Cell-type specialization is encoded by specific chromatin topologies.

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Multiplex-GAM: genome-wide identification of chromatin contacts yields insights overlooked by Hi-C.

Technology for measuring 3D genome topology is increasingly important for studying gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of genome architecture mapping (GAM), a ligation-free technique that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. When examining the strongest contacts detected by either method, we find that only one-third of these are shared. The strongest contacts specifically found in GAM often involve 'active' regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain 'inactive' regions. Our work shows that active genomic regions are involved in extensive complex contacts that are currently underestimated in ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.

tRNA-Related Sequences Trigger Systemic mRNA Transport in Plants.

In plants, protein-coding mRNAs can move via the phloem vasculature to distant tissues, where they may act as non-cell-autonomous signals. Emerging work has identified many phloem-mobile mRNAs, but little is known regarding RNA motifs triggering mobility, the extent of mRNA transport, and the potential of transported mRNAs to be translated into functional proteins after transport. To address these aspects, we produced reporter transcripts harboring tRNA-like structures (TLSs) that were found to be enriched in the phloem stream and in mRNAs moving over chimeric graft junctions. Phenotypic and enzymatic assays on grafted plants indicated that mRNAs harboring a distinctive TLS can move from transgenic roots into wild-type leaves and from transgenic leaves into wild-type flowers or roots; these mRNAs can also be translated into proteins after transport. In addition, we provide evidence that dicistronic mRNA:tRNA transcripts are frequently produced in Arabidopsis thaliana and are enriched in the population of graft-mobile mRNAs. Our results suggest that tRNA-derived sequences with predicted stem-bulge-stem-loop structures are sufficient to mediate mRNA transport and seem to be necessary for the mobility of a large number of endogenous transcripts that can move through graft junctions.

Extensive folding variability between homologous chromosomes in mammalian cells.

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, mediated by Polycomb repression. We show that histone genes are expressed with allelic imbalance in mESCs, are involved in haplotype-specific chromatin contact marked by H3K27me3, and are targets of Polycomb repression through conditional knockouts of Ezh2 or Ring1b. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.

An integrated view of the structure and function of the human 4D nucleome.

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

SplamiR--prediction of spliced miRNAs in plants.

MOTIVATION: MicroRNAs (miRNAs) are important regulators of biological processes in plants and animals. Recently, miRNA genes have been discovered, whose primary transcripts are spliced and which cannot be predicted directly from genomic sequence. Hence, more sophisticated programs for the detection of spliced miRNAs are required. RESULTS: Here, we present the first method for the prediction of spliced miRNAs in plants. For a given genomic sequence, SplamiR creates a database of complementary sequence pairs, which might encode for RNAs folding into stem-loop structures. Next, in silico splice variants of database sequences with complementarity to an mRNA of interest are classified as to whether they could represent miRNAs targeting this mRNA. Our method identifies all known cases of spliced miRNAs in rice, and a previously undiscovered miRNA in maize which is supported by an expressed sequence tag (EST). SplamiR permits identification of spliced miRNAs for a given target mRNA in many plant genomes. AVAILABILITY: The program is freely available at http://www.uni-jena.de/SplamiR.html.

Endogenous Arabidopsis messenger RNAs transported to distant tissues.

The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants? Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.

Give It AGO: The Search for miRNA-Argonaute Sorting Signals in Arabidopsis thaliana Indicates a Relevance of Sequence Positions Other than the 5'-Position Alone.

The specific recognition of miRNAs by Argonaute (AGO) proteins, the effector proteins of the RNA-induced silencing complex, constitutes the final step of the biogenesis of miRNAs and is crucial for their target interaction. In the genome of Arabidopsis thaliana (Ath), 10 different AGO proteins are encoded and the sorting decision, which miRNA associates with which AGO protein, was reported to depend exclusively on the identity of the 5'-sequence position of mature miRNAs. Hence, with only four different bases possible, a 5'-position-only sorting signal would not suffice to specifically target all 10 different AGOs individually or would suggest redundant AGO action. Alternatively, other and as of yet unidentified sorting signals may exist. We analyzed a dataset comprising 117 Ath-miRNAs with clear sorting preference to either AGO1, AGO2, or AGO5 as identified in co-immunoprecipitation experiments combined with sequencing. While mutual information analysis did not identify any other single position but the 5'-nucleotide to be informative for the sorting at sufficient statistical significance, significantly better than random classification results using Random Forests nonetheless suggest that additional positions and combinations thereof also carry information with regard to the AGO sorting. Positions 2, 6, 9, and 13 appear to be of particular importance. Furthermore, uracil bases at defined positions appear to be important for the sorting to AGO2 and AGO5, in particular. No predictive value was associated with miRNA length or base pair binding pattern in the miRNA:miRNA* duplex. From inspecting available AGO gene expression data in Arabidopsis, we conclude that the temporal and spatial expression profile may also contribute to the fine-tuning of miRNA sorting and function.