Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity

Human samples

Fresh frozen and pulverized human liver samples were purchased from Sekisui XenoTech. Samples were obtained from normal (lot #: H1281, H1296, H1310 and H1336) and non-alcoholic steatohepatitis (lot #: H0847, H0958, H1027 and H1060) donors, with appropriate consent and ethical approval obtained by Sekisui XenoTech. Detailed information about donors and biopsies characterization can be found at https://www.xenotech.com/in-vitro-test-systems/tissue-samples/tissue-preparations/ under “see donor list for availability of hepatocytes” and the associated data sheets.

Cell culture and palmitate treatment

FL83B mouse hepatocytes (ATCC®, #CRL-2390™) were grown in F-12K medium (Thermo Fisher Scientific, #21127030) supplemented with 10% fetal bovine serum (growth medium). Cells were maintained at 37 °C, 95% O2 and 5% CO2.

A stock solution of 25 mM sodium palmitate (PAL) (Sigma-Aldrich, #P9767) was prepared in serum-free F-12K media containing 1% fatty acid free bovine serum albumin (BSA) (Sigma-Aldrich, #A8806), which was incubated at 70 °C for 30 min shaking at 1000 rpm before use. Next, cells were incubated in 0, 200 and 400 µM PAL diluted in growth medium for 24 h. One percent BSA diluted in growth medium was used as control.

Cytotoxicity assay

Cytotoxicity was measured with CellTox™ Green Cytotoxicity Assay (Promega, #G8742), according to the manufacturer’s instructions.

Intracellular lipid staining

Cells were incubated with a staining solution containing 1 μg/ml of Hoechst 33342 (Thermo Fisher Scientific, #H3570) and 500 ng/ml of Nile Red (Sigma-Aldrich, #N3013) in phosphate buffered saline (PBS) for 20 min covered from light at 37 °C and 5% CO2. Next, staining solution was removed and cells were washed twice with PBS. Fresh PBS was added before measuring Nile Red (excitation/emission = 488/550 nm) and Hoechst 33342 (excitation/emission = 350/461 nm) fluorescence with a microplate reader. Nile Red signal (lipids) was normalized to Hoechst 33342 signal (DNA) to account for differences in cell number.

Transient transfections

Transfection of FL83B cells was performed using Opti-MEM™ (Thermo Fisher Scientific, #31985070) and polyethylenimine (Polysciences, # 23966). Plasmids and polyethylenimine were diluted in Opti-MEM™, following which they were mixed in a 1:3 ratio of μg DNA:μg polyethylenimine and incubated for 20 min at room temperature before adding to the cells. Cells were transfected 24 h after seeding with 0.1 or 1 μg per well of a 96 and 12 well plate, respectively, of pcDNA 3.1, pAd-Klf4 (Addgene, # 19770, gift from Konrad Hochedlinger), pFLAG/HA/mSp3 (VectorBuilder, #VB200204-1104aty), pFLAG/HA/mTfeb (VectorBuilder, #VB200204-1105cfn), pFLAG/HA/mEgr2 (VectorBuilder, #VB200204-1106upe), pFLAG/HA/mNfya (VectorBuilder, #VB200204-1107xne) or pFLAG/HA/mNfyb (VectorBuilder, #VB200204-1108efg) for a total of 48 h. Palmitate treatment was started 24 h after transfection.

Generation of knockout cells

Gene knockout (KO) was achieved by using the CRISPR-Cas9 system. To KO the mouse Tcf4 gene, FL83B cells were transfected as described above with the following plasmids: p-hCas9-mTcf4-gRNA#32572-mTcf4-gRNA#30405 (VectorBuilder, #VB200206-1079fgw) and p-hCas9-Scramble-gRNA1-Scramble-gRNA2 (VectorBuilder, #VB200206-1080bvj) as non-targeting control. Deletion of the mouse Mafk gene was performed using the Edit-R gene editing system (Horizon Discovery). First, stable Cas9 expression was induced by transducing FL83B cells with lentiCas9-Blast (Addgene, #52962-LV, gift from Feng Zhang) lentivirus at a multiplicity of infection (MOI) of 0.5 in growth medium containing 10 µg/ml of polybrene (Sigma-Aldrich, #107689). Next, cells were selected with 6 μg/ml of blasticidin (Thermo Fisher Scientific, #A1113903) and a monoclonal cell line was obtained via limiting dilution. Cas9 expressing FL83B cells were subsequently transduced with Mafk (#VSGM10144-246720110) or non-targeting control (#VSGC10215) lentiviral sgRNA at an MOI of 0.5 in growth medium containing 10 µg/ml of polybrene. Seventy two hours after Tcf4 or Mafk targeting, cells were selected with 1 μg/ml of puromycin (Thermo Fisher Scientific, #A1113803) to generate a polyclonal population of KO cells for downstream experiments.

RNA purification and quantitative PCR (qPCR)

Liver tissue and cells were lysed in 1 ml of TRI Reagent (Sigma #T9424) and incubated for 5 min at room temperature. Aqueous phase was obtained with chloroform following the manufacturer’s instructions, following which RNA was purified and reverse transcribed using Direct-zol™ RNA MiniPrep (Zymo Research, #R2050) and iScript™ cDNA Synthesis Kit (Bio-Rad, #1708891), respectively. Relative changes in mRNA content was quantified by qPCR on a StepOnePlus system (Applied Biosystems) using Fast SYBR™ Green Master Mix (Thermo Fisher Scientific, #4385612). The ΔΔCT method was used for analysis, with TATA binding protein (Tbp) as endogenous control.

RNA sequencing (RNA-seq)

Libraries from human liver tissue and wild type FL83B cells were prepared with TruSeq Stranded Total RNA Library Prep Gold (Illumina, #20020599), single-read sequencing was performed using the NextSeq 500 (Illumina). Libraries from knockout FL83B cells and their corresponding non-targeting controls were prepared with TruSeq Stranded mRNA Library Kit (Illumina, #20020595), pair-end sequencing was performed using the NovaSeq 6000 (Illumina). All data was analysed on the Galaxy platform (https://usegalaxy.eu/). Reads were trimmed with Trim Galore! (Galaxy version 0.4.3.1) and quality was assessed using FastQC (Galaxy version 0.72 + galaxy1). Reads were aligned to the hg38 or mm10 version of the human and mouse genome, respectively, using STAR (Galaxy version 2.7.7a) (Table S8). Strand specificity and read counting was performed with Infer Experiment (Galaxy version 2.6.4.1) and featureCounts (Galaxy version 2.0.1), respectively. Next, we used DESeq2 (Galaxy version 2.11.40.6 + galaxy1) for differential expression analysis (fold change ≥ 1.5, p-value < 0.05 for human liver tissue and q-value < 0.05 for FL83B cells) and the resulting data was annotated with Annotate DESeq2/DEXSeq output tables (Galaxy version 1.1.0). Overlap between different datasets was determined with Venny (version 2.1, https://bioinfogp.cnb.csic.es/tools/venny/) and volcano plots generated with Volcano Plot (Galaxy Version 0.0.3). Generation of circular plots and Gene Ontology (GO) analysis was performed with Metascape (https://metascape.org/gp/index.html#/main/step1)31. Transcription factors activity analysis was achieved with ISMARA (https://ismara.unibas.ch/mara/), where z-value ≥ 1.5 was consider as significant. We used DESeq2 normalized counts to generate heat maps and for hierarchical clustering using Morpheus (https://clue.io/morpheus).

Mass spectrometry analysis of whole cell proteome

Fifteen milligrams of powdered human liver tissue were lysed in 200 μl of lysis buffer [8 M Urea, 50 mM Tris–HCl pH7.5, 150 mM NaCl and 1 × Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, #87786)], vortexed at max speed for 15 s and incubated for 30 min at 4 °C with shaking at 14,000 rpm. Next, samples were sonicated (amplitude 100%, Cycle 0.5) four times for 30 s in a vial tweeter sonicator with 2 min pause on ice between cycles. Samples were then centrifuged for 13,000g for 10 min at 4 °C, supernatant was transferred to a new tube and the pellet was homogenized in 50 μl of lysis buffer. Following an additional centrifugation at 13,000g for 10 min at 4 °C, supernatant was combined with the previous supernatant. Samples were centrifuged a final time at 13,000g for 10 min at 4 °C and supernatant was used for mass spectrometry. On the other hand, mouse hepatocytes were seeded in 6 well plates and harvested in 80 μl of lysis buffer per well (1% sodium deoxycholate (SDC), 0.1 M TRIS, 10 mM TCEP, pH = 8.5), following lysis with 10 cycles of sonication (Bioruptor, Diagnode). All samples were reduced for 10 min at 95 °C and alkylated at 15 mM chloroacetamide for 30 min at 37 °C. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50 w/w; Promega, V5113) for 12 h at 37 °C. Tryptic digests were acidified (pH < 3) using TFA and cleaned up using iST cartridges (PreOmics, P.O.00027) according to the manufacturer’s instructions. Samples were dried under vacuum and stored at − 20 °C.

Sample aliquots comprising 25 μg of peptides were labelled with isobaric tandem mass tags (TMT 10-plex, Thermo Fisher Scientific, #90110) as described previously32. Shortly, peptides were re-suspended in 20 μl labelling buffer (2 M urea, 0.2 M HEPES, pH 8.3) and 5 μl of each TMT reagent were added to the individual peptide samples followed by a 1 h incubation at 25 °C, shaking at 500 rpm. To quench the labelling reaction, 1.5 μl aqueous 1.5 M hydroxylamine solution was added and samples were incubated for another 10 min at 25 °C shaking at 500 rpm followed by pooling of all samples. The pH of the sample pool was increased to 11.9 by adding 1 M phosphate buffer (pH 12) and incubated for 20 min at 25 °C shaking at 500 rpm to remove TMT labels linked to peptide hydroxyl groups. Subsequently, the reaction was stopped by adding 2 M hydrochloric acid until a pH < 2 was reached. Finally, peptide samples were further acidified using 5% TFA, desalted using Sep-Pak Vac 1cc (50 mg) C18 cartridges (Waters, #WAT054960) according to the manufacturer’s instructions and dried under vacuum.

TMT-labelled peptides were fractionated by high-pH reversed phase separation using a XBridge Peptide BEH C18 column (3.5 µm, 130 Å, 1 mm × 150 mm; Waters, #186003562) on an Agilent 1260 Infinity HPLC system. Peptides were loaded on column in buffer A (20 mM ammonium formate in water, pH 10) and eluted using a two-step linear gradient from 2 to 10% in 5 min and then to 50% buffer B (20 mM ammonium formate in 90% acetonitrile, pH 10) over 55 min at a flow rate of 42 µl/min. Elution of peptides was monitored with a UV detector (215 nm, 254 nm) and a total of 36 fractions were collected, pooled into 12 fractions using a post-concatenation strategy as previously described33 and dried under vacuum.

Dried peptides were re-suspended in 0.1% aqueous formic acid and subjected to LC–MS/MS analysis using a Q Exactive HF Mass Spectrometer fitted with an EASY-nLC 1000 (Thermo Fisher Scientific) and a custom-made column heater set to 60 °C. Peptides were resolved using a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin; Dr. Maisch, r119.aq.) at a flow rate of 0.2 μl min−1. The following gradient was used for separation of murine peptides: from 5% B to 15% B over 10 min to 30% B over 60 min to 45% B over 20 min to 95% B over 2 min followed by 18 min at 95% B, whereas the following gradient was used for separation of human peptides: from 5% B to 15% B over 14 min to 30% B over 80 min to 45% B over 26 min to 95% B over 2 min followed by 18 min at 95% B. Buffer A was 0.1% formic acid in water and buffer B was 80% acetonitrile, 0.1% formic acid in water.

The mass spectrometer was operated in DDA mode with a total cycle time of approximately 1 s. Each MS1 scan was followed by high-collision-dissociation (HCD) of the 10 most abundant precursor ions with dynamic exclusion set to 30 s. For MS1, 3e6 ions were accumulated in the Orbitrap over a maximum time of 100 ms and scanned at a resolution of 120,000 FWHM (at 200 m/z). MS2 scans were acquired at a target setting of 1e5 ions, maximum accumulation time of 100 ms and a resolution of 30,000 FWHM (at 200 m/z). Singly charged ions and ions with unassigned charge state were excluded from triggering MS2 events. The normalized collision energy was set to 35%, the mass isolation window was set to 1.1 m/z and one microscan was acquired for each spectrum.

The acquired raw-files were converted to the mascot generic file (mgf) format using the msconvert tool [part of ProteoWizard, version 3.0.4624 (2013-6-3)] and searched using MASCOT either against a murine database (consisting of 49434 forward and reverse protein sequences downloaded from Uniprot on 20141124) or a human database (consisting of 40832 forward and reverse protein sequences downloaded from Uniprot on 20181213) and 390 commonly observed contaminants. The precursor ion tolerance was set to 10 ppm and fragment ion tolerance was set to 0.02 Da. The search criteria were set as follows: full tryptic specificity was required (cleavage after lysine or arginine residues unless followed by proline), 3 missed cleavages were allowed, carbamidomethylation (C) and TMT6plex (K and peptide N-terminus) were set as fixed modification and oxidation (M) as a variable modification. Next, the database search results were imported into the Scaffold Q+ software (version 4.3.2, Proteome Software Inc.) and the protein false discovery rate was set to 1% based on the number of decoy hits. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Acquired reporter ion intensities in the experiments were employed for automated quantification and statistical analysis using a modified version of our in-house developed SafeQuant R script v2.332. This analysis included adjustment of reporter ion intensities, global data normalization by equalizing the total reporter ion intensity across all channels, summation of reporter ion intensities per protein and channel, calculation of protein abundance ratios and testing for differential abundance using empirical Bayes moderated t-statistics. The calculated p-values were corrected for multiple testing using the Benjamini–Hochberg method, with significance defined as fold change ≥ 1.3, p-value < 0.05 for human liver tissue and q-value < 0.05 for FL83B cells. Volcano plots were generated on the Galaxy platform (https://usegalaxy.eu/) with Volcano Plot (Galaxy Version 0.0.3). Generation of circular plots and Gene Ontology (GO) analysis was performed with Metascape (https://metascape.org/gp/index.html#/main/step1)31. Overlap between different datasets was determined with Venny (version 2.1, https://bioinfogp.cnb.csic.es/tools/venny/).

Assay for transposase-accessible chromatin by sequencing (ATAC-seq)

Nuclei from human liver (15 mg) were isolated using with the Nuclei EZ Prep kit (Sigma-Aldrich, #NUC101) and a glass dounce homogenizer (25 strokes with lose pastel and 25 strokes with tight pastel in ice-cold Nuclei EZ Lysis Buffer). Nuclei were filter through a 40 μm cell strainer and counted before ATAC-seq. On the other hand, Fl83B mouse hepatocytes were seeded in 60 mm plates 24 h before BSA or PAL treatment. Nuclei from human liver and mouse hepatocytes were then used for ATAC-seq and library preparation as previously described34. Pair-end sequencing of the libraries was performed using the Illumina NextSeq 500. All data was analysed on the Galaxy platform (https://usegalaxy.eu/). Reads were trimmed with Cutadapt (Galaxy version: 1.16.5) and quality was assessed using FastQC (Galaxy version 0.72 + galaxy1). Reads were aligned to the hg38 or mm10 version of the human and mouse genome, respectively, using Bowtie2 (Galaxy version 2.3.4.3 + galaxy0) (Table S8). Low quality reads (phred < 30) were filtered out with Filter (Galaxy Version 2.4.1) and duplicated reads were removed with MarkDuplicates (Galaxy version 2.18.2.2). As an additional quality control, we used CollectInsertSizeMetrics (Galaxy Version 2.18.2.1) and plotFingerprint (Galaxy Version 3.5.1.0.0) (Fig. S4). Next, we used Genrich (Galaxy version 0.5 + galaxy2) for peak calling (q-value < 0.05) by pooling individual BAM files (replicates) of the same experimental group, while ChIPseeker (Galaxy version, #1.18.0 + galaxy1) was used to annotate peaks. The overlap between different ATAC-seq datasets was performed with bedtools Intersect intervals (Galaxy version 2.29.0). Heat map and density plots of ATAC-seq peaks were created with plotHeatmap (Galaxy version 3.0.2.0) and plotProfile (Galaxy version 3.1.2.0.0), respectively. Data was visualized on the Integrated Genome Browser-9.1.435 to generate representative genome browser figures, for which BAM files were merged with Merge BAM Files (Galaxy version 1.2.0) and then normalized using bamCoverage (Galaxy version 3.0.2.0). CentriMo (version 5.1.0, https://meme-suite.org/meme/doc/centrimo.html) was used to perform motif enrichment analysis (E-value threshold < 0.05)36. Generation of circular plots and protein–protein interaction network analysis was performed with Metascape (https://metascape.org/gp/index.html#/main/step1)31. GO analysis was performed with DAVID 6.8 (https://david.ncifcrf.gov/). Transcription factor activity analysis was performed with Cis-Regulatory Element Motif Activities (CREMA; https://crema.unibas.ch/crumara/). Finally, clustering of transcription factor motifs based in their consensus sequence was performed using STAMP (http://www.benoslab.pitt.edu/stamp/)37.

Ethics statement

Human liver tissue was collected with appropriate informed consent and ethical approval obtained by Sekisui XenoTech. Cell culture experiments, downstream processing of cell and human samples, and other experimental protocols and methods were performed according to the current institutional guidelines and regulations of the Biozentrum, University of Basel. Work with biosafety level 1 and 2 specimen was approved by the Swiss Federal Office of Public Health and performed according to institutional and federal guidelines.

Statistics

All qPCR, cytotoxicity and intracellular lipid assays were performed at least three independent times each in triplicate. Number of replicates per experiment is indicated in the figure legend when appropriate. Values are expressed as mean ± SD. Statistical significance was determined with unpaired two-tailed t tests, with significance considered with a p < 0.05. RNA-seq, ATAC-seq and mass spectrometry experiments were performed once with three to four biological replicates as indicated in figure legends. Statistical analysis of these experiments is described above in their corresponding sections.

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