AGCAGGTGAAAACAAGCTGAATT TAGTTAATAAAGAAAAAGGCCACAACAT chr6:72,171,129-72,171,179

miR-30a/miR-30c-2 C2 TGAGGTAGAGTGGAAACTGGAGAGA AACTTAAAAAAAAATTCTTCCATCCTTCT chr6:72,175,815-72,175,865

miR-30a/miR-30c-2 C3 AGTGGCATCTTAAAGCAGCACAC TTTTTCCCTTTTGCATTTTGAGA chr6:72,178,504-72,178,554

miR-30a/miR-30c-2 C4 GCACGAATGAATATAAAAACACCAGA AAGTGCTAAAGCTATGGTTGACTGC chr6:72,181,043-72,181,093

miR-30a/miR-30c-2 C5 AGCTGCCTTGGCGTCAGTAA GAAGGATTGAAAATAGCTACTGTGTTCA chr6:72,185,502-72,185,552

miR-30a/miR-30c-2 C6 CCCAATCAGGTGTCGGAAAG CTATTGGCTACACTCCCGGG chr6:72,187,355-72,187,405

miR-30d/miR-30b C GCTCCCTCGCCTTTAGTTTGA GCTCTCCCTCAGACACACTGG chr8:135,913,684-135,913,734

miR-30d/miR-30b N CCCTCGTCATACTATGGCACG ACTTCAAGATCATGCTACTGGGC chr8:135,916,115-135,916,165

miR-30e/miR-30c-1 U TACCATCAGCAGAGGCAGTCA AGTGCATTAGGTAACAAGCGCA chr1:40,825,582-40,825,632

miR-30e/miR-30c-1 S GTCGCCCCTTCCCAATTC TGCGCAGAAGCTGTGCTC chr1:40,826,360-40,826,410

miR-30e/miR-30c-1 D TGGCCTGGCAGGTACTTTG GTGTCCCCCATTCCC chr1:40,827,102-40,827,157

miR-34a C1 GACGGGACAGCGGCATC CCCACCTGGTCCTCTTTCCT chr1:9,176,596-9,176,646

miR-34a C2 GGACTCCCGCAAAATCTCC CTTCTCGGTGACCACGCAG chr1:9,176,406-9,176,456

miR-34a C3 AACATTTTGTTGCTTCTTGGAAATT AATTGTGTAGCCTCCGTAAGGG chr1:9,176,176-9,176,226

miR-34a N1 CCTCCACGGTGGAGATGCT GTTGCTTTTTCCTGTCCCCA chr1:9,192,066-9,192,116

miR-34a N2 AAAGCTGCAGTGTCCAAATTCTC CTGATGTCGGTGACAGTGGG chr1:9,196,246-9,196,296

miR-34a N3 GGCAGGACCCGAAATAAGAAG CACCATTTGGGTGCAGGG chr1:9,196,970-9,197,020

miR-146a C GTGCCGAGGAGGGATCTAGAA CCTGCACGCTAACCCTCTCT chr5:159,827,695-159,827,745

miR-146a N AGATTGCTTCCTGAGAGTAGACAACA GTTAACTGAATTACTGGGTTGGAGC chr5:159,824,970-159,825,020

miR-150 C1 CAGAAACTGCACACCCACTCC GCTGGTTCTCTACTGCCCCC chr19:54,696,108-54,696,158

miR-150 C2 GGGCTGCTGTGTTTACAACAAC CAATCAGGGAGGAAACCGG chr19:54,696,266-54,696,317

miR-150 C3 CAAAGAGCAAGTTTAAAAGACCCC GGTGGAAGGCCTGTCAAGAG chr19:54,696,411-54,696,461

miR-150 C4 ACAGGTTATTTGATAACCCAAGGAGA GGAACCCGCTGACCTAGGA chr19:54,696,814-54,696,864

miR-150 C5 GTACCAGGGTCTGAGCCCAG CATGGCCCTGTCTCCCAAC chr19:54,695,881-54,695,932

miR-150 C6 AGCAGCAGCCTCCCACAG CGTGACTGGAGACCCCAGTT chr19:54,707,714-54,707,764

miR-150 N CTATGGACGCCCTGTGTGC TTAGAGGCTTCAGCAGGCCA chr19:54,700,660-54,700,711

miR-497/miR-195 C1 GGCTTTGGGCGGGAGT CTCTTCTGGGTCCTTGTAGGGAT chr17:6,866,331-6,866,381

miR-497/miR-195 C2 GCAGGACAATGGAAGGAAACC GTACGGAGAGGGCGGATATG chr17:6,866,500-6,866,550

miR-497/miR-195 C3 AGGCCTTCCGACGACTCAG GTTAGGGATATCGAGGTTGGCA chr17:6,866,806-6,866,856

miR-497/miR-195 C4 CCATCTGGAGAGCGAGGGA GGGTGAACGCCTGGGTCT chr17:6,866,981-6,867,031

miR-497/miR-195 N TCCGTCTTTTGCCTGCCTC AAATTGGCATCGGGACAGAG chr17:6,863,862-6,863,912

let-7a-1/let-7f-1/let-7d C TCCGTCGCCATTTTATTTCG CATTCTGCCCACCCGCT chr9:94,008,251-94,008,301

let-7a-1/let-7f-1/let-7d N AGAAGTTTCCGATGAACATATGAAGA AGCACTATGAGCCCTTCTGACAT chr9:94,006,470-94,006,520

let-7g C GTTTTCGCGGAACACCTTAGC ACCGACAGCGTGTTGCG chr3:52,287,358-52,287,408

let-7g N CTGTCGGGAAGTGAACACACC CATGGACCAAAATATGGCATCAT chr3:52,289,423-52,289,473

miR-99a/let-7c/miR-125b-2 C1 TGCACCTATTGTGTCCCTGC ACAGTGGCCAATCGGCA chr21:16,364,637-16,364,687

miR-99a/let-7c/miR-125b-2 C2 CACCCACTTCTTACCAAGAACTCC GCTTTAAGTTGTTCACCCTCAAGTTA chr21:16,488,479-16,488,529

miR-99a/let-7c/miR-125b-2 N AGTTTCACTGCTTCATTCTAAATCCTG CAATGTTTTCCATGTTGGATCAAA chr21:16,487,985-16,488,035

CDKN1A (Fig. 2c) CAGATTTGTGGCTCACTTCGTG CCTGCGTTGGTGCGCT chr6:36,754,186-36,754,236

negative (Fig. 2c) AAACCACCCATCCAGAAGGG CGTGGCAGCACTCGTAAGACT chr1:204,366,822-204,366,872

Primer sequences for characterization of the miR-29b-2/29c primary transcript

Amplicon Forward primer sequence(5'-3')

Reverse primer sequence(5'-3')

5' RACE CGACTGGAGCACGAGGACACTGA GTCAACCCTCTGCATACCCATCTCC

5' nested RACE GGACACTGACATGGACTGAAGGAGTA ATAAAAAGTTTTGGGAGCCCTGAGC

3' RACE AGAGCTGCTGCTGCTGATACTGC GCTGTCAACGATACGCTACGTAACG

3' nested RACE TGGGGACAACAGATTTGCATTGA CGCTACGTAACGGCATGACAGTG

Primer sequences for characterization of the miR-29b-1/29a primary transcript

Amplicon Forward primer sequence(5'-3')

Reverse primer sequence(5'-3')

5' RACE CGACTGGAGCACGAGGACACTGA TCCAAGAACTCACACATTCAGGCAAA

5' nested RACE GGACACTGACATGGACTGAAGGAGTA GTCTGCCGTGACAGTTCAGTAGGAG

3' RACE CTCCTACTGAACTGTCACGGCAGAC GCTGTCAACGATACGCTACGTAACG

3' nested RACE GTATGGATTCATTGCCAGGAGCTG CGCTACGTAACGGCATGACAGTG

Primer sequences for characterization of the miR-146a primary transcript

Amplicon Forward primer sequence(5'-3')

Reverse primer sequence(5'-3')

5' RACE CGACTGGAGCACGAGGACACTGA GCTGAGGATACACATCGGCTTTTC

5' nested RACE GGACACTGACATGGACTGAAGGAGTA CTCCTCGTTGTGCTACTGTCTCCTG

3' RACE TTCAGCTGGGATATCTCTGTCATCG GCTGTCAACGATACGCTACGTAACG

3' nested RACE GGGCTTGAGGACCTGGAGAGAGT CGCTACGTAACGGCATGACAGTG

Primer sequences for miRNA cloning

miRNA transcription unit Forward primer sequence(5'-3')

Reverse primer sequence(5'-3')

miR-15a/miR-16-1 ACCGCTCGAGGGCACAGAATGGACTTCAG ATACCGCTCGAGATGGCTTTTCCCCTTCAGAT

miR-22 ACCGCTCGAGCATGCCCTGCTCAGATCTTT ATACCGCTCGAGCTCTCCAACTTGCCCAAAAC

miR-26a-2 ATACCGCTCGAGCGGCAGGGTGTCTGTCTAGT ATACCGCTCGAGCAGGCTTCCAATGGATCAGT

miR-29b-1/miR-29a ACCGCTCGAGGCATGCTCTCCCATCAATA ATACCGCTCGAGACCACATGCAATTCAGGTCA

miR-30b ATACCGCTCGAGGATCCTGAATGCTGTGCCTGTTCTTT ATACCGCTCGAGATCCCTGCCAGCTAGACAA

miR-34a ATACCGCTCGAGCCTCCTGCATCCTTTCTTT ATACCGCTCGAGCCTGTGCCTTTTTCCTTCC

miR-146a ATACCGCTCGAGAGAGATCCACCCACATCAGC ATACCGCTCGAGCCTGAGACTCTGCCTTCTG

miR-150 ATACCGCTCGAGGAGTGGGTGTGCAGTTTCT ATACCGCTCGAGAGCGCACCAGAGGATATGT

miR-195/miR-497 ATACCGCTCGAGTCCCCTGAGCTGAGTTCCTA ATACCGCTCGAGATTTCCCTCTCAGCTTCGTG

let-7a-1/let-7f-1 ATACCGCTCGAGGAGCGGATTCAGATAACCA ATACCGCTCGAGCAGGACCTGACCTTGGACAT