Yy1 sirna

CD4-naïve T cells were transfected with two different YY1 siRNA (Dharmacon, #1 A-011796-16 and #2 A-011796-17), YY1 siRNA/pre-miR-181a (Ambion, #AM17100, ID:PM10421), YY1 siRNA/siDUSP6 (Dharmacon, A-003964-14), YY1/siSIRT1 (Dharmacon, A-003540-17), and control siRNA (D-001910-01), respectively, using the P3 Primary cell 4D-Nucleofector Kit for the Lonza 4D-Nucleofector System (Lonza). Transfected cells were cultured in RPMI-1640 media supplemented with 10% FBS for 48 h and then cross-linked with 1 µg/ml CD3/CD28 antibody (Ab). ERK phosphorylation was measured by Western blot. To assess CD69 expression, cells were seeded into plates coated with 1 µg/ml anti-CD3/CD28 Ab. CD69 expression was assessed on an LSR Fortessa cell analyzer (BD Biosciences) after 24 h. To assess IL-2 production, cells were cultured with anti-CD3/CD28 Dynabeads (cell to bead ratio 3:1) for 24 h before restimulated with 2.5 ng/ml PMA and 500 ng/ml ionomycin in the presence of Golgi plug (BD Biosciences, 555029) for additional 4 h. Cells were harvested, fixed, and permeabilized followed by staining with the Alexa-Fluor-488-conjugated anti-human IL-2 antibodies and analyzed on the Fortessa.

Naïve CD4 T cells were isolated from PBMC using EasySep Human Naive CD4⁺ T cell Enrichment Kit (STEMCELL Technologies, Cat. # 19555). One million cells were transfected with control siRNA (Dharmacon, D-001910-01) or YY1 siRNA (Dharmacon, A-011796-16) using the P3 Primary cell 4D-Nucleofector Kit and the Lonza 4D-Nucleofector System (Lonza). Transfected cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) for 48 h until collection. Libraries were prepared using the NuGEN Ovation Whole Blood Kit according to the manufacturer’s instructions and sequenced on an Illumina 2500 HiSeq.
RNA-sequencing (RNA-seq) reads were aligned to hg19 using STAR 2.5.3a aligner⁶² , followed by generation of transcript-level counts using featureCounts^{63 (link)} package. Normalization and identification of differentially enriched genes was performed using edgeR^{64 (link),65 (link)} and limma packages from R Bioconductor and modeled to identify differences in paired samples in control and YY1-knockdown groups.

The pri-miR-181a/b1 enhancer region was cloned into the pGL4.27 Luc2p/minP vector (Promega) as described^{28 (link)}. Thirty nanograms of pri-miR-181a/b1 Luc2p/Peak 1 was co-transfected with 1 ng Renilla luciferase reporter pRL-TK (Promega) into HEK293T cells, or Luc2p empty vector (negative control). For TCF1 loss-of-function on pri-miR-181a/b1 enhancer activity, TCF7 siRNA (Dharmacon A-019735-13-0005) or non-targeting siRNA (Dharmacon D-001910-01-05) were co-transfected; YY1 siRNA (Dharmacon A-011796-16-0005) was transfected as a positive control. For gain-of-function, different amounts of pCDNA3-HA-TCF7 (0, 30 or 100 ng, Addgene #40620) or pCDNA3-CTNNB1 plasmids (0, 30, 100 ng, Addgene #16828) were co-transfected. As for GSK3β inhibitor treatments, 1 µM BIO or 10 µM SB216763 was added 12 h after transfection. Cells were collected 48 h later, and enhancer activity was determined using the Dual Luciferase Reporter Assay System (Promega, E1910) as described by the manufacturer’s protocol.