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Long non-coding RNAs that regulate development – Tales from the dark matter of the genome. Professor Harvey Lodish Whitehead Institute for Biomedical Research Departments of Biology and Biological Engineering, MIT As metazoans evolved the number of encoded proteins remained roughly constant whilst the genome size exploded Genome size bp (millions) Number of encoded proteins Number of chromosomes Drosophila melanogaster 168 13,781 4 C elegans 100 20,424 6 Zebrafish 1505 19,929 25 Xenopus tropicalis 1700 ~21,000 10 Chicken Mouse 1050 3420 14,923 22,085 39 20 Homo sapiens 3279 21,077 23 Arabidopsis 134 27,416 5 Non-coding transcripts constitute a large fraction of the mammalian transcriptome Protein Coding Transcripts Non-coding Transcripts The ENCODE Project Consortium, 2007 The composition of non-coding transcripts in the mammalian transcriptome rRNA, snRNAs miRNAs snoRNAs tRNAs Putative LncRNAs Huttenhofer et al, 2005 Like mRNAs, many LncRNAs are transcribed by RNA Polymerase II, capped, polyadenylated, and spliced. But many remain in the nucleus. LncRNAs fall into multiple subclasses LncRNAs fall into multiple subclasses LncRNAs are involved in several important biological processes X chromosome inactivation: Xist Epigenetic modification: HOTAIR Genomic imprinting: H19, Air Enhancers for neighboring genes: ncRNA-7a p53 signaling pathway: lincRNA-p21 To be discovered X chromosome inactivation Epigenetic Regulation by Long Noncoding RNAs Jeannie T. Lee Science 14 December 2012: 1435-1439 Overview of eukaryotic transcriptional control One way of inactivating large segments of DNA by forming “closed” heterochromatin: methylation of cytosine residues in CG sequences; Methylation is maintained during DNA replication A second way of inactivating large segments of DNA by forming “closed” heterochromatin: Methylation of histone 3 lysine residue 9: Histone H3 lysine 9 methylation is maintained during chromosome replication. X chromosome inactivation LncRNAs are thought to scaffold protein complexes and can regulate gene expression in multiple ways 2 1 3 microRNA sponge RNA decoy RNP component lncRNA lncRNA lncRNA 1 2 3 4 mRNA 1 2 3 4 5 6 4 3 2 lncRNA lncRNA mRNA 4 Recruitment of chromatin modifiers 5 Translation inhibition 6 Splicing modulation 7 Degradation 1 LncRNAs are thought to scaffold protein complexes and can regulate gene expression in multiple ways 2 1 3 microRNA sponge RNA decoy RNP component lncRNA lncRNA lncRNA 1 2 3 4 mRNA 1 2 3 4 5 6 4 3 2 lncRNA lncRNA mRNA 4 Recruitment of chromatin modifiers 5 Translation inhibition 6 Splicing modulation 7 Degradation 1 Some general conclusions about LncRNAs • Most are expressed in very few copies per cell – generally ~2 - ~100 • Many but not all LncRNAs are enriched in the nucleus • Many are highly induced or repressed during specific developmental stages and are regulated by lineageimportant transcription factors. • Many are essential for differentiation or function of specific cell types. LncRNAs regulate many steps in hematopoietic differentiation LT-HSC H19 Pluripotent stem cells ST-HSC MPP Multipotent progenitors CMP CLP GMP MEP HOTAIRM1 Committed precursors lincRNA-EPS DLEU2 elncRNA-EC1,3 lincRNA-EC2,4,5,8,9 alncRNA-EC1,2,3,7 NeST EGO lincRNA-Cox2 DLEU2 LincR-Ccr2-5’AS lincRNA -Cox2 Mature cells Mast cell RBC Megakaryocyte Platelets Eosinophil Neutrophil Monocyte/ Macrophage B cell T cell Dendritic cell NK cell Erythropoietin is the singular hormone that prevents apoptosis of CFU-E progenitors, allowing their terminal proliferation and differentiation Resolution of E 14.5 fetal liver erythroid progenitors and erythroblasts Scale bar: 20 µm. In vitro culture of purified murine fetal liver CFU-E progenitors recapitulates normal terminal erythroid proliferation, differentiation, and enucleation Purified TER119-negative Day 14 fetal liver cells were cultured in vitro for one day on fibronectin-coated plates in medium containing serum and EPO. EPO was removed at the end of one day. The differentiation profiles of cultured cells were examined after 1 and 2 days by flow cytometry and BenzidineGiemsa staining. After two days in culture, the number of cells increased 20- fold and progenitors/ early erythroblasts (R1 and R2 cells) differentiated into hemoglobinized R3 R5 cells. The differentiation profile displayed by flow cytometry analysis correlated well with that shown by Benzidine-Giemsa stain. Scale bar: 20 µm. Deep sequencing to identify erythroid- specific lncRNAs Progenitors Erythroblasts Mouse E14 fetal liver: FILTERING ASSEMBLY BFU-E CFU-E Ter119+ Poly(A)+ RNA Total RNA RNA-seq ssRNA-seq Mapping to genome (TopHat) De novo transcript assembly (Cufflinks) Reliable transcript model filters (> 0.1 max coverage; > 200bp; >1 exon) Coding potential filters (pCSF<100; no PFAM domain; no BLASTx hit; CPC<0) Known coding transcript filter (no sense overlap with known mRNA exons) 802 lncRNAs (657 loci) Juan Alvarez and Wenqian Hu Not all fetal liver lncRNAs have already been annotated – many are erythroid specific ENSEMBL, UCSC & Refseq lncRNAs Erythroid lncRNAs 1648 608 194 A LncRNA with anti-apoptotic activity during the terminal differentiation of erythroid cells ESlncRNA is highly specific to the terminal differentiating erythroid cells ESlncRNA is a bona fide nuclear non-coding transcript ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis Ectopic expression of ESlncRNA protects erythroid progenitors from apoptosis caused by Epo starvation ESlncRNA modulates apoptosis in part through repressing Pycard Single molecule fluorescence in situ hybridization (FISH) shows that ESlncRNA is localized to the erythroblast nucleus Juan Alvarez Relative ESlncRNA level ESlncRNA is highly specific to Ter- 119 positive terminally differentiating erythroid cells ESlncRNA is dramatically induced during terminal erythroid differentiation, at the same stage as ~400 erythroid important genes Primary structure of ESlncRNA as determined by 5’ and 3’ RACE In erythroid cells the ESlncRNA gene contains “active” chromatin modifications and the promoter is occupied by the erythroid- important transcription factors Scl/Tal and GATA1 Experimental approaches for loss-offunction studies Total Fetal Liver Cells Lineage Depletion (Ter119, CD3, B220, CD11b, Gr-1) Lin- Cells Retrovirus Infection (shRNAs) Transduced Cells 1 Day Culture in Maintenance Medium Switch to Differentiation Medium (+ Epo) Molecular & Phenotypic Assays ESlncRNA knock-down results in inhibition of terminal erythroid proliferation ESlncRNA knock-down results in cell death Conclusions ESlncRNA is highly specific to terminal differentiating erythroid cells ESlncRNA is a bona fide nuclear non-coding transcript ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis Experimental approach for gain-of-function studies Total Fetal Liver Cells Lineage Depletion (Ter119, CD3, B220, CD11b, Gr-1) Lin- Cells Retrovirus Infection (Ectopic Expression of ESlncRNA) Transduced Cells Culture in the absence of Epo Cell Death Analysis by Flow Cytometry Ectopic expression of ESlncRNA in erythroid progenitor cells is at a physiological level Ectopic expression of ESlncRNA prevents apoptosis caused by Epo-starvation Ectopic expression of ESlncRNA prevents apoptosis caused by Epo-starvation Vector ESlncRNA Ectopic expression of ESlncRNA results in repression of many apoptotic genes including Pycard Erythroid-specific ESlncRNA regulates apoptosis in erythropoiesis ESlncRNA is highly specific to the terminal differentiating erythroid cells ESlncRNA is a bona fide nuclear non-coding transcript ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis Ectopic expression of ESlncRNA protects erythroid progenitors from apoptosis caused by Epo starvation ESlncRNA modulates apoptosis in part through repressing Pycard Fetal liver lincRNAs exhibit an extremely high degree of cell and tissue specificity Fetal liver lincRNAs exhibit an extremely high degree of cell and tissue specificity Dynamic expression of distinct lncRNA subclasses during erythropoiesis Selection of 12 new candidate erythroid lncRNAs: !"#$%$"&'()#*+,-.(/01(23#*&%0#")(!4"1"*&'1%5"&%0#( • Induced during terminal erythroid differentiation • Promoter bound by key erythroid transcription factors • Conserved in placental mammals ()*+,--./0# 1,23456./0# 7/0-,+856./0# $%&'# !"# %459,0654#:5::54-# !"#$%$"&'()#*+,-.(/01(23#*&%0#")(!4"1"*&'1%5"&%0#( Selection of candidate lncRNAs: • Abundant in and specific for erythroid cells !"##$%&'(%)"*")"+,& In situ single molecule RNA FISH: Most candidate lncRNAs are enriched in the nucleus Knockdown of most candidate lncRNA does not block glycophorin (Ter119) induction But knockdown of most candidate lncRNA blocks later red cell development, as measured by enucleation Conclusion: Multiple Types of Long Non-coding RNAs Regulate Red Blood Cell Development • We combined genome-wide surveys of expression, chromatin state and transcription factor occupancy in differentiating primary mouse fetal liver erythroid cells to systematically characterize each lncRNA subclass. • Binding of the key erythroid transcription factors GATA1 and TAL1 at lncRNA and mRNA promoters is typically accompanied by gain of H3K4me2 along with transcriptional activation. • We focused on lncRNAs from each subclass that are abundantly upregulated during terminal differentiation, including novel erythroid-specific lncRNAs conserved in humans and that are predominantly nuclear. Loss-of-function studies revealed that 12 of these lncRNAs are necessary for the proper maturation of enucleated erythroblasts. • Together, these results indicate that lncRNAs of diverse genomic origins are important components of the regulatory networks underlying development of the erythroid lineage. Pipeline for de novo lncRNA discovery in adipocytes BAT = brown adipose tissue iWAT = inguinal fat tissue eWAT = epididymal fat tissue Almost all of the sequenced lncRNAs are expressed in a highly cell- and tissue- specific fashion About half of the sequenced mRNAs are expressed in a highly cell- and tissue- specific fashion About half of the sequenced mRNAs are expressed in a highly cell- and tissue- specific fashion Wenqian Hu and Juan Alvarez-Dominguez A proud 31 year tradition continues: July 27, 2013