*3.5. Chromatin Immunoprecipitation*

Chromatin immunoprecipitation (ChIP) was performed according to a protocol from the Or Gozani lab at Stanford University (http://www.stanford.edu/group/gozani) [37], with some modifications. For details, see the Supplementary Material.

#### **4. Conclusions**

We report here that miR-17-92 cluster expression from the intronic A/T-rich promoter region, although critically depending on c-Myc binding, includes some specific contribution of sequences within ~0.7 kb upstream of the mature miR-17-5p coding sequence. Our reporter expression data suggest multiple TSSs within this A/T-rich region, although the transcription initiation region predicted by Ozsolak *et al.* [16], ~0.2 kb downstream of the c-Myc E3 box, may well be the major one. E3 site-independent transcription initiation within ~0.7 kb upstream of the mature miR-17-5p coding sequence was more pronounced in K562 *versus* HeLa cells (Figure 1), indicating cell type-specific differences in cluster expression from the intronic promoter region. By RNAi and ChIP, we establish that Pim-1 acts in concert with c-Myc at the E3 site to activate transcription from the intronic promoter region.

#### **Acknowledgments**

We are grateful to Lisa Schemberger, Nicole Bürger and Moana Klein for technical assistance, Marcus Lechner for statistical analyses and Markus Gößringer for fruitful discussions. The work was supported by grants of the Fritz Thyssen Stiftung (reference no. 10.06.1.186 to A.G. and R.K.H.) and the German Cancer Aid (Deutsche Krebshilfe, grants 106992 and 109260 to A.G., R.K.H. and A.A.).

#### **Conflict of Interest**

The authors declare no conflict of interest.

#### **Appendix**

#### *Cell Culture*

Cells (K562 and HeLa) were cultivated under standard conditions in a humidified atmosphere (37 °C, 5% CO2) supplemented with RPMI 1640 (K562) or IMDM (HeLa) containing 10% FCS (PAA, Cölbe, Germany).

#### *Chromatin Immunoprecipitation (ChIP)*

2 × 107 K562 cells in 13 mL RPMI medium were crosslinked with 1% formaldehyde for 10 min at 37 °C. Reactions were stopped by adjusting to 0.125 M glycine, and cells were collected by centrifugation at 400*g* for 5 min at room temperature. For cell lysis, cells were resuspended in 750 µL RIPA-buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% NP40, 0.1% SDS, 0.2 mM PMSF) supplemented with the complete Mini Protease Inhibitor Mix from Roche (Mannheim, Germany) according to the manufacturer's instructions. Lysed cells were sonicated in a Branson Sonifier 250 (duty cycle 50%, output control 2, for 3.5 min, on ice water) (Heinemann, Schwäbisch Gmünd, Germany) and centrifuged at 16,000*g* for 10 min at 4 °C. The supernatant was pre-cleared with 10 µL of blocked *Staphylococcus aureus* cells (Pansorbin® Cells, Calbiochem/Merck, Darmstadt, Germany) for 15 min at 4 °C on a rotor wheel. After a second centrifugation step (16,000*g*, 5 min, room temperature), the supernatant was split into two samples (each ~350 µL, representing + and − specific antibody (AB)), which were adjusted to buffer D (16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X 100, 0.01% SDS) and a total volume of 500 µL. 1 µg (1 to 5 µL) of the respective antibody was added to "+AB" samples, whereas the same volume buffer D was added to "−AB" samples. At this point, also the "Mock" control was prepared, consisting of 500 µL buffer D.

"+AB", "−AB " and "Mock" samples were then incubated for at least 3 h at 4 °C. The following antibodies were used: monoclonal anti-c-Myc (sc-40) and anti-Pim-1 (sc-13513) (Santa Cruz Biotechnology (Heidelberg, Germany), anti-Phospho HP1γ (Ser83) polyclonal antibody (2600S) (Cell Signaling Technology, Danvers, MA, USA). In the case of mouse monoclonal antibodies (c-Myc and Pim-1), samples were additionally incubated for 1 h with a second monoclonal goat anti-mouse IgG antibody (sc-2005, Santa Cruz Biotechnology, Heidelberg, Germany). Immunoprecipitation was initiated by adding 10 µL of Pansorbin® cells (see above) to the "+AB", "−AB" and "Mock" samples, followed by incubation for 15 min at room temperature. Samples were centrifuged (16,000× *g*, 3 min, room temperature); the supernatant of the "−AB" sample was saved, later serving as the input control. Pellets were washed twice with dialysis buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA) and four times with IP-wash buffer (100 mM Tris-HCl pH 9.0, 500 mM LiCl, 1% NP40, 1% deoxycholate). Antibody-bound material was eluted from Pansorbin® cells by adding 150 µL elution buffer (50 mM NaHCO3, 1% SDS), vortexing for 3 s, and centrifugation (16,000 *g*, 3 min, room temperature). The supernatant was collected and the procedure was repeated. Reverse crosslinking and RNA digestion was performed in 280 µL buffer [0.3 M NaCl, and 1 µL RNase A (10 mg/mL)] for 5 h at 67 °C. Chromatin was precipitated with ethanol, followed by a Proteinase K digest (Thermo Fisher Scientific, Bremen, Germany). DNA was purified by phenol/chloroform extraction and ethanol precipitation in the presence of 0.3 M NaOAc, pH 5.2. PCR amplification using Taq DNA polymerase and the co-immunoprecipitated DNA as template was done under the following conditions: 2 min at 95 °C in the absence of enzyme, followed by 35 amplification cycles of 45 s at 95 °C/45 s at 60 °C/45 s at 72 °C using the following primers:

A1 forward 5'-AAA GGC AGG CTC GTC GTT G A1 reverse 5'-CGG GAT AAA GAG TTG TTT CTC CAA A2 forward 5'-ACA TGG ACT AAA TTG CCT TTA AAT G A2 reverse 5'-AAT CTT CAG TTT TAC AAG GTG ATG A3 forward 5'-ACT GCA GTG AAG GCA CTT GT A3 reverse 5'-TGC CAG AAG GAG CAC TTA GG A4 forward 5'-CCA ATA ATT CAA GCC AAG CAA A4 reverse 5'-AAA TAG CAG GCC ACC ATC AG A5 forward 5'-GCC CAA TCA AAC TGT CCT GT A5 reverse 5'-CGG GAC AAG TGC AAT ACC AT

#### *Transfection of Reporter Constructs*

For transfection of the suspension cell line K562, cells were washed in medium without serum, followed by electroporation in a BioRad Gene Pulser XCell (München, Germany) with a single pulse in a 4 mm cuvette, using 5 µg of the respective pGL3 derivative plasmid per million cells. 48 h after transfection, cells were washed in PBS, lysed and prepared for luciferase reporter assay measurements.

#### *Transfection of siRNAs*

Transfection of the suspension cell line K562 was performed by electroporation. After a washing step in medium without serum, 10<sup>6</sup> cells were mixed with 1 µg of siRNA (VR1 siRNA, Pim-1 siRNA, c-Myc siRNA or E2F3 siRNA). For the double knockdown experiments, 1 µg of each siRNA, respectively, was used. Cells were electroporated at 330 V with a single pulse in a BioRad Gene Pulser XCell (München, Germany) using a 4 mm cuvette. After electroporation, K562 cells were resuspended in medium containing 10% FCS and cultivated in a humidified atmosphere at 37 °C for 24 h. Cells were washed with PBS and prepared for total RNA extraction.

Transfection of HeLa cells was done using the transfection agent Lipofectamine™ 2000 (Life Technologies Invitrogen, Darmstadt, Germany). One day before transfection, 8 × 104 cells were seeded into 24-well plates and cultivated under standard conditions. SiRNA complexes (VR1 siRNA, Pim-1 siRNA, c-Myc siRNA or E2F3 siRNA) were prepared according to the manufacturer's protocol. To perform single knockdown experiments, 40 pmol of the respective siRNA were used. In case of the double knockdown experiments, 20 pmol of each siRNA were mixed in Opti-MEM® 1 (Life Technologies Invitrogen, Darmstadt, Germany). 4 to 6 h after transfection, the medium was replaced with IMDM containing 10% FCS. Cells were cultivated 48 h under standard conditions until preparation for total RNA extraction.

#### *RNA Preparation and Quantitative Real-Time PCR*

For total RNA isolation, transfected cells (K562 or HeLa) were lysed (vortexing or mixing by pipetting up and down) in 750 µL lysis solution (0.8 M guanidinium-thiocyanate, 0.4 M ammonium-thiocyanate, 0.1 M sodium acetate pH 5.0, 5% glycerin, 38% phenol pH 4.5–5.0 (Roth®-Aqua-Phenol, Roth, Karlsruhe, Germany), 1 pellet 8-hydroxychinolin). Then, 200 µL of chloroform was added and phases were separated by centrifugation. The aqueous phase was mixed with 2 volumes of isopropanol, followed by incubation for 15 min at room temperature and centrifugation. The air-dried RNA pellet was dissolved in RNase-free water and incubated for 30 min at 37 °C with 1 U DNase I per µg RNA in 100 µL 1× DNase I buffer (DNase I, Thermo Fisher Scientific, Schwerte, Germany) according to the manufacturer's instructions. Then, another identical aliquot of DNase I was added, followed by incubation at 37 °C for another 30 min. Samples were extracted with an equal volume of Roth®-Aqua-Phenol (see above), followed by extraction of the aqueous phase with chloroform and isopropanol precipitation as above. RNA pellets were finally washed with 75% ethanol, air-dried and redissolved in 10 µL RNase-free water. 0.5 to 1 µg of total RNA were reverse-transcribed with RevertAid H Minus RT Polymerase (Thermo Fisher Scientific) according to the manufacturer's protocol. For determination of KD efficiencies a random hexamer primer was used to generate cDNA samples. In case of calculating pri-mir-17-92 levels the gene-specific reverse primer specified below was used for cDNA synthesis. Quantitative RT-PCR was performed in duplicate in a BioRad iQ™5 (BioRad, München, Germany) with the Absolute qPCR SYBR Green Capillary Mix (Thermo Scientific AbGene, Hamburg, Germany); cDNAs were diluted 1:5 or 1:10 and 4 µL of the reaction mixture used for determining RNA transcription levels. Quantitative PCR assays for miRNA detection were conducted as follows: Thermo-Start™ DNA polymerase was activated for 15 min at 95 °C followed by 55 amplification cycles of 10 s at 95 °C/20 s at 60 °C/12 s at 72 °C. Subsequently, melting curves of the PCR products were generated: samples were cooled from 95 to 65 °C (20 °C per s), kept at 65 °C for 20 s, followed by heating steps of 1 °C per cycle up to 95 °C and kept for 10 s at 95 °C.

Quantitative RT-PCR assays for mRNA detection were changed as follows: Thermo-Start™ DNA polymerase was activated for 15 min at 95 °C followed by 55 amplification cycles with a denaturation step for 10 s, primer annealing for 10 s at 55 °C and amplification at 72 °C for 10 s. Subsequently, a melting curve was generated for the PCR products; samples cooled from 95 to 65 °C (20 °C per s), kept at 65 °C for 20 s, followed by heating steps of 1 °C per cycle up to 95 °C and kept for 10 s at 95 °C.

The mRNA and pri-miR-17-92 levels were calculated from the crossing points by the 2^-∆∆*C*<sup>T</sup> method [38] using β-Actin mRNA or 5S rRNA as internal controls. Knockdown efficiency was quantitated by qRT-PCR and only those experiments showing more than 65% reduction in protein levels were used for quantification of pri-miR-17-92 levels. All primers for qPCR measurements were purchased from Metabion (Martinsried, Germany) and designed using the software tool Universal ProbeLibrary (Roche Applied Biosystems, Mannheim, Germany). A list of all primer sequences according to the human sequences is shown underneath:

Pri-miR-17-92: forward primer 5'-CAT CTA CTG CCC TAA GTG CTC CTT and reverse primer 5'-GCT TGG CTT GAA TTA TTG GAT GA; 5S rRNA: 5'-TCT CGT CTG ATC TCG GAA GC and 5'-AGC CTA CAG CAC CCG GTA TT;

c-Myc mRNA: forward primer 5'-CCT TGC AGC TGC TTA GAC and reverse primer 5'-GAG TCG TAG TCG AGG TCA T; E2F3 mRNA: forward primer 5'-GAG ACT GAA ACA CAC AGT CC and reverse primer 5'-CCT GAG TTG GTT GAA GCC; Pim-1 mRNA: forward primer 5'-ATC AGG GGC CAG GTT TTC T and reverse primer 5'-GGG CCA AGC ACC ATC TAA T; Actin mRNA: forward primer 5'-CCA ACC GCG AGA AGA TGA and reverse primer 5'-CCA GAG GCG TAC AGG GAT AG.
