HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers.

Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

Author Correction: High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

In Fig. 4e of this Article, the labels for 'Control' and 'HFD' were reversed ('Control' should have been labelled blue rather than purple, and 'HFD' should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for 'V' and 'GW' were reversed ('V' should have been labelled blue rather than purple, and 'GW' should have been labelled purple instead of blue). The original figure has been corrected online.

Growth-inhibitory and tumor- suppressive functions of p53 depend on its repression of CD44 expression.

The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44's expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells.

High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

Human iPSC-derived microglia assume a primary microglia-like state after transplantation into the neonatal mouse brain.

Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease.

A molecular wound response program associated with regeneration initiation in planarians.

Planarians are capable of regenerating any missing body part and present an attractive system for molecular investigation of regeneration initiation. The gene activation program that occurs at planarian wounds to coordinate regenerative responses remains unknown. We identified a large set of wound-induced genes during regeneration initiation in planarians. Two waves of wound-induced gene expression occurred in differentiated tissues. The first wave includes conserved immediate early genes. Many second-wave genes encode conserved patterning factors required for proper regeneration. Genes of both classes were generally induced by wounding, indicating that a common initial gene expression program is triggered regardless of missing tissue identity. Planarian regeneration uses a population of regenerative cells (neoblasts), including pluripotent stem cells. A class of wound-induced genes was activated directly within neoblasts, including the Runx transcription factor-encoding runt-1 gene. runt-1 was required for specifying different cell types during regeneration, promoting heterogeneity in neoblasts near wounds. Wound-induced gene expression in neoblasts, including that of runt-1, required SRF (serum response factor) and sos-1. Taken together, these data connect wound sensation to the activation of specific cell type regeneration programs in neoblasts. Most planarian wound-induced genes are conserved across metazoans, and identified genes and mechanisms should be important broadly for understanding wound signaling and regeneration initiation.

Dynamic changes in ORC localization and replication fork progression during tissue differentiation.

BACKGROUND: Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. RESULTS: We exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. CONCLUSIONS: These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.

Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2.

Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyzes trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3, but the significance of this remains unclear. Here, we identify a class of short RNAs, approximately 50-200 nucleotides in length, transcribed from the 5' end of polycomb target genes in primary T cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription, and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis, and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.

MERAV: a tool for comparing gene expression across human tissues and cell types.

The oncogenic transformation of normal cells into malignant, rapidly proliferating cells requires major alterations in cell physiology. For example, the transformed cells remodel their metabolic processes to supply the additional demand for cellular building blocks. We have recently demonstrated essential metabolic processes in tumor progression through the development of a methodological analysis of gene expression. Here, we present the Metabolic gEne RApid Visualizer (MERAV, http://merav.wi.mit.edu), a web-based tool that can query a database comprising ∼4300 microarrays, representing human gene expression in normal tissues, cancer cell lines and primary tumors. MERAV has been designed as a powerful tool for whole genome analysis which offers multiple advantages: one can search many genes in parallel; compare gene expression among different tissue types as well as between normal and cancer cells; download raw data; and generate heatmaps; and finally, use its internal statistical tool. Most importantly, MERAV has been designed as a unique tool for analyzing metabolic processes as it includes matrixes specifically focused on metabolic genes and is linked to the Kyoto Encyclopedia of Genes and Genomes pathway search.

A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells.

Throughout the plant and animal kingdoms specific cell types become polyploid, increasing their DNA content to attain a large cell size. In mammals, megakaryocytes (MKs) become polyploid before fragmenting into platelets. The mammalian trophoblast giant cells (TGCs) exploit their size to form a barrier between the maternal and embryonic tissues. The mechanism of polyploidization has been investigated extensively in Drosophila, in which a modified cell cycle--the endocycle, consisting solely of alternating S and gap phases--produces polyploid tissues. During S phase in the Drosophila endocycle, heterochromatin and specific euchromatic regions are underreplicated and reduced in copy number. Here we investigate the properties of polyploidization in murine MKs and TGCs. We induced differentiation of primary MKs and directly microdissected TGCs from embryonic day 9.5 implantation sites. The copy number across the genome was analyzed by array-based comparative genome hybridization. In striking contrast to Drosophila, the genome was uniformly and integrally duplicated in both MKs and TGCs. This was true even for heterochromatic regions analyzed by quantitative PCR. Underreplication of specific regions in polyploid cells is proposed to be due to a slower S phase, resulting from low expression of S-phase genes, causing failure to duplicate late replicating genomic intervals. We defined the transcriptome of TGCs and found robust expression of S-phase genes. Similarly, S-phase gene expression is not repressed in MKs, providing an explanation for the distinct endoreplication parameters compared with Drosophila. Consistent with TGCs endocycling rather than undergoing endomitosis, they have low expression of M-phase genes.

Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.

Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.

Developmental control of gene copy number by repression of replication initiation and fork progression.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Extensive alternative polyadenylation during zebrafish development.

The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3' UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially increased and improved existing 3' UTR annotations, resulting in confidently identified 3' UTRs for >79% of the annotated protein-coding genes in zebrafish. mRNAs from most zebrafish genes undergo alternative CPA, with those from more than a thousand genes using different dominant 3' UTRs at different stages. These included one of the poly(A) polymerase genes, for which alternative CPA reinforces its repression in the ovary. 3' UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3' UTRs are highly expressed in the ovary, yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3' UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At 2 h post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3' UTRs provide a resource for studying gene regulation during vertebrate development.

Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes.

We investigated the influence of normal cell phenotype on the neoplastic phenotype by comparing tumors derived from two different normal human mammary epithelial cell populations, one of which was isolated using a new culture medium. Transformation of these two cell populations with the same set of genetic elements yielded cells that formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. While one cell type (HMECs) yielded squamous cell carcinomas, the other cell type (BPECs) yielded tumors closely resembling human breast adenocarcinomas. Transformed BPECs gave rise to lung metastases and were up to 10(4)-fold more tumorigenic than transformed HMECs, which are nonmetastatic. Hence, the pre-existing differences between BPECs and HMECs strongly influence the phenotypes of their transformed derivatives.

Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into multiple mesoderm lineages in the course of normal tissue homeostasis or during injury. We have previously shown that MSCs migrate to sites of tumorigenesis, where they become activated by cancer cells to promote metastasis. However, the molecular and phenotypic attributes of the MSC-induced metastatic state of the cancer cells remained undetermined. Here, we show that bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones. We also show that LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, we find that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations. Collectively, our studies highlight a critical role for LOX in cancer metastasis and indicate that the signaling pathways controlling stroma-induced EMT are distinct from pathways regulating the development of cancer stem cells.

A regulatory program for excretory system regeneration in planarians.

Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. Here, we identify a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. We determined that planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, we identified novel nephridia genes. Our results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin.

Lipolysis-stimulated lipoprotein receptor (LSR) is the host receptor for the binary toxin Clostridium difficile transferase (CDT).

Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea and pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for increased morbidity and mortality of CDI, produce the binary actin-ADP ribosylating toxin Clostridium difficile transferase (CDT) in addition to the Rho-glucosylating toxins A and B. CDT depolymerizes the actin cytoskeleton, increases adherence and colonization of Clostridia by induction of microtubule-based cell protrusions and, eventually, causes death of target cells. Using a haploid genetic screen, we identified the lipolysis-stimulated lipoprotein receptor as the membrane receptor for CDT uptake by target cells. Moreover, we show that Clostridium perfringens iota toxin, which is a related binary actin-ADP ribosylating toxin, enters target cells via the lipolysis-stimulated lipoprotein receptor. Identification of the toxin receptors is essential for understanding of the toxin uptake and provides a most valuable basis for antitoxin strategies.