TFEB regulates murine liver cell fate during development and regeneration

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.


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Data analysis
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Nunzia Pastore, Andrea Ballabio 03/04/2020 Zen black was used for image acquisition in Zeiss axiocam MR microscope. FACSDiva was used for the acquisition of FACS data.
CRISPRscan was used for the design of sgRNA sequences. Flow-Jo software was used for the analysis of FACS data. ImageJ software was used for image analysis. Bbduk software (bbmap suite) was used to trimm sequence reads. STAR 2.6.0a on mm10 reference assembly was used for sequence alignment. Htseq count 0.9.1 and Ensemble assembly were used to determine the expression levels of genes. EdgeR was used for differential expression analysis. Bioconductor R package Affy was used to process and normalize microarray data. Bioconductor R package clusterProfiler was used for enrichment analysis. MatchPWM algorithm with Biostrings package was used for the analysis of TFEB binding sites. GraphPad Prism v7 was used for statistical analysis.
The data that support the findings of this study are available from the corresponding authors upon reasonable request. Sequencing data are available in NCBIs Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE35015. Data were not excluded from the analyses.
Replication numbers are indicated in the figure legends.
No randomization was possible for animal experiments since mice were selected by genotype. All control animals were littermate controls. For experiments involving cells, all control and treatment wells were plated at the same time.
Blinding was not possible for mouse experiments since experimental groups were determined based on the different genotypes. Automated methods of data recording and analysis were adopted when possible to prevent user bias. Validation from manufacturer related to application in our study: validated and approved for western blot, ELISA, immunohistochemistry and immunofluorescence. Validation from our study: gives band at expected molecular weight in WB analysis. Rb-anti-Sox9 (Millipore AB5535), #Citations: 152 (Merk link): https://www.merckmillipore.com/IT/it/product/Anti-Sox9-Antibody,MM_NF-AB5535?ReferrerURL=https%3A%2F%2Fwww.google.com%2F#anchor_REF. Validation from manufacturer related to application in our study: validated and approved for western blot, ChIP, ChIP-seq, immunohistochemistry and immunofluorescence. Validation from our study: gives band at expected molecular weight in WB analysis and gives expected results in a control slide for IF analysis. Rb-anti-RFP (Abcam ab62341), #Citations: 151 (Abcam link): https://www.abcam.com/rfp-antibody-ab62341references.html#active-tab. Validation from manufacturer related to application in our study: validated and approved for western blot, immunohistochemistry, immunoprecipitation and immunofluorescence. Validation from our study: gives band at expected molecular weight in WB analysis and gives expected results in a control slide for IF analysis.
Mouse Hepatoblasts were isolated from mouse embryos at stage E13.5 Primary mouse hepatocytes were isolated from 2-months old mice Cells were authenticated by analysis of specific cell markers.
Primary cells used in the study were not tested for mycoplasma.
No commonly used cell lines were used in the study