Polyomavirus mutation that confers a cell-specific cis advantage for viral DNA replication.

The structural and biological properties of a polyomavirus mutant selected in Friend erythroleukemic cells were investigated. The growth efficiency of this mutant (PyFL78) was compared with that of the parental PyA2 strain by a growth competition assay in Friend erythroleukemic and 3T3 (or 3T6) cell lines. The results reveal that PyFL78 displays a cis-acting growth advantage over the PyA2 parental strain in Friend erythroleukemic cells but not in 3T3 or 3T6 cells. This cell-specific cis advantage is shown to be due to modifications within the polyomavirus noncoding regulatory region.

Lysine-specific demethylase LSD1 regulates autophagy in neuroblastoma through SESN2-dependent pathway.

Autophagy is a physiological process, important for recycling of macromolecules and maintenance of cellular homeostasis. Defective autophagy is associated with tumorigenesis and has a causative role in chemotherapy resistance in leukemia and in solid cancers. Here, we report that autophagy is regulated by the lysine-specific demethylase LSD1/KDM1A, an epigenetic marker whose overexpression is a feature of malignant neoplasia with an instrumental role in cancer development. In the present study, we determine that two different LSD1 inhibitors (TCP and SP2509) as well as selective ablation of LSD1 expression promote autophagy in neuroblastoma cells. At a mechanistic level, we show that LSD1 binds to the promoter region of Sestrin2 (SESN2), a critical regulator of mTORC1 activity. Pharmacological inhibition of LSD1 triggers SESN2 expression that hampers mTORC1 activity, leading to enhanced autophagy. SESN2 overexpression suffices to promote autophagy in neuroblastoma cells, while loss of SESN2 expression reduces autophagy induced by LSD1 inhibition. Our findings elucidate a mechanism whereby LSD1 controls autophagy in neuroblastoma cells through SESN2 transcription regulation, and we suggest that pharmacological targeting of LSD1 may have effective therapeutic relevance in the control of autophagy in neuroblastoma.

Transcription activation by targeted recruitment of the RNA polymerase II CTD phosphatase FCP1.

Human FCP1 in association with RNAP II reconstitutes a highly specific CTD phosphatase activity and is required for recycling RNA polymerase II (RNAP II) in vitro. Here we demonstrate that targeted recruitment of FCP1 to promoter templates, through fusion to a DNA-binding domain, stimulates transcription. We demonstrate that a short region at the C-terminus of the FCP1 protein is required and sufficient for activation, indicating that neither the N-terminal phosphatase domain nor the BRCT domains are required for transcription activity of DNA-bound FCP1. In addition, we demonstrate that the C-terminus region of FCP1 suffices for efficient binding in vivo to the RAP74 subunit of TFIIF and is also required for the exclusive nuclear localization of the protein. These findings suggest a role for FCP1 as a positive regulator of RNAP II transcription.

Class A helix-loop-helix proteins are positive regulators of several cyclin-dependent kinase inhibitors' promoter activity and negatively affect cell growth.

The class A of basic helix-loop-helix (bHLH) proteins are ubiquitously expressed transcription factors playing a pivotal role in the regulation of cell growth and differentiation. We determined that enforced expression of all four different mammalian members of this family, E12, E47, E2-2, and HEB, suppresses the cell colony-forming efficiency of several cell lines. To gain insights into the mechanisms by which class A bHLH factors affect cell growth, we have investigated their role in the transcriptional regulation of cyclin-dependent kinase inhibitors. We found that p21CIP1/ WAF1, p15INK4B, and p16INK4B promoter sequences contain E-boxes that render these genes competent for class A bHLH-mediated transcriptional activation and Id-mediated repression. The mechanism underlying the class A bHLH-mediated inhibition of cell growth does not involve an arrest of G1 progression in 293T cells. In fact, contrary to what has been found in 3T3 NIH fibroblasts, we found that enhanced expression of class A bHLH proteins led to a decreased proliferation rate by promoting cell death associated with the induction of apoptosis. These findings highlight the role of the class A bHLH proteins as general negative regulators of cell proliferation through a mechanism(s) that involves both enhancement of several cyclin-dependent kinase inhibitor genes expression and promotion of cell death.

Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo.

The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II. The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA. Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo. Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect. We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound. Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation.

Differential transcriptional regulation of c-myc promoter through the same DNA binding sites targeted by Sp1-like proteins.

Sp1 and Sp3 are closely related members of a gene family encoding proteins with very similar structural features. The zinc finger DNA binding domains of Sp1 and Sp3 are highly conserved and they bind to GC and GT box with comparable affinities. To begin to delineate the specific roles of these two members of the Sp1-like gene family, here we have analysed the DNA binding specificity and their effects on activation of human c-myc promoter. We found that both proteins bind to the same sites of c-myc promoter, upstream to both the P1 and P2 initiation sites. Cotransfection experiments, in mammalian and insect cells, indicated that Sp1 trans-activate c-myc promoter, whereas Sp3 did not. In addition, enforced expression of Sp3 repressed Sp1-mediated activation of c-myc. Finally, we found that Sp1 and E2F-1/DP-1 cooperatively trans-activate c-myc promoter. In contrast enforced expression of Sp3 fails to repress E2F-1/DP-1-mediated activation.

Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation.

OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the TATA-binding protein (TBP). DESIGN: HIV long terminal repeat reporter plasmids were cotransfected into mammalian and Drosophila insect cells with expression vectors encoding Tat and vectors encoding TBP either alone or linked to an heterologous DNA-binding domain. METHODS: The activity of the different reporters was compared in the presence or absence of Tat or TBP, or both, upon cotransfections into human and Drosophila insect cell lines. RESULTS: Tat protein is unable to transactivate enhancerless HIV-1 minimal promoter bearing only the TATA box and TAR. Artificial recruitment of human TBP (hTBP) to the enhancerless HIV minimal promoter was found to trigger gene expression and coexpression of Tat resulted in a marked synergy. Tat protein cooperated with DNA-bound hTBP by inducing high levels of processive viral transcripts. Synergy between Tat and hTBP was also observed when both factors were targeted to a promoter DNA template. The functional cooperation between TBP and Tat was further demonstrated using the Drosophila Schneider SL2 cells. In these cells Tat protein alone was ineffective; however, coexpression of Drosophila TBP and Tat resulted in a trans-activating response region-dependent synergistic transactivation of basal transcription. CONCLUSION: The strong synergy between TBP and Tat in the absence of any DNA-bound activator suggests that Tat stimulates transcription in an activator-independent manner most likely by a functional interaction with general transcription factors that occurs after TBP recruitment. Thus, efficient recruitment of TBP represents a limiting step for Tat transactivation.

Inhibition of Tat transactivation by the RNA polymerase II CTD-phosphatase FCP1.

OBJECTIVES: To asses the role of the RNAPII carboxy-terminal domain (CTD) phosphatase FCP1 on HIV-1 Tat-mediated transactivation. DESIGN: Construction of expression vectors encoding FCP1 phosphatase and analysis of their functions on Tat activity. METHODS: Basal and Tat-mediated transactivation of HIV-1 long terminal repeat (LTR)-driven transcription was compared, by transient transfections, in the presence of FCP1 phosphatase. Protein interactions were analysed by in vitro binding assays. RESULTS: FCP1 specifically and effectively represses Tat transactivation but not HIV-1 LTR-basal transcription. Protein interaction assays demonstrated that FCP1 specifically and directly binds Tat in vitro. CONCLUSION: The specific and efficient inhibitory function of FCP1 highlights the important role of this CTD-phosphatase in Tat-mediated transactivation, and it suggests that FCP1 might represent a specific target for modulation of Tat activity in infected cells.

Functional interaction between the HIV-1 Tat transactivator and the inhibitory domain of the Oct-2 cellular transcription factor.

OBJECTIVES: To examine the interaction of the HIV Tat protein and the inhibitory domain of the cellular transcription factor Oct-2. DESIGN: HIV reporter plasmids were co-transfected with expression vectors encoding wild-type and mutant Tat proteins and vectors encoding the inhibitory domain either alone or linked to a DNA-binding domain. METHODS: The activity of the different reporters was compared in the presence or absence of Tat or the inhibitory domain, or both. RESULTS: In the presence of the Tat protein, the Oct-2 inhibitory domain enhances the activation of Tat-responsive promoters by Tat. This effect is not observed when either the inhibitory domain or Tat cannot bind to the target gene, but is observed when one or both of the factors can bind. No activation is observed when the Oct-2 domain is co-transfected with Tat mutants that lack the Tat activation domain, indicating that the Oct-2 domain cannot substitute for the Tat activation domain. CONCLUSION: These data indicate that the Oct-2 inhibitory domain can functionally interact with Tat to form a complex with enhanced transactivating ability.

The CDK9-associated cyclins T1 and T2 exert opposite effects on HIV-1 Tat activity.

OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the cyclin T1 and T2 proteins which, in association with cyclin dependent kinase (CDK)9, are the regulatory subunits of the TAK/P-TEFb cellular complex strictly required for Tat transactivation. DESIGN: HIV-1 long terminal repeat (LTR) reporter plasmid was co-transfected into human and rodent cells with expression vectors encoding Tat and vectors encoding the cyclins T1, T2a and T2b, respectively. METHODS: Tat-mediated transactivation of HIV-1 LTR-driven transcription was compared in the presence or absence of different cyclins T (T1, T2a and T2b), upon co-transfections into human and rodent cell lines. Protein interactions were analysed by in vitro binding assays. RESULTS: It was found that Tat function in rodent cells is enhanced by co-expression of cyclin T1 but not cyclin T2. The N-terminal region (amino acids 1-290) of cyclin T1 is sufficient for this function and for binding to Tat and CDK9. Cyclin T2 binds to CDK9 but not to Tat. Moreover, enforced expression of cyclin T2 inhibits cyclin T1-mediated enhancement of Tat in rodent cells and it represses Tat activity in human cells. CONCLUSION: Efficient Tat transactivation in rodent cells occurs in the presence of human cyclin T1 but not in the presence of cyclin T2; overexpression of cyclin T2 inhibits Tat function in both rodent and human cells.

Transcriptional regulation by the Sp family proteins.

Sp1 is one of the very first cellular transcription factors to be identified and cloned in virtue of its binding to a G-rich motif in the SV40 early promoter. Sp1 protein binds to the G-rich sequences present in a variety of cellular and viral promoters and stimulates their transcriptional activity. Recently, a number of other GC and/or GT box-binding factors homologous to Sp1 have been isolated, namely Sp2, Sp3 and Sp4, and the two more distantly related factors, BTEB and BTEB2. The discovery of this family highlights a previously unknown level of complexity of transcriptional regulation of promoters containing GC and/or GT box motifs. This review focuses primarily on strategies aimed to elucidate the transcription properties of the Sp1-like factors and discusses the experimental problems inherent in the attempt to define their respective functions.

Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II.

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. Here, we demonstrate that cyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, we found that only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, we found that the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, our data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo.

The ZNF35 human zinc finger gene encodes a sequence-specific DNA-binding protein.

We developed a rapid method to determine DNA-binding sites for putative DNA-binding proteins. This procedure has been successfully used to define a specific consensus site for the human ZNF35 zinc finger gene. ZNF35 encodes a 58-kDA polypeptide containing 11 consecutive finger motifs located at the amino terminus, and an acidic domain located at the carboxy terminus. These features suggest that ZNF35 is a site-specific DNA-binding protein involved in the regulation of gene expression. We have expressed the ZNF35 protein from E. coli and have employed a Southwestern-polymerase chain reaction method using random oligonucleotides to identify its high-affinity binding site. The core sequence for the ZNF35 protein-binding site is 5'-C/GC/GAAG/TA-3'.

Sequences both 5' and 3' to the transcription initiation site contribute to the ability of a mouse H-2 gene to respond to type I interferon.

To investigate the cis-acting DNA elements that are involved in the regulation of class I major histocompatibility complex genes by interferon, several promoter fragments of the H-2Kk gene were linked to the reporter chloramphenicol acetyl transferase (CAT) gene, and the CAT expression was analyzed in stable transfected cell lines. The functional activities of progressive deletions of the 5'-flanking region of the H-2Kk gene linked to the CAT gene have allowed us to define a discrete cis-acting DNA region necessary for interferon-mediated stimulation. Moreover, the H-2Kk gene transcribed by the nonregulated SV40 early promoter was also found to be under interferon regulation. Thus interferon enhancement of the H-2Kk gene expression appears to be mediated by two cis-acting elements, one located in the 5'-flanking region and the other by sequences downstream from the transcription initiation site.

The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells.

The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation.

Negative and positive transcriptional control during cell proliferation (Review).

A significant amount of experimental evidence has demonstrated that progression of the cell cycle in mammalian cells is associated with periodic transcriptional activation/ repression of growth-regulatory genes. We summarize our current knowledge and views on the role of the critical cell cycle regulators such as the retinoblastoma proteins in transcription repression and their functional connections with various different transcription factors. In addition, we discuss the role of oncogenes such as TIF1 alpha, PML and RFL which belong to a characteristic subgroup of RING finger proteins that contain the RING finger (C3HC4 zinc finger) the B-boxes and a putative coiled-coil (RBCC configuration) as mediators of transcription repression.

LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription.

Myc is a transcription factor that significantly contributes to cancer progression by modulating the expression of important genes through binding to a DNA sequence, CACGTG, called E-box. We find that on Myc binding to chromatin, the lysine-demethylating enzyme, LSD1, triggers a transient demethylation of lysine 4 in the histone H3. In addition, we demonstrate that Myc binds and recruits LSD1 to the E-box chromatin and the formation of this complex is stimulated by cAMP-PKA. Demethylation by LSD1 produces H(2)O(2), which locally oxidizes guanine and induces the recruitment of 8-oxoguanine-DNA glycosylase (OGG1) and of the nuclease Ape1 on the E-box chromatin. Inhibition of oxidation or silencing of LSD1, OGG1 or Ape1 significantly reduce transcription and inhibit mRNA accumulation of Myc-target genes. Collectively, these data highlight the role of transient LSD1-mediated demethylation of H3K4 leading to local DNA oxidation as driving force in the assembly of the Myc-induced transcription initiation complex.

Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

Differential role for Sp1/Sp3 transcription factors in the regulation of the promoter activity of multiple cyclin-dependent kinase inhibitor genes.

Cyclin-dependent kinase inhibitors play a significant role in cell cycle progression and in cellular differentiation and their expression is regulated in different cellular settings. GC-rich regions in the promoter sequences of the cyclin-dependent kinase inhibitor genes p15INK4B and p21CIP1/WAF1 mediate the transcriptional response of these genes to extracellular stimuli. Similar GC-rich sequences in the promoter of the p15INK4A and p16INK4B gene can be targeted for transcriptional inactivation by methylation of cytosine residues. GC-rich regions represent putative target sites for binding of the ubiquitously expressed Sp1 and Sp3 transcription factors. Using a combination of functional and biochemical studies, we analyzed the potential role of the Sp1 and Sp3 factors in the regulation of CDKI p15, p16, and p21 promoter activities. Using transient reporter gene assays, we determined that Sp1 is a strong activator of these promoters, whereas Sp3 functions as a weak transactivator. We have identified multiple protein-binding sites in the proximal promoter sequences of these genes by footprinting analysis. Some of these sites are bound by Sp1 and Sp3, as demonstrated by gel-shift experiments using Sp1/Sp3-specific antibodies, permitting the demonstration that a differential role exists for Sp1 and Sp3 in the regulation of the activity of these promoters.

Transcriptional control by cell-cycle regulators: a review.

In eukaryotes, progression of the cell cycle is associated with periodic transcription activation/repression of growth-regulatory genes. We summarize here current knowledge and views on the role of critical cell-cycle regulators such as the retinoblastoma pocket family members and cyclin-dependent kinases in the regulation of gene transcription. In particular, we discuss here the role of specific cyclin-dependent kinase complexes in the regulation of basal transcription. Although the functional connections between transcription and cell-cycle regulators is far from being understood, recent progress has been made in connecting cell-cycle progression to dedicated components of the RNA polymerase II transcription apparatus complex.