GRO-seq in mouse Epiblast-primed, Primitive endoderm-primed, and differentiated endoderm cells
ABSTRACT: The identification of changes in transcriptional regulation during priming and differentiation of embryonic stem (ES) cells towards the endoderm lineage. Specific populations of ES cells, either primed or committed to endoderm, were isolated and subjected to global nuclear run on sequencing (GRO-Seq). The hHex-Venus (HV) reporter ES cell line, HVJu5.1 (Canham et al., 2010) was used to isolate HV- and HV+ ES cells. Primed ES cells were identified based on the expression of the HV marker in addition to the cell surface marker of undifferentiated ES cells, SSEA-1, (the lower and upper 25% of SSEA-1+, HV expressing cells). When challenged to differentiate, HV- ES cells are primed towards an epiblast fate, while HV+ ES cells are primed towards primitive endoderm. However, these populations are considered primed, rather than committed, as they will readily interconvert when re-introduced into standard ES cell culture conditions. ES cells were grown in self-renewing conditions (GMEM, LIF, 10% FCS, plated on gelatin coated dishes). Endoderm was obtained by differentiating ES cells in medium without the cytokine LIF for 5 days. The HV+, SSEA-1- differentiated fraction was then sorted and represents an early stage in endoderm differentiation. Three subpopulations were sorted by flow cytometry from clone HVJu5.1 of the HV ES cell line as such: HV-,SSEA-1+; HV+SSEA-1+; HV+, SSEA-1-; where HV = Hex-Venus expression. Two biological replicates were collected per sample. Nuclei were isolated from each population and GRO-seq was carried out.
Project description:Quantitation of polyA mRNA levels in subpopulations of the HexVenus reporter (clone HV5.1) mouse ES cell line growing under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry. The aim was to identify genes which are differentially expressed between FACS-sorted HV-S+ and HV+S+ primed mESC populations. Overall design: mESCs carrying a fluorescently tagged Hhex-Venus (HV) reporter construct were FACS sorted into Ssea1+HV- and Ssea1+HV+ populations. RNA was isolated from these populations immediately following FACS sorting and hybridised to Agilent 44k expression arrays. This process was performed as two independent biological replicates.
Project description:We report that ES cells cultured in ground state (2i and 2i/LIF) culture conditions are heterogeneous and show heterogeneus expression of extraembryonic markers. Using a highly sensitive reporter for the endoderm marker Hex we can sort Hex high and low populations from either serum/LIF or 2i/LIF and demonstrate that they have different functional properties. Here we explored the transcriptional basis of these functional differences and noted that Hex low (HV-) and Hex high (HV+) populations showed more distinct expression profiles in 2i/LIF than in serum/LIF. Additionally in 2i/LIF the HV+ population showed an upregulation of extraembryonic markers (such as trophoblast stem cell specific genes) and also imprinted genes compared to the HV- population, which is not observed when these populations are sorted from serum/LIF. We also analysed the transcriptional effect of LIF in 2i by analysing unsorted ES cells cultured in either 2i alone or 2i with LIF. We observed that the addition of LIF led to an upregulation of extraembryonic markers but did not effect the expression of pluripotency genes, other than Klf4. Additionally, the most significantly upregulated genes from 2i/LIF cultured ES cells compared to 2i cultured ES cells showed the greatest correlation to placental tissue when compared to the GNF tissue specific expression database. This analysis, alongside functional experiments, suggested that HV+ ES cells in 2i/LIF corresponded to an extraembryonically primed population of cells and that the addition of LIF supported this population. RNA-seq of sorted Hex low and high expressing ES cell populations cultured in serum/LIF or 2i/LIF as well as unsorted ES cells from 2i or 2i/LIF.
Project description:X-linked Chromatin Immunoprecipitation (ChIP) for the histone modification H3K27me3 was performed on subpopulations of the HexVenus reporter mouse ES cell line (clone HV1.5) grown under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry prior to further processing specific to each method. The aim was to determine if subtle changes in priming gene transcription were associated with changes in the magnitude of H3K27me3. Overall design: mESCs carrying a fluorescently tagged Hhex-Venus (HV) reporter construct were FACS sorted and immediately cross-linked with formaldehyde and then stored at -80°C prior to performing ChIP. ChIP was performed on two independent biological replicates for both the primed and re-sorted populations in addition to a single replicate for the minor HV+S- (spontaneously differentiated) population. Sequencing libraries were generated for each sample and deep sequenced.
Project description:Chromatin Immunoprecipitation (ChIP) for the histone modification H3K4me3 and H3K27me3 and Genome Wide Run-on seq (GROseq) were performed on subpopulations of the HexVenus reporter mouse ES cell line (clone HV1.5) growing under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry prior to further processing specific to each method. The aim was to determine if differential gene expression between these populations is primarily mediated at the level of transcription and if these changes are concomitant with changes in chromatin state. Overall design: mESCs carrying a fluorescently tagged Hhex-Venus (HV) reporter construct were FACS sorted into Ssea1+HV- and Ssea1+HV+ populations. Nuclei were prepared from these populations immediately following FACS sorting and then further processed as per the relevant protocol. Two independent biological replicates were prepared for each sample and sequencing libraries were the pooled prior to sequencing or combined bioinformatically following sequencing as outlined for each sample.
Project description:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. We evaluated ribosome occupancy of mRNAs in embryonic stem cells and Epiblast-Like Aggregates by means of polysome profiling with a modified Translating Ribosome Affinity Purification protocol (TRAP).
Project description:Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3b signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters.Upon withdrawal of 2i/LIF, naïve mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalency acquisition on lineage regulatory genes. The feasibility for establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in rodent ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. Four chromatin marks H3K4me1, H3K4me3, H3K27ac and H3K27me3 were measured from 3 cell lines: C1 and WIBR3 (naïve and conventional/primed stem cells), and BGO1 (only naïve stem cells).
Project description:Sequencing of the 3’ end of poly(A)+ RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3’ region extraction and deep sequencing (3’READS), to address mispriming issues that often plague 3’ end sequencing. Here we report a new version, named 3’READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)+ RNA and to remove bulk of the poly(A) tail, 3’READS+ generates RNA fragments with an optimal number of terminal As that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3’READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes, and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (~50%). 3’READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3’ end counting, especially when the amount of input total RNA is limited. Nine 3'READS+ libraries were made with different amounts (100 ng, 200 ng, 400 ng, 1000 ng, 5000 ng, 15000 ng) of input Hela RNA.
Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5’-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-seq experiments (two biological replicates) were performed in nuclei from many wild-type states and a dosage compensation mutant