Project description:The metazoan genome is compartmentalized in areas of highly interacting chromatin known as topologically associating domains (TADs). TADs are demarcated by boundaries mostly conserved across cell types and even across species. However, a genome-wide characterization of TAD boundary strength in mammals is still lacking. In this study, we first use fused two-dimensional lasso as a machine learning method to improve Hi-C contact matrix reproducibility, and, subsequently, we categorize TAD boundaries based on their insulation score. We demonstrate that higher TAD boundary insulation scores are associated with elevated CTCF levels and that they may differ across cell types. Intriguingly, we observe that super-enhancers are preferentially insulated by strong boundaries. Furthermore, we demonstrate that strong TAD boundaries and super-enhancer elements are frequently co-duplicated in cancer patients. Taken together, our findings suggest that super-enhancers insulated by strong TAD boundaries may be exploited, as a functional unit, by cancer cells to promote oncogenesis.
Project description:CTCF plays an important role in 3D genome organization by adjusting the strength of chromatin insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in a tandem array with a complex divergent or convergent orientation. Here, using Pcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in mice in vivo or CBS elements in cultured cells in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intra-TAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries in developmental gene regulation and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.
Project description:Tumor mutational burden (TMB) is a promising predictive biomarker for cancer immunotherapy. Patients with a high TMB have better responses to immune checkpoint inhibitors. Currently, the gold standard for determining TMB is whole-exome sequencing (WES). However, high cost, long turnaround time, infrastructure requirements, and bioinformatics demands have prevented WES from being implemented in routine clinical practice. Panel-sequencing-based estimates of TMB have gradually replaced WES TMB; however, panel design biases could lead to overestimation of TMB. To stratify TMB-high patients better without sequencing all genes and avoid overestimating TMB, we focused on DNA damage repair (DDR) genes, in which dysfunction may increase somatic mutation rates. We extensively explored the association between the mutation status of DDR genes and TMB in different cancer types. By analyzing the mutation data from The Cancer Genome Atlas, which includes information for 33 different cancer types, we observed no single DDR gene/pathway in which mutation status was significantly associated with high TMB across all 33 cancer types. Therefore, a computational algorithm was proposed to identify a cancer-specific gene set as a surrogate for stratifying patients with high TMB in each cancer. We applied our algorithm to skin cutaneous melanoma and lung adenocarcinoma, demonstrating that the mutation status of the identified cancer-specific DDR gene sets, which included only 9 and 14 genes, respectively, was significantly associated with TMB. The cancer-specific DDR gene set can be used as a cost-effective approach to stratify patients with high TMB in clinical practice.
Project description:Recent genome-wide association studies corroborate classical research on developmental programming indicating that obesity is primarily a neurodevelopmental disease strongly influenced by nutrition during critical ontogenic windows. Epigenetic mechanisms regulate neurodevelopment; however, little is known about their role in establishing and maintaining the brain's energy balance circuitry. We generated neuron and glia methylomes and transcriptomes from male and female mouse hypothalamic arcuate nucleus, a key site for energy balance regulation, at time points spanning the closure of an established critical window for developmental programming of obesity risk. We find that postnatal epigenetic maturation is markedly cell type and sex specific and occurs in genomic regions enriched for heritability of body mass index in humans. Our results offer a potential explanation for both the limited ontogenic windows for and sex differences in sensitivity to developmental programming of obesity and provide a rich resource for epigenetic analyses of developmental programming of energy balance.
Project description:Soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) is the most destructive pest affecting soybeans [Glycine max (L.) Merr.] in the U.S. To date, only two major SCN resistance alleles, rhg1 and Rhg4, identified in PI 88788 (rhg1) and Peking (rhg1/Rhg4), residing on chromosomes (Chr) 18 and 8, respectively, have been widely used to develop SCN resistant cultivars in the U.S. Thus, some SCN populations have evolved to overcome the PI 88788 and Peking derived resistance, making it a priority for breeders to identify new alleles and sources of SCN resistance. Toward that end, 461 soybean accessions from various origins were screened using a greenhouse SCN bioassay and genotyped with Illumina SoySNP50K iSelect BeadChips and three KASP SNP markers developed at the Rhg1 and Rhg4 loci to perform a genome-wide association study (GWAS) and a haplotype analysis at the Rhg1 and Rhg4 loci. In total, 35,820 SNPs were used for GWAS, which identified 12 SNPs at four genomic regions on Chrs 7, 8, 10, and 18 that were significantly associated with SCN resistance (P < 0.001). Of those, three SNPs were located at Rhg1 and Rhg4, and 24 predicted genes were found near the significant SNPs on Chrs 7 and 10. KASP SNP genotyping results of the 462 accessions at the Rhg1 and Rhg4 loci identified 30 that carried PI 88788-type resistance, 50 that carried Peking-type resistance, and 58 that carried neither the Peking-type nor the PI 88788-type resistance alleles, indicating they may possess novel SCN resistance alleles. By using two subsets of SNPs near the Rhg1 and Rhg4 loci obtained from SoySNP iSelect BeadChips, a haplotype analysis of 461 accessions grouped those 58 accessions differently from the accessions carrying Peking or PI 88788 derived resistance, thereby validating the genotyping results at Rhg1 and Rhg4. The significant SNPs, candidate genes, and newly characterized SCN resistant accessions will be beneficial for the development of DNA markers to be used for marker-assisted breeding and developing soybean cultivars carrying novel sources of SCN resistance.
Project description:Epigenetic research has rapidly evolved into a dynamic field of genome biology. Chromatin regulation has been proved to be an essential aspect for all genomic processes, including DNA repair. Chromatin structure is modified by enzymes and factors that deposit, erase, and interact with epigenetic marks such as DNA and histone modifications, as well as by complexes that remodel nucleosomes. In this review we discuss recent advances on how the chromatin state is modulated during this multi-step process of damage recognition, signaling, and repair. Moreover, we examine how chromatin is regulated when different pathways of DNA repair are utilized. Furthermore, we review additional modes of regulation of DNA repair, such as through the role of global and localized chromatin states in maintaining expression of DNA repair genes, as well as through the activity of epigenetic enzymes on non-nucleosome substrates. Finally, we discuss current and future applications of the mechanistic interplays between chromatin regulation and DNA repair in the context cancer treatment.
Project description:Saturation mutagenesis--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory and protein-coding nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or "safe-harbour" integration fail to capture the native context of the endogenous chromosomal locus. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.