Genomic Prevalence of Heterochromatic H3K9me2 and Transcription Do Not Discriminate Pluripotent from Terminally Differentiated Cells

Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change, many genes show altered expression upon differentiation. Quantitative sequencing of transcripts demonstrates however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global basal level transcription in stem cells. This suggests that cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity

Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions.

Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms

Epigenome plasticity during cellular differentiation

Tight control of gene expression is crucial to govern cell function and identity at any developmental stage. Epigenetic modifications of chromatin have emerged as important determinants for chromatin structure and gene expression. The aim of this work was to test the hypothesis that epigenetic mechanisms contribute to the establishment and maintenance of cell type specific gene expression patterns and to delimiting the developmental potential of somatic cells. Towards this goal we defined genome-wide targets of epigenetic reprogramming during neuronal differentiation of mouse embryonic stem cells. DNA methylation, which is a potent and stable repressive modification, is increasing during differentiation of embryonic stem cells into neurons. Many de novo methylation targets encode pluripotency-associated and germline specific genes and only few appear to be specific for alternative lineages. Polycomb-mediated repression, a distinct epigenetic repression pathway, was previously shown to be essential for embryonic patterning and maintaining developmental potential in stem cells. Unlike DNA methylation, Polycomb targets are very dynamic during neuronal differentiation. Repression is resolved at activated genes while novel targets appear at both the multipotential neuronal progenitor state and the terminally differentiated neuron state. As in stem cells, many Polycomb targets in neuronal progenitor cells will be activated upon further differentiation. Polycomb could therefore serve as a general safe-guard system for genes that can be activated at later stages but need to be tightly controlled to avoid precocious and uncontrolled cell fate changes. In summary, there are at least two distinct epigenetic modes of repression, which nonetheless might crosstalk for target specification. Stable repression of the pluripotency program is conferred by DNA methylation. In turn, Polycomb mediates a more transient repression mechanism with cell type and developmental stage specific targets. Together, this argues that epigenetic mechanisms contribute to cellular differentiation and development via stabilizing gene expression programs initiated by transcription factors. Hence, epigenetic mechanisms could be viewed as additional regulatory layer for balancing gene regulation in order to confer robustness to cellular states and gene expression programs rather than as key drivers for setting up such cell type specific gene expression patterns

Genetics and epigenetics: stability and plasticity during cellular differentiation.

Stem cells and multipotent progenitor cells face the challenge of balancing the stability and plasticity of their developmental states. Their self-renewal requires the maintenance of a defined gene-expression program, which must be stably adjusted towards a new fate upon differentiation. Recent data imply that epigenetic mechanisms can confer robustness to steady state gene expression but can also direct the terminal fate of lineage-restricted multipotent progenitor cells. Here, we review the latest models for how changes in chromatin and DNA methylation are regulated during cellular differentiation. We further propose that targets of epigenetic repression share common features in the sequences of their regulatory regions, thereby suggesting a co-evolution of epigenetic pathways and classes of cis-acting elements

Nonsense-associated alternative splicing of T-cell receptor β genes: No evidence for frame dependence

Mutations that generate premature translation-termination codons (PTCs) often result in production of alternatively spliced mRNAs. While in many cases, the PTC-causing mutation was found to affect splicing directly by disrupting an exonic splicing enhancer, induction of alternative splicing of TCR-βpre-mRNA has been reported to be specific for mutations that prematurely terminate the open reading frame. During testing of a cyto-nuclear feedback model that would have explained how cytoplasmic translation could influence nuclear splicing of TCR-βtranscripts, control experiments questioned the frame dependence of the nonsense-associated altered splicing (NAS) of TCR-βpre-mRNA. A subsequent detailed analysis of alternatively spliced TCR-βmRNA expressed from different minigene constructs with nonsense, silent, or frame-shift mutations at various positions revealed no correlation between truncation of the reading frame and production of alternatively spliced mRNA. Our study thus contradicts the previously reported PTC specificity of TCR-βNAS and points out the need for systematically testing the PTC specificity in other cases where NAS has been observed

Nonsense-associated alternative splicing of T-cell receptor beta genes : no evidence for frame dependence

Mutations that generate premature translation-termination codons (PTCs) often result in production of alternatively spliced mRNAs. While in many cases, the PTC-causing mutation was found to affect splicing directly by disrupting an exonic splicing enhancer, induction of alternative splicing of TCR-beta pre-mRNA has been reported to be specific for mutations that prematurely terminate the open reading frame. During testing of a cyto-nuclear feedback model that would have explained how cytoplasmic translation could influence nuclear splicing of TCR-beta transcripts, control experiments questioned the frame dependence of the nonsense-associated altered splicing (NAS) of TCR-beta pre-mRNA. A subsequent detailed analysis of alternatively spliced TCR-beta mRNA expressed from different minigene constructs with nonsense, silent, or frame-shift mutations at various positions revealed no correlation between truncation of the reading frame and production of alternatively spliced mRNA. Our study thus contradicts the previously reported PTC specificity of TCR-beta NAS and points out the need for systematically testing the PTC specificity in other cases where NAS has been observed

Methylated DNA immunoprecipitation (MeDIP).

Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine. The resulting enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR to assess the methylation state of individual regions. Alternatively, MeDIP can be combined with large-scale analysis using microarrays as a genome-wide experimental readout. This protocol has been applied to generate comprehensive DNA methylation profiles on a genome-wide scale in mammals and plants, and further to identify abnormally methylated genes in cancer cells