Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence.

To determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder

Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation

Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXaintermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xiabnormalities seen in primed hESCs, it may provide cells better suited for downstream applications

Determinants of Unique DNA Methylation, Histone Modification, and Nucleosome Occupany at CpG Islands

In an attempt to understand DNA methylation in various contexts, we have examined chromatin modification at enhancers and CpG islands. At both DNA features, we find the binding of transcription factors is the major determinant of methylation status.At the enhancer for the tissue-specific inflammatory gene Il12b, we attempted to isolate the DNA sequence necessary for the establishment of a low methylation window usually present in most cell types. We cloned Il12b enhancer deletions into a bacterial artificial chromosome that could recapitulate native chromatin when stably transfected into murine ES cells, but were unable to remove the low methylation window without deleting the full enhancer sequence. The Il12b enhancer is uniquely methylated in embryonic stem cells compared to all other cell types; it has higher methylation than usual and responds to certain changes in growth conditions. DNA methylation increases globally during stem cell differentiation, but DNA methylation at the Il12b enhancer remains constant in successfully differentating cells. Finally, we take advantage of variable Il12b enhancer methylation in embryonic stem cells to demonstrate that, following differentiation to a macrophage fate, moderate enhancer methylation does not prevent Il12b expression.To understand what factors influence the well studied low DNA methylation, histone 3 lysine 4 trimethylation (H3K4me3), and low nucleosome occupancy at CpG islands, we cloned CpG rich DNA into bacterial artificial chromosomes which were stably transfected into ES cells. Analysis of the integrated BACs revealed that CpG island features are each controlled through separate mechanisms. We determined several properties of CpG island features based on experimental deletions and fusions of a small CpG island and the 601 positioning sequence. Protection from DNA methylation at CpG islands can occur either by binding of a specific transcription factor, or by a size threshold mechanism in murine ES cells. H3K4me3 marking requires low DNA methylation, but unmethylated CpGs are not sufficient to recruit high levels. Nucleosome density is influenced by transcription factor binding and sequence positioning determinants, but is unaffected by low DNA methylation and moderate H3K4me3 levels.We expanded our analysis of CpG islands to include all CpG rich regions in the human genome, which were computationally determined based on our own criteria. Using available chromatin datasets, we assayed the effect of nucleotide content on CpG island features. We found that CpG density and island size correlated with high levels of CpG island features. However, by far the strongest determinant of CpG island features was association with a promoter. Promoter CpG rich regions were strongly biased to accumulate high levels of all CpG island features, which could not be explained by nucleotide content. Instead, we showed that promoter CpG islands have much higher transcription factor binding then other CpG islands in the genome, and high binding is correlated with lower DNA methylation and higher H3K4me3. Finally, we observed a difference in the DNA methylation at human and mouse CpG islands. High CpG density mouse CpG islands are much more susceptible to demethylation.This multifaceted study elucidates many previously undefined relationships between transcription factors and chromatin properties. These findings will be beneficial to describing the complex mechanisms that drive regulation of cell fate and gene expression

Determinants of Unique DNA Methylation, Histone Modification, and Nucleosome Occupany at CpG Islands

In an attempt to understand DNA methylation in various contexts, we have examined chromatin modification at enhancers and CpG islands. At both DNA features, we find the binding of transcription factors is the major determinant of methylation status.At the enhancer for the tissue-specific inflammatory gene Il12b, we attempted to isolate the DNA sequence necessary for the establishment of a low methylation window usually present in most cell types. We cloned Il12b enhancer deletions into a bacterial artificial chromosome that could recapitulate native chromatin when stably transfected into murine ES cells, but were unable to remove the low methylation window without deleting the full enhancer sequence. The Il12b enhancer is uniquely methylated in embryonic stem cells compared to all other cell types; it has higher methylation than usual and responds to certain changes in growth conditions. DNA methylation increases globally during stem cell differentiation, but DNA methylation at the Il12b enhancer remains constant in successfully differentating cells. Finally, we take advantage of variable Il12b enhancer methylation in embryonic stem cells to demonstrate that, following differentiation to a macrophage fate, moderate enhancer methylation does not prevent Il12b expression.To understand what factors influence the well studied low DNA methylation, histone 3 lysine 4 trimethylation (H3K4me3), and low nucleosome occupancy at CpG islands, we cloned CpG rich DNA into bacterial artificial chromosomes which were stably transfected into ES cells. Analysis of the integrated BACs revealed that CpG island features are each controlled through separate mechanisms. We determined several properties of CpG island features based on experimental deletions and fusions of a small CpG island and the 601 positioning sequence. Protection from DNA methylation at CpG islands can occur either by binding of a specific transcription factor, or by a size threshold mechanism in murine ES cells. H3K4me3 marking requires low DNA methylation, but unmethylated CpGs are not sufficient to recruit high levels. Nucleosome density is influenced by transcription factor binding and sequence positioning determinants, but is unaffected by low DNA methylation and moderate H3K4me3 levels.We expanded our analysis of CpG islands to include all CpG rich regions in the human genome, which were computationally determined based on our own criteria. Using available chromatin datasets, we assayed the effect of nucleotide content on CpG island features. We found that CpG density and island size correlated with high levels of CpG island features. However, by far the strongest determinant of CpG island features was association with a promoter. Promoter CpG rich regions were strongly biased to accumulate high levels of all CpG island features, which could not be explained by nucleotide content. Instead, we showed that promoter CpG islands have much higher transcription factor binding then other CpG islands in the genome, and high binding is correlated with lower DNA methylation and higher H3K4me3. Finally, we observed a difference in the DNA methylation at human and mouse CpG islands. High CpG density mouse CpG islands are much more susceptible to demethylation.This multifaceted study elucidates many previously undefined relationships between transcription factors and chromatin properties. These findings will be beneficial to describing the complex mechanisms that drive regulation of cell fate and gene expression

Species-Specific Relationships between DNA and Chromatin Properties of CpG Islands in Embryonic Stem Cells and Differentiated Cells.

CpG islands often exhibit low DNA methylation, high histone H3 lysine 4 trimethylation, low nucleosome density, and high DNase I hypersensitivity, yet the rules by which CpG islands are sensed remain poorly understood. In this study, we first evaluated the relationships between the DNA and the chromatin properties of CpG islands in embryonic stem cells using modified bacterial artificial chromosomes. Then, using a bioinformatic approach, we identified strict CpG-island density and length thresholds in mouse embryonic stem and differentiated cells that consistently specify low DNA methylation levels. Surprisingly, the human genome exhibited a dramatically different relationship between DNA properties and DNA methylation levels of CpG islands. Further analysis allowed speculation that this difference is accommodated in part by evolutionary changes in the nucleotide composition of orthologous promoters. Thus, a change in the rules by which CpG-island properties are sensed may have co-evolved with compensatory genome adaptation events during mammalian evolution

Species-Specific Relationships between DNA and Chromatin Properties of CpG Islands in Embryonic Stem Cells and Differentiated Cells.

CpG islands often exhibit low DNA methylation, high histone H3 lysine 4 trimethylation, low nucleosome density, and high DNase I hypersensitivity, yet the rules by which CpG islands are sensed remain poorly understood. In this study, we first evaluated the relationships between the DNA and the chromatin properties of CpG islands in embryonic stem cells using modified bacterial artificial chromosomes. Then, using a bioinformatic approach, we identified strict CpG-island density and length thresholds in mouse embryonic stem and differentiated cells that consistently specify low DNA methylation levels. Surprisingly, the human genome exhibited a dramatically different relationship between DNA properties and DNA methylation levels of CpG islands. Further analysis allowed speculation that this difference is accommodated in part by evolutionary changes in the nucleotide composition of orthologous promoters. Thus, a change in the rules by which CpG-island properties are sensed may have co-evolved with compensatory genome adaptation events during mammalian evolution

Oct4:Sox2 binding is essential for establishing but not maintaining active and silent states of dynamically regulated genes in pluripotent cells

Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states

Efficient Many-To-Many Point Matching in One Dimension

Appears in Graphs and Combinatorics, vol. 23 (2007), supplement, Computational Geometry and Graph Theory. The Akiyama-Chvatal Festschrift. The original publication is available at www.springerlink.com. Abstract. Let S and T be two sets of points with total cardinality n. The minimum-cost many-to-many matching problem matches each point in S to at least one point in T and each point in T to at least one point in S, such that sum of the matching costs is minimized. Here we examine the special case where both S and T lie on the line and the cost of matching s ∈ S to t ∈ T is equal to the distance between s and t. In this context, we provide an algorithm that determines a minimum-cost many-to-many matching in O(n log n) time, improving the previous best time complexity of O(n 2) for the same problem. 1

Efficient many-To-many point matching in one dimension

SCOPUS: ar.jinfo:eu-repo/semantics/publishe