Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes.

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens.

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

The TetR-type MfsR protein of the integrative and conjugative element (ICE) ICEclc controls both a putative efflux system and initiation of ICE transfer.

Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR.

Lack of Smad3 signaling leads to impaired skeletal muscle regeneration.

Smad3 is a key intracellular signaling mediator for both transforming growth factor-β and myostatin, two major regulators of skeletal muscle growth. Previous published work has revealed pronounced muscle atrophy together with impaired satellite cell functionality in Smad3-null muscles. In the present study, we have further validated a role for Smad3 signaling in skeletal muscle regeneration. Here, we show that Smad3-null mice had incomplete recovery of muscle weight and myofiber size after muscle injury. Histological/immunohistochemical analysis suggested impaired inflammatory response and reduced number of activated myoblasts during the early stages of muscle regeneration in the tibialis anterior muscle of Smad3-null mice. Nascent myofibers formed after muscle injury were also reduced in number. Moreover, Smad3-null regenerated muscle had decreased oxidative enzyme activity and impaired mitochondrial biogenesis, evident by the downregulation of the gene encoding mitochondrial transcription factor A, a master regulator of mitochondrial biogenesis. Consistent with known Smad3 function, reduced fibrotic tissue formation was also seen in regenerated Smad3-null muscle. In conclusion, Smad3 deficiency leads to impaired muscle regeneration, which underscores an essential role of Smad3 in postnatal myogenesis. Given the negative role of myostatin during muscle regeneration, the increased expression of myostatin observed in Smad3-null muscle may contribute to the regeneration defects.

Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA.

Clustering of alphavbeta3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC(1) astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCalpha implicated PKCalpha and RhoA activation in these events. Therefore, combined interaction of the astrocyte alphavbeta3-integrin-syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCalpha- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1-integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Bacterial transcriptional regulators for degradation pathways of aromatic compounds.

Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways.

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

LRF-mediated Dll4 repression in erythroblasts is necessary for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.

Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus.

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.