*Plasmid Construction and Seed Mutagenesis*

All primers, which were used for plasmid construction and seed mutagenesis, are listed below (restriction sites in italics):

pGL3 1.5 kb: 5'-ATA T*AG ATC T*TG CCG CCG GGA AAC GGG TT and reverse primer R1 5'-ATA T*AA GCT T*CC ATA CAA ATT CAG CAT AAT CCC TAA TGG;

pGL3 625 bp: 5'-ATA TAG ATC TCT TTA GAC AAT GTA CCT TTT CTG and reverse primer R1 (see above);

pGL3 339 bp: 5'-ATA T*AG ATC T*GT GGA AGC CAG AAG AGG AGG A and reverse primer R1 (see above);

pGL3 279 bp: 5'-ATA TAG ATC TGG TAC ACA TGG ACT AAA TTG CC and reverse primer R1 (see above);

pGL3 197 bp: 5'-ATA T*AG ATC T*CT CTA TGT GTC AAT CCA TTT GGG AG and reverse primer R1 (see above);

pGL3 250 bp: 5'-ATA T*AG ATC T*GT GGA AGC CAG AAG AGG AGG A and reverse primer R3 5'- ATA T*AA GCT T*GC CTT AAG AAT TCT TTA CAG AAG GC;

pGL3 190 bp: 5'-ATA T*AG ATC T*GG TAC ACA TGG ACT AAA TTG CC and reverse primer R3 (see above);

pGL3 108 bp: 5'-ATA T*AG ATC T*CT CTA TGT GTC AAT CCA TTT GGG AG and reverse primer R3 (see above);

pGL3 339 inv: 5'-ATA T*AA GCT T*GT GGA AGC CAG AAG AGG AGG A and 5'-ATA T*AG ATC T*CC ATA CAA ATT CAG CAT AAT CCC TAA TGG.

#### *Bioinformatical Promoter Analysis*

For promoter analyses of the intronic A/T-rich region, a sequence of 1490 bp (human), beginning at the functional c-Myc site (E3) and ending at the 5'-end of the first mature miRNA sequence, miR-17-5p (for details, see Supplementary Figure S1), was analyzed with several web-based promoter prediction tools. The following tools were used to predict promoter elements or putative TSSs:

*Neural Network promoter prediction:* (http://www.fruitfly.org/seq\_tools/promoter.html) [39]; *McPromoter006:* (http://tools.genome.duke.edu/generegulation/McPromoter/) [40]; *Promoter 2.0 Prediction Server:* (http://www.cbs.dtu.dk/services/Promoter/) [41]; *PromPredict:* (http://nucleix.mbu.iisc.ernet.in/prompredict/prompredict.html) [42].

Putative TSSs were predicted and calculated using the software available at http://rulai.cshl.org/tools/genefinder/CPROMOTER/human.htm [43].

All promoter prediction tools predicted several different promoter elements in the 1.5 kb region upstream of miR-17-5p and only the web-based tool Promoter 2.0 failed. The calculated promoter predictions of the indicated tools did not match in any sequence region, giving the assumption that the A/T-rich 1.5 kb intronic region in front of the miR-17-92 coding sequence has only weak promoter activity itself. Due to the fact that this intronic region has an overall high A/T-content, nearly all software programs were able to detect putative promoter regions.

#### *Statistical Analysis*

Statistical analyses were done using the software R [44]. *p*-Values were calculated with the Welch Two Sample Test.

#### **Supplementary Figures**

**Figure S1.** Relevant sequence region of C13*orf*25, including the CpG island harboring the host gene promoter, the A/T-rich region, and the miR-17-92 cluster; sequence and position of the last exon 4 of C13*orf*25 is indicated at the end. Shown sequences are based on the NCBI reference sequence NG\_032702.1 and the GRCh37/hg19 assembly [25]. The boundaries of the CpG island, important previously identified regulatory elements, mature miRNA coding sequences and relevant primer sequences are highlighted in the sequence and annotated at the margins.



**Figure S1.** *Cont.*

**Figure S2.** Quantitative RT-PCR of the pri-mir-17-92 transcription levels in the human cell lines K562, HeLa and HUH7 (hepato cellular carcinoma cells). 2^-ΔΔpri-mir-17-92 values are normalized against 5S rRNA and obtained from at least 3 independent experiments (+/−S.E.M.). The amount of pri-mir-17-92 transcript in K562 cells was set to 1.

**Figure S3.** (**A**) Schematic representation of the intronic A/T-rich region preceding the miR-17-92 coding sequence. The region A1 defines the genomic sequence 0.1 kb downstream of the functional c-Myc binding site (E3 box) that was amplified in ChIP analyses. A2 covers a segment immediately upstream of the miRNA-coding region; A3–A5 are located along the coding sequence of the human miR-17-92 cluster. The length (bp) of each amplicon is indicated at the top; (**B**) ChIP analysis of the regions A1 to A5 in K562 cells, using an antibody specific for HP1γ or RNA polymerase II (only A2 analyzed). +AB, with antibody; −AB, without antibody; Mock, buffer only without cell lysate; Input, supernatant of the "−AB" sample after immunoprecipitation and centrifugation (for details, see Supplementary Material); (**C**) ChIP analysis of the A2 region in HeLa cells using the antibody specific for HP1γ.

**Figure S4.** Effect of an siRNA-mediated Pim-1 knockdown on promoter activity of the ~1.5 kb reporter construct in HeLa cells. RLU values were derived from 5 independent triplicate experiments (+/−S.D.). RLU values for the c ontrol (left bar, transfected with the reporter plasmid but in the absence of a siRNA) were set to 100%. Lipofectamine transfection of HeLa cells was done as described under Supplementary methods, with the following alterations: 2 × 105 cells were used, and 40 pmol (0.6 µg) siRNA plus 0.5 µg of the reporter plasmid were combined in 50 µL Opti-MEM ® I medium and mixed with 1.5 µL Lipofectamine™ 2000 in 50 µL Opti-MEM ® I medium. The resulting mixture (~100 µL) was incubated for 20 min at room temperature to allow complex formation before addition to the cells. For the control (left bar), the siRNA was omitted.

### **Supplementary Table**

**Table S1.** Quantification of the pri-miR-17-92 levels and the c-Myc, E2F3 and Pim-1 mRNA levels after siRNA-dependent knockdown by qRT-PCR. Expression 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. To determine the c-Myc, E2F3 and Pim-1 knockdown efficiencies, expression levels were normalized to the levels obtained by transfection of K562- or HeLa cells with an unrelated siRNA directed against the vanilloid receptor (siVR1).





### **References**



Reprinted from *IJMS*. Cite as: Nagata, Y.; Shimizu, E.; Hibio, N.; Ui-Tei, K. Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA. *Int. J. Mol. Sci.* **2013**, *14*, 11171-11189.
