Ribosome impairment regulates intestinal stem cell identity via ZAKɑ activation

Abstract

The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5-negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity.

Fig. 1. Inhibition of translation drives stem cell identity changes.

a Western blot analysis confirming reduced Raptor and pS6 (Ser235/236) protein levels in cells from 3 different animals treated with 4-Hydroxytamoxifen. β-actin and Ponceau serve as loading control. b Incorporation of ³⁵S-methionine shows decreased protein synthesis in Rptor^fl/fl organoids compared to WT. Mean and standard deviation are shown (n = 4 biological replicates each accessed in technical duplicates). p values were determined using a two-tailed t-test. c Representative images showing morphological differences in organoids upon Rptor loss, compared to WT. Pictures were acquired using 5x (left panel) and 20x (right panel) magnifications. d RT-qPCR analysis of individual genes related to stem (Lgr5, Mex3a and Axin2), differentiation (Lyz1, Muc2 and Alpi), and fetal-like state (Tacstd2, Sca1, Spp1 and Cnx43) of Rptor^fl/fl organoids compared to WT, using Hprt as a reference. Mean and standard error of the mean are shown (n = 3 biological replicates each accessed in technical triplicates). p values were determined using a two-tailed t-test. e Representative 3D-reconstructed confocal images of wild type and Rptor^fl/fl organoids show significant reduction of adult stem cell marker OLFM4 (left panel, green) and activation of fetal markers SCA1 (middle panel, green) and TACSTD2 (right panel, green). Dapi is used to visualize the nuclei (magenta). Scale bar is 50 µm. f OCR and ECAR analyses reveal decreased respiration and increased glycolysis in Rptor^fl/fl organoids compared to WT. Mean and standard deviation are shown (n = 2 biological replicates each accessed in technical quadriplicates). Refer to Supp. Fig. 1 for quantifications. g Gene Set Enrichment Analysis based on RNASeq differential expression data comparing Rptor^fl/fl organoids to WT (n = 4 from 2 biological replicates for each). Enrichment is shown for transcriptional signatures related to stemness, glycolysis, mTORC1 signaling and fetal-like state. p values were determined using the clusterProfiler package.

Fig. 2. Metabolism is translationally regulated in fetal-like ISCs.

a Experimental workflow of the RiboSeq experiment comparing the translatome of Rptor^fl/fl organoids to WT. Crypt cultures were generated from VillinCre^ERT2RPL22.HA mice both in the presence and absence of Raptor. Tagged ribosomes were pulldown using an HA antibody and ribosome protected fragments were sequenced and mapped to the genome. b Western blot analysis confirming efficient pulldown of HA-tagged RPL22 ribosomes. RPL7 co-immunoprecipitation is used as an indicator of intact ribosomes. β-actin serves as a loading control. Experiments were done in one biological replicate. c Gene Set Enrichment Analysis based on differential translation efficiency data comparing Rptor^fl/fl organoids to stem cell enriched cultures (n = 3). Enrichment is shown for transcriptional signatures related to oxidative phosphorylation and glycolysis. p values were determined using the clusterProfiler package. d Experimental workflow of the RiboSeq experiment comparing the translatome of intestinal stem cells from Rptor^fl/fl to WT mice. Ribosomes were tagged specifically in the instestinal stem cells of mice using an Lgr5Cre^ERT2 promoter, both in the presence and absence of Raptor. 24 h after tamoxifen induction, the ribosomes are recombined with the HA tag exclusively in the intestinal stem cells, allowing the capture and further study of their translatome by RiboSeq. e Imunohistochemistry staining of Lgr5Cre^ERT2RPL22.HA intestines show that HA staining is restricted to ISCs. Stainings were done in 3 biological replicates. Scale bar: 20 μm. f Gene Set Enrichment Analysis based on differential ribosomal occupancy data comparing the intestinal stem cells of Rptor^fl/fl and WT mice (n = 3 biological replicates). Enrichment is shown for transcriptional signatures related to glycolysis and fetal-like state. p values were determined using the clusterProfiler package. g Distribution of RPFs along transcripts show an accumulation of reads in the stop codon in the Lgr5Cre^ERT2Rptor^fl/flRPL22.HA mice (blue) compared with the Lgr5Cre^ERT2RPL22.HA (gray). Barplot depicts RPF abundance change (log2FC) between the two conditions and lines show total RPF abundance (%), for which RPFs are grouped based on their A-site position with stop-codon as reference.

Fig. 3. Ribosome impairment drives metabolic changes and the fetal signature via ZAKα activation.

a RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids treated with cycloheximide at either a low (0.015 ug/ml) or high dose (100 ug/ml) for 30 min. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. b RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids cultured in media without glutamine or leucine for 1 h. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. c Western blot confirming activation of ZAKα in Rptor^fl/fl and low-dose cycloheximide treated organoids (upper panel) and organoids deprived from glutamine and leucine (bottom panel) compared to WT. Red arrows indicate phosphorylated ZAKα, black arrows indicate the ZAKα levels, green stars indicate non-specific bands. Ponceau serves as a loading control. Blots were done on two animals. d RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids treated with low-dose cycloheximide (0.015 ug/ml) for 30 min in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. e OCR and ECAR analyses reveal that deleting ZAKα rescues the metabolic changes caused by low-dose cycloheximide (0.015 ug/ml). Mean and SD are shown (n = 1 biological replicate accessed in technical quadriplicates). f RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT and Rptor^fl/fl organoids in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test. g ECAR analysis show that ZAKα deletion rescues the metabolic changes caused by Rptor deletion. Mean and SD are shown (n = 1 biological replicate accessed in technical quadriplicates). h RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids grown in glutamine-depleted media, in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test.

Fig. 4. ZAKα activates the SRC-YAP axis during ribosome impairment and results in ISCs identity switch in vivo during leucine deprivation.

A Volcano plot depicting differentially enriched interactors of ZAKα in Rptor^fl/fl organoids, using RIME-MS (n = 2 biological replicates). An empty vector tagged FLAG bait is used as a control. Significance when p-value ≼ 0,05 (t-test, two-tailed). B Immunoprecipitation of ZAKα-FLAG confirms interaction with SRC in organoids treated with cycloheximide (0.015 ug/ml) and deprived from glutamine. EV_FLAG is used as a control. One biological animal was used. C Quantification of the western blot shows enrichment of ZAKα-SRC binding in organoids treated with cycloheximide (0.015 ug/ml) and deprived from glutamine, compared with wild type cells. D Western blot showing activation of SRC in Rptor^fl/fl, low dose cycloheximide and glutamine-deprived organoids compared to wild type. This activation is dependent on ZAKα activity, as inhibition with vemurafenib abolishes SRC phosphorylation (T416). β-actin serves as a loading control. Blue panel has a higher exposure. Blots were done on one animal. E Western blot showing activation of YAP in Rptor^fl/fl, low dose cycloheximide and glutamine-deprived organoids compared to wild type. This activation is dependent on SRC, as inhibition with dasatinib abolishes YAP phosphorylation (Y357). β-actin serves as a loading control. Blue panel has a higher exposure. Blots were done on one animal. F Gene Set Enrichment Analysis based on RNASeq differential expression data comparing Rptor^fl/fl organoids to WT (n = 4 from 2 biological replicates for each). Enrichment is shown for transcriptional signatures related to YAP-target genes. p-values were determined using the clusterProfiler package. G RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT and Rptor^fl/florganoids treated with inhibitors for SRC (dasatinib, 100 nM, 24 h) and YAP (verteporfin, 3 uM for 24 h). Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test. H Experimental workflow of the dietary interventions performed in WT and ZAK KO mice. I RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of colons of WT and ZAK KO mice. Hprt is used as a reference. Mean and SEM are shown (n = 5 (WT) and 9 (ZAK KO) biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test.