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.

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.

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.

Control of RNA polymerase II activity by dedicated CTD kinases and phosphatases.

The elongation phase of eukaryotic transcription by RNA polymerase II (RNAPII) is an important target for regulation of gene expression. An interplay of positive and negative elongation factors determines the elongation activity of RNAPII in different promoters. The phosphorylation status of the carboxyl-terminal-domain (CTD) of the larger subunit of RNAPII appears to be the regulatory focus of different factors regulating mRNA processivity. The emerging model of the transcription cycle proposes that the phosphorylation state of the CTD is dynamic during elongation with different forms predominating at different stages of transcription. Shortly after initiation RNA polymerase II comes under the control of negative elongation factors and enters abortive elongation. Escape from the action of these negative controls requires the action of at least one positive elongation factor identified in the P-TEFb complex composed of the Cyclin-Dependent Kinase CDK9 and its regulatory subunit cyclin T. Finally, the requirement of CTD phosphatase activity, identified in the FCP1 protein, has been invoked as necessary to recycle the hypophosphorylated form of the RNA polymerase II competent to reinitiate the transcription cycle.

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.

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.

Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus.

We have isolated cDNA clones of the human c-myb mRNA that contain approximately 3.4 kilobases of the approximately 3.8-kilobase mRNA sequence. Nucleotide sequence analysis shows that the c-myb mRNA contains an open reading frame of 1920 nucleotides, which could encode a 72-kDa protein. The cDNA nucleotide sequence and the predicted amino acid sequence of the c-myb protein are highly homologous to the corresponding chicken and mouse proteins. In particular, a region toward the NH2 terminus of the protein containing a 3-fold tandem repeat of 51 residues is evolutionarily conserved and is the only region of homology with the Drosophila c-myb protein. This region may represent a functionally important structure, most likely the DNA-binding domain. cDNA clones have been used to isolate genomic clones and to define a preliminary intron/exon organization of the c-myb gene. Identification of 5' and 3' coding and noncoding exons indicates that the human c-myb locus spans a 40-kilobase region.

Mutational analysis of the human endogenous ERV9 proviruses promoter region.

ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. To define all the elements required for the correct transcription activity of the ERV9 promoter and to establish a precise correlation between the elements important for basal transcription, we have systematically analyzed the in vivo and in vitro transcriptional activity of many different ERV9 promoter mutants, including a series of linker-scanning mutations across the promoter region. We report here that the ERV9 promoter contains two elements controlling the selection of the correct start sites, a TATA box and an Inr-like region; the concerted action of both elements is necessary for faithful transcription. Finally, using a series of GAL4 protein fusion constructs in cotransfection experiments, we demonstrated that various transcription factors can synergistically induce a high level of transcription when bound to an ERV9 DNA promoter.

Sp3 represses transcription when tethered to promoter DNA or targeted to promoter proximal RNA.

Sp3 is a member of the Sp family of transcription factors, and it binds to the GC box with an affinity and specificity comparable with that of Sp1. Previous studies have shown that Sp3 repressed Sp1-mediated transcriptional activation, suggesting that Sp3 is an inhibitory member of the Sp family. The experiments described here demonstrate that Sp3 contains a portable repression domain that can function independently from the zinc finger DNA-binding domain. We found that the amino-terminal region of Sp3 tethered to a promoter DNA by connecting to a heterologous DNA-binding protein domain represses transcriptional activation by different positive regulators. Moreover, we determined that Sp3 targeted to a promoter-proximal RNA sequence acts as a transcriptional repressor. Taken together, our results suggest that Sp3 functions as a repressor by protein-protein interaction with components of the general transcription complex.

Retinoblastoma protein tethered to promoter DNA represses TBP-mediated transcription.

The retinoblastoma (RB) tumour suppressor protein negatively regulates cell proliferation by modulating transcription of growth-regulatory genes. Recruitment of Rb to promoters, by association with E2F complex or by fusion with heterologous DNA-binding domains, demonstrated that Rb represses directly transcription. Recent studies also suggest that the RB protein is able to repress gene transcription mediated by the RNA polymerase I and III. Since the TATA-binding protein (TBP) is an important component for transcription mediated by all three RNA polymerases, we have analysed the functional interaction between Rb and TBP in vivo in the context of RNA pol II-driven transcription. We demonstrated that in mammalian cells Rb tethered to promoter represses TBP-mediated activation in vivo, and Rb-mediated repression is reversed in the presence of the inhibition of histone deacetylase activity by trichostatin A (TSA).

Recruitment of human TBP selectively activates RNA polymerase II TATA-dependent promoters.

An increasing body of evidence suggests that eukaryotic activators stimulate polymerase II transcription by facilitating the assembly of the functional basal machinery at the promoter. Here we describe experiments that provide added support for the idea that recruitment of TATA-binding protein (TBP) is a rate-limiting step for transcription activation in mammalian cells. We found that, in human cell lines, recruitment of TBP to a promoter, as a GAL4-TBP fusion protein, can provide a substantial activation of transcription. Activation mediated by the hTBP, tethered to promoter DNA, is strictly dependent upon the presence of a functional TATA element, and it directs faithful transcription initiation. Interestingly, GAL4-hTBP activation was not observed from initiator (Inr) -dependent TATA-less promoters. These results suggest that TBP binding to DNA is not a rate-limiting step for the initial stages of TFIID recruitment to initiator-dependent TATA-less promoters. Finally, we provide evidence that synergy between GAL4-hTBP and defined transcription domains is restricted to activators, such as VP16 and Tat, which are likely to function at steps subsequent to the TFIID recruitment. These findings strengthen the idea that recruitment of TBP represents an important mechanism of activation of TATA-dependent promoters, and on the other hand, they suggest that TBP-DNA interactions are largely dispensable for specific transcription of initiator dependent TATA-less promoters.

Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains.

Sp3 is a member of the Sp family of transcription factors and binds to DNA with affinity and specificity comparable to that of Sp1. We demonstrate that Sp3 is a bifunctional transcription factor that can both activate and repress transcription. Gene fusion experiments in mammalian cells demonstrate that the Sp3 activation potential is distributed over an extensive glutamine-rich N-terminal region, whereas the repressor activity has been mapped in a 72-amino acid region located at the 5' of the zinc finger DNA-binding domain. We demonstrated that the repression activity is strictly dependent on the context of the DNA-binding sites bound by Sp3. We found that Sp3 represses transcription of promoters bearing multiple GAL4 DNA-binding sites, whereas it activates isogenic reporters containing a single GAL4-binding site. Transfection experiments in Drosophila cells that lack endogenous Sp activity demonstrated that Sp3 does not possess an active repression domain that can function in insect cells, rather it is a weak transcriptional activator of the c-myc promoter. Our results strongly suggest that Sp3 is a dual-function regulator whose activity is dependent upon both the promoter and the cellular context.

Constitutive and IL-6-induced nuclear factors that interact with the human C-reactive protein promoter.

Transcription of the human C-reactive protein (CRP) gene is induced by interleukin-6 (IL-6) during acute inflammation. Important information for inducible CRP expression is located within the 90 bases preceding the transcriptional start site. We show that the CRP promoter contains two adjacent binding sites (beta and alpha) that interact with at least two hepatocyte-specific nuclear proteins, H-APF-1 and H-APF-2. Point mutations that abolish or reduce binding drastically affect the level of CRP gene expression. Binding to beta is identical when extracts from uninduced or IL-6-induced Hep3B cells are used. On the contrary, both quantitative and qualitative changes in the alpha binding can be detected with extracts from uninduced cells or from cells treated with IL-6 or IL-6 + cycloheximide. A synthetic promoter based on the multimerization of the beta-binding domain, but not of the alpha-domain, is highly inducible when transfected in hepatoma cells. These results are discussed in relation to the structure of the promoter region of other acute phase inducible genes.