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Quinclorac (QNC) is a highly selective, hormonal, and low-toxic herbicide with a long duration. And the growth and development of subsequent crops are easily affected by QNC accumulated in the soil. In this paper, a QNC-degrading strain D was isolated and screened from the rice paddy soil. Through morphology, physiological and biochemical tests and 16Sr DNA gene analysis, strain D was identified asCellulosimicrobium cellulanssp. And the QNC degradation characteristics of strain D were studied. Under the optimal culture conditions, the QNC-degrading rate was 45.9% after culturing for 21 days. The QNC-degrading efficiency of strain D in the field was evaluated by a simulated pot experiment. The results show that strain D can promote the growth of rice and QNC-degrading effectively. This research could provide a new bacterial species for microbial degradation of QNC and lay a theoretical foundation for further research on QNC remediation.ImportanceAt present, some QNC-degrading bacteria have been isolated from different environments, but there are no reports ofCellulosimicrobium cellulanssp. bacterial that could degrade QNC. In this study, a new QNC-degradation strain was selected from the paddy soil. The degradation characteristics of strain D were studied in detail. The results shown that strain D had a satisfactory quinclorac-degrading efficiency. Two degradation products of QNC by strain D were identified by HPLC-Q-TOF/MS: 3-pyridylacetic acid (138.0548 m/z) and 3-ethylpyridine (108.0805 m/z), which have not been reported before. The strain D had a potential ability of quinclorac-degrading effectively in the quinclorac-polluted paddy field environment.

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4 + T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.

The human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci—almost always bound by CTCF and cohesin—lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid. Mapping the nucleome in 4D during cohesin loss and recovery reveals that cohesin degradation eliminates loop domains but has only modest transcriptional consequences. Keywords: cohesion; genome architecture; loop extrusion; chromatin loops; superenhancers; gene regulation; nuclear compartments; Hi-C; 4D Nucleome; CTCF

SUMMARYThe human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci - almost always bound by CTCF and cohesin – lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loci in different compartments that had been in the same loop domain become more segregated. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. Cohesin restoration quickly reverses these effects, consistent with a model where loop extrusion is rapid.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA-seq profiles from activated CD4+ T cells of up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, in patterns consistent with the 3D organization of chromosomes measured by in situ Hi-C in T cells. 15% of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak through disrupting binding sites for transcription factors important for T cell differentiation and activation. These ATAC quantitative trait nucleotides (ATAC-QTNs) have the largest effects on co-accessible peaks, are associated with gene expression from the same aliquot of cells, are rarely affecting core binding motifs, and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis- regulatory elements, in isolation or in concert, to influence gene expression in primary immune cells that play a key role in many human diseases.

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the “Barr body.” Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called “superdomains,” such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called “superloops.” DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

Procalcitonin (PCT) is expressed in nonthryoidal tissues of humans during severe infections. Serum PCT levels are measured to diagnose and guide therapy, and there is some evidence that PCT may also contribute to the pathogenesis of sepsis. We tested whether disruption of the gene encoding PCT in mice affected the course of sepsis. Mice with exons 2–5 of the gene encoding calcitonin/calcitonin gene–related polypeptide α (Calca) knocked out and congenic C57BL/6J control mice were challenged with aerosolized Streptococcus pneumoniae or Pseudomonas aeruginosa, or injected intraperitoneally with S. pneumoniae. There were no significant differences in the survival of knockout and control mice in the two pneumonia models, and no significant differences in weight loss, splenic bacterial counts, or blood leukocyte levels in the peritoneal sepsis model. To verify disruption of the Calca gene in knockout mice, the absence of calcitonin in the serum of knockout mice and its presence and inducibility in control mice were confirmed. To evaluate PCT expression in nonthyroidal tissues of control mice, transcripts were measured in multiple organs. PCT transcripts were not significantly expressed in liver or spleen of control mice challenged with aerosolized P. aeruginosa or intraperitoneal endotoxin, and were expressed in lung only at low levels, even though serum IL-6 rose 3,548-fold. We conclude that mice are not an ideal loss-of-function model to test the role of PCT in the pathogenesis of sepsis because of low nonendocrine PCT expression during infection and inflammation. Nonetheless, our studies demonstrate that nonendocrine PCT expression is not necessary for adverse outcomes from sepsis.

The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.

DNA methylation is a critical regulator of cis-regulatory elements that can impact the distribution of epigenetic regulators and transcription factors. We have mapped DNA methylation changes in human CD34+ cells and downstream progeny to detect changes in DNA methylation with differentiation. Some large regions of low DNA methylation emerge with differentiation, which we call dynamic (d-) canyons. In order to determine whether these correlate with changes in the 3D genome, we generated 6 high-resolution Hi-C contact maps from CD34+CD38- cells, Adult bone marrow CD34+ cells, 7 days cultured CD34+ cells, CD71+CD36+CD235+ Erythroid cells, CD4+ T cells and AML patient blast cells. We generated ~ 1 billion mapped reads in order to create each 3D map of the genome at kilobase resolution. We identified several sites of d-canyons which correlate with changes in Hi-C loops. To study the functional role of d-canyons, we performed CRISPR/CAS9 mediated genome engineering targeting three sites of interest in CD34+ progenitors. We designed sgRNAs to delete several d-canyon regions that exhibited cell-type specific methylation and dynamic H3K27ac marking. First, we targeted a novel putative regulatory region of the RUNX1 gene, a critical master regulator of hematopoiesis that is frequently mutated in human leukemia. From genome-wide DNA methylation profiling, we identified a d-canyon located in the first intron of RUNX1, which overlaped with a H3K27ac peak in human CD34+CD38- hematopoietic progenitor cells. DNA methylation in this region is further depleted in T cells and increased in AML cells, suggesting a role for regulating RUNX1 in specific cell types. To study its function, we designed 2 sgRNAs to delete a 1.8 kb d-canyon. CRISPR/Cas9-mediated deletion of this region resulted in ablation of RUNX1 expression in cord blood hematopoietic stem cells and a significant decrease of engraftment activity in NSG mice along with an increase of erythroid colony forming ability in in vitro assays. In addition, we identified d-canyons upstream of the master regulators GATA2 and in the HOXA cluster. We deleted 1.7kb d-canyon upstream of GATA2 gene, finding that it resulted in increased self-renewal of CD34+CD38- cells in NSG xenografts. In contrast, when we deleted one a d-canyon located ~2Mb upstream of the HOXA cluster, within a HiC loop signal present only in CD34+CD38- cells, we observed in decreased CD34+CD38- cell self-renewal but a significant increase of CD34+CD38+ differentiated progenitors in an in vitro culture system, as well as in NSG mice. Colony forming assay showed decreased colony size and numbers. Finally, we identified a d-canyon associated with TCF3, also known as E2A, a transcription factor involved in B and T cell lineage differentiation. We designed 4 sgRNAs to delete 1.5kb d-canyon edge regions within the second intron of TCF3. Removal of this region resulted in a significant decrease of CD19+ B cells, but an increase of CD3+ T cells in NSG mice. Taken together, these results suggest the functional importance of d-canyons for orchestrating genome architecture and fate decisions of hematopoietic stem cells. These findings advance our understanding of the relationship between DNA methylation changes and loop interactions, providing new insights into the potential impact of potential aberrant DNA methylation and chromatin structure. Disclosures No relevant conflicts of interest to declare.