Small Nucleolar RNAs in the Dlk1-Gtl2 Imprinting Locus Maintains LT-HSC Self-renewal by Regulation of rRNA Modification and Translation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE166518 | GEO | 2024/06/03
REPOSITORIES: GEO
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Project description:Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the cleavage and chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. To explore function of snoRNAs in SNORD113-114 cluster in HSCs, we established maternally KO mice by CRISPR-Cas9 technology. Here, we used RiboMeth-seq to examine rRNA 2’-O-me modification changes influenced by SNORD113-114 cluster KO.
2024-06-03 | GSE166515 | GEO
Project description:Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. To explore function of snoRNAs in SNORD113-114 cluster in HSCs, we established maternally KO mice by CRISPR-Cas9 technology. Here, we used a modified small RNA-seq method to examine snoRNA profile in KO mice.
2024-06-03 | GSE166517 | GEO
Project description:Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the cleavage and chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. To explore function of snoRNAs in SNORD113-114 cluster in HSCs, we established maternally KO mice by CRISPR-Cas9 technology. Here, we used scRNA-seq to examine HSC RNA profiles influenced by SNORD113-114 cluster KO.
2024-06-03 | GSE166516 | GEO
Project description:Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the cleavage and chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. To explore function of snoRNAs in SNORD113-114 cluster in HSCs, we established maternally KO mice by CRISPR-Cas9 technology. Here, we used RNA-seq to examine polysome RNA profiles in KO mice.
2024-06-03 | GSE166514 | GEO
Project description:The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.
| S-EPMC5545934 | biostudies-literature
Project description:The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Delta5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Delta5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Delta5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.
| S-EPMC1609179 | biostudies-literature
Project description:BackgroundMicroRNAs play an important functional role in post-transcriptional gene regulation. One of the largest known microRNA clusters is located within the imprinted Dlk1/Gtl2 region on human chromosome 14 and mouse chromosome 12. This cluster contains more than 40 microRNA genes that are expressed only from the maternal chromosome in mouse.ResultsTo shed light on the function of these microRNAs and possible crosstalk between microRNA-based gene regulation and genomic imprinting, we performed extensive in silico analyses of the microRNAs in this imprinted region and their predicted target genes.Bioinformatic analysis reveals that these microRNAs are highly conserved in both human and mouse. Whereas the microRNA precursors at this locus mostly belong to large sequence families, the mature microRNAs sequences are highly divergent. We developed a target gene prediction approach that combines three widely used prediction methods and achieved a sufficiently high prediction accuracy. Target gene sets predicted for individual microRNAs derived from the imprinted region show little overlap and do not differ significantly in their properties from target genes predicted for a group of randomly selected microRNAs. The target genes are enriched with long and GC-rich 3' UTR sequences and are preferentially annotated to development, regulation processes and cell communication. Furthermore, among all analyzed human and mouse genes, the predicted target genes are characterized by consistently higher expression levels in all tissues considered.ConclusionOur results suggest a complex evolutionary history for microRNA genes in this imprinted region, including an amplification of microRNA precursors in a mammalian ancestor, and a rapid subsequent divergence of the mature sequences. This produced a broad spectrum of target genes. Further, our analyses did not uncover a functional relation between imprinted gene regulation of this microRNA-encoding region, expression patterns or functions of predicted target genes. Specifically, our results indicate that these microRNAs do not regulate a particular set of genes. We conclude that these imprinted microRNAs do not regulate a particular set of genes. Rather, they seem to stabilize expression of a variety of genes, thereby being an integral part of the genome-wide microRNA gene regulatory network.
| S-EPMC2500034 | biostudies-literature
Project description:Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.
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Project description:The imprinted delta like 1 homolog (DLK1) - thyroxine deiodinase type III (DIO3) locus regulates development and growth. Its imprinting regulation involves two differentially methylated regions (DMRs), intergenic-DMR (IG-DMR) and maternally expressed gene 3-DMR (Meg3-DMR). In mice, a maternal deletion of the IG-DMR leads to LOI in the locus, proving that the IG-DMR is a cis-acting imprinting control region of the locus. However, the Meg3-DMR overlaps with the promoter, exon 1 and intron 1 of the Meg3 gene. Because deletion of the Meg3-DMR inactivates the Meg3 gene, their roles in imprinting regulation of Meg3-DMR mice is unknown. Therefore, we generated two mouse models: Meg3Δ(1-4) and Meg3Δ(2-4), respectively targeting exons 1-4 and exons 2-4 of the Meg3 gene. A maternal deletion of Meg3Δ(1-4) caused embryonic death and LOI in both embryos and placentas, but did not affect methylation status of the IG-DMR. In contrast, mice carrying a maternal deletion of Meg3Δ(2-4) were born normally and did not have LOI. These data indicate that it is the Meg3-DMR, not the Meg3 gene, which regulates imprinting of the Dlk1-Dio3 locus.
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Project description:Differential DNA methylation plays a critical role in the regulation of imprinted genes. The differentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, influencing the expression of genes across the imprinting cluster. In contrast, DNA methylation patterns are more labile at secondary differentially methylated regions which are established at imprinted loci during post-implantation development. To investigate the nature of these more variably methylated secondary differentially methylated regions, we adopted a hairpin linker bisulfite mutagenesis approach to examine CpG dyad methylation at differentially methylated regions associated with the murine Dlk1/Gtl2 imprinting cluster on both complementary strands.We observed homomethylation at greater than 90% of the methylated CpG dyads at the IG-DMR, which serves as the imprinting control element. In contrast, homomethylation was only observed at 67-78% of the methylated CpG dyads at the secondary differentially methylated regions; the remaining 22-33% of methylated CpG dyads exhibited hemimethylation.We propose that this high degree of hemimethylation could explain the variability in DNA methylation patterns at secondary differentially methylated regions associated with imprinted loci. We further suggest that the presence of 5-hydroxymethylation at secondary differentially methylated regions may result in hemimethylation and methylation variability as a result of passive and/or active demethylation mechanisms.
| S-EPMC5480104 | biostudies-literature