Cell culture and transfections
HEK293T cells were grown in DMEM with 10% (v/v) FCS (Capricon Scientific), 2 mM L-Glutamine (Capricon Scientific), and 1% (v/v) Pen Strep (GE Healthcare) at 37°C and 5% CO2. Stable cell lines were established using an optimized Sleeping Beauty Transposon System (28). 50 ng of SB transposase vector SB100X and 1 µg of the respective plasmid(s) were applied with Metafectene Pro® (Biontex) (pSBtet::MLL-AF4, pSBtet::MLL-AF4m, pSBtet::AF4-MLL, pSBtet::TAFIC::GFP). After 24 hours, cells were subjected to either Puromycin (AF4-MLL, 2µg/ml) or Blasticidine (MLL-AF4, MLL-AF4m; 15µg/ml). The cells were incubated with selection markers for 3–10 days. Transgene induction was carried for at least 48h with 1µg/ml Doxycycline. Another cell line was created by stable transfection of the pSBtet-P vector (without Luciferase) and used throughout the experiments as mock control.
Plasmid Constructions
The plasmid encoding the MTM-HA-hum-pSer gene was kindly provided by Akihiko Yokoyama (Tokyo, Japan; 2), and was used to replace to human pSer domain by the murine counterpart to obtain the MTM-HA-mur-pSer construct. Both constructs were cloned via Sfi1 sites into pSBtet-P.
The TAFIC::GFP construct was designed by fusing the open reading frames of TAFIC with that one of super-folder GFP (pET29BH4:10xHis-TEV-sGFP was a gift from Dr. Jan Hering, Frankfurt, Germany) to obtain the final constructs. This construct was cloned via Sfi1 sites into pSBtet-B. TAFIC is central part of SL1 and was already shown to bind strongest to the pSer domain of AF4 (1), and thus, was used in all experiments to represent SL1 binding to MA4 or MA4m.
The MA4::mCh and MA4m::mCh constructs were designed by eliminating the terminal stop codon and fusing the open reading frame to the mCherry open reading frame. The two final constructs were cloned into pSBtet-P.
The rRNA promoter sequence was cloned by PCR from the human genome with the 2 oligonucleotides pHrRNA.F (5'-CACCTCGAGCGCGATCCTTTCTGGAGAGTCCC-3') and pHrRNA.R (5'-AAGCGAATTCGACGAGAACGCCTGACACGCAC-3'), digested with XhoI and EcoRI and cloned as a 754 bp long DNA fragment into the pGL3-IRES-Basic (Addgene) to obtain a Pol I Luciferase reporter plasmid. pGL3-IRES served as negative control. A SV40-Renilla Luciferase construct served as internal standard for all experiments. The ribosomal promoter element contained the upstream element (binding site for UBF) and the core element (binding site for UBF and SL1), as well as the transcriptional start site.
Rna Extraction, Cdna Synthesis And Rt-pcr Experiments
RNA extraction, cDNA synthesis and RT-PCR experiments
In all 6 stable cell lines, transgene induction was carried by using 1µg/ml Doxycycline for 48 hours. Total RNA was isolated by using RNeasy® Mini Kit (Qiagen) and cDNA synthesis were performed using SuperScript® II (Invitrogen). All isolated RNAs were quality checked (Agilent Bioanalyzer) and final concentrations were determined. Equal amounts of total RNA were used throughout all experiments, and all experiments were performed with 3 biological replicates. Primers used for RT-PCR analyses are as follows: A4M.F 5’-TCCGGCCCATGGATGGTCAAGATCAGGC-3’, A4M.R 5’-TTGTGGAAGGGCTCACAACAGACTTGGC-3’, MA4.F 5’-ACCTACCCCATCAGCAAGAGAGGATCCTGC-3’, MA4.R 5’-GCCATGAATGGGTCATTTCCTTCAGAATCT-3’, Af4.pSer.F 5’-CGTCTCCATGCTGGAGGACGACCTGCAGCTCAG-3’ and Af4.pSer.R 5’AGAATGCTCCTGGTCACTGCTGCCCTCAGCGACA-3’. Target gene transcription was quantified in Q-PCR experiments with the following primers: HOXA9.F (5’-CAATGCTGAGAATGAGAGCGG-3’), HOXA9.R (5’-TGTATAGGGGCACCGCTTTTT-3’); HOXB4_RT.F (5’-CCTGGATGCGCAAAGTTCAC-3’), HOXB4_RT.R (5’-CCTTCTCCAGCTCCAAGACC-3’), GAPDH.F 5’-GGTCACCAGGGCTGCTTTTA-3’, GAPDH.R 5’-CGTTCTCAGCCTTGACGGTG-3’, qPCR_45SrRNA_28S.F (5’-CGATCTATTGAAAGTCAGCCCTCGACACAAGG-3’) and qPCR_45SrRNA_3’ETS.R (5’-CGGTCGGCGGGAGAGGCCGGGAGGGAGGAAGACGAACG-3’).
Differential Gene Expression Profiling By Mace-seq
For the MACE-Seq experiments, all cell lines were treated with 1 µg/ml Doxycycline for 48h with and total RNA were isolated from transfected cell lines. After testing the correct expression of transgenes, differential gene expression (DGE) profiles were obtained by MACE (Massive Analysis of cDNA Ends) - Seq experiments following the manufacturer protocol (GenXPro, Frankfurt, Germany). Three biological replicates of each cell line were compared with 3 biological replicates of mock-transfected cells. The MACE-libraries were prepared at GenXPro GmbH using the Massive Analysis of cDNA Ends (MACE) Library Preparation Kit (v2.0) from GenXPro GmbH. First, cDNA was generated using Oligo(dT) primers with distinct Oligo IDs per sample for subsequent pooling of up to 24 samples. After pooling, cDNA was fragmentated to an average size of 200 bp using the sonicator Biorupter Plus (Diagenode, Belgium). The distribution of cDNA fragment sizes was monitored using the automated microfluidic electrophoresis station LabChip GXII Touch HT platform (PerkinElmer, USA). The Poly(A) containing cDNA fragments were purified using solid phase reversible immobilization (SPRI) beads (Agencourt AMPure XP, USA), end repaired and ligated to distinct 8-base pair UMI Adapters (also called TrueQuant adapters). Then, the library containing labelled and fragmentated cDNA was amplified by PCR, purified by SPRI beads (Agencourt AMPure XP, USA) and strand-specific sequenced using the HiSeq2500 (Illumina, USA).
Bioinformatic analysis was performed according to the analysis pipeline for MACE libraries by GenXPro GmbH. Unique Oligo IDs and UMIs on each transcript allowed initial demultiplexing and subsequent removal of PCR-duplicates. The remaining reads were trimmed for high-quality as well as adapter-free sequences and aligned to the human reference genome (Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13) using Bowtie 2. Resulting output data were implemented in the database program FileMaker for further analysis. All data received from the Bioconductor software from the MACE-Seq experiments were incorporated into a FILEMAKER database program. In addition, we used the following server for further data analysis: Heatmapper (http://www.heatmapper.ca/expression/) for heatmap analyses and VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/) for volcano plots.
Antibodies Used Throughout This Study
The following antibodies have been used throughout this study: anti-β-Catenin (Cell Signaling, #8480), anti HA-Peroxidase (Sigma Aldrich, #34071100), anti rabbit IgG-Peroxidase (Abcam, ab6721; secondary antibody, Western Blot), anti mouse IgG-Peroxidase (Abcam, ab97023; secondary antibody, Western Blot), anti GFP(Abcam, ab290), anti UBF (Santa Cruz, sc-13125), anti mCherry (Abcam, ab125096), anti β-Actin-Peroxidase (Sigma Aldrich, A3854), anti RNA Polymerase II (Diagenode, AC-055-100), anti mouse IgG-Alexa Fluor®586 (Abcam, ab175473; secondary antibody, IHC), anti p53 (Santa Cruz, sc-47698), respectively.
Cell Fixation And Immunofluorescence Staining And Detection
HEK 293T cells lines were cultivated on Poly-D-Lysin pretreated glass chamber slides and transgene expression was induced for 48 h with 1µg/ml Doxycycline. Next, cells were washed with PBS containing 1 mM CaCl2 and 0,5 mM MgCl2 and then fixed for 20 min in cell fixing solution (3,7 % Formaldehyde (v/v) in PBS + 1 mM CaCl2, 0,5 mM MgCl2) following quenching in 50 mM Glycin in PBS + 1 mM CaCl2, 0,5 mM MgCl2 for 5 min. After repeated washing with PBS, cells were permeabilised for 15 min in a permeabilisation solution (0,2 % Triton™ X-100, 0,1 % SDS in PBS + 1 mM CaCl2, 0,5 mM MgCl2).
In case of immunostaining, glass slides with fixed cells on the surface were blocked in a Coplin Jar with TBST with 5 % BSA for 1h and afterwards incubated in TBST diluted primary antibody o/n at 4°C. The next day cells were washed with TBST and incubated in TBST diluted secondary antibody for 1h at RT. After repeated washing with TBST stained cells were embedded in Duolink® In Situ Mounting Medium with DAPI (Sigma Aldrich) and analysed with the fluorescence microscope Observer Z1 (Carl Zeiss).
For Quantification of UBF protein levels 1 x 104 cells of each stable transfected HEK 293T cell line were seeded in triplicates in a 96-well plate and incubated for 48 h with Doxycycline. After the fixation procedure as mentioned above, an antibody incubation was carried out in TBST o/n and for 1 h respectively. After washing with TBST, 50 µL of HCS NuclearMask™ Blue Stain (H10325, Thermo Fisher Scientific) was added per well and incubated 30 min at RT protected from light. Afterwards, all wells were washed again and 100 µl of TBST was added before measuring flourescense signals at the Varioskan Flash plate reader (Thermo Fisher Scientific). The analysis was performed by normalization to DAPI and mock.
Q-pcr Experiments
All quantitative PCR analyses were performed with the StepOnePlus™ System (Applied Biosystems). All measurements were normalized to the Ct values of GAPDH of mock transfected cells and were analyzed in triplicates. The results were evaluated by the comparative ΔΔCt method.
Viabilitätsassay
For the determination of cell viability 1x106 HEK 293T cells were seeded into 10 cm cell culture dishes and transgene expression was induced for 48 h with 1µg/ml Doxycycline. Cells were detached by Accutase® (Capricorn) treatment and an Aliquot was mixed with Acridine Orange and DAPI containing Solution (Chemometec) and analysed with the Nucleocounter NC3000™ (Chemometec) according to manufactors instructions.
Luciferase Reporter Assay
The rRNA promoter acitvity was measured using the Dual-Luciferase® Reporter Assay System“ from Promega. 4x105 HEK 293T cells were seeded in a 6-well plate in triplicates. The expression of transgenes was induced by the addition of 1µg/ml Doxycycline for 48 h. 24 h prior to analysis, cells were transiently transfected with reporter and control vectors. Measurement of Luciferase activities was performed according to manufactors instructions.
Western Blot
5x105 cells of each HEK 293T cell line were cultivated in 6-well plates for 48 h with 1µg/ml Doxycycline for induction of transgene expression. Afterwards cells were lysed for 45 min in 50 µl lysis buffer (1% Triton X-100 (v/v), 1% Deoxycholat (w/v), 1x protease inhibitor cocktail (Roche) at 4°C. Cell lysates were obtained after centrifugation at 13.000 rpm for 10 min. Whole cell lysate was loaded onto a 10 % SDS Gel. Seperated proteins were transferred onto a PVDF membrane using the standard protocol for Trans-Blot TURBO system (BioRad). After blocking in TBST + 5 % BSA for 1 h at RT, membranes were incubated in primary antibody o/n at 4°C. The next day membranes were washed in TBST and incubated in secondary antibody for 1 h at RT following detection using the Clarity™ ECL Western substrate and Chemi DOC™XRS + Imager (Biorad).
Co Immunoprecipitation
1x107 HEK 293T cells were seeded into a 15 cm cell culture dish with 1µg/ml Doxycycline for induction of transgene expression for 48 h. The Medium was discarded, cells were washed with ice cold PBS, resuspended in lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100 (v/v), 0,5 % NP40 (v/v), 1x Protease Inhibitor Cocktail (Roche)) and incubated for 30 min on ice. After centrifugation at 1000 rpm for 5 min at 4°C the cell lysate was transferred into a new 1,5 ml reaction tube. Protein concentration was determined with Pierce™ BCA Protein Assay (Thermo Fisher Scietific) and 200 µg protein was used per IP which was performed according to NEB protocol.
Chromatin Immunoprecipitation Experiments
ChIP experiments were performed using the Abcam protocol. Stably transfected HEK 293T cells (1 x 107 cells on a 145-mm cell culture plate) were transgene-induced with 1µg/ml Doxycycline for 48 h. For double fixation, the cells were incubated with 2 mM di(N-succinimidyl)glutarate for 45 minutes and 1 % (v/v) formaldehyde for 10 minutes. Sheared chromatin was incubated with magnetic A/G beads and antibodies overnight following precipitation. Quantitative PCR analysis was performed with the percent input method from ThermoFisher Scientific by using the following primers: rRNA.Prom.for (5’-GGCTGCGATGGTGGCGTTTTTGG-3’) and rRNA.Prom.rev (5’-GGACAGCGTGTCAGCAATAACCCG-3’).
Click It Protein Synthesis Assay
The analysis of protein biosynthesis in HEK 293T celllines was performed with 2 x 104 cells in each Poly-D-Lysin pretreated 96-well plates after 48h induction with 1µg/ml Docxcyclin with the „Click-iT™ HPG Alexa Fluor™ 594 Protein Synthesis Assay“(Invitrogen) according to manufactors instructions. Experiments were performed with 6 biological replicates per cell line.