A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes.

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

Transcription Factor AP4 Mediates Cell Fate Decisions: To Divide, Age, or Die.

Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) is a basic-helix-loop-helix-leucine-zipper transcription factor that was first identified as a protein bound to SV40 promoters more than 30 years ago. Almost 15 years later, AP4 was characterized as a target of the c-Myc transcription factor, which is the product of a prototypic oncogene that is activated in the majority of tumors. Interestingly, AP4 seems to represent a central hub downstream of c-Myc and N-Myc that mediates some of their functions, such as proliferation and epithelial-mesenchymal transition (EMT). Elevated AP4 expression is associated with progression of cancer and poor patient prognosis in multiple tumor types. Deletion of AP4 in mice points to roles of AP4 in the control of stemness, tumor initiation and adaptive immunity. Interestingly, ex vivo AP4 inactivation results in increased DNA damage, senescence, and apoptosis, which may be caused by defective cell cycle progression. Here, we will summarize the roles of AP4 as a transcriptional repressor and activator of target genes and the contribution of protein and non-coding RNAs encoded by these genes, in regulating the above mentioned processes. In addition, proteins interacting with or regulating AP4 and the cellular signaling pathways altered after AP4 dysregulation in tumor cells will be discussed.

Intron Retained, Transcript Detained: Intron Retention as a Hallmark of the Quiescent Satellite Cell State.

Molecular signatures defining quiescence in muscle satellite cells (mSCs) remain enigmatic. In this issue of Developmental Cell, Yue et al. adapted an in vivo fixation approach to isolate dormant mSCs from healthy muscle. Characterizing the transcriptome from these cells, they identified intron retention as a novel hallmark of mSC quiescence.

Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated ß-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells.

Effects of three-layered nanodisk size on cell detection sensitivity of plasmon resonance biosensors.

The double resonance plasmonic biosensors based on Au nanodisks (NDs) with a thin SiO2 spacer between the top and bottom Au layers were employed to detect MCF-7 breast cancer cells. The hybridized modes between the localized surface plasmon resonance of Au NDs and the gap coupling resonance of NDs with the Au film underneath have been observed. These multiple metallic layer NDs exhibit higher sensitivity than the common single metallic layer NDs. The extinction spectra showed double resonance bands that could be tailored by varying the ND size. Three sizes of multiple layer NDs ranging from 60 to 200 nm diameter (dia.) were generated and their refractive sensitivity to the surrounding media were analyzed for cell detection. Nanodisks with 120 dia. showed the highest refractive sensitivity up to 230 nm/refractive index unit. These sensors could be used to detect a broad range of MCF-7 cells from a low cell concentration down of 1.0×10(3)cells/ml up to a high cell concentration of 1.7×10(7) cells/ml.

Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth.

BACKGROUND: Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection. RESULTS: In this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo. CONCLUSIONS: We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

Luminescent ruthenium(II) complex bearing bipyridine and N-heterocyclic carbene-based C∧N∧C pincer ligand for live-cell imaging of endocytosis.

Luminescent ruthenium(II)-cyanide complex with N-heterocyclic carbene pincer ligand C(∧)N(∧)C = 2,6-bis(1-butylimidazol-2-ylidene)pyridine and 2,2'-bipyridine (bpy) shows minimal cytotoxicity to both human breast carcinoma cell (MCF-7) and human retinal pigmented epithelium cell (RPE) in a wide range of concentration (0.1-500 µM), and can be used for the luminescent imaging of endocytosis of the complex in these cells.

Effects of nanoparticle size and cell type on high sensitivity cell detection using a localized surface plasmon resonance biosensor.

A localized surface plasmon resonance (LSPR) effect was used to distinguish cell concentration on ordered arrays of Au nanoparticles (NPs) on glass substrates. Human-derived retinal pigment epithelial RPE-1 cells with flatter bodies and higher confluency were compared with breast cancer MCF-7 cells. Nanosphere lithography was used to form Au NPs with average diameters of 500 and 60 nm in order to compare cell detection range, resonance peak shift, and cell concentration sensitivity. A larger cell concentration range was detected on the larger 500 nm Au NPs compared to 60 nm Au NPs (8.56 × 10(3)-1.09 × 10(6) vs. 3.43 × 10(4)-2.73 × 10(5)cells/ml). Resonance peak shift could distinguish RPE-1 from MCF-7 cells on both Au NPs. RPE-1 cells consistently displayed larger resonance peak shifts compared to MCF-7 cells until the detection became saturated at higher concentration. For both types of cells, higher concentration sensitivity in the range of ~10(4)-10(6)cells/ml was observed on 500 nm compared to 60 nm Au NPs. Our results show that cells on Au NPs can be detected in a large range and at low concentration. Optimal cell sensing can be achieved by altering the dimensions of Au NPs according to different cell characteristics and concentrations.

Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli.

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.