James R. Lundblad

The Human T-cell Leukemia Virus-1 Transcriptional Activator Tax Enhances cAMP-responsive Element-binding Protein (CREB) Binding Activity through Interactions with the DNA Minor Groove

Tax-1, the transcriptional activation protein of human T-cell leukemia virus-1, increases transcription from the human T-cell leukemia virus-1 long terminal repeat and specific cellular promoters through interactions with cellular DNA-binding proteins. The Tax response elements (TxREs) of the long terminal repeat resemble cAMP response elements (CREs), the target of cAMP-responsive element-binding protein (CREB). CREB binds the TxRE with reduced affinity; however, the interaction is specifically enhanced by Tax. Using a fluorescence quenching method, we determined that CREB dimerizes in the absence of DNA, and that Tax does not enhance dimerization. DNA footprinting of the TxRE with 1,10-phenanthroline-copper complex demonstrates that Tax contacts DNA and extends the footprint of CREB to GC-rich sequences flanking the core CRE-like element. The minor groove-binding drug chromomycin A3, but not distamycin A, disrupted Tax-enhanced CREB binding to the TxRE. Substitution of the guanine-rich sequences flanking the core of the TxRE with inosine residues also blocked the Tax effect. Finally, the IC-substituted TxRE binds CREB with increased affinity, suggesting flanking DNA influences the binding of CREB to the core CRE-like element. These data indicate that Tax does not regulate DNA binding of CREB by altering dimerization, but rather enhances DNA binding by additionally interacting with the minor groove of flanking DNA sequences.

Differential Activation of Viral and Cellular Promoters by Human T-cell Lymphotropic Virus-1 Tax and cAMP-responsive Element Modulator Isoforms

We have previously proposed that cAMP-responsive element-binding protein (CREB) activity is stimulated by human T-cell lymphotropic virus-1 (HTLV-1) Tax through two mechanisms that are differentially dependent upon CREB phosphorylation. We have tested this model by examining how Tax affects transcriptional activation mediated by the cAMP-responsive element (CRE) modulator (CREM). The CREM proteins are highly homologous to CREB, particularly in their DNA-binding domains and the kinase-inducible domain (KID), a region that interacts with the coactivator CREB-binding protein (CBP) in a phosphorylation-dependent manner. Despite this similarity, most CREM isoforms are transcriptional repressors. CREMα lacks the glutamine-rich domains found in CREB that are essential for transcriptional activation. We show that the normally repressive CREMα activates the HTLV-1 and cellular CREs in the presence of Tax; activation of the viral element is phosphorylation-independent, and activation of the cellular CRE is phosphorylation-dependent. CREMΔ(C-G) lacks both the KID and the glutamine-rich regions. This isoform activates the HTLV-1 long terminal repeat in a phosphorylation-independent manner, but does not activate the cellular CRE. This study suggests that Tax, interacting with the basic/zipper region of CREM, recruits CBP to the viral promoter. Tax activation of the cellular CRE depends on the KID and its ability to interact with CBP in a phosphorylation-dependent manner.

The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest.

Poly(ADP)-ribose polymerase (PARP) inhibitors modify the enzymatic activity of PARP1/2. When certain PARP inhibitors are used either alone or in combination with DNA damage agents they may cause a G2/M mitotic arrest and/or apoptosis in a susceptible genetic context. PARP1 interacts with the cell cycle checkpoint proteins Ataxia Telangectasia Mutated (ATM) and ATM and Rad3-related (ATR) and therefore may influence growth arrest cascades. The PARP inhibitor PJ34 causes a mitotic arrest by an unknown mechanism in certain cell lines, therefore we asked whether PJ34 conditionally activated the checkpoint pathways and which downstream targets were necessary for mitotic arrest. We found that PJ34 produced a concentration dependent G2/M mitotic arrest and differentially affected cell survival in cells with diverse genetic backgrounds. p53 was activated and phosphorylated at Serine15 followed by p21 gene activation through both p53-dependent and -independent pathways. The mitotic arrest was caffeine sensitive and UCN01 insensitive and did not absolutely require p53, ATM or Chk1, while p21 was necessary for maintaining the growth arrest. Significantly, by using stable knockdown cell lines, we found that neither PARP1 nor PARP2 was required for any of these effects produced by PJ34. These results raise questions and cautions for evaluating PARP inhibitor effectiveness, suggesting whether effects should be considered not only on PARPs diverse ADP-ribosylation independent protein interactions but also on homologous proteins that may be producing either overlapping or distinct effect.

C-terminal binding protein and poly(ADP)ribose polymerase 1 contribute to repression of the p21 waf1/cip1 promoter

Transcriptional repression by the C-terminal binding protein (CtBP) is proposed to require nicotinamide adenine dinucleotide dehydrogenase (NAD(H). Previous studies have implicated CtBP in transcriptional repression of the p21waf1/cip1 gene. Similarly, the NAD-dependent poly(adenosine diphosphate)ribose polymerase 1 (PARP1) may affect p21 expression via its NAD-dependent enzymatic activity; we therefore asked if PARP1 and CtBP were functionally linked in regulating p21 transcription. We found that restraint of basal p21 transcription requires both CtBP and PARP1. PARP inhibition attenuated activation of p21 transcription by both p53-independent and p53-dependent processes, in a CtBP-dependent manner. CtBP1+2 or PARP1+2 knockdown partially activated p21 gene expression, suggesting relief of a corepressor function dependent on both proteins. We localized CtBP-responsive repression elements to the proximal promoter region, and found ZBRK1 overexpression could also overcome DNA damage-dependent, but not p53-dependent activation through this region. By chromatin immunoprecipitation we find dismissal of CtBP from the proximal promoter following DNA-damage, and that PARP1 associates with a CtBP corepressor complex in nuclear extracts. We propose a model in which both CtBP and PARP functionally interact in a corepressor complex as components of a molecular switch necessary for p21 repression, and following DNA damage signals activation of p21 transcription by corepressor dismissal and co-activator recruitment.

Nicotinamide Adenine Dinucleotide Induced Multimerization of the Co-repressor CtBP1 relies on a Switching Tryptophan

Background: C-terminal binding protein 1 (CtBP1) assembles into a tetrameric transcriptional co-repressor but how it directs gene expression is not clear. Results: CtBP1 requires NAD(H) for transition into multimers. Its biochemical activities are separable from transcriptional repression. Conclusion: Tryptophan 318 permits CtBP1 to first dimerize and then tetramerize after the binding of NAD(H). Significance: Clarification of how CtBP1 tetramerizes will permit development of CtBP inhibitors to target oncogenesis. The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD+ binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp318) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD+ binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp318 switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.

Structural Determinants of CtBP Function

The structural characteristics of the CtBP family of transcriptional corepressors suggest an additional role for coenzyme nicotinamide adenine dinudeotide in the repression of gene expression. Remarkably, CtBP orthologues are unique among transcriptional regulators in that they display striking primary sequence and structural similarity to the D-isomer specific 2-hydroxyacid dehydrogenase class of enzymes. Recent structural studies of rat CtBP/BARS and human CtBPl provide insight into the role of pyridine dinucleotide binding in regulation of CtBP quaternary structure, and corepression activity through association with -PXDLS-containing targets.