Search

Summary T cell development is restricted to the thymus and is dependent on high levels of Notch signaling induced within the thymic microenvironment. To understand Notch function in thymic restriction, we investigated the basis for target gene selectivity in response to quantitative differences in Notch signal strength, focusing on the chromatin architecture of genes essential for T cell differentiation. We find that high Notch signal strength is required to activate promoters of known targets essential for T cell commitment, including Il2ra, Cd3ε, and Rag1, which feature low CpG content (LCG) and DNA inaccessibility in hematopoietic stem progenitor cells. Our findings suggest that promoter DNA inaccessibility at LCG T lineage genes provides robust protection against stochastic activation in inappropriate Notch signaling contexts, limiting T cell development to the thymus., Highlights • Notch target gene promoters differentially respond to Notch signal strength • T cell commitment targets feature low CpG content and DNA inaccessibility in HSPCs • Promoter DNA inaccessibility protects T cell target genes from stochastic activation • LCG promoters act as safeguards of Notch-induced differentiation, Bernstein and colleagues investigate target gene selectivity in response to quantitative differences in Notch signal strength. Their findings suggest that low CpG content and DNA inaccessibility at the promoters of essential T cell commitment targets provide robust protection against stochastic gene activation in inappropriate Notch signaling contexts, ensuring that T cell development is limited to the Notch ligand-rich thymus.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (i) gene-rich and SINE-rich euchromatin, (ii) gene-poor and LINE/LTR-rich heterochromatin and (iii) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show using FISH and 4C-seq technologies that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespectively of their chromosomal context thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes. ispartof: Genome Research vol:27 issue:6 pages:922-933 ispartof: location:United States status: published

In metazoans, the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes, by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps, we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this “A/T rule” in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition.

Background Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. Methods We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. Results Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. Conclusions Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).

SummaryReporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.

Mutations in the A-type lamins A and C, two major components of the nuclear lamina, cause a large group of phenotypically diverse diseases collectively referred to as laminopathies. These conditions often involve defects in chromatin organization. However, it is unclear whether A-type lamins interact with chromatin in vivo and whether aberrant chromatin–lamin interactions contribute to disease. Here, we have used an unbiased approach to comparatively map genome-wide interactions of gene promoters with lamin A and progerin, the mutated lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome (HGPS) in mouse cardiac myoytes and embryonic fibroblasts. We find that lamin A-associated genes are predominantly transcriptionally silent and that loss of lamin association leads to the relocation of peripherally localized genes, but not necessarily to their activation. We demonstrate that progerin induces global changes in chromatin organization by enhancing interactions with a specific subset of genes in addition to the identified lamin A-associated genes. These observations demonstrate disease-related changes in higher order genome organization in HGPS and provide novel insights into the role of lamin–chromatin interactions in chromatin organization. Electronic supplementary material The online version of this article (doi:10.1007/s00412-012-0376-7) contains supplementary material, which is available to authorized users.

Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome – NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome – NL interactions.

The three-dimensional organization of chromosomes within the nucleus and its dynamics during differentiation are largely unknown. To visualize this process in molecular detail, we generated high-resolution maps of genome-nuclear lamina interactions during subsequent differentiation of mouse embryonic stem cells via lineage-committed neural precursor cells into terminally differentiated astrocytes. This reveals that a basal chromosome architecture present in embryonic stem cells is cumulatively altered at hundreds of sites during lineage commitment and subsequent terminal differentiation. This remodeling involves both individual transcription units and multigene regions and affects many genes that determine cellular identity. Often, genes that move away from the lamina are concomitantly activated; many others, however, remain inactive yet become unlocked for activation in a next differentiation step. These results suggest that lamina-genome interactions are widely involved in the control of gene expression programs during lineage commitment and terminal differentiation.