Is there a causal link between PTEN deficient tumors and immunosuppressive tumor microenvironment?

The PTEN tumor suppressor is the second most commonly inactivated gene across cancer types. While it's role in PI3K/AKT and DNA damage pathways are clear, increasing evidences suggest that PTEN may also promote anti-tumor immunity. PTEN-deficient tumors are characterized by (i) reduced levels of cytotoxic T cells, helper T cells and NK cells, (ii) elevated pro-oncogenic inflammatory cytokines like CCL2 and (iii) increased levels of immunosuppressive cells such as MDSCs and Tregs. An intriguing possibility is that link between PTEN and anti-tumor immunity is mediated by the interferon signaling pathway. In this review, we summarize the evidences for the mechanistic link between PTEN deficiency and immunosuppressive tumor microenvironment and the interferon signaling pathway. We further discuss how the link between these pathways can be exploited for development of personalized immunotherapy for patients with PTEN deficient tumors.

Copy number variation and selection during reprogramming to pluripotency.

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

SET8 methyltransferase activity during the DNA double-strand break response is required for recruitment of 53BP1.

DNA double-strand breaks (DSBs) activate a signaling pathway known as the DNA damage response (DDR) which via protein-protein interactions and post-translational modifications recruit signaling proteins, such as 53BP1, to chromatin flanking the lesion. Depletion of the SET8 methyltransferase prevents accumulation of 53BP1 at DSBs; however, this phenotype has been attributed to the role of SET8 in generating H4K20 methylation across the genome, which is required for 53BP1 binding to chromatin, prior to DNA damage. Here, we report that SET8 acts directly at DSBs during the DNA damage response (DDR). SET8 accumulates at DSBs and is enzymatically active at DSBs. Depletion of SET8 just prior to the induction of DNA damage abrogates 53BP1's accumulation at DSBs, suggesting that SET8 acts during DDR. SET8's occupancy at DSBs is regulated by histone deacetylases (HDACs). Finally, SET8 is functionally required for efficient repair of DSBs specifically via the non-homologous end-joining pathway (NHEJ). Our findings reveal that SET8's active role during DDR at DSBs is required for 53BP1's accumulation.

Evolution of chromosome organization driven by selection for reduced gene expression noise.

The distribution of genes on eukaryotic chromosomes is nonrandom, but the reasons behind this are not well understood. The commonly observed clustering of essential genes is a case in point. Here we model and test a new hypothesis. Essential proteins are unusual in that random fluctuations in abundance (noise) can be highly deleterious. We hypothesize that persistently open chromatin domains are sinks for essential genes, as they enable reduced noise by avoidance of transcriptional bursting associated with chromatin remodeling. Simulation of the model captures clustering and correctly predicts that (i) essential gene clusters are associated with low nucleosome occupancy (ii) noise-sensitive nonessential genes cluster with essential genes (iii) nonessential genes of similar knockout fitness are physically linked (iv) genes in domains rich in essential genes have low noise (v) essential genes are rare subtelomerically and (vi) essential gene clusters are preferentially conserved. We conclude that different noise characteristics of different genomic domains favors nonrandom gene positioning. This has implications for gene therapy and understanding transgenic phenotypes.

Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells.

Mutations in human induced pluripotent stem cells (iPSCs) pose a risk for their clinical use due to preferential reprogramming of mutated founder cell and selection of mutations during maintenance of iPSCs in cell culture. It is unknown, however, if mutations in iPSCs are due to stress associated with oncogene expression during reprogramming. We performed whole exome sequencing of human foreskin fibroblasts and their derived iPSCs at two different passages. We found that in vitro passaging contributed 7% to the iPSC coding point mutation load, and ultradeep amplicon sequencing revealed that 19% of the mutations preexist as rare mutations in the parental fibroblasts suggesting that the remaining 74% of the mutations were acquired during cellular reprogramming. Simulation suggests that the mutation intensity during reprogramming is ninefold higher than the background mutation rate in culture. Thus the factor induced reprogramming stress contributes to a significant proportion of the mutation load of iPSCs.

Systemic Influences of Mammary Cancer on Monocytes in Mice.

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer.

Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome.

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

The impact of the nucleosome code on protein-coding sequence evolution in yeast.

Coding sequence evolution was once thought to be the result of selection on optimal protein function alone. Selection can, however, also act at the RNA level, for example, to facilitate rapid translation or ensure correct splicing. Here, we ask whether the way DNA works also imposes constraints on coding sequence evolution. We identify nucleosome positioning as a likely candidate to set up such a DNA-level selective regime and use high-resolution microarray data in yeast to compare the evolution of coding sequence bound to or free from nucleosomes. Controlling for gene expression and intra-gene location, we find a nucleosome-free "linker" sequence to evolve on average 5-6% slower at synonymous sites. A reduced rate of evolution in linker is especially evident at the 5' end of genes, where the effect extends to non-synonymous substitution rates. This is consistent with regular nucleosome architecture in this region being important in the context of gene expression control. As predicted, codons likely to generate a sequence unfavourable to nucleosome formation are enriched in linker sequence. Amino acid content is likewise skewed as a function of nucleosome occupancy. We conclude that selection operating on DNA to maintain correct positioning of nucleosomes impacts codon choice, amino acid choice, and synonymous and non-synonymous rates of evolution in coding sequence. The results support the exclusion model for nucleosome positioning and provide an alternative interpretation for runs of rare codons. As the intimate association of histones and DNA is a universal characteristic of genic sequence in eukaryotes, selection on coding sequence composition imposed by nucleosome positioning should be phylogenetically widespread.

Single-cell RNA sequencing of human breast tumour-infiltrating immune cells reveals a γδ T-cell subtype associated with good clinical outcome.

The association of increased levels of tumour-infiltrating gamma-delta (γδ) T cells with favorable prognosis across many cancer types and their ability to recognize stress antigens in an MHC unrestricted manner has led to an increased interest in exploiting them for cancer immunotherapy. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood γδ T cells from healthy adult donors and from fresh tumour biopsies of breast cancer patients. We identified five γδ T cells subtypes in blood and three subtypes of γδ T cells in breast tumour. These subtypes differed in the expression of genes contributing to effector functions such as antigen presentation, cytotoxicity, and IL17A and IFNγ production. Compared with the blood γδ T cells, the breast tumour-infiltrating γδ T cells were more activated, expressed higher levels of cytotoxic genes, yet were immunosuppressed. One subtype in the breast tumour that was IFNγ-positive had no obvious similarity to any of the subtypes observed in the blood γδ T cell and was the only subtype associated with improved overall survival of breast cancer patients. Taken together, our study has identified markers of subtypes of human blood γδ T cells and uncovered a tumour-infiltrating γδ T cells subtype associated improved overall cancer survival.

Chromatin remodelling is a major source of coexpression of linked genes in yeast.

In diverse organisms, neighbouring genes in the genome tend to be positively coexpressed more than expected by chance. When the similarity of transcription regulation is controlled for, adjacent genes have much higher coexpression rates than unlinked genes, supporting a role for chromatin modelling. Consequently, many incidences of low-to-moderate level coexpression of linked genes might well be spurious rather than an indication of functional coordination. These results have implications for gene therapy and for understanding gene order evolution, suggesting that chromosomal proximity alone is adequate to achieve some level of coexpression.

Spines and neurite branches function as geometric attractors that enhance protein kinase C action.

Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.

scID Uses Discriminant Analysis to Identify Transcriptionally Equivalent Cell Types across Single-Cell RNA-Seq Data with Batch Effect.

The power of single-cell RNA sequencing (scRNA-seq) stems from its ability to uncover cell type-dependent phenotypes, which rests on the accuracy of cell type identification. However, resolving cell types within and, thus, comparison of scRNA-seq data across conditions is challenging owing to technical factors such as sparsity, low number of cells, and batch effect. To address these challenges, we developed scID (Single Cell IDentification), which uses the Fisher's Linear Discriminant Analysis-like framework to identify transcriptionally related cell types between scRNA-seq datasets. We demonstrate the accuracy and performance of scID relative to existing methods on several published datasets. By increasing power to identify transcriptionally similar cell types across datasets with batch effect, scID enhances investigator's ability to integrate and uncover development-, disease-, and perturbation-associated changes in scRNA-seq data.

SET8 localization to chromatin flanking DNA damage is dependent on RNF168 ubiquitin ligase.

The DNA damage response (DDR) associated post-translational modifications recruit chromatin remodelers, signaling proteins such as 53BP1 and repair factors to chromatin flanking DNA double strand breaks (DSBs) to promote its repair. Although localization of both RNF168 ubiquitin ligase and SET8 methyltransferase at DSBs is essential for 53BP1's recruitment to DSBs, it is unclear if they do so via the same pathways. Here we report that RNF168 mediates SET8's recruitment to DSBs. Depletion of cellular pool of ubiquitin through proteasome inhibition abolished RNF168 and SET8's localization to DNA damage. Knockdown of RNF8 or RNF168 abolished SET8's recruitment to DNA damage. Moreover, RNF168 and SET8 form stable complexes in vivo. Based on these results we propose a model in which SET8, which despite being a pan-chromatin binding protein, can accumulate several folds at chromatin flanking DSBs through tethering to other proteins that specifically localize to chromatin regions with specific modifications.

Stratus not altocumulus: a new view of the yeast protein interaction network.

Systems biology approaches can reveal intermediary levels of organization between genotype and phenotype that often underlie biological phenomena such as polygenic effects and protein dispensability. An important conceptualization is the module, which is loosely defined as a cohort of proteins that perform a dedicated cellular task. Based on a computational analysis of limited interaction datasets in the budding yeast Saccharomyces cerevisiae, it has been suggested that the global protein interaction network is segregated such that highly connected proteins, called hubs, tend not to link to each other. Moreover, it has been suggested that hubs fall into two distinct classes: "party" hubs are co-expressed and co-localized with their partners, whereas "date" hubs interact with incoherently expressed and diversely localized partners, and thereby cohere disparate parts of the global network. This structure may be compared with altocumulus clouds, i.e., cotton ball-like structures sparsely connected by thin wisps. However, this organization might reflect a small and/or biased sample set of interactions. In a multi-validated high-confidence (HC) interaction network, assembled from all extant S. cerevisiae interaction data, including recently available proteome-wide interaction data and a large set of reliable literature-derived interactions, we find that hub-hub interactions are not suppressed. In fact, the number of interactions a hub has with other hubs is a good predictor of whether a hub protein is essential or not. We find that date hubs are neither required for network tolerance to node deletion, nor do date hubs have distinct biological attributes compared to other hubs. Date and party hubs do not, for example, evolve at different rates. Our analysis suggests that the organization of global protein interaction network is highly interconnected and hence interdependent, more like the continuous dense aggregations of stratus clouds than the segregated configuration of altocumulus clouds. If the network is configured in a stratus format, cross-talk between proteins is potentially a major source of noise. In turn, control of the activity of the most highly connected proteins may be vital. Indeed, we find that a fluctuation in steady-state levels of the most connected proteins is minimized.

Evolutionary and physiological importance of hub proteins.

It has been claimed that proteins with more interaction partners (hubs) are both physiologically more important (i.e., less dispensable) and, owing to an assumed high density of binding sites, slow evolving. Not all analyses, however, support these results, probably because of biased and less-than reliable global protein interaction data. Here we provide the first examination of these issues using a comprehensive literature-curated dataset of well-substantiated protein interactions in Saccharomyces cerevisiae. Whereas use of less reliable yeast two-hybrid data alone can reject the possibility that local connectivity correlates with measures of dispensability, in higher quality datasets a relatively robust correlation is observed. In contrast, local connectivity does not correlate with the rate of protein evolution even in reliable datasets. This perhaps surprising lack of correlation with evolutionary rate appears in part to arise from the fact that hub proteins do not have a higher density of residues associated with binding. However, hub proteins do have at least one other set of unusual features, namely rapid turnover and regulation, as manifest in high mRNA decay rates and a large number of phosphorylation sites. This, we suggest, is an adaptation to minimize unwanted activation of pathways that might be mediated by adventitious binding to hubs, were they to actively persist longer than required at any given time point. We conclude that hub proteins are more important for cellular growth rate and under tight regulation but are not slow evolving.

Spatial regulation and the rate of signal transduction activation.

Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement.

Depletion of somatic mutations in splicing-associated sequences in cancer genomes.

BACKGROUND: An important goal of cancer genomics is to identify systematically cancer-causing mutations. A common approach is to identify sites with high ratios of non-synonymous to synonymous mutations; however, if synonymous mutations are under purifying selection, this methodology leads to identification of false-positive mutations. Here, using synonymous somatic mutations (SSMs) identified in over 4000 tumours across 15 different cancer types, we sought to test this assumption by focusing on coding regions required for splicing. RESULTS: Exon flanks, which are enriched for sequences required for splicing fidelity, have ~ 17% lower SSM density compared to exonic cores, even after excluding canonical splice sites. While it is impossible to eliminate a mutation bias of unknown cause, multiple lines of evidence support a purifying selection model above a mutational bias explanation. The flank/core difference is not explained by skewed nucleotide content, replication timing, nucleosome occupancy or deficiency in mismatch repair. The depletion is not seen in tumour suppressors, consistent with their role in positive tumour selection, but is otherwise observed in cancer-associated and non-cancer genes, both essential and non-essential. Consistent with a role in splicing modulation, exonic splice enhancers have a lower SSM density before and after controlling for nucleotide composition; moreover, flanks at the 5' end of the exons have significantly lower SSM density than at the 3' end. CONCLUSIONS: These results suggest that the observable mutational spectrum of cancer genomes is not simply a product of various mutational processes and positive selection, but might also be shaped by negative selection.

Human somatic cells deficient for RAD52 are impaired for viral integration and compromised for most aspects of homology-directed repair.

Homology-directed repair (HDR) maintains genomic integrity by eliminating lesions such as DNA double-strand breaks (DSBs), interstrand crosslinks (ICLs) and stalled replication forks and thus a deficiency in HDR is associated with genomic instability and cancer predisposition. The mechanism of HDR is best understood and most rigorously characterized in yeast. The inactivation of the fungal radiation sensitive 52 (RAD52) gene, which has both recombination mediator and single-strand annealing (SSA) activities in vitro, leads to severe HDR defects in vivo. Confusingly, however, the inactivation of murine and chicken RAD52 genes resulted in mouse and chicken cells, respectively, that were largely aphenotypic. To clarify this issue, we have generated RAD52 knockout human cell lines. Human RAD52-null cells retain a significant level of SSA activity demonstrating perforce that additional SSA-like activities must exist in human cells. Moreover, we confirmed that the SSA activity associated with RAD52 is involved in, but not absolutely required for, most HDR subpathways. Specifically, a deficiency in RAD52 impaired the repair of DNA DSBs and intriguingly decreased the random integration of recombinant adeno-associated virus (rAAV). Finally, an analysis of pan-cancer genome data from The Cancer Genome Atlas (TCGA) revealed an association between aberrant levels of RAD52 expression and poor overall survival in multiple cancers. In toto, our work demonstrates that RAD52 contributes to the maintenance of genome stability and tumor suppression in human cells.