Genes to Cells最新文献

The small GTPases RhoA and Cdc42 and their effector proteins play crucial roles in neutrophil chemotaxis. However, endogenous localization and regulation of these proteins have remained largely unknown. Here, we show, using a trichloroacetic acid fixation method, that endogenous RhoA and Cdc42 are preferentially accumulated at the F-actin-rich leading edge (pseudopod) during chemotaxis of human neutrophil-like PLB-985 cells in response to the chemoattractant C5a. Interestingly, the enrichment of RhoA is impaired by knockdown of Cdc42, indicating a positive regulation by Cdc42. Depletion of Cdc42 or RhoA each induces the formation of multiple pseudopods, confirming their significance in cell polarization with an organized actin network at the front. The Rho-associated kinase ROCK is also recruited to the leading edge during chemotaxis in a manner dependent on not only RhoA and Cdc42 but also aPKC, a Cdc42-interacting kinase that can also bind to ROCK. ROCK promotes phosphorylation of the myosin light chain at the front, possibly regulating pseudopod contractility. Knockdown of aPKC suppresses neutrophil chemotaxis by disturbing pseudopod orientation without forming multiple protrusions. An incorrectly oriented pseudopod is also observed in ROCK-depleted cells. Thus, aPKC, as well as RhoA and Cdc42, likely regulates neutrophil chemotaxis partly by recruiting ROCK to the leading edge for correct directionality.

Cis-regulatory elements (cREs) play a crucial role in regulating gene expression and determining cell differentiation and state transitions. To capture the heterogeneous transitions of cell states associated with these processes, detecting cRE activity at the single-cell level is essential. However, current analytical methods can only capture the average behavior of cREs in cell populations, thereby obscuring cell-specific variations. To address this limitation, we proposed an attention-based deep neural network framework that integrates DNA sequences, genomic distances, and single-cell multi-omics data to detect cREs and their activities in individual cells. Our model shows higher accuracy in identifying cREs within single-cell multi-omics data from healthy human peripheral blood mononuclear cells than other existing methods. Furthermore, it clusters cells more precisely based on predicted cRE activities, enabling a finer differentiation of cell states. When applied to publicly available single-cell data from patients with glioma, the model successfully identified tumor-specific SOX2 activity. Additionally, it revealed the heterogeneous activation of the ZEB1 transcription factor, a regulator of epithelial-to-mesenchymal transition-related genes, which conventional methods struggle to detect. Overall, our model is a powerful tool for detecting cRE regulation at the single-cell level, which may contribute to revealing drug resistance mechanisms in cell sub-populations.

Astrocytes, the most prevalent type of glial cells, have been found to play a crucial part in numerous physiological functions. By offering metabolic and structural support, astrocytes are vital for the proper functioning of the brain and regulating information processing and synaptic transmission. Astrocytes located in the medial prefrontal cortex (mPFC) are highly responsive to environmental changes and have been associated with the development of brain disorders. One of the primary mechanisms through which the brain responds to environmental factors is epitranscriptome modification. M6-methyladenosine methylation is the most prevalent internal modification of eukaryotic messenger RNA (mRNA), and it significantly impacts transcript processing and protein synthesis. However, the effects of m6A on astrocyte transcription and function are still not well understood. Our research demonstrates that ALKBH5, an RNA demethylase of m6A found in astrocytes, affects gene expression in the mPFC. These findings suggest that further investigation into the potential role of astrocyte-mediated m6A methylation in the mPFC is warranted.

The dysfunction of the innate immune system is well-described as a clinical characteristic of COVID-19. While several groups have reported human endogenous retroviruses (ERVs) as enhancing factors of immune reactivity, characterization of the COVID-19-specific ERVs has not yet been sufficiently conducted. Here, we revealed the transcriptome profile of more than 500 ERV subfamilies and innate immune response genes in eight different cohorts of platelet, peripheral blood mononuclear cells (PBMCs), lung, frontal cortex of brain, ventral midbrain, pooled human umbilical vein endothelial cells (pHUVECs), placenta, and cardiac microvascular endothelial cells (HCMEC) from COVID-19 patients (total; n = 124) and normal samples (total; n = 53) using publicly available datasets. While upregulation of ERV subfamilies was found in platelets, PBMCs, and placenta, the immune reactivity was confined to only platelets and PBMCs. It is noteworthy that the evolutionary ages of the upregulated ERV subfamilies detected in platelets and PBMCs were younger than other ERV subfamilies, but the tendency was not seen in the upregulated ERV subfamilies in placenta. The results suggest that only evolutionarily young ERVs can function as enhancing factors of the immune reactivity in COVID-19 patients. The finding should be instrumental in understanding the COVID-19 immunopathology.

Tumor development often requires cellular adaptation to a unique, high metabolic state; however, the molecular mechanisms that drive such metabolic changes in TFE3–rearranged renal cell carcinoma (TFE3-RCC) remain poorly understood. TFE3-RCC, a rare subtype of RCC, is defined by the formation of chimeric proteins involving the transcription factor TFE3. In this study, we analyzed cell lines and genetically engineered mice, demonstrating that the expression of the chimeric protein PRCC-TFE3 induced a hypoxia-related signature by transcriptionally upregulating HIF1α and HIF2α. The upregulation of HIF1α by PRCC-TFE3 led to increased cellular ATP production by enhancing glycolysis, which also supplied substrates for the TCA cycle while maintaining mitochondrial oxidative phosphorylation. We crossed TFE3-RCC mouse models with Hif1α and/or Hif2α knockout mice and found that Hif1α, rather than Hif2α, is essential for tumor development in vivo. RNA-seq and metabolomic analyses of the kidney tissues from these mice revealed that ketone body production is inversely correlated with tumor development, whereas de novo lipid synthesis is upregulated through the HIF1α/SREBP1-dependent mechanism in TFE3-RCC. Our data suggest that the coordinated metabolic shift via the PRCC-TFE3/HIF1α/SREBP1 axis is a key mechanism by which PRCC-TFE3 enhances cancer cell metabolism, promoting tumor development in TFE3-RCC.

Single-cell RNA-sequencing (scRNA-seq) is a powerful method to comprehensively overlook gene expression profiles of individual cells in various tissues, providing fundamental datasets for classification of cell types and further functional analyses. Here we adopted scRNA-seq analysis for the zebrafish olfactory sensory neurons which respond to water-borne odorants and pheromones to elicit various behaviors crucial for survival and species preservation. Firstly, a single-cell dissociation procedure of the zebrafish olfactory rosettes was optimized by using cold-active protease, minimizing artifactual neuronal activation. Secondly, various cell types were classified into distinct clusters, based on the expressions of well-defined marker genes. Notably, we validated non-overlapping expressions of different families of olfactory receptors among the clusters of olfactory sensory neurons. Lastly, we succeeded in estimating candidate olfactory receptors responding to a particular odor stimulus by carefully scrutinizing correlated expressions of immediate early genes. Thus, scRNA-seq is a useful measure for the analysis of olfactory sensory neurons not only in classifying functional cell types but also in identifying olfactory receptor genes for given odorants and pheromones.