Constitutive Transcription Factors Research Articles

GIBBERELLIN PERCEPTION SENSOR 2 reveals genesis and role of cellular GA dynamics in light-regulated hypocotyl growth.

The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.

Abstract 2156: IK-930 is a novel TEAD inhibitor for the treatment of cancers harboring mutations in the Hippo signal transduction pathway

Abstract The Hippo pathway is critical to cancer progression, biogenesis, metastasis, and therapeutic resistance. Many cancer indications have a high frequency of mutations in the Hippo pathway. These pro-tumor mutations lead to constitutive TEAD transcription factor activation, which drives gene expression involved in cell growth and pro-survival signaling. To target cancers harboring mutations in the Hippo pathway, we have discovered and are developing IK-930, a novel small molecule that selectively inhibits TEAD-dependent transcription by directly blocking autopalmitoylation. IK-930 prevents this critical post-translational modification that is required for the functional interaction of TEAD with two transcriptional activators, YAP1 and TAZ (WWTR1). IK-930 inhibits in vitro proliferation of Hippo pathway-deficient cancer cell lines, but not Hippo pathway wild type cells. In human mesothelioma xenografts, IK-930 downregulates TEAD-dependent genes. Daily oral administration of IK-930 resulted in antitumor activity in Hippo-dysregulated mesothelioma xenograft models. In EGFR or KRAS mutated tumors, IK-930 enhanced apoptosis and in vivo antitumor activity in combination with EGFR and MEK inhibitors, respectively. IK-930 is inactive in a broad panel of kinases, receptors, and transporters, furthering evidence of selectivity. Multispecies pharmacokinetic analysis and additional in vitro ADME properties imply favorable pharmacokinetic properties, with a low potential for clinically significant drug-drug interactions. To identify indications that may be dependent on TEAD we evaluated tumor types for the incidence of gene alterations in the Hippo pathway, as well as for YAP1 and TAZ activation. YAP1 and TAZ activation was assessed by evaluating nuclear protein expression in tumor tissue microarrays. These analyses showed high YAP1 nuclear expression in tumors with frequent genetic alterations, that may help guide the development of IK-930. Mesothelioma ranks top among tumor types evaluated, due to high prevalence of YAP1 nuclear expression and Hippo pathway genetic alterations, which are predominantly mutations and copy number alterations in the tumor suppressor NF2. In summary, Hippo pathway dysregulation has been implicated in the etiology of multiple tumor types, including mesothelioma. IK-930 is a selective and potent TEAD inhibitor expected to enter the clinic in early 2022. Our analysis pointed to tumors with genetic alterations driving aberrant Hippo signaling. IK-930 demonstrates efficacy in tumor models representing these indications and beneficial combination activity with other targeted therapies. Taken together, these data have informed the clinical development plan for IK-930. Citation Format: Benjamin S. Amidon, Marta Sanchez-Martin, Wilmin Bartolini, Sakeena Syed, Karen McGovern, Lan Xu, Jeffrey Ecsedy, X. Michelle Zhang, Alex Constan, Alfredo C. Castro. IK-930 is a novel TEAD inhibitor for the treatment of cancers harboring mutations in the Hippo signal transduction pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2156.

IRF2 regulates cellular survival and Lenvatinib-sensitivity of hepatocellular carcinoma (HCC) through regulating β-catenin

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Lenvatinib oral chemotherapy is approved as a first-line treatment of patients with unresectable HCC. The efficacy and therapeutic duration of lenvatinib are limited by drug resistance, and the mechanism is unclear. IRF2 is a constitutive transcription factor associated with the development of various cancers by regulating cancer cell growth, apoptosis, and drug resistance. However, the potential role of IRF2 in lenvatinib resistance in HCC has not been explored. In this study, we found that IRF2 promoted proliferation, inhibited apoptosis, and increased lenvatinib resistance of HCC cells by regulating β-catenin expression. Silencing IRF2 downregulated the expression of β-catenin, while overexpressing IRF2 upregulated β-catenin. Moreover, the expression of β-catenin and IRF2 was positively correlated in HCC tissues. Inhibiting β-catenin with XAV-939 effectively abrogated β-catenin expression caused by lenvatinib treatment. These findings identify an important function of IRF2 in HCC and demonstrate a mechanism of lenvatinib resistance of HCC cells. Targeting IRF2 may be a potential strategy to improve the therapeutic effect of lenvatinib on HCC.

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Present status of Catharanthus roseus monoterpenoid indole alkaloids engineering in homo- and hetero-logous systems.

Catharanthus roseus synthesizes one of the most structurally, chemically and biologically active phytomolecules monoterpenoids indole alkaloids (MIAs) with having a wide range of pharmaceutical activities. Being the sole source of antineoplastic MIAs vinblastine and vincristine C. roseus has become one of the most valued plant. The low in planta availability of these MIAs and unavailability of alternative chemical synthesis system has enhanced their demand and equally let to the exorbitant market cost. To bridge this gap alternative production systems have been investigated using MIAs metabolic engineering (ME) in the homologous and heterologous systems. The availability of improved recombinant technologies along with genomics and metabolomics tools has opened the door of tremendous new potentials of ME. To encash these potentials of ME for MIAs pathway, efforts were made by expressing constitutive structure biosynthesis enzymes, transporters, and transcription factors of C. roseus MIAs biosynthesis in both homologous and heterologous systems. Here we review the knowledge of C. roseus MIAs pathway metabolic engineering in homologous and heterologous systems, gained in the past 35years of C. roseus research.

ERR\u03b1 suppression enhances the cytotoxicity of the MEK inhibitor trametinib against colon cancer cells

BackgroundERRα, a constitutive transcription factor that regulates energy metabolism, plays an important role in the progression of various tumours. However, its role in cell survival and proliferation and its implication in targeted therapy in colon cancer remains elusive.MethodsThe expression of ERRα in colon cancer tissues and cell lines was detected by using western blotting and immunohistochemistry. A wound healing assay and a transwell assay were performed to examine the migration and invasion of the colon cancer cells. A cell viability assay, clonogenic assay, western blot assay and the dual-luciferase reporter assay were employed to study the interaction between trametinib (inhibitor of MEK) and EGF treatment. Flow cytometry, western blotting, quantitative reverse-transcription polymerase chain reaction and xenograft studies were used to identify whether the combination of trametinib and simvastatin had a synergistic effect.ResultsERRα positively regulated the cell proliferation, migration and invasion of colon cancer cells, and the suppression of ERRα completely reduced the EGF treatment-induced proliferation of colon cancer cells. Further investigation showed that trametinib partially restrained the up-regulation of ERRα induced by the EGF treatment, and ERRα inhibition increased the sensitivity of colon cancer cells to trametinib. At last, we combined trametinib with simvastatin, a common clinically used drug with a new reported function of transcriptional activity inhibition of ERRα, and found that this combination produced a synergistic effect in inhibiting the proliferation and survival of colon cancer cells in vitro as well as in vivo.ConclusionsThe present data indicated that ERRα acted as an oncogene in colon cancer cells, and the combined targeting of ERRα and MEK might be a promising therapeutic strategy for colon cancer treatment.

Transcription factor c-Maf is essential for IL-10 gene expression in B cells.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. c-Maf has been suggested as an essential transcriptional factor for IL-10 production in CD4+ T cells and macrophages. However, it remains unclear whether c-Maf participates in IL-10 expression in B cells. In this study, we investigated the role of c-Maf in the transcriptional regulation of IL-10 in regulatory B cells, as well as the underlying molecular mechanism. We found that c-Maf was constitutively expressed in resting B cells. c-Maf expression was upregulated in the presence of LPS and dose-dependently enhanced IL-10 production following binding to the IL-10 promoter. Moreover, a lower expression of c-Maf and decreased production of regulatory B (Breg) cells were detected in mice with collagen-induced arthritis (CIA), which may contribute to the pathological changes. Taken together, these data demonstrate that c-Maf is an indispensable yet constitutive transcription factor for IL-10 gene expression in LPS-activated B cells.

Molecular strategies to increase yeast iron accumulation and resistance

All eukaryotic organisms rely on iron as an essential micronutrient for life because it participates as a redox-active cofactor in multiple biological processes. However, excess iron can generate reactive oxygen species that damage cellular macromolecules. The low solubility of ferric iron under physiological conditions increases the prevalence of iron deficiency anemia. A common strategy to treat iron deficiency consists of dietary iron supplementation. The baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, but also as a feed supplement. In response to iron deficiency, the yeast Aft1 transcription factor activates cellular iron acquisition. However, when constitutively active, Aft1 inhibits growth probably due to iron toxicity. In this report, we have studied the consequences of using hyperactive AFT1 alleles, including AFT1-1UP, to increase yeast iron accumulation. We first characterized the iron sensitivity of cells expressing different constitutively active AFT1 alleles. We rescued the high iron sensitivity conferred by the AFT1 alleles by deleting the sphingolipid signaling kinase YPK1. We observed that the deletion of YPK1 exerts different effects on iron accumulation depending on the AFT1 allele and the environmental iron. Moreover, we determined that the impairment of the high-affinity iron transport system partially rescues the high iron toxicity of AFT1-1UP-expressing cells. Finally, we observed that AFT1-1UP inhibits oxygen consumption through activation of the RNA-binding protein Cth2. Deletion of CTH2 partially rescues the AFT1-1UP negative respiratory effect. Collectively, these results contribute to understand how the Aft1 transcription factor functions and the multiple consequences derived from its constitutive activation.

Transcription Factors in Breast Cancer-Lessons From Recent Genomic Analyses and Therapeutic Implications.

Multiplatform genomic analyses have identified 93 frequently altered genes in breast cancer. Of these, as many as 49 genes are directly or indirectly involved in transcription. These include constitutive and inducible DNA-binding transcription factors (DB-TFs, 13 genes), corepressors/coactivators (14 genes), epigenetic (10), and mediator/splicing/rRNA (3) factors. At least nine additional genes are immediate upstream regulators of transcriptional cofactors. G:profiler analysis reveals that these alterations affect cell cycle, development/differentiation, steroid hormone, and chromatin modification pathways. A notable observation is that DB-TFs that mediate major oncogenic signaling (e.g., WNT, receptor tyrosine kinase (RTK), NOTCH, and HIPPO), which switch from default repression (signal OFF) to transcriptional activation (signal ON), are not altered in breast cancer. Instead, corepressors (e.g., pRb for E2F1 downstream of various proliferation signals) or upstream factors (e.g., APC and AXIN for TCF, downstream of canonical WNT signaling) are lost, or coactivators (e.g., NOTCH1/2 for CSL/RBPJk) are induced. In contrast, constitutive (MYC, TBX3) and signal-induced (TP53, FOXA1) DB-TFs that do not mediate default repression are directly altered in breast cancer. Some of these TFs have been implicated in the establishment of super-enhancers and positive transcriptional elongation. In addition, oncogenic transcription is induced by mutations affecting regulatory elements or chromatin conformation that create new TF-binding sites in promoters and enhancers of oncogenic genes to promote tumorigenesis. Here we review these diverse oncogenic alterations in TFs in BC and discuss implications for therapy.

Yin-Yang 1 and Yin-Yang 2 exert opposing effects on the promoter activity of interleukin 4.

Interleukin (IL)-4 acts on T cells as a growth and activation factor, and promotes the differentiation of type 2 T helper cells. In T cells, expression of the gene encoding IL-4 is regulated by inducible or constitutive factors. Yin-Yang (YY)-1 is one of constitutive transcription factors binding to the IL-4 promoter. The recently identified YY2 protein is similar to YY1, with both sharing high levels of homology in their zinc finger motifs. However, the role of YY2 in T cells is unclear. YY1 and YY2 were constitutively expressed in EL4 T cells, and their expression was not dependent on stimulation. IL-4 promoter (-741/+56 fragment) activity was enhanced by YY1, but inhibited by YY2. The enhanced IL-4 promoter activity by YY1 was reduced by simultaneous expression of YY2. In addition, the DNA binding affinity of YY1 to the IL-4 promoter was adversely affected by YY2. Our results suggest that YY1 and YY2 exert opposing effects on the IL-4 promoter as they compete for the same DNA binding sites.

Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites

As the only source for the low-abundance antitumor agents vinblastine and vincristine, Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement. The biosynthesis of these monoterpenoid indole alkaloids (MIAs) is a complex multistep enzymatic network that is tightly regulated by developmental and environmental factors. Here we review the knowledge achieved in the past 30 years of the MIA pathway in C. roseus, from genetic to metabolic aspects. Two early precursor pathways and a late mono-/bis-indole alkaloid pathway have been largely elucidated and established, as well as their intercellular and subcellular compartmentation. Many genes encoding constitutive structural biosynthetic enzymes, transcription factors, and transporters involved in these pathways have been cloned, characterized and applied in metabolic engineering strategies to improve the MIA production. However, genetic modification in the pathway in C. roseus resulted in complicated changes of both secondary and primary metabolism, affecting not only the MIA pathway but also other pathways. Research at metabolic level is required to increase the knowledge on the genetic regulation of the whole metabolic network connected to the MIA biosynthesis. Nuclear magnetic resonance-based metabolomics (metabolic profiling, fingerprinting and flux analyses) in combination with other “omics” have been implemented in studies of C. roseus for pathway elucidation, including among others, understanding stress response, cross talk between pathways, and diversion of carbon fluxes, with the aim to fully unravel the MIA biosynthesis, its regulation and the function of the alkaloids in the plant from a systems biology point of view.

Hippocampal dynamics of synaptic NF-kappa B during inhibitory avoidance long-term memory consolidation in mice

Since the discovery that long-term memory is dependent on protein synthesis, several transcription factors have been found to participate in the transcriptional activity needed for its consolidation. Among them, NF-kappa B is a constitutive transcription factor whose nuclear activity has proven to be necessary for the consolidation of inhibitory avoidance in mice. This transcription factor has a wide distribution in the nervous system, with a well-reported presence in dendrites and synaptic terminals. Here we report changes in synaptosomal NF-kappa B localization and activity, during long-term memory consolidation. Activity comparison of synaptosomal and nuclear NF-kappa B, indicates different dynamics for both localizations. In this study we identify two pools of synaptosomal NF-kappa B, one obtained with the synaptoplasm (free fraction) and the second bound to the synaptosomal membranes. During the early steps of consolidation the first pool is activated, as the membrane associated transcription factor fraction increases and concomitantly the free fraction decreases. These results suggest that the activation of synaptic NF-kappa B and its translocation to membranes are part of the consolidation of long-term memory in mice.

Rapid regulation of microRNA following induction of long-term potentiation in vivo.

Coordinated regulation of gene expression is essential for consolidation of the memory mechanism, long-term potentiation (LTP). Triggering of LTP by N-methyl-D-aspartate receptor (NMDAR) activation rapidly activates constitutive and inducible transcription factors, which promote expression of genes responsible for LTP maintenance. As microRNA (miRNA) coordinate expression of genes related through seed sites, we hypothesize that miRNA contribute to the regulation of the LTP-induced gene response. MiRNA function primarily as negative regulators of gene expression. As LTP induction promotes a generalized rapid up-regulation of gene expression, we predicted a complementary rapid down-regulation of miRNA levels. Accordingly, we carried out global miRNA expression profiling in the rat dentate gyrus 20 min post-LTP induction in vivo. Consistent with our hypothesis, we found a large number of differentially expressed miRNA, the majority down-regulated. Detailed analysis of miR-34a-5p and miR-132-3p revealed this down-regulation was transient and NMDAR-dependent, whereby block of NMDARs released an activity-associated inhibitory mechanism. Furthermore, down-regulation of mature miR-34a-5p and miR-132-3p occurred solely by post-transcriptional mechanisms, occurring despite an associated up-regulation of the pri-miR-132 transcript. To understand how down-regulation of miR-34a-5p and miR-132-3p intersects with the molecular events occurring following LTP, we used bioinformatics to identify potential targets. Previously validated targets included the key LTP-regulated genes Arc and glutamate receptor subunits. Predicted targets included the LTP-linked kinase, Mapk1, and neuropil-associated transcripts Hn1 and Klhl11, which were validated using luciferase reporter assays. Furthermore, we found that the level of p42-Mapk1, the protein encoded by the Mapk1 transcript, was up-regulated following LTP. Together, these data support the interpretation that miRNA, in particular miR-34a-5p and miR-132-3p, make a surprisingly rapid contribution to synaptic plasticity via dis-inhibition of translation of key plasticity-related molecules.

Rho protein GTPases and their interactions with NFκB: crossroads of inflammation and matrix biology.

The RhoGTPases, with RhoA, Cdc42 and Rac being major members, are a group of key ubiquitous proteins present in all eukaryotic organisms that subserve such important functions as cell migration, adhesion and differentiation. The NFκB (nuclear factor κB) is a family of constitutive and inducible transcription factors that through their diverse target genes, play a major role in processes such as cytokine expression, stress regulation, cell division and transformation. Research over the past decade has uncovered new molecular links between the RhoGTPases and the NFκB pathway, with the RhoGTPases playing a positive or negative regulatory role on NFκB activation depending on the context. The RhoA–NFκB interaction has been shown to be important in cytokine-activated NFκB processes, such as those induced by TNFα (tumour necrosis factor α). On the other hand, Rac is important for activating the NFκB response downstream of integrin activation, such as after phagocytosis. Specific residues of Rac1 are important for triggering NFκB activation, and mutations do obliterate this response. Other upstream triggers of the RhoGTPase–NFκB interactions include the suppressive p120 catenin, with implications for skin inflammation. The networks described here are not only important areas for further research, but are also significant for discovery of targets for translational medicine.

Expanding the Boolean Logic of the Prokaryotic Transcription Factor XylR by Functionalization of Permissive Sites with a Protease-Target Sequence

The σ54-dependent prokaryotic regulator XylR implements a one-input/one-output actuator that transduces the presence of the aromatic effector m-xylene into transcriptional activation of the cognate promoter Pu. Such a signal conversion involves the effector-mediated release of the intramolecular repression of the N-terminal A domain on the central C module of XylR. On this background, we set out to endow this regulator with additional signal-sensing capabilities by inserting a target site of the viral protease NIa in permissive protein locations that once cleaved in vivo could either terminate XylR activity or generate an effector-independent, constitutive transcription factor. To find optimal protein positions to this end, we saturated the xylR gene DNA with a synthetic transposable element designed for randomly delivering in-frame polypeptides throughout the sequence of any given protein. This Tn5-based system supplies the target gene with insertions of a selectable marker that can later be excised, leaving behind the desired (poly) peptides grafted into the protein structure. Implementation of such knock-in-leave-behind (KILB) method to XylR was instrumental to produce a number of variants of this transcription factor (TF) that could compute in vivo two inputs (m-xylene and protease) into a single output following a logic that was dependent on the site of the insertion of the NIa target sequence in the TF. Such NIa-sensitive XylR specimens afforded the design of novel regulatory nodes that entered protease expression as one of the signals recognized in vivo for controlling Pu. This approach is bound to facilitate the functionalization of TFs and other proteins with new traits, especially when their forward engineering is made difficult by, for example, the absence of structural data.

Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus

BackgroundDifferential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer’s disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15).ResultsTreatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer’s disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα.ConclusionsOur results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.

Human TNFα-induced protein 3-interacting protein 1 (TNIP1) promoter activation is regulated by retinoic acid receptors

Coregulator proteins play key roles in transcriptional control by members of the nuclear receptor superfamily. Previously, we demonstrated that tumor necrosis factor α (TNFα)-induced protein 3-interacting protein 1 (TNIP1) is a corepressor of agonist-bound retinoic acid receptors (RARs). Additionally, TNIP1 has been shown to repress peroxisome proliferator-activated receptors (PPAR) and NF-κB activity and interact with HIV proteins nef and matrix. TNIP1 transcriptional regulation, however, is under studied. Here we show that under permissive epigenetic conditions, TNIP1 expression is induced by all trans retinoic acid (ATRA). Within a 6000bp region of the human TNIP1 promoter we cloned, both proximal and distal promoter regions are RAR responsive with the latter having RA response elements (RAREs) recognizable by their sequence and functionality in native promoter and synthetic RARE luciferase constructs, EMSA, and ChIP assays. These findings suggest a feedback loop whereby RARs activate expression of TNIP1, which then attenuates their activity. Together with anticipated constitutive transcription factors and the previously described NF-κB-responsiveness of the proximal TNIP1 promoter, the expected combinatorial control of TNIP1 expression could likely modulate TNIP1's impact in any of its target pathways. The degree of control by RARs or other transcription factors would in turn depend on their cell-specific level of expression and/or activation from signals in the environment such as ATRA and TNFα.

Temporal Profiling of Gene Networks Associated with the Late Phase of Long-Term Potentiation In Vivo

Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo.

Tcra Enhancer Activation by Inducible Transcription Factors Downstream of Pre-TCR Signaling

The Tcra enhancer (Eα) is essential for pre-TCR-mediated activation of germline transcription and V(D)J recombination. Eα is considered an archetypical enhanceosome that acts through the functional synergy and cooperative binding of multiple transcription factors. Based on dimethylsulfate genomic footprinting experiments, there has been a long-standing paradox regarding Eα activation in the absence of differences in enhancer occupancy. Our data provide the molecular mechanism of Eα activation and an explanation of this paradox. We found that germline transcriptional activation of Tcra is dependent on constant phospholipase Cγ, as well as calcineurin- and MAPK/ERK-mediated signaling, indicating that inducible transcription factors are crucially involved. NFAT, AP-1, and early growth response factor 1, together with CREB-binding protein/p300 coactivators, bind to Eα as part of an active enhanceosome assembled during pre-TCR signaling. We favor a scenario in which the binding of lymphoid-restricted and constitutive transcription factors to Eα prior to its activation forms a regulatory scaffold to recruit factors induced by pre-TCR signaling. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors dictates the Eα function. This mechanism for enhancer activation may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.

PPARγ and NF-κB regulate the gene promoter activity of their shared repressor, TNIP1

Human TNFAIP3 interacting protein 1 (TNIP1) has diverse functions including support of HIV replication through its interaction with viral Nef and matrix proteins, reduction of TNFα-induced signaling through its interaction with NF-κB pathway proteins, and corepression of agonist-bound retinoic acid receptors and peroxisome proliferator-activated receptors (PPAR). The wide tissue distribution of TNIP1 provides the opportunity to influence numerous cellular responses in these roles and defining control of TNIP1 expression would be central to improved understanding of its impact on cell function. We cloned 6 kb of the human TNIP1 promoter and performed predictive and functional analyses to identify regulatory elements. The promoter region proximal to the transcription start site is GC-rich without a recognizable TATA box. In contrast to this proximal ~ 500 bp region, 6 kb of the promoter increased reporter construct constitutive activity over five-fold. Throughout the 6 kb length, in silico analysis identified several potential binding sites for both constitutive and inducible transcription factors; among the latter were candidate NF-κB binding sequences and peroxisome proliferator response elements (PPREs). We tested NF-κB and PPAR regulation of the endogenous TNIP1 gene and cloned promoter by expression studies, electrophoretic mobility shift assays, and chromatin immunoprecipitations. We validated NF-κB sites in the TNIP1 promoter proximal and distal regions as well as one PPRE in the distal region. The ultimate control of the TNIP1 promoter is likely to be a combination of constitutive transcription factors and those subject to activation such as NF-κB and PPAR.