Identification of functional enhancer variants associated with type I diabetes in CD4+ T cells.

Type I diabetes is an autoimmune disease mediated by T-cell destruction of ß cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ T-cell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants.

Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Optical Genome Mapping: A Machine-Based Platform in Cytogenomics.

Optical genome mapping (OGM) has generated excitement following decades of research and development. Now, commercially available technical platforms have been used to compare various other cytogenetic and cytogenomic technologies, including karyotype, microarrays, and DNA sequencing, with impressive results. In this chapter, using OGM as a case study, we advocate for a new trend in future cytogenomics, emphasizing the power of machine automation to deliver higher-quality cytogenomic data. By briefly discussing OGM, along with its major advantages and limitations, we underscore the importance of karyotype-based genomic research, from both a theoretical framework and a new technology perspective. We also call for the encouragement of further technological platform development for the future of cytogenetics and cytogenomics.

Predicting chromatin conformation contact maps.

Over the past 15 years, a variety of next-generation sequencing assays have been developed for measuring the 3D conformation of DNA in the nucleus. Each of these assays gives, for a particular cell or tissue type, a distinct picture of 3D chromatin architecture. Accordingly, making sense of the relationship between genome structure and function requires teasing apart two closely related questions: how does chromatin 3D structure change from one cell type to the next, and how do different measurements of that structure differ from one another, even when the two assays are carried out in the same cell type? In this work, we assemble a collection of chromatin 3D datasets-each represented as a 2D contact map- spanning multiple assay types and cell types. We then build a machine learning model that predicts missing contact maps in this collection. We use the model to systematically explore how genome 3D architecture changes, at the level of compartments, domains, and loops, between cell type and between assay types.

SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Epigenetic Modifications in Genome Help Remembering the Stress Tolerance Strategy Adopted by the Plant.

Genetic information in eukaryotic organisms is stored, replicated, transcribed, and inherited through the nucleus of a cell. Epigenetic modifications in the genetic material, including DNA methylation, histone modification, changes in non-coding RNA (ncRNA) biogenesis, and chromatin architecture play important roles in determining the genomic landscape and regulating gene expression. Genome architecture (structural features of chromatin, affected by epigenetic modifications) is a major driver of genomic functions/activities. Segregation of euchromatin (transcriptionally active) from heterochromatin (transcriptionally repressed chromosome) and positioning of genes in specific nuclear space in eukaryotic cells emphasise non-randomness in the organization of the genetic information. Not only does the base sequence of a gene carry the genetic information but the covalent modifications of bases, three-dimensional positioning of the genome, and chromatin loops are vital for switching on/off the gene and regulating its expression during growth/environmental stress. The epigenetic dynamics depend on the activities of writers and erasers under changing environmental conditions. The discovery of non-coding RNAs (one of the players in de novo methylation of DNA), increased DNA methylation protein (guide for the DNA demethylase), and methylation monitoring sequence (that helps keep a balance between DNA demethylation and methylation) have been some of the new developments in the era of epigenomics. To respond to environmental stimuli, plants depend on modulating gene expression through different mechanisms including biochemical, molecular, genetic, and epigenetic alterations. Studies on plants might provide better insights into epigenetic stress memory and molecular bases of adaptability to enable (epi)genome editing of crops for climate resilience and sustainable agriculture in the present era of multifaceted climate change.

Evolutionary analysis of gene ages across TADs associates chromatin topology with whole-genome duplications.

Topologically associated domains (TADs) are interaction subnetworks of chromosomal regions in 3D genomes. TAD boundaries frequently coincide with genome breaks while boundary deletion is under negative selection, suggesting that TADs may facilitate genome rearrangements and evolution. We show that genes co-localize by evolutionary age in humans and mice, resulting in TADs having different proportions of younger and older genes. We observe a major transition in the age co-localization patterns between the genes born during vertebrate whole-genome duplications (WGDs) or before and those born afterward. We also find that genes recently duplicated in primates and rodents are more frequently essential when they are located in old-enriched TADs and interact with genes that last duplicated during the WGD. Therefore, the evolutionary relevance of recent genes may increase when located in TADs with established regulatory networks. Our data suggest that TADs could play a role in organizing ancestral functions and evolutionary novelty.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.

Unveiling Alterations of Epigenetic Modifications and Chromatin Architecture Leading to Lipid Metabolic Reprogramming during the Evolutionary Trastuzumab Adaptation of HER2-Positive Breast Cancer.

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

Dose-Dependent Transcriptional Response to Ionizing Radiation Is Orchestrated with DNA Repair within the Nuclear Space.

Radiation therapy is commonly used to treat glioblastoma multiforme (GBM) brain tumors. Ionizing radiation (IR) induces dose-specific variations in transcriptional programs, implicating that they are tightly regulated and critical components in the tumor response and survival. Yet, our understanding of the downstream molecular events triggered by effective vs. non-effective IR doses is limited. Herein, we report that variations in the genetic programs are positively and functionally correlated with the exposure to effective or non-effective IR doses. Genome architecture analysis revealed that gene regulation is spatially and temporally coordinated with DNA repair kinetics. The radiation-activated genes were pre-positioned in active sub-nuclear compartments and were upregulated following the DNA damage response, while the DNA repair activity shifted to the inactive heterochromatic spatial compartments. The IR dose affected the levels of DNA damage repair and transcription modulation, but not the order of the events, which was linked to their spatial nuclear positioning. Thus, the distinct coordinated temporal dynamics of DNA damage repair and transcription reprogramming in the active and inactive sub-nuclear compartments highlight the importance of high-order genome organization in synchronizing the molecular events following IR.

Wiring the Brain by Clustered Protocadherin Neural Codes / 神经科学通报·英文版

There are more than a thousand trillion specific synaptic connections in the human brain and over a million new specific connections are formed every second during the early years of life. The assembly of these staggeringly complex neuronal circuits requires specific cell-surface molecular tags to endow each neuron with a unique identity code to discriminate self from non-self. The clustered protocadherin (Pcdh) genes, which encode a tremendous diversity of cell-surface assemblies, are candidates for neuronal identity tags. We describe the adaptive evolution, genomic structure, and regulation of expression of the clustered Pcdhs. We specifically focus on the emerging 3-D architectural and biophysical mechanisms that generate an enormous number of diverse cell-surface Pcdhs as neural codes in the brain.