Indigenous Peoples globally are part of the nutrition transition. They may be among the most extreme for the extent of dietary change experienced in the last few decades. In this paper, we report survey data from 44 representative communities from 3 large cultural areas of the Canadian Arctic: the Yukon First Nations, Dene/Métis, and Inuit communities. Dietary change was represented in 2 ways: 1) considering the current proportion of traditional food (TF) in contrast to the precontact period (100% TF); and 2) the amount of TF consumed by older vs. younger generations. Total diet, TF, and BMI data from adults were investigated. On days when TF was consumed, there was significantly less (P < 0.01) fat, carbohydrate, and sugar in the diet, and more protein, vitamin A, vitamin D, vitamin E, riboflavin, vitamin B-6, iron, zinc, copper, magnesium, manganese, phosphorus, potassium, and selenium. Vitamin C and folate, provided mainly by fortified food, and fiber were higher (P < 0.01) on days without TF for Inuit. Only 10-36% of energy was derived from TF; adults>> 40 y old consistently consumed more (P < 0.05) TF than those younger. Overall obesity (BMI>> or = 30 kg/m(2)) of Arctic adults exceeded all-Canadian rates. Measures to improve nutrient-dense market food (MF) availability and use are called for, as are ways to maintain or increase TF use.

In normal pregnancy, there is a marked increase in the procoagulant activity in maternal blood characterized by elevation of factors VII, X, VIII, fibrinogen and von Willebrand factor, which is maximal around term. This is associated with an increase in prothrombin fragments (PF1+2) and thrombin-antithrombin complexes. There is a decrease in physiological anticoagulants manifested by a significant reduction in protein S activity and by acquired activated protein C (APC) resistance. The overall fibrinolytic activity is impaired during pregnancy, but returns rapidly to normal following delivery. This is largely due to placental derived plasminogen activator inhibitor type 2 (PAI-2), which is present in substantial quantities during pregnancy. D-dimer, a specific marker of fibrinolysis resulting from breakdown of cross-linked fibrin polymer by plasmin, increases as pregnancy progresses. Overall, there is a 4- to 10-fold increased thrombotic risk throughout gestation and the postpartum period. Local haemostasis at the placental throphoblast level is characterized by increased tissue factor (TF) expression and low expression of the inhibitor TFPI. Microparticles derived from maternal endothelial cells and platelets, and from placental throphoblasts may contribute to the procoagulant effect. Local anticoagulant mechanisms on placental throphoblasts are important for counterbalance of the procoagulant milieu. Disruption of anticoagulant mechanisms, for example, autoantibodies, to annexin V may increase pregnancy complications in patients with antiphospholipid antibodies (APLA).

Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.

We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of "The costimulatory signal during T cell activation" Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient's age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4(+)CD28(+) T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.

Sepsis is often associated with haemostatic changes ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by widespread microvascular thrombosis and subsequent consumption of platelets and coagulation proteins, eventually causing bleeding manifestations. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. The latter, along with the micro-organism and its derivatives are now believed to drive the major changes responsible for massive thrombin formation and fibrin deposition, namely 1) the aberrant expression of the TF by different cells (especially monocytes-macrophages), 2) the impairment of physiological anticoagulant pathways, orchestrated mainly by dysfunctional endothelial cells (ECs) and 3) the suppression of fibrinolysis due to overproduction of plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). The ensuing microvascular thrombosis and ischemia are thought to contribute to tissue injury and multiple organ dysfunction syndrome (MODS). Recent evidence indicates that extracellular nuclear materials released from activated and especially apoptotic or necrotic cells, e.g. High Mobility Group Box-1 (HMGB-1) and histones, are endowed with cell toxicity, proinflammatory and clot-promoting properties and thus, during sepsis, they may represent late mediators that propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenesis of DIC and MODS may have implications for the development of new therapeutic agents potentially useful for the management of severe sepsis.

BACKGROUND

We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT(1)-AA) directed at the AT(1) receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT(1)-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT(1)-AA cause vascular smooth muscle cells (VSMC) to express TF.

RESULTS

IgG from preeclamptic patients containing AT(1)-AA was purified with anti-human IgG columns. AT(1)-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT(1) receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal-related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT(1)-AA specificity by coimmunoprecipitating the AT(1) receptor with AT(1)-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT(1)-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT(1)-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect.

CONCLUSIONS

We conclude that AT(1)-AA and Ang II both stimulate the AT(1) receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT(1)-AA on human cells that could contribute to the pathogenesis of preeclampsia.

Transcription factor (TF) proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations). We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the comparative study of transcriptional regulation, and the work presented here will form the basis for tool and method development for future transcriptional modeling studies.

Cis-regulatory elements (CREs) control gene expression by recruiting transcription factors (TFs) and other DNA binding proteins. We aim to understand how individual nucleotides contribute to the function of CREs. Here we introduce CRE analysis by sequencing (CRE-seq), a high-throughput method for producing and testing large numbers of reporter genes in mammalian cells. We used CRE-seq to assay >1,000 single and double nucleotide mutations in a 52-bp CRE in the Rhodopsin promoter that drives strong and specific expression in mammalian photoreceptors. We find that this particular CRE is remarkably complex. The majority (86%) of single nucleotide substitutions in this sequence exert significant effects on regulatory activity. Although changes in the affinity of known TF binding sites explain some of these expression changes, we present evidence for complex phenomena, including binding site turnover and TF competition. Analysis of double mutants revealed complex, nucleotide-specific interactions between residues in different TF binding sites. We conclude that some mammalian CREs are finely tuned by evolution and function through complex, nonadditive interactions between bound TFs. CRE-seq will be an important tool to uncover the rules that govern these interactions.

Transcription factors (TFs) regulate the expression of genes at the transcriptional level. Modification of TF activity dynamically alters the transcriptome, which leads to metabolic and phenotypic changes. Thus, functional analysis of TFs using 'omics-based' methodologies is one of the most important areas of the post-genome era. In this mini-review, we present an overview of Arabidopsis TFs and introduce strategies for the functional analysis of plant TFs, which include both traditional and recently developed technologies. These strategies can be assigned to five categories: bioinformatic analysis; analysis of molecular function; expression analysis; phenotype analysis; and network analysis for the description of entire transcriptional regulatory networks.

Previous studies have mapped the visuotopic organization of visual areas from V1 through V4 in the occipital cortex and of area TE in the temporal cortex, but the cortex in between, at the occipito-temporal junction, has remained relatively unexplored. To determine the visuotopic organization of this region, receptive fields were mapped at 1,200 visually responsive sites on 370 penetrations in the ventral occipital and temporal cortex of five macaques. We identified a new visual area, roughly corresponding to cytoarchitectonic area TEO, located between the ventral portion of V4 and area TE. Receptive fields in TEO are intermediate in size between those in V4 and TE and have a coarse visuotopic organization. Collectively, receptive fields in TEO appear to cover nearly the entire contralateral visual field. The foveal and parafoveal representation of TEO is located laterally on the convexity of the inferior temporal gyrus, and the peripheral field is represented medially on the ventral surface of the hemisphere, within and medial to the occipitotemporal sulcus. Beyond the medial border of TEO, within cyteoarchitectonic area TF, is another visually responsive region, which we have termed VTF; this region may also have some crude visual topography. Bands of constant eccentricity in TEO appear to be continuous with those in V2, V3v, and V4. The upper field representation in TEO is located adjacent to that in ventral V4, with a representation of the horizontal meridian forming the boundary between the two areas. The lower field representation in TEO is located just anterior to the upper field but is smaller. In contrast to the orderly representation of eccentricity in TEO, we found little consistent representation of polar angle, other than the separation of upper and lower fields. The results of injecting anatomical tracers in two animals suggest that TEO is an important link in the pathway that relays visual information from V1 to the inferior temporal cortex. TEO is thus likely to play an important role in pattern perception.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibody-induced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL antibodies. The identification of TF as an important mediator of C5a-induced oxidative burst in neutrophils in aPL-induced fetal injury provides a new target for therapy to prevent pregnancy loss in the antiphospholipid syndrome.

Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1beta mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1beta-deficient subline Hepa1C4, as well as antibodies raised against HIF-1alpha and ARNT/HIF-1beta confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5' HBS was more efficient in binding HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.

We studied the molecular mechanism of greater efficacy of paclitaxel-loaded nanoparticles (Tx-NPs) following conjugation to transferrin (Tf) ligand in breast cancer cell line. NPs were formulated using biodegradable polymer, poly(lactic-co-glycolide) (PLGA), with encapsulated Tx and conjugated to Tf ligand via an epoxy linker. Tf-conjugated NPs demonstrated greater and sustained antiproliferative activity of the drug in dose- and time-dependent studies compared to that with drug in solution or unconjugated NPs in MCF-7 and MCF-7/Adr cells. The mechanism of greater antiproliferative activity of the drug with conjugated NPs was determined to be due to their greater cellular uptake and reduced exocytosis compared to that of unconjugated NPs, thus leading to higher and sustained intracellular drug levels. The increase in antiproliferative activity of the drug with incubation time in MCF-7/Adr cells with Tf-conjugated NPs suggests that the drug resistance can be overcome by sustaining intracellular drug retention. The intracellular disposition characteristics of Tf-conjugated NPs following their cellular uptake via Tf receptors could have been different from that of unconjugated NPs via nonspecific endocytic pathway, thus influencing the NP uptake, their intracellular retention, and hence the therapeutic efficacy of the encapsulated drug.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

Intravascular infusion is the most popular route for therapeutic multipotent mesenchymal stromal/stem cell (MSC) delivery in hundreds of clinical trials. Meta-analysis has demonstrated that bone marrow MSC infusion is safe. It is not clear if this also applies to diverse new cell products derived from other sources, such as adipose and perinatal tissues. Different MSC products display varying levels of highly procoagulant tissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (IBMIR). Suitable strategies for assessing and controlling hemocompatibility and optimized cell delivery are crucial for the development of safer and more effective MSC therapies.

Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.

Transcription factors (TFs) are essential regulators of gene expression, and mutated TF genes have been shown to cause numerous human genetic diseases. Yet to date, no single, comprehensive database of human TFs exists. In this work, we describe the collection of an essentially complete set of TF genes from one depiction of the human ORFeome, and the design of a microarray to interrogate their expression. Taking 1468 known TFs from TRANSFAC, InterPro, and FlyBase, we used this seed set to search the ScriptSure human transcriptome database for additional genes. ScriptSure's genome-anchored transcript clusters allowed us to work with a nonredundant high-quality representation of the human transcriptome. We used a high-stringency similarity search by using BLASTN, and a protein motif search of the human ORFeome by using hidden Markov models of DNA-binding domains known to occur exclusively or primarily in TFs. Four hundred ninety-four additional TF genes were identified in the overlap between the two searches, bringing our estimate of the total number of human TFs to 1962. Zinc finger genes are by far the most abundant family (762 members), followed by homeobox (199 members) and basic helix-loop-helix genes (117 members). We designed a microarray of 50-mer oligonucleotide probes targeted to a unique region of the coding sequence of each gene. We have successfully used this microarray to interrogate TF gene expression in species as diverse as chickens and mice, as well as in humans.

We previously mapped the type 2 diabetes mellitus-2 locus (T2dm2), which affects fasting insulin levels, to distal chromosome 19 in a leptin-deficient obese F2 intercross derived from C57BL/6 (B6) and BTBR T+ tf/J (BTBR) mice. Introgression of a 7-Mb segment of the B6 chromosome 19 into the BTBR background (strain 1339A) replicated the reduced insulin linked to T2dm2. The 1339A mice have markedly impaired insulin secretion in vivo and disrupted islet morphology. We used subcongenic strains derived from 1339A to localize the T2dm2 quantitative trait locus (QTL) to a 242-kb segment comprising the promoter, first exon and most of the first intron of the Sorcs1 gene. This was the only gene in the 1339A strain for which we detected amino acid substitutions and expression level differences between mice carrying B6 and BTBR alleles of this insert, thereby identifying variation within the Sorcs1 gene as underlying the phenotype associated with the T2dm2 locus. SorCS1 binds platelet-derived growth factor, a growth factor crucial for pericyte recruitment to the microvasculature, and may thus have a role in expanding or maintaining the islet vasculature. Our identification of the Sorcs1 gene provides insight into the pathway underlying the pathophysiology of obesity-induced type 2 diabetes mellitus.