Transcriptional Components Research Articles

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30-40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

Antiangiogenic and Antimitotic Effects of Aspirin in Hypoxia–Reoxygenation Modulation of the LOX-1-NADPH Oxidase Axis as a Potential Mechanism

Hypoxia–reoxygenation (HR) is a primary driver of angiogenesis in both atherogenesis and tumorigenesis. The main target of hypoxia-driven proangiogenic signaling is adherens junctions responsible for contact inhibition of endothelial cells. We analyzed the effects of hypoxia (8–12 hours) followed by a brief period of reoxygenation (2 hours) (HR) on angiogenesis and integrity of adherens junction in cultured human umbilical vein endothelial cells as well as the effects of aspirin on modulation of human umbilical vein endothelial cells' response to HR. Cells exposed to HR displayed considerable enhancement of tube formation (angiogenesis) on matrigel. Immunocytostaining of near-confluent cells revealed that HR caused disruption of adherens junctions and internalization of their components VE-cadherin, p120 catenin, and b-catenin. Additionally, HR resulted in the appearance of binucleated cells, and VE-cadherin in colocalization with b-catenin was found to be positioned between the separating nuclei. Presence of aspirin (acetylsalicylic acid, 1 mM) resulted in preservation of adherens junctions on the cellular membrane and prevented angiogenesis as well as mitosis. HR caused upregulation LOX-1, the p47(phox) subunit of NADPH, while reducing transcription of endothelial nitric oxide synthase. Aspirin had no effect on endothelial nitric oxide synthase and canceled the transcriptional activation of the LOX-1 and p47(phox) subunit of NADPH oxidase. Based on these data, we hypothesize that aspirin preserves the integrity of adherens junctions and thus blunts angiogenic response to HR through downregulation of LOX-1 and the LOX-1-mediated p47(phox) component of NADPH oxidase transcription, thus preventing NADPH oxidase assembly and function.

PGC-1α regulates a HIF2α-dependent switch in skeletal muscle fiber types

The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) coordinates a broad set of transcriptional programs that regulate the response of skeletal muscle to exercise. However, the complete transcriptional network controlled by PGC-1α has not been described. In this study, we used a qPCR-based screen of all known transcriptional components (Quanttrx) to identify transcription factors that are quantitatively regulated by PGC-1α in cultured skeletal muscle cells. This analysis identified hypoxia-inducible factor 2 α (HIF2α) as a major PGC-1α target in skeletal muscle that is positively regulated by both exercise and β-adrenergic signaling. This transcriptional regulation of HIF2α is completely dependent on the PGC-1α/ERRα complex and is further modulated by the action of SIRT1. Transcriptional profiling of HIF2α target genes in primary myotubes suggested an unexpected role for HIF2α in the regulation of muscle fiber types, specifically enhancing the expression of a slow twitch gene program. The PGC-1α-mediated switch to slow, oxidative fibers in vitro is dependent on HIF2α, and mice with a muscle-specific knockout of HIF2α increase the expression of genes and proteins characteristic of a fast-twitch fiber-type switch. These data indicate that HIF2α acts downstream of PGC-1α as a key regulator of a muscle fiber-type program and the adaptive response to exercise.

A multi-level study of recombinant Pichia pastoris in different oxygen conditions

BackgroundYeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism.ResultsThe physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking.ConclusionsThis systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Alkaline-stress response in Glycine soja leaf identifies specific transcription factors and ABA-mediated signaling factors

Transcriptome of Glycine soja leaf tissue during a detailed time course formed a foundation for examining transcriptional processes during NaHCO(3) stress treatment. Of a total of 2,310 detected differentially expressed genes, 1,664 genes were upregulated and 1,704 genes were downregulated at various time points. The number of stress-regulated genes increased dramatically after a 6-h stress treatment. GO category gene enrichment analysis revealed that most of the differentially expressed genes were involved in cell structure, protein synthesis, energy, and secondary metabolism. Another enrichment test revealed that the response of G. soja to NaHCO(3) highlights specific transcription factors, such as the C2C2-CO-like, MYB-related, WRKY, GARP-G2-like, and ZIM families. Co-expressed genes were clustered into ten classes (P < 0.001). Intriguingly, one cluster of 188 genes displayed a unique expression pattern that increases at an early stage (0.5 and 3h), followed by a decrease from 6 to 12h. This group was enriched in regulation of transcription components, including AP2-EREBP, bHLH, MYB/MYB-related, C2C2-CO-like, C2C2-DOF, C2C2, C3H, and GARP-G2-like transcription factors. Analysis of the 1-kb upstream regions of transcripts displaying similar changes in abundance identified 19 conserved motifs, potential binding sites for transcription factors. The appearance of ABA-responsive elements in the upstream of co-expression genes reveals that ABA-mediated signaling participates in the signal transduction in alkaline response.

Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α

Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Multiple functions of Ldb1 required for β-globin activation during erythroid differentiation

AbstractLdb1 and erythroid partners SCL, GATA-1, and LMO2 form a complex that is required to establish spatial proximity between the β-globin locus control region and gene and for transcription activation during erythroid differentiation. Here we show that Ldb1 controls gene expression at multiple levels. Ldb1 stabilizes its erythroid complex partners on β-globin chromatin, even though it is not one of the DNA-binding components. In addition, Ldb1 is necessary for enrichment of key transcriptional components in the locus, including P-TEFb, which phosphorylates Ser2 of the RNA polymerase C-terminal domain for efficient elongation. Furthermore, reduction of Ldb1 results in the inability of the locus to migrate away from the nuclear periphery, which is necessary to achieve robust transcription of β-globin in nuclear transcription factories. Ldb1 contributes these critical functions at both embryonic and adult stages of globin gene expression. These results implicate Ldb1 as a factor that facilitates nuclear relocation for transcription activation.

Molecular Signatures of the Primitive Prostate Stem Cell Niche Reveal Novel Mesenchymal-Epithelial Signaling Pathways

BackgroundSignals between stem cells and stroma are important in establishing the stem cell niche. However, very little is known about the regulation of any mammalian stem cell niche as pure isolates of stem cells and their adjacent mesenchyme are not readily available. The prostate offers a unique model to study signals between stem cells and their adjacent stroma as in the embryonic prostate stem cell niche, the urogenital sinus mesenchyme is easily separated from the epithelial stem cells. Here we investigate the distinctive molecular signals of these two stem cell compartments in a mammalian system.Methodology/Principal FindingsWe isolated fetal murine urogenital sinus epithelium and urogenital sinus mesenchyme and determined their differentially expressed genes. To distinguish transcripts that are shared by other developing epithelial/mesenchymal compartments from those that pertain to the prostate stem cell niche, we also determined the global gene expression of epidermis and dermis of the same embryos. Our analysis indicates that several of the key transcriptional components that are predicted to be active in the embryonic prostate stem cell niche regulate processes such as self-renewal (e.g., E2f and Ap2), lipid metabolism (e.g., Srebp1) and cell migration (e.g., Areb6 and Rreb1). Several of the enriched promoter binding motifs are shared between the prostate epithelial/mesenchymal compartments and their epidermis/dermis counterparts, indicating their likely relevance in epithelial/mesenchymal signaling in primitive cellular compartments. Based on differential gene expression we also defined ligand-receptor interactions that may be part of the molecular interplay of the embryonic prostate stem cell niche.Conclusions/SignificanceWe provide a comprehensive description of the transcriptional program of the major regulators that are likely to control the cellular interactions in the embryonic prostatic stem cell niche, many of which may be common to mammalian niches in general. This study provides a comprehensive source for further studies of mesenchymal/epithelial interactions in the prostate stem cell niche. The elucidation of pathways in the normal primitive niche may provide greater insight into mechanisms subverted during abnormal proliferative and oncogenic processes. Understanding these events may result in the development of specific targeted therapies for prostatic diseases such as benign prostatic hypertrophy and carcinomas.

From epoxycarotenoids to ABA: The role of ABA 8′-hydroxylases in drought-stressed maize roots

The ability of plants to withstand drought, a potentially major constraint to yield and production, is influenced by abscisic acid (ABA). ABA is synthesized in the cytosol from plastid carotenoid pathway derived precursors, and later inactivated by the action of ABA hydroxylases. Endogenous accumulation of ABA is controlled by both its synthesis and catabolism. Enzymatic activity of ABA 8′-hydroxylase (ABA8Ox), also referred to as CYP707A, is considered one of the key steps in modulating ABA levels that control numerous physiological processes. To investigate the role of this enzyme, maize ABA8Ox gene family members were identified. ABA8Ox gene expression was then analyzed in different tissues and roots during the drought-stress response in maize. These genes were found to be expressed in all tissues, with a high degree of specificity to each tissue and some degree of overlap. Maize ABA8Ox1a and ABA8Ox1b were shown to be the major transcript components for regulating ABA catabolism in drought-stressed roots. Phylogenetic and gene-structure analyses were performed to extend the implications and infer the cause of ABA catabolism in other cereal crops.

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Transcription factors in plants and ABA dependent and independent abiotic stress signalling

Plants face variable environmental stresses that negatively affect plant growth and productivity. The multiplicity of responses is an important aspect of the complexity of stress signalling. Abscisic acid (ABA) is a broad-spectrum phytohormone involved not only in regulating stomatal opening, growth and development but also in coordinating various stress signal transduction pathways in plants during abiotic stresses. The both ABA-dependent and ABA-independent signal transduction pathways from stress signal perception to gene expression involve different transcription factors such as DREB, MYC/MYB, AREB/ABF, NAM, ATAF1,2, CUC and their corresponding cis-acting elements DRE, MYCRS/MYBRS, ABRE, NACRS. Genetic analysis of ABA mutants has given insight that ABA-dependent and ABA-independent pathways for osmotic stress and cold stress interact and converge. This review focuses on ABA-dependent and ABA-independent transcriptional components and cascades, their specificity and crosstalk in stress gene regulation.

Foxp1/2/4-NuRD Interactions Regulate Gene Expression and Epithelial Injury Response in the Lung via Regulation of Interleukin-6

To determine the underlying mechanism of Foxp1/2/4-mediated transcriptional repression, a yeast two-hybrid screen was performed that identified p66beta, a transcriptional repressor and component of the NuRD chromatin-remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as overexpression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, whereas loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins, and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine interleukin-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin-remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.

An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells

SummaryTranscription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-β, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.

TORC: A New Twist on Corticotropin-Releasing Hormone Gene Expression

CRH is the principal hypothalamic neurohormone responsible for hypothalamic-pituitary adrenal (HPA) axis activity (1). CRH also serves as an important transsynaptic signal in various neurocircuits in the brain that regulate behavioral and emotional responses to stress (2). Thus, CRH appears to be a key molecule in the coordination of behavioral and neuroendocrine responses to stressful events. Dysregulation of CRH neuronal function is associated with stress-related psychiatric disorders, and abnormal CRH neuropeptide production levels appear to underlie some of this neuronal dysregulation (3). Consequently, there is much interest in determining the regulation of CRH gene expression, with a particular eye on identifying new regulatory proteins that may point to candidate genes for disease susceptibility (4). Studies find that there is high basal CRH gene expression within the medial parvocellular portion of the hypothalamic paraventricular nucleus. However, CRH gene expression is also rapidly increased by various excitatory stimuli that lead to HPA axis activation (5, 6). The coupling of neuronal excitation with CRH gene induction appears to be largely driven by protein kinase A (PKA)-mediated activation of the transcription factor cAMP response element binding protein (CREB) (7, 8). But some recent findings have thrown a wrench in this straightforward understanding of CRH gene expression. Liu et al. (9) noted that, in a hypothalamic-derived neuronal cell line (4B cells) and hypothalamic primary neuronal cultures, treatment with either the phorbol ester phorbol-12-myristate-13-acetate (PMA; protein kinase C activator) or forskolin (adenylate cyclase activator) led to CREB activation (phosphorylation of CREB ser133). Paradoxically, however, only forskolin treatment was sufficient to stimulate CRH gene promoter activity and CRH gene expression, even though this stimulatory activity required CREB activation (9). A related phenomenon has been observed in vivo: microinfusion of forskolin but not phorbol ester in the rat paraventricular nucleus led to increased CRH gene expression, even though both drugs led to increased HPA axis hormone secretion (10). These findings prompted Liu et al. (9) to consider the possible role of a CREB coregulatory factor in the regulation of CRH gene expression. Such a factor would have to meet three conditions. It would have to be rapidly activated by stimulatory intercellular signals, activated by forskolin but not PMA, and dependent on CREB for CRH gene transactivation capability. In the current issue of Endocrinology, Liu et al. (11) explored the possibility that the recently discovered transducer of regulated CREB activity (TORC), which is also known as CREB-regulated transcription coactivator, could be such a factor. Two groups independently discovered this evolutionarily conserved family of CREB coactivators (TORC1, TORC2, and TORC3) by use of high-throughput screening strategies for proteins that potentiated CREB-dependent gene expression (12, 13). Subsequent studies in various peripheral tissue cell lines have found that TORC has a number of interesting biochemical features. These features not only account for TORC’s ability to interact with CREB protein and thereby permit CREB-dependent gene expression, but they also provide for diverse ways in which TORC can be activated or deactivated. TORC binds to the dimerization and DNA binding domain of CREB. Although this interaction does not appear to alter CREB’s DNA binding capability, it enhances CREB’s association with TAFII130, a component of the general transcription

96 Hsp90 inhibition with 17AAG can sensitize human breast cancer cells to taxol

Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins (BIPs). In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (MRJ), Hsp90, and Hsp70. Hsp90 is a known target of HDAC6 and reversible acetylation is one of the mechanisms that is implicated in regulation of Hsp90 chaperone complex activity. BRMS1 interacted with class II HDACs, HDAC 4, 5, and 6. We further found that BRMS1 is stabilized by Hsp90, and its turnover is proteasome dependent. The stability of BRMS1 protein may be important in maintaining the functional role of BRMS1 in metastasis suppression.

Activation of a Poised RNAPII-Dependent Promoter Requires Both SAGA and Mediator

A growing number of promoters have key components of the transcription machinery, such as TATA-binding protein (TBP) and RNA polymerase II (RNAPII), present at the promoter prior to activation of transcription. Thus, while transcriptional output undergoes a dramatic increase between uninduced and induced conditions, occupancy of a large portion of the transcription machinery does not. As such, activation of these poised promoters depends on rate-limiting steps after recruitment of TBP and RNAPII for regulated expression. Little is known about the transcription components required in these latter steps of transcription in vivo. To identify components with critical roles in transcription after recruitment of TBP in Saccharomyces cerevisiae, we screened for loss of gene expression activity from promoter-tethered TBP in >100 mutant strains deleted for a transcription-related gene. The assay revealed a dramatic enrichment for strains containing deletions in genes encoding subunits of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and Mediator. Analysis of an authentic postrecruitment-regulated gene (CYC1) reveals that SAGA occupies the promoter under both uninduced and induced conditions. In contrast, Mediator is recruited only after transfer to inducing conditions and correlates with activation of the preloaded polymerase at CYC1. These studies indicate the critical functions of SAGA and Mediator in the mechanism of activation of genes with rate-limiting steps after recruitment of TBP.

Transcriptional control of preadipocyte determination by Zfp423

The worldwide epidemic of obesity has increased the urgency of developing a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors 1, but the molecular basis for preadipocyte determination is not understood. Using a novel method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of PPARγ in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. ShRNA-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte PPARγ expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is strikingly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates PPARγ expression, in part, through amplification of the BMP signaling pathway, an effect dependent on the SMAD binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Comprehensive survey of developmental genes in the pea aphid, Acyrthosiphon pisum: frequent lineage-specific duplications and losses of developmental genes.

Aphids exhibit unique attributes, such as polyphenisms and specialized cells to house endosymbionts, that make them an interesting system for studies at the interface of ecology, evolution and development. Here we present a comprehensive characterization of the developmental genes in the pea aphid, Acyrthosiphon pisum, and compare our results to other sequenced insects. We investigated genes involved in fundamental developmental processes such as establishment of the body plan and organogenesis, focusing on transcription factors and components of signalling pathways. We found that most developmental genes were well conserved in the pea aphid, although many lineage-specific gene duplications and gene losses have occurred in several gene families. In particular, genetic components of transforming growth factor beta (TGFbeta) Wnt, JAK/STAT (Janus kinase/signal transducer and activator of transcription) and EGF (Epidermal Growth Factor) pathways appear to have been significantly modified in the pea aphid.

Genomic Approaches Uncover Increasing Complexities in the Regulatory Landscape at the Human SCL (TAL1) Locus

The SCL (TAL1) transcription factor is a critical regulator of haematopoiesis and its expression is tightly controlled by multiple cis-acting regulatory elements. To elaborate further the DNA elements which control its regulation, we used genomic tiling microarrays covering 256 kb of the human SCL locus to perform a concerted analysis of chromatin structure and binding of regulatory proteins in human haematopoietic cell lines. This approach allowed us to characterise further or redefine known human SCL regulatory elements and led to the identification of six novel elements with putative regulatory function both up and downstream of the SCL gene. They bind a number of haematopoietic transcription factors (GATA1, E2A LMO2, SCL, LDB1), CTCF or components of the transcriptional machinery and are associated with relevant histone modifications, accessible chromatin and low nucleosomal density. Functional characterisation shows that these novel elements are able to enhance or repress SCL promoter activity, have endogenous promoter function or enhancer-blocking insulator function. Our analysis opens up several areas for further investigation and adds new layers of complexity to our understanding of the regulation of SCL expression.