Wide-Scale Analysis of Human Functional Transcription Factor Binding Reveals a Strong Bias towards the Transcription Start Site

We introduce a novel method to screen the promoters of a set of genes with shared biological function, against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. The gene sets were obtained from the functional Gene Ontology (GO) classification; for each set and motif we optimized the sequence similarity score threshold, independently for every location window (measured with respect to the TSS), taking into account the location dependent nucleotide heterogeneity along the promoters of the target genes. We performed a high throughput analysis, searching the promoters (from 200bp downstream to 1000bp upstream the TSS), of more than 8000 human and 23,000 mouse genes, for 134 functional Gene Ontology classes and for 412 known DNA motifs. When combined with binding site and location conservation between human and mouse, the method identifies with high probability functional binding sites that regulate groups of biologically related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were put to several experimental tests. By allowing a "flexible" threshold and combining our functional class and location specific search method with conservation between human and mouse, we are able to identify reliably functional TF binding sites. This is an essential step towards constructing regulatory networks and elucidating the design principles that govern transcriptional regulation of expression. The promoter region proximal to the TSS appears to be of central importance for regulation of transcription in human and mouse, just as it is in bacteria and yeast.Comment: 31 pages, including Supplementary Information and figure

Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation.

Cancer-associated fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a stress-responsive transcriptional repressor, is down-modulated in skin cancer stromal cells, and Atf3 knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of IL6 induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous cancer-stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to cancer- and stroma-focused intervention

Mechanisms and models of somatic cell reprogramming

Whitehead Institute for Biomedical Research (Jerome and Florence Brill Graduate Student Fellowship)National Institutes of Health (U.S.) (US NIH grant RO1-CA087869)National Institutes of Health (U.S.) (US NIH grant R37-CA084198)National Science Foundation (U.S.) (NSF Graduate Research Fellowship)National Institutes of Health (U.S.) ((NIH) Kirschstein National Research Service Award,1 F32 GM099153-01A1)Vertex Pharmaceuticals Incorporated (Vertex Scholar

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways

DNA demethylation pathways: Additional players and regulators.

DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments

Generation of integration-free neural progenitor cells from cells in human urine

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.postprin

SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors

Cancer stem cells display extremely large evolvability alternating plastic and rigid networks as a potential mechanism Network models, novel therapeutic target strategies, and the contributions of hypoxia, inflammation and cellular senescence

Cancer is increasingly perceived as a systems-level, network phenomenon. The major trend of malignant transformation can be described as a two-phase process, where an initial increase of network plasticity is followed by a decrease of plasticity at late stages of tumor development. The fluctuating intensity of stress factors, like hypoxia, inflammation and the either cooperative or hostile interactions of tumor inter-cellular networks, all increase the adaptation potential of cancer cells. This may lead to the bypass of cellular senescence, and to the development of cancer stem cells. We propose that the central tenet of cancer stem cell definition lies exactly in the indefinability of cancer stem cells. Actual properties of cancer stem cells depend on the individual "stress-history" of the given tumor. Cancer stem cells are characterized by an extremely large evolvability (i.e. a capacity to generate heritable phenotypic variation), which corresponds well with the defining hallmarks of cancer stem cells: the possession of the capacity to self-renew and to repeatedly re-build the heterogeneous lineages of cancer cells that comprise a tumor in new environments. Cancer stem cells represent a cell population, which is adapted to adapt. We argue that the high evolvability of cancer stem cells is helped by their repeated transitions between plastic (proliferative, symmetrically dividing) and rigid (quiescent, asymmetrically dividing, often more invasive) phenotypes having plastic and rigid networks. Thus, cancer stem cells reverse and replay cancer development multiple times. We describe network models potentially explaining cancer stem cell-like behavior. Finally, we propose novel strategies including combination therapies and multi-target drugs to overcome the Nietzschean dilemma of cancer stem cell targeting: "what does not kill me makes me stronger"

Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification