Significant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions.

Memory T cells provide long-lasting defense responses through their ability to rapidly reactivate, but how they efficiently “recall” an inflammatory transcriptional program remains unclear. Here, we show that human CD4 ⁺ memory T helper 2 (T _H 2) cells carry a chromatin landscape synergistically reprogrammed at both one-dimensional (1D) and 3D levels to accommodate recall responses, which is absent in naive T cells. In memory T _H 2 cells, recall genes were epigenetically primed through the maintenance of transcription-permissive chromatin at distal (super)enhancers organized in long-range 3D chromatin hubs. Precise transcriptional control of key recall genes occurred inside dedicated topologically associating domains (“memory TADs”), in which activation-associated promoter-enhancer interactions were preformed and exploited by AP-1 transcription factors to promote rapid transcriptional induction. Resting memory T _H 2 cells from patients with asthma showed premature activation of primed recall circuits, linking aberrant transcriptional control of recall responses to chronic inflammation. Together, our results implicate stable multiscale reprogramming of chromatin organization as a key mechanism underlying immunological memory and dysfunction in T cells.

Depression is the most prevalent psychiatric disorder with a complex and elusive etiology that is moderately heritable. Identification of genes would greatly facilitate the elucidation of the biological mechanisms underlying depression, however, its complex etiology has proved to be a major bottleneck in the identification of its genetic risk factors, especially in genome-wide association-like studies. In this study, we exploit the properties of a genetic isolate and its family-based structure to explore whether relatively rare exonic variants influence the burden of depressive symptoms in families. Using a multistep approach involving linkage and haplotype analyses followed by exome sequencing in the Erasmus Rucphen Family (ERF) study, we identified a rare (minor allele frequency (MAF)=1%) missense c.1114C>T mutation (rs115482041) in the RCL1 gene segregating with depression across multiple generations. Rs115482041 showed significant association with depressive symptoms (N=2393, β_T-allele=2.33, P-value=1 × 10⁻⁴) and explained 2.9% of the estimated genetic variance of depressive symptoms (22%) in ERF. Despite being twice as rare (MAF<0.5%), c.1114C>T showed similar effect and significant association with depressive symptoms in samples from the independent population-based Rotterdam study (N=1604, β_T-allele=3.60, P-value=3 × 10⁻²). A comparison of RCL1 expression in human and mouse brain revealed a striking co-localization of RCL1 with the layer 1 interlaminar subclass of astrocytes found exclusively in higher-order primates. Our findings identify RCL1 as a novel candidate gene for depression and offer insights into mechanisms through which RCL1 may be relevant for depression.

Background: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function—the storage, expression, and replication of genetic information—is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence.Results: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30–100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above.Conclusions: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow “architectural sequencing” at a genome mechanics level to understand the inseparable systems genomic properties.