Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ T cell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

The ETS transcription factor PU.1 plays an essential role in blood cell development. Its precise expression pattern is governed by cis-regulatory elements (CRE) acting at the chromatin level. CREs mediate the fine-tuning of graded levels of PU.1, deviations of which can cause acute myeloid leukemia. In this review, we perform an in-depth analysis of the regulation of PU.1 expression in normal and malignant hematopoiesis. We elaborate on the role of trans-acting factors and the biomolecular interplays in mediating local chromatin dynamics. Moreover, we discuss the current understanding of CRE bifunctionality exhibiting enhancer or silencer activities in different blood cell lineages and future directions toward gene-specific chromatin-targeted therapeutic development.

Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation.

Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.

BACKGROUND: Recognition of enhancer-promoter Interactions (EPIs) is crucial for human development. EPIs in the genome play a key role in regulating transcription. However, experimental approaches for classifying EPIs are too expensive in terms of effort, time, and resources. Therefore, more and more studies are being done on developing computational techniques, particularly using deep learning and other machine learning techniques, to address such problems. Unfortunately, the majority of current computational methods are based on convolutional neural networks, recurrent neural networks, or a combination of them, which don\'t take into consideration contextual details and the long-range interactions between the enhancer and promoter sequences. A new transformer-based model called EPI-Trans is presented in this study to overcome the aforementioned limitations. The multi-head attention mechanism in the transformer model automatically learns features that represent the long interrelationships between enhancer and promoter sequences. Furthermore, a generic model is created with transferability that can be utilized as a pre-trained model for various cell lines. Moreover, the parameters of the generic model are fine-tuned using a particular cell line dataset to improve performance.
RESULTS: Based on the results obtained from six benchmark cell lines, the average AUROC for the specific, generic, and best models is 94.2%, 95%, and 95.7%, while the average AUPR is 80.5%, 66.1%, and 79.6% respectively.
CONCLUSIONS: This study proposed a transformer-based deep learning model for EPI prediction. The comparative results on certain cell lines show that EPI-Trans outperforms other cutting-edge techniques and can provide superior performance on the challenge of recognizing EPI.

BACKGROUND: Enhancers are important gene regulatory elements that promote the expression of critical genes in development and disease. Aberrant enhancer can modulate cancer risk and activate oncogenes that lead to the occurrence of various cancers. However, the underlying mechanism of most enhancers in cancer remains unclear. Here, we aim to explore the function and mechanism of a crucial enhancer in melanoma.
METHODS: Multi-omics data were applied to identify an enhancer (enh17) involved in melanoma progression. To evaluate the function of enh17, CRISPR/Cas9 technology were applied to knockout enh17 in melanoma cell line A375. RNA-seq, ChIP-seq and Hi-C data analysis integrated with luciferase reporter assay were performed to identify the potential target gene of enh17. Functional experiments were conducted to further validate the function of the target gene ETV4. Multi-omics data integrated with CUT&Tag sequencing were performed to validate the binding profile of the inferred transcription factor STAT3.
RESULTS: An enhancer, named enh17 here, was found to be aberrantly activated and involved in melanoma progression. CRISPR/Cas9-mediated deletion of enh17 inhibited cell proliferation, migration, and tumor growth of melanoma both in vitro and in vivo. Mechanistically, we identified ETV4 as a target gene regulated by enh17, and functional experiments further support ETV4 as a target gene that is involved in cancer-associated phenotypes. In addition, STAT3 acts as a transcription factor binding with enh17 to regulate the transcription of ETV4.
CONCLUSIONS: Our findings revealed that enh17 plays an oncogenic role and promotes tumor progression in melanoma, and its transcriptional regulatory mechanisms were fully elucidated, which may open a promising window for melanoma prevention and treatment.

Macrophages elicit immune responses to pathogens through induction of inflammatory genes. Here, we examined the role of three variants of the SWI/SNF nucleosome remodeling complex-cBAF, ncBAF, and PBAF-in the macrophage response to bacterial endotoxin (lipid A). All three SWI/SNF variants were prebound in macrophages and retargeted to genomic sites undergoing changes in chromatin accessibility following stimulation. Cooperative binding of all three variants associated with de novo chromatin opening and latent enhancer activation. Isolated binding of ncBAF and PBAF, in contrast, associated with activation and repression of active enhancers, respectively. Chemical and genetic perturbations of variant-specific subunits revealed pathway-specific regulation in the activation of lipid A response genes, corresponding to requirement for cBAF and ncBAF in inflammatory and interferon-stimulated gene (ISG) activation, respectively, consistent with differential engagement of SWI/SNF variants by signal-responsive transcription factors. Thus, functional diversity among SWI/SNF variants enables increased regulatory control of innate immune transcriptional programs, with potential for specific therapeutic targeting.

An age-dependent increase in IFN-γ expression by intestinal intraepithelial lymphocytes (IELs) contributes to the acquisition of resistance to infection by pathogens. However, how IELs acquire the ability to produce IFN-γ remains to be elucidated. Here, we report that IELs in the small intestine acquire the ability to rapidly produce IFN-γ at two distinct life stages. TCRαβ+ IELs (αβIELs) started producing IFN-γ at 4 weeks of age, within 1 week after weaning. In contrast, TCRγδ+ IELs (γδIELs) started producing IFN-γ at 7 weeks of age. In mice lacking Eγ4, an enhancer of the TCRγ locus (Eγ4-/- mice), Thy-1+ Vγ5+ γδIELs, a major subpopulation of γδIELs, were specifically reduced and their ability to produce IFN-γ was severely impaired, whereas Vγ2+ γδIELs normally produced IFN-γ. In Eγ4-/- mice, TCR expression levels were reduced in Vγ5+ γδIEL precursors in the thymus but unchanged in the Vγ5+ IELs. Nevertheless, TCR responsiveness in Vγ5+ γδIELs was impaired in Eγ4-/- mice, suggesting that the TCR signal received in the thymus may determine TCR responsiveness and the ability to produce IFN-γ in the gut. These results suggest that αβIELs and γδIELs start producing IFN-γ at different life stages and that the ability of Vγ5+ γδIELs to produce IFN-γ in the gut may be predetermined by TCR signaling in IEL precursors in the thymus.