TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury

Abstract

Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah-/- mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type-specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.

The dynamic chromatin architecture of the regenerating liver

“…We identified 16,043 differentially accessible regions between quiescent and repopulating hepatocytes ( Figure 2B, Table 2), of which 1,244 displayed increased accessibility in 1-week and 1,266 increased accessibility in 4week repopulating hepatocytes, while 2,058 regions showed decreased accessibility in week 1 and 2,036 decreased in week 4. Hierarchical clustering of the differentially accessible sites showed a clear separation of repopulating and quiescent hepatocytes ( Figure 2C), corroborating with previous transcriptome studies that 1-week and 4-week repopulating hepatocytes have a similar expression profile distinct from quiescent 8 hepatocytes 15 . Replicates also clustered within the same condition, illustrating the reproducibility between biological replicates.…”

“…This is achieved by expressing the GFP-tagged nuclear envelope protein SUN1-GFP with FAH in Fah -/mice, followed by sorting of GFP-positive nuclei from repopulating hepatocytes and ATAC-seq 20 . We identify promoter accessibility changes corresponding to upregulation of cell cycle pathways and downregulation of metabolic pathways, corroborating previous gene expression studies 12,15 . Integrative expression level and chromatin accessibility analysis suggests that gene activation is primarily associated with increased promoter accessibility, while inactivation is correlated with closure of selected promoters and enhancers.…”

“…Establishing the relationship of chromatin structure, transcriptional regulators, and the effects on gene expression is therefore vital in elucidating the transcriptional control governing the regenerative process. To date, most studies have relied on transcriptomic studies to document gene expression changes in the regenerating liver [11][12][13][14][15] , while two others that focused on histone modifications 16,17 . However, these processes are downstream of chromatin reorganization and therefore do not capture the dynamic crosstalk of chromatin accessibility and transcriptional regulation.…”

“…However, these processes are downstream of chromatin reorganization and therefore do not capture the dynamic crosstalk of chromatin accessibility and transcriptional regulation. To identify transcriptomic changes specific to repopulating hepatocytes, we previously employed the translating ribosome affinity purification (TRAP) 18 method followed by high-throughput RNA-sequencing (TRAP-seq) that coexpresses the GFP-tagged ribosomal protein, GFP-L10A, with FAH followed by affinity purification with anti-GFP antibodies to isolate translating mRNAs only from repopulating hepatocytes 15 . To 6 discern the dynamic chromatin patterns that underlie liver repopulation, we now implement the 'isolation of nuclei tagged in specific cell types' (INTACT) 19 method to isolate nuclei only from repopulating hepatocytes.…”

A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver

“…To examine transcripts induced in FAH-positive repopulating hepatocytes for putative miRNA binding sites, we utilized our previously published TRAP-Seq data set generated using the Fah -/mouse model. 2 Transcripts in repopulating hepatocytes were assigned to one of four clusters using k-means clustering based on temporal changes in expression level ( Figure 4A). The 3'UTRs of transcripts of cluster four (n=3,071), which displayed elevated expression at four weeks after the initiation of liver injury, were significantly enriched for the seed recognition sequences of several miRNAs identified in our screen as strong regulators of repopulation.…”