The Effect of Transposable Element Insertions on Gene Expression Evolution in Rodents

Background:Many genomes contain a substantial number of transposable elements (TEs), a few of which are known to be involved in regulating gene expression. However, recent observations suggest that TEs may have played a very important role in the evolution of gene expression because many conserved non-genic sequences, some of which are know to be involved in gene regulation, resemble TEs. Results:Here we investigate whether new TE insertions affect gene expression profiles by testing whether gene expression divergence between mouse and rat is correlated to the numbers of new transposable elements inserted near genes. We show that expression divergence is significantly correlated to the number of new LTR and SINE elements, but not to the numbers of LINEs. We also show that expression divergence is not significantly correlated to the numbers of ancestral TEs in most cases, which suggests that the correlations between expression divergence and the numbers of new TEs are causal in nature. We quantify the effect and estimate that TE insertion has accounted for ~20% (95% confidence interval: 12% to 26%) of all expression profile divergence in rodents. Conclusions:We conclude that TE insertions may have had a major impact on the evolution of gene expression levels in rodents

Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

BACKGROUND: Uncovering epigenetic states by chromatin immunoprecipitation and microarray hybridization (ChIP-chip) has significantly contributed to the understanding of gene regulation at the genome-scale level. Many studies have been carried out in mice and humans; however limited high-resolution information exists to date for non-mammalian vertebrate species. PRINCIPAL FINDINGS: We report a 2.1-million feature high-resolution Nimblegen tiling microarray for ChIP-chip interrogations of epigenetic states in zebrafish (Danio rerio). The array covers 251 megabases of the genome at 92 base-pair resolution. It includes ∼15 kb of upstream regulatory sequences encompassing all RefSeq promoters, and over 5 kb in the 5' end of coding regions. We identify with high reproducibility, in a fibroblast cell line, promoters enriched in H3K4me3, H3K27me3 or co-enriched in both modifications. ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes. H3K4me3- and/or H3K27me3-enriched genes are associated with distinct transcriptional status and are linked to distinct functional categories. CONCLUSIONS: We have designed and validated for the scientific community a comprehensive high-resolution tiling microarray for investigations of epigenetic states in zebrafish, a widely used developmental and disease model organism

Inherent Signals in Sequencing-Based Chromatin-ImmunoPrecipitation Control Libraries

The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.First, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.We found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights

Suv4-20h Histone Methyltransferases Promote Neuroectodermal Differentiation by Silencing the Pluripotency-Associated Oct-25 Gene

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state

Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells

February 17, 2011The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome[superscript 1, 2, 3, 4, 5], resulting in altered patterns of gene expression[superscript 2, 6, 7, 8, 9]. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs)[superscript 10, 11] that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells, suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches, we found that one such lincRNA (lincRNA-RoR) modulates reprogramming, thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells

SICTIN: Rapid footprinting of massively parallel sequencing data

BACKGROUND: Massively parallel sequencing allows for genome-wide hypothesis-free investigation of for instance transcription factor binding sites or histone modifications. Although nucleotide resolution detailed information can easily be generated, biological insight often requires a more general view of patterns (footprints) over distinct genomic features such as transcription start sites, exons or repetitive regions. The construction of these footprints is however a time consuming task. METHODS: The presented software generates a binary representation of the signals enabling fast and scalable lookup. This representation allows for footprint generation in mere minutes on a desktop computer. Several different input formats are accepted, e.g. the SAM format, bed-files and the UCSC wiggle track. CONCLUSIONS: Hypothesis-free investigation of genome wide interactions allows for biological data mining at a scale never before seen. Until recently, the main focus of analysis of sequencing data has been targeted on signal patterns around transcriptional start sites which are in manageable numbers. Today, focus is shifting to a wider perspective and numerous genomic features are being studied. To this end, we provide a system allowing for fast querying in the order of hundreds of thousands of features

Mapping Dynamic Histone Acetylation Patterns to Gene Expression in Nanog-depleted Murine Embryonic Stem Cells

Embryonic stem cells (ESC) have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac) on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in an concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.Comment: accepted at PLoS Computational Biolog

Q&A: ChIP-seq technologies and the study of gene regulation

10.1186/1741-7007-8-56BMC Biology85

Virus-free induction of pluripotency and subsequent excision of reprogramming factors

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors1-7. However, such iPS cells contain a large number of viral vector integrations1,8, any one of which could cause unpredictable genetic dysfunction. While c-Myc is dispensable for reprogramming9,10, complete elimination of the other exogenous factors is also desired since ectopic expression of either Oct4 or Klf4 can induce dysplasia11,12. Two transient transfection reprogramming methods have been published to address this issue13,14. However, the efficiency of either approach is extremely low, and neither has thus far been applied successfully to human cells. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc​,​ Klf4​,​ Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimeric mice. When the single vector reprogramming system was combined with a piggyBac transposon15,16 we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models

Fgfr3 Is a Transcriptional Target of Ap2δ and Ash2l-Containing Histone Methyltransferase Complexes

Polycomb (PcG) and trithorax (trxG) proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2δ that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2δ and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2δ and Ash2l, and we have identified evolutionarily conserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2δ-Ash2l complex, we analyzed several promoters for the presence of Ap2δ and Ash2l by chromatin immunoprecipitation (ChIP). Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2δ-Ash2l complex. Additionally, we found that Ap2δ is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3). Thus, we have identified several candidate targets of complexes containing Ap2δ and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes