The floral repressors TEMPRANILLO1 and 2 modulate salt tolerance by regulating hormonal components and photo-protection in Arabidopsis.

Members of the plant specific RAV family of transcription factors regulate several developmental and physiological processes. In the model plant Arabidopsis thaliana, the RAV TEMPRANILLO 1 (TEM1) and TEM2 control important phase changes such as the juvenile to adult and the vegetative to reproductive transitions. Besides their known regulatory function in plant development, a transcriptomics analysis of transgenic plants overexpressing TEM1 also revealed overrepresentation of Gene Ontology (GO) categories related to abiotic stress responses. Therefore, to investigate the biological relevance of these TEM-dependent transcriptomic changes and elucidate whether TEMs contribute to the modulation of plant growth in response to salinity, we analyzed the behavior of TEM gain and loss of function mutants subjected to mild and high salt stresses at different development stages. With respect to increasing salinity, TEM overexpressing plants were hypersensitive whereas the tem1 tem2 double mutants were more tolerant. Precisely, tem1 tem2 mutants germinated and flowered faster than the wild-type plants under salt stress conditions. Also, tem1 tem2 plants showed a delay in salt-induced leaf senescence, possibly as a consequence of downregulation of jasmonic acid biosynthesis genes. Besides a shorter life cycle and delayed senescence, tem1 tem2 mutants appeared to be better suited to withstand oxidative stress as they accumulated higher levels of α-tocopherol (an important antioxidant metabolite) and displayed a slower degradation of photosynthetic pigments. Taken together, our studies suggest novel and crucial roles for TEM in adaptive growth as they modulate plant development in response to environmental changes such as increasing soil salinity.

The Master Regulator Protein BAZ2B Can Reprogram Human Hematopoietic Lineage-Committed Progenitors into a Multipotent State.

Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1-/- murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B, from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.

A Positive Feedback Loop of BBX11-BBX21-HY5 Promotes Photomorphogenic Development in Arabidopsis.

Light is the most important environmental factor affecting many aspects of plant development. In this study, we report that B-box protein 11 (BBX11) acts as a positive regulator of red light signaling. BBX11 loss-of-function mutant seedlings display significantly elongated hypocotyls under conditions of both red light and long day, whereas BBX11 overexpression causes markedly shortened hypocotyls under various light states. BBX11 binds to the HY5 promoter to activate its transcription, while both BBX21 and HY5 associate with the promoter of BBX11 to positively regulate its expression. Taken together, our results reveal positive feedback regulation of photomorphogenesis consisting of BBX11, BBX21, and HY5, thus substantiating a transcriptional regulatory mechanism in the response of plants to light during normal development.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis.

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

DREAM complex suppresses DNA methylation maintenance genes and precludes DNA hypermethylation.

The DNA methyltransferases MET1 and CMT3 are known to be responsible for maintenance of DNA methylation at symmetric CG and CHG sites, respectively, in Arabidopsis thaliana. However, it is unknown how the expression of methyltransferase genes is regulated in different cell states and whether change in expression affects DNA methylation at the whole-genome level. Using a reverse genetic screen, we identified TCX5, a tesmin/TSO1-like CXC domain-containing protein, and demonstrated that it is a transcriptional repressor of genes required for maintenance of DNA methylation, which include MET1, CMT3, DDM1, KYP and VIMs. TCX5 functions redundantly with its paralogue TCX6 in repressing the expression of these genes. In the tcx5 tcx6 double mutant, expression of these genes is markedly increased, thereby leading to markedly increased DNA methylation at CHG sites and, to a lesser extent, at CG sites at the whole-genome level. Furthermore, our whole-genome DNA methylation analysis indicated that the CG and CHG methylation level is lower in differentiated quiescent cells than in dividing cells in the wild type but is comparable in the tcx5/6 mutant, suggesting that TCX5/6 are required for maintenance of the difference in DNA methylation between the two cell types. We identified TCX5/6-containing multi-subunit complexes, which are known as DREAM in other eukaryotes, and demonstrated that the Arabidopsis DREAM components function as a whole to preclude DNA hypermethylation. Given that the DREAM complexes are conserved from plants to animals, the preclusion of DNA hypermethylation by DREAM complexes may represent a conserved mechanism in eukaryotes.

Arabidopsis JMJ29 is involved in trichome development by regulating the core trichome initiation gene GLABRA3.

Plant trichomes are large single cells that are organized in a regular pattern and play multiple biological functions. In Arabidopsis, trichome development is mainly governed by the core trichome initiation regulators, including the R2R3 type MYB transcript factor GLABRA 1 (GL1), bHLH transcript factors GLABRA 3/ENHANCER OF GLABRA 3 (GL3/EGL3), and the WD-40 repeat protein TRANSPARENT TESTA GLABRA 1 (TTG1), as well as the downstream trichome regulator GLABRA 2 (GL2). GL1, GL3/EGL3, and TTG1 can form a trimeric activation complex to activate GL2, which is required for the trichome initiation and maintenance during cell differentiation. Arabidopsis JMJ29 is a JmjC domain-containing histone demethylase belonging to the JHDM2/KDM3 group. Members of the JHDM2/KDM3 group histone demethylases are mainly responsible for the H3K9me1/2 demethylation. In the present study, we found that the trichome density on leaves and inflorescence stems is significantly decreased in jmj29 mutants. The expression of the core trichome regulators GL1, GL2, and GL3 is decreased in jmj29 mutants as well. Furthermore, JMJ29 can directly target GL3 and remove H3K9me2 on the GL3 locus. Collectively, we found that Arabidopsis JMJ29 is involved in trichome development by directly regulating GL3 expression. These results provide further insights into the molecular mechanism of epigenetic regulation in Arabidopsis trichome development.

CEH-60/PBX regulates vitellogenesis and cuticle permeability through intestinal interaction with UNC-62/MEIS in Caenorhabditis elegans.

The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis.

The RNA polymerase II preinitiation complex. Through what pathway is the complex assembled?

The general transcription factors required for the assembly of the RNA polymerase II preinitiation complex at TATA-dependent promoters are well known. However, recent studies point to two quite distinct pathways for assembly of these components into functional transcription complexes. In this review, the two pathways are compared and potential implications for gene regulatory mechanisms are discussed.

RNA polymerase II transcription: structure and mechanism.

A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters.

Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell-specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4-10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome.

A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing.

Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

[Role of general transcription factors and the TFTC complex in transcription activation in vivo as revealed with a model of the hsp70 gene].

General transcription factors (GTFs) were tested for the presence on the promoter of the Drosophila melanogaster hsp70 gene in vivo. TBP, TBP-associated TAF proteins, TFIIB, TFIIF (RAP30), TFIIH (XPB), the TFTC complex (GCN5 and TRRAP), and a Mediator complex subunit (MEDI 3) were detected on the promoter before heat induction. Heat exposure significantly reduced the contents of TBP, TAF proteins, TFIIB, and TFIIF (RAP30), while these proteins were detected in ecdysone-inducible loci. It was assumed on the basis of these findings that a special mechanism induces transcription from the hsp70 promoter and that the apparent presence or absence of GTFs does not always reflect the transcriptional status of a gene.

Prediction of the general transcription factors associated with RNA polymerase II in Plasmodium falciparum: conserved features and differences relative to other eukaryotes.

BACKGROUND: To date, only a few transcription factors have been identified in the genome of the parasite Plasmodium falciparum, the causative agent of malaria. Moreover, no detailed molecular analysis of its basal transcription machinery, which is otherwise well-conserved in the crown group of eukaryotes, has yet been reported. In this study, we have used a combination of sensitive sequence analysis methods to predict the existence of several parasite encoded general transcription factors associated with RNA polymerase II. RESULTS: Several orthologs of general transcription factors associated with RNA polymerase II can be predicted among the hypothetical proteins of the P. falciparum genome using the two-dimensional Hydrophobic Cluster Analysis (HCA) together with profile-based search methods (PSI-BLAST). These predicted orthologous genes encoding putative transcription factors include the large subunit of TFIIA and two candidates for its small subunit, the TFIIE beta-subunit, which would associate with the previously known TFIIE alpha-subunit, the TFIIF beta-subunit, as well as the p62/TFB1 subunit of the TFIIH core. Within TFIID, the putative orthologs of TAF1, TAF2, TAF7 and TAF10 were also predicted. However, no candidates for TAFs with classical histone fold domain (HFD) were found, suggesting an unusual architecture of TFIID complex of RNA polymerase II in the parasite. CONCLUSION: Taken together, these results suggest that more general transcription factors may be present in the P. falciparum proteome than initially thought. The prediction of these orthologous general transcription factors opens the way for further studies dealing with transcriptional regulation in P. falciparum. These alternative and sensitive sequence analysis methods can help to identify candidates for other transcriptional regulatory factors in P. falciparum. They will also facilitate the prediction of biological functions for several orphan proteins from other apicomplexan parasites such as Toxoplasma gondii, Cryptosporidium parvum and Eimeria.

Teaching resources. Nuclear transactivators and repressors.

This Teaching Resource provides lecture notes and slides for a class covering the mechanism and regulation of eukaryotic gene transcription machinery and is part of the course "Cell signaling systems: a course for graduate students." This lecture begins with an overview of eukaryotic gene transcription and provides discussions regarding the current models of mechanisms of transcription by RNA polymerase II (Pol II). Recent views on the action of Mediator will also be discussed. Finally, a detailed study will be presented to provide evidence for a role for enhancer-promoter communication in gene activation.

Reduced infiltration and increased apoptosis of leukocytes at sites of inflammation by systemic administration of a membrane-permeable IkappaBalpha repressor.

OBJECTIVE: NF-kappaB activation is associated with several inflammatory disorders, including rheumatoid arthritis (RA), making this family of transcription factors a good target for the development of antiinflammatory treatments. Although inhibitors of the NF-kappaB pathway are currently available, their specificity has not been adequately determined. IkappaBalpha is a physiologic inhibitor of NF-kappaB and a potent repressor experimentally when expressed in a nondegradable form. We describe here a novel means for specifically regulating NF-kappaB activity in vivo by administering a chimeric molecule comprising the super-repressor IkappaBalpha (srIkappaBalpha) fused to the membrane-transducing domain of the human immunodeficiency virus Tat protein (Tat-srIkappaBalpha). METHODS: The Wistar rat carrageenan-induced pleurisy model was used to assess the effects of in vivo administration of Tat-srIkappaBalpha on leukocyte infiltration and on cytokine and chemokine production. RESULTS: Systemic administration of Tat-srIkappaBalpha diminished infiltration of leukocytes into the site of inflammation. Analysis of the recruited inflammatory cells confirmed uptake of the inhibitor and reduction of the NF-kappaB activity. These cells exhibited elevated caspase activity, suggesting that NF-kappaB is required for the survival of leukocytes at sites of inflammation. Analysis of exudates, while showing decreases in the production of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-1beta, also revealed a significant increase in the production of the neutrophil chemoattractants cytokine-induced neutrophil chemoattractant 1 (CINC-1) and CINC-3 compared with controls. This result could reveal a previously unknown feedback mechanism in which infiltrating leukocytes may down-regulate local production of these chemokines. CONCLUSION: These results provide new insights into the etiology of inflammation and establish a strategy for developing novel therapeutics by regulating the signaling activity of pathways known to function in RA.

Cleavage, but not read-through, stimulation activity is responsible for three biologic functions of transcription elongation factor S-II.

Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites. In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites. This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in the in vitro systems. Therefore, Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions. Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid. 6-Azauracil also induced IMD2 gene expression in both these mutant strains and the wild type. Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37 degrees C, a restrictive temperature for a strain bearing double null mutations of spt4 and S-II. In contrast, strains bearing S-II mutations defective in both cleavage and read-through stimulation had phenotypes similar to those of an S-II null mutant. However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain. These results suggest that cleavage, but not read-through, stimulation activity is responsible for all three biologic functions of S-II (i.e. suppression of 6-azauracil sensitivity, induction of the IMD2 gene, and suppression of temperature sensitivity of spt4 null mutant).

Promoting elongation with transcript cleavage stimulatory factors.

Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals. A number of elongation factors have been identified that enhance the rate or efficiency of transcription. One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex. These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes. High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors. However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes.

Transcriptional activation through the vitamin D receptor in osteoblasts.

Osteoblasts are bone-forming cells that play an essential role in the development and maintenance of a mineralized bone extracellular matrix and they are target cells for vitamin D. Osteoblasts express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates the expression of osteoblastic-specific genes such as osteocalcin and osteopontin. VDR is a ligand-inducible transcription factor which heterodimerizes with retinoid X receptor (RXR) and binds as a heterodimer to vitamin D-responsive elements (VDREs) in the promoter region of vitamin-D responsive genes, ultimately leading to their increased transcription. Important structural aspects of the VDR and the role that each functional domain plays in mediating VDR action in the context of the osteoblast are discussed. A summary of the potential molecular mechanisms involved in VDR-activated transcription highlighting the importance of interactions between the VDR and general transcription factors (GTFs), TBP-associated factors (TAFIIs), and nuclear receptor coactivator and corepressor proteins are reviewed. These interactions have a role in linking the VDR-RXR heterodimer to the transcriptional pre-initiation complex (PIC) and in regulating the transcription of vitamin D-dependent genes. In addition, recent findings suggest that these interactions are important for regulating the accessibility to promoters by modifying the acetylation state of histones. The complex interplay that occurs between VDR and these various factors to determine the overall transcriptional activity of vitamin D-responsive genes will be summarized.