Disruption of the Mammalian Ccr4-Not Complex Contributes to Transcription-Mediated Genome Instability.

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.

Neighbor-specific gene expression revealed from physically interacting cells during mouse embryonic development.

Development of multicellular organisms is orchestrated by persistent cell-cell communication between neighboring partners. Direct interaction between different cell types can induce molecular signals that dictate lineage specification and cell fate decisions. Current single-cell RNA-seq technology cannot adequately analyze cell-cell contact-dependent gene expression, mainly due to the loss of spatial information. To overcome this obstacle and resolve cell-cell contact-specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PIC-seq) and assessed them alongside similar single-cell transcriptomes derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of the PIC-seq data identified gene expression signatures that were dependent on the presence of specific neighboring cell types. Our computational predictions, validated experimentally, demonstrated that neural progenitor (NP) cells upregulate Lhx5 and Nkx2-1 genes, when exclusively interacting with definitive endoderm (DE) cells. Moreover, there was a reciprocal impact on the transcriptome of DE cells, as they tend to upregulate Rax and Gsc when in contact with NP cells. Using individual cell transcriptome data, we formulated a means of computationally predicting the impact of one cell type on the transcriptome of its neighboring cell types. We have further developed a distinctive spatial-t-distributed stochastic neighboring embedding to display the pseudospatial distribution of cells in a 2-dimensional space. In summary, we describe an innovative approach to study contact-specific gene regulation during embryogenesis.

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. METHODS: Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRß), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. RESULTS: We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5-6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRß, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRß+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. CONCLUSION: We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

Differentially mutated subnetworks discovery.

PROBLEM: We study the problem of identifying differentially mutated subnetworks of a large gene-gene interaction network, that is, subnetworks that display a significant difference in mutation frequency in two sets of cancer samples. We formally define the associated computational problem and show that the problem is NP-hard. ALGORITHM: We propose a novel and efficient algorithm, called DAMOKLE, to identify differentially mutated subnetworks given genome-wide mutation data for two sets of cancer samples. We prove that DAMOKLE identifies subnetworks with statistically significant difference in mutation frequency when the data comes from a reasonable generative model, provided enough samples are available. EXPERIMENTAL RESULTS: We test DAMOKLE on simulated and real data, showing that DAMOKLE does indeed find subnetworks with significant differences in mutation frequency and that it provides novel insights into the molecular mechanisms of the disease not revealed by standard methods.

Single Cell RNA-Sequencing for the Study of Atherosclerosis.

Atherosclerosis is a major cause of coronary artery disease and stroke. A massive and new type of data has finally arrived in the field of atherosclerosis: single cell RNA sequencing (scRNAseq). Recently, scRNAseq has been successfully applied to the study of atherosclerosis to identify previously uncharacterized cell populations. scRNAseq is an effective approach to evaluate heterogeneous cell populations by measuring the transcriptomic profiles at the single cell level. Besides the studies of atherosclerosis, scRNAseq is being employed in various areas of biology, including cancer research and organ development. In order to analyze these new massive datasets, various analytic approaches have been developed. This review aims to enhance the understanding of this new technology by exploring how the single cell transcriptome has been applied to the study of atherosclerosis and further discuss potential analysis of using scRNAseq.