The Role And Mechanism Of CUL4B In Intestinal Homeostasis Maintenance And Fate Decision Of Intestinal Stem Cells

The small intestine involves in multiple biological processes such as digestion,nutrient absorption,metabolic homeostasis,regeneration,microbial barrier,and mucosal immunity.The mammalian intestine is consist of crypt-villi units and self-renews at a rapid turnover rate.Intestinal stem cells(ISCs)are located at the bottom of crypts and responsible for intestinal epithelial renewal.ISCs maitain self-renewal and differentiate into multiple cell fates through characteristic asymmetric divisions.ISCs play an important role in the self-renewal of tract development and intestinal homeostasis.ISCs ruel continuous self-renewal and produce differentiated progenies including rapidly proliferating transit-amplifying(TA)cells,absorptive lineages such as enterocytes,secretory lineages such as Paneth cells,Enteroendocrine cells,Goblet cells,and other cells(Tuft and M cells).The stemness and the cell fates of ISCs are tightly controlled by various signals.Among them,a increased gradient of Wnt signals restricted to the bottom of crypt fuel the continued renewal,ISC stemness and regeneration.Epithelial deletion of Tcf7l2(TCF4),the downstream transcription factor of Wnt pathway,results in a rapid loss of Lgr5+cells.Conversely,overexpression of Dickkopf1(Dkk1),the secreted Wnt inhibitor,leads to loss of crypts.The activation of Wnt signal together with repression of Notch leads to the differentiation of ISCs towards Paneth cells(PCs).The activation of Notch signals together with the turning off of Wnt signals promotes ISCs to differentiate into the lineage of absorptive cells.BMP signals are activated in villi instead of crypts.Either the conditional knockout of BMP receptor BMPR1A or overexpression of the BMP inhibitor Noggin,promotes the formation of ectopic crypts in mice.These signals construct a complex regulatory network to modulate intestinal function and ISCs fate.Further research is still required to clarify new factors and understand regulatory molecular mechanism.The protein encoded by CUL4B gene belongs to the Cullin family.The members of this family form the CULLIN-RING ligase which are the largest E3 ubiquitin ligases in mammals.It involves in regulating multiple biological activities such as cell cycle,transcription,signal transduction,growth and development,viral infections,and DNA damage repair.As the scaffold protein,CUL4B is linked to the adaptor protein by the N-terminus by DDB1 and binds to the RING protein by its C-terminus to form a CRL4B complex.CRL4B complex catalyze the transfer of ubiquitin from E2 enzymes to the substrate proteins for its subsequent ubiquitination modification.CUL4B is highly homologous with CUL4A but has a unique nuclear localization sequence(NLS)that distinguishes it from CUL4A in multiple functions.We and other group have found that the disruption of Cul4b in mice results in early embryonic lethality.The number of mature sperm in the epididymis of Cul4b knockout mice decreased with abnormal morphology.Meanwhile,specific knockout of Cul4b in islet delta cells led to a decrease in insulin secretion and then caused glucose intolerance.CUL4B is involved in the regulation of multiple signaling pathways.CUL4B positively regulated Wnt/β-catenin pathway by protecting β-catenin from GSK3-mediated protein degradation in Hepatocellular Carcinoma(HCC)through nuclear-based transcriptional regulation.CRL4B complex reduced the substrate PPARγ through ubiquitination and affected adipocyte differentiation.Cul4b knockout mice were more prone to obesity.However,no research has addressed the role of CUL4B in intestine regulation.Therefore,in this paper,we will use gene knockout mice combined with intestinal organoid models to explore the expression characteristics of CUL4B in intestine and clarify its role in regulation of intestinal homeostasis and ISCs fate.We aim to further analyze the molecular mechanism of the regulation.Part Ⅰ:The Role of CUL4B in Intestinal HomeostasisIn this part,we established Cul4b intestinal knockout mice model,and preliminarily analyzed the role of CUL4B in intestinal development and self-renewal.We obtained the following results:1.CUL4B was highly expressed in intestinal crypt stem cell compartment.CUL4B was highly expressed at the bottom of crypts where located the stem cell compartment.Interestingly,unlike many cell types in other mammalian tissues where CUL4B was predominantly localized in the nucleus,CUL4B was mainly detected in the cytoplasm of intestine.The cytoplasm location of CUL4B was not changed in organoids treated with Leptomycin B.In accordance with it,co-staining showed the co-express of CUL4B with Lgr5+,the marker of ISCs,and Lyz+,the marker of Paneth cells.CUL4B expression was obviously elevated in organoids cultured in stem cell-enriched expansion medium(EM)than those in differetiation medium(DM).2.Deletion of Cul4b led to reduced self-renewal of ISCs.Cul4b intestinal epithelialspecific knockout mice pVillin-Cre(KOIEC),CAG-CreERT2(KOCAG)and Lgr5-EGFP-IRESCreERT2(KOLgr5)were constructed.Knockdown efficiency of KOIEC mice was confirmed at the mRNA and protein levels.Epithelial deficiency of CUL4B had no effect on mouse body weight but led to decreased intestinal weight,shortened villi,and reduction of crypt cell number.We observed elongated crypts and villi in the transgenic mice.BrdU labeling assay and Lgr5+tdTomato confirmed a slower rate of self-renewal of small intestines of Cul4b intestinal knockout mice.By GO,KEGG and GSEA analysis,we also found downregulation of Ribosome and DNA replication-related pathways in CUL4B-deficient tissues and organoids by mRNA sequencing.The staining of PCNA showed a slower proliferation in KOIEC mice.3.CUL4B deficiency influenced the number of ISCs and differentiated cell types.By immunofluorescence/immunohistochemical staining,in situ hybridization,and Real-time Quantitative PCR of ISCs and differentiated cells,we found that CUL4B-deficient resulted in a reduction of ISCs,Paneth cells,Goblet cells,and Enteroendocrine cells,and an increase of the number of Enterocyte.mRNA sequencing analysis also showed the enrichment of PPAR signaling pathway and enhanced intestinal absorption function in CUL4B deficiency mice.We did not observe obvious changes of CUL4B on intestines with age.Consist with the phenotype of intestine,the number of colon stem cells,Paneth cells and Goblet cells were also reduced in the colon of KOIEC mice.4.Confirming the role of CUL4B in intestinal regulation by in vitro organoid model.We isolated crypts from KOIEC and WT mice to culture them in DM and EM.Organoids derived from KOIEC mice displayed reduced budding efficiency during passaging.Decreased size and formation efficiency were also observed in KOIEC organoids cultured in EM by single cell assay.Meanwhile immunofluorescent staining of proliferative signal,Ki67 was obviously reduced.We observed a significant reduction of Paneth cells per bud in KOIEC organoids.UEA-1 staining confirmed the loss of Paneth cells.In contrast,increased number of Paneth cells per bud was detected in CUL4B overexpression organoids.In summary,CUL4B was highly expressed in intestinal crypt stem cell compartment and expressed in cytoplasm specifically in intestine,and CUL4B deficiency influenced intestinal development and self-renewal.Part Ⅱ:CUL4B promotes Wnt/β-catenin signals,ISCs self-renewal and cell fate decisionThe results above showed that CUL4B maintained the stemness of ISCs and promoted cell commitment toward secretory progenitors and their terminally differentiation into Paneth cells,Goblet cells and Enteroendocrine cells.Meanwhile,terminally differentiated lineage of enterocytes differentiated from absorptive progenitors was repressed.On the one hand,Wnt signals were essential for maintenance of ISCs self-renewal and the formation of secretory progenitor cells.On the other hand,activating Wnt signaling with repressing Notch signaling promoted stem progenitor cells to differentiate into Paneth cells.Combined with mRNA sequencing,we comprehensively analyzed the pathway affected by CUL4B and utililized biochemical and molecular methods to obtains the following results:1.CUL4B promoted the Wnt/β-catenin pathway expression.By mRNA-Seq we found that the differential expression genes(DEGs)that changed in both Cul4b knockout mice intestine and organoid were overlapped with the DEGs in Ctnnb1 knockout mice and Apc knockout mice tissues.We examined the effect of CUL4B deficiency on the expression of the Wnt/β-catenin target genes.qRT-PCR confirmed that lack of CUL4B resulted in a significant decrease of Wnt target genes.Western Blot showed that decreased β-catenin,p-GSK3β(Ser9)and activating form of p-β-catenin(S33/S37/T41)in Cul4b knockout mice.Consistently,they were increased in CUL4B transgenic intestine.Through GSEA analysis and real-time quantitative PCR verification,we found that CUL4B positively regulated downstream target gene of MYC.2.Altered Wnt pathway rescued the phenotypes in Cul4b knockout and transgenic mice intestine.We found that the addition of GSK3β-inhibitor,CHIR99021 and BIO,could efficiently rescue the decreased budding formation,decreased proliferation and the reduction of β-catenin and its target genes.Furthermore,treating CUL4B overexpression organoids with Wnt inhibitor IWP-2 significantly attenuated increased budding formation and expression of Wnt target genes.3.Secreted Wnt3a by Paneth cells was essential for CUL4B in intestinal maintenance.We collected supernatant from WT and KOIEC organoids and treated WT organoids with the supernatant.Compared with the organoids cultured in WT supernatant,the organoids cultured in KOIEC supernatant grew more slowly.qRT-PCR confirmed the down-regulation of Wnt target genes in organoids treated with KOIEC supernatant.Furthermore,the Wnt luciferase reporter system and TOP/FOP Flash Dual Fluorescent Reporter Gene System were used to detect the Wnt activity.The reporter cells treated with supernatant from KOIEC organoids,but not supernatant from KOLgr5 organoids revealed a significantly reduced Wnt activity.Consistently,the addition of recombinant Wnt3a rescued the reduction of budding formation and Wnt pathway in KOIEC organoids.It was further found that there was no significant difference in the budding rate and the number of Paneth cells per bud between DM cultured KOLgr5 and WT organoids.Decreased size was also observed in KOLgr5 organoids cultured in EM in single cell assay.However,the statistical analysis of the single-cell clone formation rate of primary organoids showed that there was no significant difference in the formation ability of KOLgr5 and WT organoids.In summary,we found that CUL4B regulated the Wnt/β-catenin pathway to promote ISCs self-renewal and niche regulation.The alteration of the Wnt pathway rescued the intestinal phenotype caused by CUL4B knockout or overexpression.Wnt3a secreted by Paneth cells was essential for the function of CUL4B in the intestine.Part Ⅲ:CUL4B promotes Wnt signaling and regulates intestinal function by degrading IRGM1We have demonstrated CUL4B was mainly detected in the cytoplasm of intestine.To search for the ubiquitylated substrates degraded by CUL4B through 26S proteasome pathway.We identified CRL4B targets intestinal IRGM1 for degradation by non-standard quantitative proteomics and ubiquitination modification non-standard quantitative omics.We explored how CUL4B participated in the intestinal regulation through IRGM1 to determine whether it affected the Wnt signaling pathway.We obtain the following results:1.IRGM1 was the ubiquitylated degradation substrate of CRL4B complex.We selected 12 candidate substrates from proteins of increased expression and decreased ubiquitinated proteins in Cul4b-deficient crypts.We identified that immunity-related GTPase subfamily protein(IRGM1)was significantly upregulated both in isolated crypts and cultured organoids derived from KOIEC and KOLgr5 mice by WB.2.CUL4B affected the number and function of Paneth cells by degrading IRGM1.The reduced number of Paneth cells and transcript levels of lysosome and AMPs in Cul4b KOIEC organoids was rescued by deletion of Irgml in a dose dependent manner.Decreased organoid formation,budding efficiency and cell numbers per bud were rescued after deletion of Irgm1 as well.3.CUL4B promoted the self-renewal of the Wnt pathway and stem cells by degrading IRGM1.Infection of Irgm1 knockdown lentivirus significantly upregulated the transcription of Wnt target genes(Lgr5 and Sox9)and Wnt luciferase activity in a dose-dependent manner.Interfering Irgm1 efficiently attenuated the reduction of other Wnt target genes and β-catenin and p-GSK3β(Ser9)by Western Blot showed.4.The preliminary research on the role of CUL4B in the regulation of intestinal microbes and the reaction with pathogenic microorganism infection.Paneth cells influenced intestinal microbiota and microbial responses.By 16S RNA sequencing,we found CUL4B did not affect fecal microbiota diversity of mouse fecal samples.We constructed an acute colon-cecal inflammation model in mice by Salmonella typhimurium infection.Mice lacking CUL4B developed more severe pathogen-induced inflammatory responses.But CUL4B deficiency of intestinal organoids did not affect pathogenic microorganism-induced intestinal inflammation because lack of immunocells.In this research,by constructing intestinal epithelial cell knockout,inducible total knockout and inducible ISCs Cul4b knockout mice models,as well as in vitro organoid models,we found that CUL4B promoted Wnt signaling pathway by degrading IRGM1,maintenance of intestinal homeostatic function,stem cell self-renewal and cell fate determination.