AT-rich Sequences Research Articles

Crystal structure of the HMGA AT-hook 1 domain bound to the minor groove of AT-rich DNA and inhibition by antikinetoplastid drugs

High mobility group (HMG) proteins are intrinsically disordered nuclear non-histone chromosomal proteins that play an essential role in many biological processes by regulating the expression of numerous genes in eukaryote cells. HMGA proteins contain three DNA binding motifs, the “AT-hooks”, that bind preferentially to AT-rich sequences in the minor groove of B-form DNA. Understanding the interactions of AT-hook domains with DNA is very relevant from a medical point of view because HMGA proteins are involved in different conditions including cancer and parasitic diseases. We present here the first crystal structure (1.40 Å resolution) of the HMGA AT-hook 1 domain, bound to the minor groove of AT-rich DNA. In contrast to AT-hook 3 which bends DNA and shows a larger minor groove widening, AT-hook 1 binds neighbouring DNA molecules and displays moderate widening of DNA upon binding. The binding affinity and thermodynamics of binding were studied in solution with surface plasmon resonance (SPR)-biosensor and isothermal titration calorimetry (ITC) experiments. AT-hook 1 forms an entropy-driven 2:1 complex with (TTAA)2-containing DNA with relatively slow kinetics of association/dissociation. We show that N-phenylbenzamide-derived antikinetoplastid compounds (1–3) bind strongly and specifically to the minor groove of AT-DNA and compete with AT-hook 1 for binding. The central core of the molecule is the basis for the observed sequence selectivity of these compounds. These findings provide clues regarding a possible mode of action of DNA minor groove binding compounds that are relevant to major neglected tropical diseases such as leishmaniasis and trypanosomiasis.

TaWRKY55-TaPLATZ2 module negatively regulate saline-alkali stress tolerance in wheat.

Saline-alkaline soils are a major environmental problem that limit plant growth and crop productivity. Plasma membrane H+-ATPases and the salt overly sensitive (SOS) signaling pathway play important roles in plant responses to saline-alkali stress. However, little is known about the functional genes and mechanisms regulating the transcription of H+-ATPases and SOS pathway genes under saline-alkali stress. In the present study, we identified that the plant AT-rich sequence and zinc-binding (TaPLATZ2) transcription factor are involved in wheat response to saline-alkali stress by directly suppressing the expression of TaHA2/TaSOS3. The knockdown of TaPLATZ2 enhances salt and alkali stress tolerance, while overexpression of TaPLATZ2 leads to salt and alkali stress sensitivity in wheat. In addition, TaWRKY55 directly upregulated the expression of TaPLATZ2 during saline-alkali stress. Through knockdown and overexpression of TaWRKY55 in wheat, TaWRKY55 was shown to negatively modulate salt and alkali stress tolerance. Genetic analyses confirmed that TaPLATZ2 functions downstream of TaWRKY55 in response to salt and alkaline stresses. These findings provide a TaWRKY55-TaPLATZ2-TaHA2/TaSOS3 regulatory module that regulates wheat responses to saline-alkali stress.

Identification and Functional Characterization of Abiotic Stress Tolerance-Related PLATZ Transcription Factor Family in Barley (Hordeum vulgare L.).

Plant AT-rich sequence and zinc-binding proteins (PLATZs) are a novel category of plant-specific transcription factors involved in growth, development, and abiotic stress responses. However, the PLATZ gene family has not been identified in barley. In this study, a total of 11 HvPLATZs were identified in barley, and they were unevenly distributed on five of the seven chromosomes. The phylogenetic tree, incorporating PLATZs from Arabidopsis, rice, maize, wheat, and barley, could be classified into six clusters, in which HvPLATZs are absent in Cluster VI. HvPLATZs exhibited conserved motif arrangements with a characteristic PLATZ domain. Two segmental duplication events were observed among HvPLATZs. All HvPLATZs were core genes present in 20 genotypes of the barley pan-genome. The HvPLATZ5 coding sequences were conserved among 20 barley genotypes, whereas HvPLATZ4/9/10 exhibited synonymous single nucleotide polymorphisms (SNPs); the remaining ones showed nonsynonymous variations. The expression of HvPLATZ2/3/8 was ubiquitous in various tissues, whereas HvPLATZ7 appeared transcriptionally silent; the remaining genes displayed tissue-specific expression. The expression of HvPLATZs was modulated by salt stress, potassium deficiency, and osmotic stress, with response patterns being time-, tissue-, and stress type-dependent. The heterologous expression of HvPLATZ3/5/6/8/9/10/11 in yeast enhanced tolerance to salt and osmotic stress, whereas the expression of HvPLATZ2 compromised tolerance. These results advance our comprehension and facilitate further functional characterization of HvPLATZs.

Unveiling the multifaceted interactions of antitumor drug mitoxantrone with ct-DNA through biophysical and in silico studies

Mitoxantrone, an anthraquinone derivative, is a widely used anticancer drug with its well-known ability to engage in complex interactions with DNA. Although known for its intercalating ability, the enigma surrounding its binding modes with DNA persists. The existing corpus of literature primarily focuses on mitoxantrone-DNA interactions with short DNA sequences, thereby yielding insights into its interactive nature is limited to this specific sequence. This study aims to elucidate the diverse modes with which mitoxantrone interacts with calf thymus DNA using a combination of spectroscopy, calorimetry and in silico studies. The findings from spectroscopic, calorimetric and molecular dynamic results in correlation with existing literature, unveil a fascinating narrative: mitoxantrone intercalates at lower concentrations but promotes condensation at higher concentrations. Although intercalation with side chains positioned in the minor/major groove is the major binding mode in GC-rich sequences, molecular modelling studies hint at an alternative binding mode in AT-rich sequences where it exclusively displays pure electrostatic interaction. These findings underscore the pivotal role of both drug structure and base sequence in dictating binding mode and affinity. Such insights not only deepen the understanding of structure-activity relationships but also hold promise for guiding future drug design strategies.

Dissection of core promoter syntax through single nucleotide resolution modeling of transcription initiation.

How the DNA sequence of cis-regulatory elements encode transcription initiation patterns remains poorly understood. Here we introduce CLIPNET, a deep learning model trained on population-scale PRO-cap data that predicts the position and quantity of transcription initiation with single nucleotide resolution from DNA sequence more accurately than existing approaches. Interpretation of CLIPNET revealed a complex regulatory syntax consisting of DNA-protein interactions in five major positions between -200 and +50 bp relative to the transcription start site, as well as more subtle positional preferences among transcriptional activators. Transcriptional activator and core promoter motifs work non-additively to encode distinct aspects of initiation, with the former driving initiation quantity and the latter initiation position. We identified core promoter motifs that explain initiation patterns in the majority of promoters and enhancers, including DPR motifs and AT-rich TBP binding sequences in TATA-less promoters. Our results provide insights into the sequence architecture governing transcription initiation.

Utility of special AT-rich sequence-binding protein 2 (SATB2) immunohistochemistry as a marker for secondary perianal paget disease.

A panel-based approach using immunohistochemistry (IHC) is currently used for subtyping perianal Paget disease (PPD) in the absence of a synchronous neoplasm. Special AT-rich Sequence Binding Protein 2 (SATB2) has been established as a sensitive and specific marker for lower gastrointestinal tract carcinomas. We evaluated its performance as a marker of secondary PPD. A panel of IHCs including CK7, CK20, GCDFP-15, CDX2, and SATB2 were performed on fifteen cases of PPD (identified between 1991-2001) and seven cases of primary vulvar Paget disease with perianal involvement. Eight cases (53%) were classified as secondary PPD based on the presence of a synchronous (n = 7) or a metachronous neoplasm (n = 1). There was no differential staining for CK7 (positive in 7/7 primary vs. 7/8 secondary PPD; P = 1.00) and CK20 (positive in 4/7 primary vs. 8/8 secondary PPD; P = .08). GCDFP-15 was positive in 5/7 cases of primary PPD while negative in all cases of secondary PPD (P = .01). CDX2 was positive in all cases of secondary PPD (P = .001) while SATB2 was positive in 7/8 cases of secondary PPD (P = .01). Both CDX2 and SATB2 were positive in 1/7 cases of primary PPD. The addition of an IHC panel in conjunction with clinical/imaging findings can help definitively classify PPD as either primary or secondary in most cases. We show that SATB2 has comparable performance to CDX2 and can be a helpful additional tool.

Binding to the Other Side: The AT-Hook DNA-Binding Domain Allows Nuclear Factors to Exploit the DNA Minor Groove.

The "AT-hook" is a peculiar DNA-binding domain that interacts with DNA in the minor groove in correspondence to AT-rich sequences. This domain has been first described in the HMGA protein family of architectural factors and later in various transcription factors and chromatin proteins, often in association with major groove DNA-binding domains. In this review, using a literature search, we identified about one hundred AT-hook-containing proteins, mainly chromatin proteins and transcription factors. After considering the prototypes of AT-hook-containing proteins, the HMGA family, we review those that have been studied in more detail and that have been involved in various pathologies with a particular focus on cancer. This review shows that the AT-hook is a domain that gives proteins not only the ability to interact with DNA but also with RNA and proteins. This domain can have enzymatic activity and can influence the activity of the major groove DNA-binding domain and chromatin docking modules when present, and its activity can be modulated by post-translational modifications. Future research on the function of AT-hook-containing proteins will allow us to better decipher their function and contribution to the different pathologies and to eventually uncover their mutual influences.

Oncogenic Role of SATB2 In Vitro: Regulator of Pluripotency, Self-Renewal, and Epithelial-Mesenchymal Transition in Prostate Cancer.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer and assessed whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency-maintaining transcription factors (cMYC, OCT4, SOX2, KLF4, and NANOG), CADHERIN switch, and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of BCL-2, BSP, NANOG, MYC, XIAP, KLF4, and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival, and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-CADHERIN and inhibited the expression of N-CADHERIN, SNAIL, SLUG, and ZEB1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

Posttranscriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell- and tissue-dependent.

The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the 2 transgenes.

GhiPLATZ17 and GhiPLATZ22, zinc-dependent DNA-binding transcription factors, promote salt tolerance in upland cotton.

The utilization of transcriptome analysis, functional validation, VIGS, and DAB techniques have provided evidence that GhiPLATZ17 and GhiPLATZ22 play a pivotal role in improving the salt tolerance of upland cotton. PLATZ (Plant AT-rich sequences and zinc-binding proteins) are known to be key regulators in plant growth, development, and response to salt stress. In this study, we comprehensively analyzed the PLATZ family in ten cotton species in response to salinity stress. Gossypium herbaceum boasts 25 distinct PLATZ genes, paralleled by 24 in G. raimondii, 25 in G. arboreum, 46 in G. hirsutum, 48 in G. barbadense, 43 in G. tomentosum, 67 in G. mustelinum, 60 in G. darwinii, 46 in G. ekmanianum, and a total of 53 PLATZ genes attributed to G. stephensii. The PLATZ gene family shed light on the hybridization and allopolyploidy events that occurred during the evolutionary history of allotetraploid cotton. Ka/Ks analysis suggested that the PLATZ gene family underwent intense purifying selection during cotton evolution. Analysis of synteny and gene collinearity revealed a complex pattern of segmental and dispersed duplication events to expand PLATZ genes in cotton. Cis-acting elements and gene expressions revealed that GhiPLATZ exhibited salt stress resistance. Transcriptome analysis, functional validation, virus-induced gene silencing (VIGS), and diaminobenzidine staining (DAB) demonstrated that GhiPLATZ17 and GhiPLATZ22 enhance salt tolerance in upland cotton. The study can potentially advance our understanding of identifying salt-resistant genes in cotton.

Systematic identification of cargo-mobilizing genetic elements reveals new dimensions of eukaryotic diversity.

Cargo-mobilizing mobile elements (CMEs) are genetic entities that faithfully transpose diverse protein coding sequences. Although common in bacteria, we know little about eukaryotic CMEs because no appropriate tools exist for their annotation. For example, Starships are giant fungal CMEs whose functions are largely unknown because they require time-intensive manual curation. To address this knowledge gap, we developed starfish, a computational workflow for high-throughput eukaryotic CME annotation. We applied starfish to 2 899 genomes of 1 649 fungal species and found that starfish recovers known Starships with 95% combined precision and recall while expanding the number of annotated elements ten-fold. Extant Starship diversity is partitioned into 11 families that differ in their enrichment patterns across fungal classes. Starship cargo changes rapidly such that elements from the same family differ substantially in their functional repertoires, which are predicted to contribute to diverse biological processes such as metabolism. Many elements have convergently evolved to insert into 5S rDNA and AT-rich sequence while others integrate into random locations, revealing both specialist and generalist strategies for persistence. Our work establishes a framework for advancing mobile element biology and provides the means to investigate an emerging dimension of eukaryotic genetic diversity, that of genomes within genomes.

Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production

The manufacturing of mRNA vaccines relies on cell-free based systems that are easily scalable and flexible compared with the traditional vaccine manufacturing processes. Typically, standard processes yield 2 to 5 g L−1 of mRNA, with recent process optimisations increasing yields to 12 g L−1. However, increasing yields can lead to an increase in the production of unwanted by-products, namely dsRNA. It is therefore imperative to reduce dsRNA to residual levels in order to avoid intensive purification steps, enabling cost-effective manufacturing processes. In this work, we exploit sequence modifications downstream of the T7 RNA polymerase promoter to increase mRNA yields whilst simultaneously minimising dsRNA. In particular, transcription performance was optimised by modifying the sequence downstream of the T7 promoter with additional AT-rich sequences. We have identified variants that were able to produce higher amounts of mRNA (up to 14 g L−1) in 45 min of reaction. These variants exhibited up to a 30% reduction in dsRNA byproduct levels compared to a wildtype T7 promoter, and have similar EGFP protein expression. The results show that optimising the non-coding regions can have an impact on mRNA production yields and quality, reducing overall manufacturing costs.

Abstract 1259: HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration

Abstract Cancer cells undergo epigenetic reprogramming to co-opt stem cell networks and drive tumor progression, although how this occurs remains unclear. The High Mobility Group A1 (HMGA1) gene encodes an architectural transcription factor that binds to DNA at AT-rich sequences where it recruits chromatin complexes to modulate gene expression. HMGA1 is highly expressed during embryogenesis and in adult stem cells, but silenced in differentiated cells. HMGA1 becomes re-expressed in aggressive cancers where high levels portend adverse outcomes. In colorectal cancer (CRC), HMGA1 is among the genes most highly overexpressed compared to normal colon epithelium. We previously discovered that HMGA1 is required for oncogenic phenotypes in CRC cell lines by inducing genes involved in an epithelial-mesenchymal transition. Hmga1 transgenic mice also develop aberrant proliferation and polyposis of small intestinal epithelium where Hmga1 amplifies Wnt signals to enhance self-renewal. To determine how HMGA1 functions in colon epithelial homeostasis and tumorigenesis, we examined mouse models with overexpression or deficiency of Hmga1. Here, we uncover a previously unknown role for HMGA1 as an epigenetic gatekeeper of Wnt signals in colon epithelial cells, both during tumorigenesis driven by mutant APC or stress-induced tissue repair. Strikingly, loss of just a single Hmga1 allele within the colon epithelium disrupts tumorigenesis while prolonging survival in two models of colon tumorigenesis driven by APC mutation, including CDX2P-CreERTCApcfl/fl and APCMin mice inoculated with pro-carcinogenic, enterotoxigenic Bacteroides fragilis. Single cell RNA sequencing in Apc mutant crypt epithelial cells reveals that intact Hmga1 is required for expansion in the Lgr5+ stem cell pool, deep secretory cells, and tuft cells during tumorigenesis. Hmga1 deficiency impacts the stem cell pool the greatest, decreasing Lgr5+ stem cells with skewing to more proliferative, transit amplifying-like cells. Single cell transcriptomic analyses together with assays of single cell chromatin accessibility demonstrate that Hmga1 maintains accessible chromatin to activate gene loci involved in Wnt signals, including Ascl2, a stem cell master regulator, Wnt agonist receptors, and Wnt downstream effectors. Surprisingly, either depletion or overexpression of Hmga1 has no impact on colon epithelial regeneration under homeostatic conditions, although Hmga1 overexpression enhances tissue repair and survival following injury induced by inflammation or irradiation. Together, our results establish HMGA1 as an epigenetic gatekeeper of Wnt signals in colon stem cells during APC-driven tumorigenesis or injury, but not steady state homeostasis, highlighting its role as a promising therapeutic target for both cancer and regenerative medicine. Citation Format: Linda M. S. Resar, Li Z. Luo, Lingling Xian, Iliana Herrera, Shaoguang Wu, Jung-Hyun Kim, Ying Feng, Julian Calderon-Espinosa, Eric R. Fearon, Cynthia Sears. HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1259.

Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori.

Adaptation to variations in pH is crucial for the ability of Helicobacter pylori to persist in the human stomach. The acid responsive two-component system ArsRS, constitutes the global regulon that responds to acidic conditions, but molecular details of how transcription is affected by the ArsR response regulator remains poorly understood. Using a combination of DNA-binding studies, in vitro transcription assays, and H. pylori mutants, we demonstrate that phosphorylated ArsR (ArsR-P) forms an active protein complex that binds DNA with high specificity in order to affect transcription. Our data showed that DNA topology is key for DNA binding. We found that AT-rich DNA sequences direct ArsR-P to specific sites and that DNA-bending proteins are important for the effect of ArsR-P on transcription regulation. The repression of sabA transcription is mediated by ArsR-P with the support of Hup and is affected by simple sequence repeats located upstream of the sabA promoter. Here stochastic events clearly contribute to the fine-tuning of pH-dependent gene regulation. Our results reveal important molecular aspects for how ArsR-P acts to repress transcription in response to acidic conditions. Such transcriptional control likely mediates shifts in bacterial positioning in the gastric mucus layer.

Genome-Wide Analysis of the PLATZ Gene Family and Identification of Seed Development-Related Genes in Flax [Linum usitatissimum L.

ABSTRACT The plant AT-rich sequence and zinc-binding proteins (PLATZ) are a new class of zinc-dependent DNA-binding proteins that play important roles in plant growth and development. But it has not been systematically identified in flax (Linum usitatissimum L.), as an important seed crop with high nutritional value. In this study, 28 LuPLATZ proteins were identified from flax and divided into six phylogenetic groups. Fragment duplication was an important driving factor in the evolution of LuPLATZ genes. The RNA-seq results showed that the LuPLATZ genes had different expression patterns. Quantitative analysis of 16 LuPLATZ genes was performed, and the qRT-PCR results were consistent with the RNA-seq results. The expression of LuPLATZ6, LuPLATZ23, and LuPLATZ27 increased significantly during seed development. The observed change in the expression pattern suggests that these three genes could have significant regulatory functions during seed formation. These three LuPLATZ proteins were localized to the nucleus, suggesting that they could be conventional transcription factors. LuPLATZ23 and LuPLATZ27 were co-expressed with LuNAC1 and LuNAC75, suggesting an interactive relationship. This study aims to offer a theoretical foundation for high-yielding molecular breeding of flax.

Reduced representative methylome profiling of cell-free DNA for breast cancer detection.

Whole-genome methylation sequencing of cfDNA is not cost-effective for tumor detection. Here, we introduce reduced representative methylome profiling (RRMP), which employs restriction enzyme for depletion of AT-rich sequence to achieve enrichment and deep sequencing of CG-rich sequences. We first verified the ability of RRMP to enrich CG-rich sequences using tumor cell genomic DNA and analyzed differential methylation regions between tumor cells and normal whole blood cells. We then analyzed cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals to detect breast cancer by building machine learning models. RRMP captured 81.9% CpG islands and 75.2% gene promoters when sequenced to 10 billion base pairs, with an enrichment efficiency being comparable to RRBS. RRMP allowed us to assess DNA methylation changes between tumor cells and whole blood cells. Applying our approach to cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals, we developed machine learning models that could discriminate between breast cancer and non-breast cancer controls (AUC = 0.85), suggesting possibilities for truly non-invasive cancer detection. We developed a new method to achieve reduced representative methylome profiling of cell-free DNA for tumor detection.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Astrocytes of the Anterior Commissure Regulate the Axon Guidance Pathways of Newly Generated Neocortical Neurons in the Opossum Monodelphis domestica.

In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12-P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent.

Triptolide inhibits esophageal squamous cell carcinoma progression by regulating the circNOX4/miR-153-3p/SATB1 signaling pathway.

To explore the role and mechanism of triptolide in regulating esophageal squamous cell carcinoma (ESCC) progression by mediating the circular RNA (circRNA)-related pathway. The expression levels of circNOX4, miR-153-3p and special AT-rich sequence binding protein-1 (SATB1) were measured by qRT-PCR. Cell proliferation was confirmed by cell counting kit-8 assay and colony formation assay. Flow cytometry was employed to measure cell apoptosis and cell cycle process. Moreover, cell migration and invasion were detected using transwell assay. The protein levels of epithelial-mesenchymal transformation markers and SATB1 were determined by western blot analysis. Furthermore, dual-luciferase reporter assay and RIP assay were performed to confirm the interaction between miR-153-3p and circNOX4 or SATB1. Xenograft tumor models were built to verify the effects of triptolide and circNOX4 on ESCC tumor growth. CircNOX4 was highly expressed in ESCC tissues and cells, and its expression could be reduced by triptolide. Triptolide could inhibit ESCC proliferation, cell cycle process, migration, invasion, EMT process, and promote apoptosis, while these effects were reversed by circNOX4 overexpression. MiR-153-3p could be sponged by circNOX4, and the promotion effect of circNOX4 on the progression of triptolide-treated ESCC cells was abolished by miR-153-3p overexpression. SATB1 was a target of miR-153-3p. Also, SATB1 knockdown reversed the enhancing effect of miR-153-3p inhibitor on the progression of triptolide-treated ESCC cells. Triptolide reduced ESCC tumor growth by regulating the circNOX4/miR-153-3p/SATB1 axis. Triptolide could hinder ESCC progression, which was mainly achieved by regulating the circNOX4/miR-153-3p/SATB1 axis.

Effect of structural variation in the promoter region of RsMYB1.1 on the skin color of radish taproot.

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1.1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1.1. Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion- or deletion-based GWAS revealed that one large AT-rich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1.1 in the nR radishes (RsMYB1.1nR), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1.1nR in the nR radishes, the inactive RsMYB1.1nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1.1nR, resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS, RsDFR, and RsANS, in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish.