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The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer–promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer–promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types. Acute loss of CTCF disproportionately affects the transcription of genes that display promoter-proximal CTCF binding and are dependent on long-distance enhancers.

We report a molecular assay, Methyl-HiC, that can simultaneously capture the chromosome conformation and DNA methylome in a cell. Methyl-HiC reveals coordinated DNA methylation status between distal genomic segments that are in spatial proximity in the nucleus, and delineates heterogeneity of both the chromatin architecture and DNA methylome in a mixed population. It enables simultaneous characterization of cell-type-specific chromatin organization and epigenome in complex tissues.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

Some animals have the remarkable capacity for mirror self-recognition (MSR), yet any implications for self-awareness remain uncertain and controversial. This is largely because explicit tests of the two potential mechanisms underlying MSR are still lacking: mental image of the self and kinesthetic visual matching. Here, we test the hypothesis that MSR ability in cleaner fish, Labroides dimidiatus, is associated with a mental image of the self, in particular the self-face, like in humans. Mirror-naive fish initially attacked photograph models of both themselves and unfamiliar strangers. In contrast, after all fish had passed the mirror mark test, fish did not attack their own (motionless) images, but still frequently attacked those of unfamiliar individuals. When fish were exposed to composite photographs, the self-face/unfamiliar body were not attacked, but photographs of unfamiliar face/self-body were attacked, demonstrating that cleaner fish with MSR capacity recognize their own facial characteristics in photographs. Additionally, when presented with self-photographs with a mark placed on the throat, unmarked mirror-experienced cleaner fish demonstrated throat-scraping behaviors. When combined, our results provide clear evidence that cleaner fish recognize themselves in photographs and that the likely mechanism for MSR is associated with a mental image of the self-face, not a kinesthetic visual-matching model. Humans are also capable of having a mental image of the self-face, which is considered an example of private self-awareness. We demonstrate that combining mirror test experiments with photographs has enormous potential to further our understanding of the evolution of cognitive processes and private self-awareness across nonhuman animals.

DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decrease global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins. DNMT3A and DNMT3L form a complex to deposit DNA methylation in mammalian germ cells. Here, the authors report that loss-of-function of ADD domains of DNMT3A and/or DNMT3L has various impacts on DNA methylation landscapes in mouse oocytes and sperm.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures 1 . Recent surveys of mouse and human brains with single-cell transcriptomics 2 – 6 and high-throughput imaging technologies 7 , 8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis -regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis -regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans. A comprehensive analysis of gene regulatory elements in 160 distinct cell types from the mouse cerebrum.

An animal that tries to remove a mark from its body that is only visible when looking into a mirror displays the capacity for mirror self-recognition (MSR), which has been interpreted as evidence for self-awareness. Conservative interpretations of existing data conclude that convincing evidence for MSR is currently restricted to great apes. Here, we address proposed shortcomings of a previous study on MSR in the cleaner wrasse Labroides dimidiatus , by varying preexposure to mirrors and by marking individuals with different colors. We found that (1) 14/14 new individuals scraped their throat when a brown mark had been provisioned, but only in the presence of a mirror; (2) blue and green color marks did not elicit scraping; (3) intentionally injecting the mark deeper beneath the skin reliably elicited spontaneous scraping in the absence of a mirror; (4) mirror-naive individuals injected with a brown mark scraped their throat with lower probability and/or lower frequency compared to mirror-experienced individuals; (5) in contrast to the mirror images, seeing another fish with the same marking did not induce throat scraping; and (6) moving the mirror to another location did not elicit renewed aggression in mirror-experienced individuals. Taken together, these results increase our confidence that cleaner fish indeed pass the mark test, although only if it is presented in ecologically relevant contexts. Therefore, we reiterate the conclusion of the previous study that either self-awareness in animals or the validity of the mirror test needs to be revised.

INTRODUCTION: A bladder hernia associated with bladder calculi is an extremely rare condition, with no consensus established regarding its management. Herein, we report a case in which 1-stage surgical treatment was performed for a bladder hernia complicated by bladder calculi.CASE PRESENTATION: The patient presented with a swelling in the right inguinal region. CT revealed a bladder hernia accompanied by multiple bladder calculi. An inguinal incision was made, the bladder stones were removed, and hernia repair was performed using the Lichtenstein method. At 3 years postoperatively, there was no recurrence of either bladder calculi or hernia.CONCLUSIONS: For inguinal hernias accompanied by bladder calculi in which transurethral lithotripsy is challenging, open surgical fragmentation of bladder calculi and inguinal hernia repair by using the Lichtenstein method through the same surgical field may be useful approaches.

Whether animals are self-aware has important implications for our approaches to both animal cognition and animal welfare. A landmark moment in animal cognition research was when great apes passed the mark-test and demonstrated mirror self-recognition (MSR). Animals that pass the mark-test are capable of visually self-recognising and considered to be self-aware. Other taxa, including a fish, the cleaner wrasse (cleaner fish: Labroides dimidiatus ) have also now passed the mark-test, forcing a rethink of the mental and neurological requirements for MSR. Previous research has largely focused on which species can pass the mark-test, rather than the processes underlying MSR. Here, we marked mirror-naïve cleaner fish with an ecologically relevant mark resembling an ectoparasite and then undertook detailed behavioural observations after exposure to a mirror. We found that cleaner fish achieve MSR rapidly, implying self-awareness prior to mirror exposure. By observing the exact timing of MSR in individuals, we could also report previously undocumented differences in pre- and post-MSR behaviours, including post-MSR exploratory behaviour of the mirror’s reflective properties. We find remarkable parallels between the processing of MSR in humans and cleaner fish, suggesting that some aspects of self-awareness are conserved across animal taxa.

The pathogenesis of lung cancer associated with idiopathic pulmonary fibrosis (IPF) has remained largely uncharacterized. To provide insight into this condition, we undertook genomic profiling of IPF‐associated lung cancer as well as of adjacent fibrosing lung tissue in surgical specimens. Isolated DNA and RNA from 17 IPF‐associated non‐small cell lung cancer and 15 paired fibrosing lung tissue specimens were analyzed by next‐generation sequencing with a panel that targets 161 cancer‐related genes. Somatic genetic alterations were frequently identified in TP53 (n = 6, 35.3%) and PIK3CA (n = 5, 29.4%) genes in tumor samples as well as in EGFR (n = 7, 46.7%), PIK3CA (n = 5, 33.3%), ERBB3 (n = 4, 26.7%), and KDR (n = 4, 26.7%) in IPF samples. Genes related to the RAS‐RAF signaling pathway were also frequently altered in tumor (n = 7, 41.2%) and IPF (n = 3, 20.0%) samples. The number of somatic alterations identified in IPF samples was almost as large as that detected in paired tumor samples (81 vs 90, respectively). However, only 6 of the 81 somatic alterations detected in IPF samples overlapped with those in paired tumor samples. The accumulation of somatic mutations was thus apparent in IPF tissue of patients with IPF‐associated lung cancer, and the RAS‐RAF pathway was implicated in lung tumorigenesis. The finding that somatic alterations were not frequently shared between tumor and corresponding IPF tissue indicates that IPF‐associated lung cancer does not develop through the stepwise accumulation of somatic alterations in IPF. The genetic basis of idiopathic pulmonary fibrosis‐associated lung cancer remains largely unknown. We show that somatic alterations were frequently identified in fibrosing lung tissue as much as that in tumor tissue, and that genes related to the RAS‐RAF signaling pathway were frequently altered in both specimens. These findings can provide a basis for the development of targeted drugs for such tumors.