Cancer immunotherapy targeting co-inhibitory pathways by checkpoint blockade shows remarkable efficacy in a variety of cancer types. However, only a minority of patients respond to treatment due to the stochastic heterogeneity of tumor microenvironment (TME). Recent advances in single-cell RNA-seq technologies enabled comprehensive characterization of the immune system heterogeneity in tumors but posed computational challenges on integrating and utilizing the massive published datasets to inform immunotherapy. Here, we present Tumor Immune Single Cell Hub (TISCH, http://tisch.comp-genomics.org), a large-scale curated database that integrates single-cell transcriptomic profiles of nearly 2 million cells from 76 high-quality tumor datasets across 27 cancer types. All the data were uniformly processed with a standardized workflow, including quality control, batch effect removal, clustering, cell-type annotation, malignant cell classification, differential expression analysis and functional enrichment analysis. TISCH provides interactive gene expression visualization across multiple datasets at the single-cell level or cluster level, allowing systematic comparison between different cell-types, patients, tissue origins, treatment and response groups, and even different cancer-types. In summary, TISCH provides a user-friendly interface for systematically visualizing, searching and downloading gene expression atlas in the TME from multiple cancer types, enabling fast, flexible and comprehensive exploration of the TME.

GLABRA3 (GL3) encodes a bHLH protein that interacts with the WD repeat protein, TTG1. GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations. However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction. A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 (EGL3). When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background. The double bHLH mutant, gl3 egl3, has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing. Furthermore, the triple bHLH mutant, gl3 egl3 tt8, phenocopies the ttg1 mutation. Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3, interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3. These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.

The positive transcription elongation factor b (P-TEFb) contains cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9). For activating the expression of eukaryotic genes, the histidine-rich sequence in CycT1 binds the heptapeptide repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), whereupon Cdk9 phosphorylates the CTD. We found that alanine-substituted heptapeptide repeats that cannot be phosphorylated also bind CycT1. When placed near transcription units, these CTD analogs block effects of P-TEFb. Remarkably, the transcriptional repressor PIE-1 fromCaenorhabditis elegansbehaves analogously. It binds CycT1 via an alanine-containing heptapeptide repeat and inhibits transcriptional elongation. Thus, our findings reveal a new mechanism by which repressors inhibit eukaryotic transcription.

Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer-related death worldwide. Although epigenetic deregulation is known to be important for tumor progression, the molecular mechanisms in NSCLC remain unclear. Here, we found that G9A (known as EHMT2), a histone methyltransferase responsible for mono- or di-methylation of histone 3 (H3) lysine 9 (K9), is significantly upregulated in NSCLC. Knocking down G9A or pharmacological inhibition of its activity suppressed tumor cell growth, colony formation, invasion and migration. Furthermore, G9A exerts these functions by repressing CASP1 expression. Knocking down CASP1 in G9A-deficient cell restored capacities of tumor cell invasion and migration. Mechanistically, G9A silences the CASP1 promoter activity by increasing H3K9me2 around its promoter. Finally, high expression of G9A or low expression of CASP1 is correlated with poor overall survival in lung adenocarcinoma. Overall, our study uncovers a novel mechanism of G9A promoting tumor cell growth and invasion by silencing CASP1, and implies that G9A may serve as a therapeutic target in treating NSCLC.

DNA methylation is among the most stable epigenetic marks, ensuring tissue-specific gene expression in a heritable manner throughout development. Here we report that differentiated mesodermal somatic cells can confer tissue-specific changes in DNA methylation on epidermal progenitor cells after fusion in stable multinucleate heterokaryons. Myogenic factors alter regulatory regions of genes in keratinocyte cell nuclei, demethylating and activating a muscle-specific gene and methylating and silencing a keratinocyte-specific gene. Because these changes occur in the absence of DNA replication or cell division, they are mediated by an active mechanism. Thus, the capacity to transfer epigenetic changes to other nuclei is not limited to embryonic stem cells and oocytes but is also a property of highly specialized mammalian somatic cells. These results suggest the possibility of directing the reprogramming of readily available postnatal human progenitor cells toward specific tissue cell types.

Phosphorylation of Ser2 of the heptapeptide repeat of the CTD of mammalian pol II by P‐TEFb is associated with productive elongation of transcription of protein‐coding genes. Here, we show that the CTD of pol II transcribing the human U2 snRNA genes is phosphorylated on Ser2 in vivo and that both the CDK9 kinase and cyclin T components of P‐TEFb are required for cotranscriptional recognition of the 3′ box RNA 3′ end processing signal. However, inhibitors of CDK9 do not affect transcription of the U2 genes, indicating that P‐TEFb functions exclusively as an RNA processing factor in expression of these relatively short, intronless genes. We also show that inhibition of CDK9 does not adversely affect either transcription of an intron‐less, replication‐activated histone H2b gene or recognition of the histone gene‐specific U7‐dependent RNA 3′ end formation signal. These results emphasize that the role of P‐TEFb as an activator of transcription elongation can be separated from its role in RNA processing and that neither function is universally required for expression of mammalian pol II‐dependent genes.