MRNA-seq of intestinal epithelial cells isolated from Rnf20 and Rnf40 knockout mice
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Mus musculus
SUBMITTER: Robyn Laura Kosinsky
PROVIDER: E-MTAB-9405 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Intestinal epithelial cells (IECs) were isolated from the colon of Villin-CreERT2, Rnf20-flox and Rnf40-flox mice two weeks upon the Tamoxifen-induced, intestinal knockout of Rnf20 and Rnf40. ChIP-seq for H3K4me3 was performed using snap-frozen IECs.
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Project description:WAC is a known positive regulator of (macro)autophagy. WAC also forms a complex with RNF20/RNF40 to promote H2B monoubiquitination and hence to affect transcriptional regulation. This study addresses whether the WAC/RNF20/RNF40 complex regulates autophagy through effects on gene expression. WAC, RNF20 and RNF40 were knocked-down using pools of siRNAs in HEK293A cells. Each knockdown was in triplicate and the control was RISCfree siRNA. mRNA expression profiles were investigated using an Illumina HT12v4 Bead Array.
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Project description:The role of Tfr1 in non-erythroid tissues remains elusive due to the embryonic lethality of the Tfr1 global knockout mouse model. To bypass this problem, we generated a mouse model in which Tfr1 was conditionally deleted in intestinal epithelial cells (IECs). These mice developed severe IEC disruption, characterized by blunted villi, edema, loss of proliferative intervillus IECs, accumulation of lipids, and early neonatal lethality. Strikingly, a wide range of genes associated with epithelial-to-mesenchymal transition were highly upregulated in IEC lacking Tfr1. Additionally, candidate vesicular transport and sorting genes implicated in lipid absorption and trafficking were downregulated. Surprisingly, the presence of a mutant allele of Tfr1, which is unable to bind to iron-loaded transferrin, was capable of rescuing the lethality, intestinal epithelial homeostasis, and proliferation in a majority of the Tfr1 conditional knockout mice. 9 samples (3 wildtype, 3 knockout, 3 rescue) were prepared from the intestinal epithelial cells isolated from the small intestine and proximal colon.
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Project description:Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD are unknown. Histone H2B mono-ubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40 and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment=6.01, p=1.67x10-03), some of whom had abnormal laterality associated with cilia dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right asymmetry and impaired cilia motility. Rnf20, Rnf40 and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and non-ciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3. Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.
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Project description:Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD are unknown. Histone H2B mono-ubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40 and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment=6.01, p=1.67x10-03), some of whom had abnormal laterality associated with cilia dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right asymmetry and impaired cilia motility. Rnf20, Rnf40 and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and non-ciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3. Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.
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Project description:In this study, we focused on elucidating the role of Ring Finger Protein 40 (RNF40) in bone development by using a conditional knockout mouse approach during different stages of osteoblast (OB) differentiation and maturation. RNF40 forms an obligate E3 ubiquitin ligase complex together with RNF20 and mediates the monoubiquitination of lysine 120 of histone H2B (H2BK120ub1). We provide evidence that RNF40 regulates OB differentiation in a stage-dependent manner. Additionally, we show that RNF40 is required for bone cell crosstalk whereby loss of RNF40 leads to a reduction in osteoclast numbers and function through modulation of vitamin D receptor (VDR)-induced Rankl expression in OBs. Taken together, these data imply an important role of RNF40-mediated H2Bub1 in bone formation and remodeling and provide a basis for further investigation of its anti-resorptive potential for the treatment of conditions such as osteoporosis or cancer-associated osteolysis. In this study we show that RNF40 is required in earlier stages of osteoblast differentiation and is involved in bone cell crosstalk by regulating vitamin D induced Rankl expression
2021-02-24 | GSE135628 | GEO
Project description:In this study, we focused on elucidating the role of Ring Finger Protein 40 (RNF40) in bone development by using a conditional knockout mouse approach during different stages of osteoblast (OB) differentiation and maturation. RNF40 forms an obligate E3 ubiquitin ligase complex together with RNF20 and mediates the monoubiquitination of lysine 120 of histone H2B (H2BK120ub1). We provide evidence that RNF40 regulates OB differentiation in a stage-dependent manner. Additionally, we show that RNF40 is required for bone cell crosstalk whereby loss of RNF40 leads to a reduction in osteoclast numbers and function through modulation of vitamin D receptor (VDR)-induced Rankl expression in OBs. Taken together, these data imply an important role of RNF40-mediated H2Bub1 in bone formation and remodeling and provide a basis for further investigation of its anti-resorptive potential for the treatment of conditions such as osteoporosis or cancer-associated osteolysis. In this study we show that RNF40 is required in earlier stages of osteoblast differentiation and is involved in bone cell crosstalk by regulating vitamin D induced Rankl expression
2021-02-24 | GSE135627 | GEO
Project description:Gene body-associated monoubiquitination of H2B (H2Bub1) coupled with promoter-associated active histone modifications such as trimethylation H3 on lysine 4 (H3K4me3) and acetylation H3 on lysine 27 (H3K27ac) facilitate gene transcription. However, surprisingly, only a subset of genes is altered in their expression following knockdown of H2B ubiquitin-protein ligases RNF20 or RNF40 in human cells. In order to obtain a more complete genome- and transcriptome-wide understanding of the role of H2Bub1 in gene transcription, we generated tamoxifen-inducible Rnf40 knockout mouse embryonic fibroblasts (MEFs). Mapping of H2Bub1, H3K4me3, H3K27me3, and H3K27ac occupancy identified an “ascending” gene cluster including bivalent genes, which is occupied by low to moderate amounts of H2Bub1 and exhibits high variability in transcription. We provide evidence that the variation in expression is highly associated with the variations in H3K4me3, H3K27me3, and H3K27ac occupancy. Moreover, these variably marked “ascent” genes are selectively regulated in Rnf40-deficient MEFs. Consistent with previous studies, the effect of Rnf40 loss on the expression of genes within this cluster is also variable with some genes demonstrating increased while others decreased mRNA levels following Rnf40 deletion. Importantly, we identified the Ezh2 gene as an Rnf40/H2Bub1 target whose expression significantly decreased in Rnf40-deficient MEFs. Notably, we show that genes upregulated following Rnf40 deletion are enriched for H3K27me3, which is decreased following Rnf40 deletion, and these effects can be mimicked by treating with a small molecule EZH2 inhibitor. On the other hand, consistent with findings in many eukaryotic systems, genes whose expression decreases following Rnf40 deletion show an enrichment of H3K4me3 and decreased levels following deletion. Together, we provide mechanistic information by which Rnf40, presumably via H2Bub1, modulates gene expression via coordination of the active and repressive marks H3K4me3 and H3K27me3.
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