Teijiro Aso

Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II

The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.

Homocysteine Induces Programmed Cell Death in Human Vascular Endothelial Cells through Activation of the Unfolded Protein Response

Severe hyperhomocysteinemia is associated with endothelial cell injury that may contribute to an increased incidence of thromboembolic disease. In this study, homocysteine induced programmed cell death in human umbilical vein endothelial cells as measured by TdT-mediated dUTP nick end labeling assay, DNA ladder formation, induction of caspase 3-like activity, and cleavage of procaspase 3. Homocysteine-induced cell death was specific to homocysteine, was not mediated by oxidative stress, and was mimicked by inducers of the unfolded protein response (UPR), a signal transduction pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum. Dominant negative forms of the endoplasmic reticulum-resident protein kinases IRE1α and -β, which function as signal transducers of the UPR, prevented the activation of glucose-regulated protein 78/immunoglobulin chain-binding protein and C/EBP homologous protein/growth arrest and DNA damage-inducible protein 153 in response to homocysteine. Furthermore, overexpression of the point mutants of IRE1 with defective RNase more effectively suppressed the cell death than the kinase-defective mutant. These results indicate that homocysteine induces apoptosis in human umbilical vein endothelial cells by activation of the UPR and is signaled through IRE1. The studies implicate that the UPR may cause endothelial cell injury associated with severe hyperhomocysteinemia.

Interaction of Elongation Factors TFIIS and Elongin A with a Human RNA Polymerase II Holoenzyme Capable of Promoter-specific Initiation and Responsive to Transcriptional Activators

Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongationin vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

The inducible elongin A elongation activation domain: structure, function and interaction with the elongin BC complex.

The elongin (SIII) complex strongly stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along the DNA. Elongin (SIII) is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which bind stably to each other to form a binary complex that interacts with elongin A and strongly induces its transcriptional activity. The elongin (SIII) complex is a potential target for negative regulation by the von Hippel‐Lindau (VHL) tumor suppressor protein, which is capable of binding stably to the elongin BC complex and preventing it from activating elongin A. Here, we identify an elongin A domain sufficient for activation of elongation and demonstrate that it is a novel type of inducible activator that targets the RNA polymerase II elongation complex and is evolutionarily conserved in species as distantly related as Caenorhabditis elegans and man. In addition, we demonstrate that both the elongin A elongation activation domain and the VHL tumor suppressor protein interact with the elongin BC complex through a conserved elongin BC binding site motif that is essential for induction of elongin A activity by elongin BC and for tumor suppression by the VHL protein.

The RNA polymerase II elongation complex.

The initiation stage of transcription by RNA polymerase II has long been regarded as the primary site for regulation of eukaryotic gene ex‐pression. Nevertheless, a growing body of evidence reveals that the RNA polymerase II elongation com‐plex is also a major target for regulation. Biochelmical studies are implicating an increasing number of transcription factors in the regulation of elongation, and these transcription factors are being found to function by a diverse collection of mechanises. Moreover, unexpected features of the structure and catalytic mechanism of RNA polymerase II are forcing a reconsideration of long‐held views on the jnechanics of some of the most basic aspects of polymerase function. In this review, we will describe recent insights into the structures and functions of RNA polymerase II and the transcription factors that control its activity during the elongation stage of eukaryotic messenger RNA synthesis.—Aso, T., Conaway, J. W., Conaway, R. C. The RNA polpnerase II elongation complex. FASEB J. 9, 1419‐1428(1995)

PHYSICAL INTERACTION AND FUNCTIONAL ANTAGONISM BETWEEN THE RNA POLYMERASE II ELONGATION FACTOR ELL AND P53

ELL was originally identified as a gene that undergoes translocation with the trithorax-likeMLL gene in acute myeloid leukemia. Recent studies have shown that the gene product, ELL, functions as an RNA polymerase II elongation factor that increases the rate of transcription by RNA polymerase II by suppressing transient pausing. Using yeast two-hybrid screening with ELL as bait, we isolated the p53 tumor suppressor protein as a specific interactor of ELL. The interaction involves respectively the transcription elongation activation domain of ELL and the C-terminal tail of p53. Through this interaction, ELL inhibits both sequence-specific transactivation and sequence-independent transrepression by p53. Thus, ELL acts as a negative regulator of p53 in transcription. Conversely, p53 inhibits the transcription elongation activity of ELL, suggesting that p53 is capable of regulating general transcription by RNA polymerase II through controlling the ELL activity. Elevated levels of ELL in cells resulted in the inhibition of p53-dependent induction of endogenous p21 and substantially protected cells from p53-mediated apoptosis that is induced by genotoxic stress. Our observations indicate the existence of a mutually inhibitory interaction between p53 and a general transcription elongation factor ELL and raise the possibility that an aberrant interaction between p53 and ELL may play a role in the genesis of leukemias carrying MLL-ELL gene translocations.

Molecular cloning of the cDNA coding for proline-rich protein (PRP): Identity of PRP as C4b-binding protein

Proline-rich protein (PRP) is a plasma protein with a high proportion of proline residues and possessing lipid-binding properties. In order to clarify its structure, a human liver cDNA library was screened using anti-PRP antiserum. Several overlapping phage cDNA clones were isolated and the total nucleotide sequence of the cDNA, 2178 bp in length, was analyzed. The amino acid composition of PRP deduced from the cDNA was essentially the same as that reported for PRP. In a homology search, the cDNA sequence was almost completely the same as the previously reported cDNA sequence of C4b-binding protein. Furthermore, the reported molecular weights of the two proteins under both reduced and unreduced conditions were quite alike. These findings indicate that PRP is identical with C4bp.

Transcription syndromes and the role of RNA polymerase II general transcription factors in human disease.

Messenger RNA synthesis is a major site for the regulation of gene expression. Eukaryotic messenger RNA synthesis is catalyzed by multisubunit RNA polymerase II (1–3) and proceeds via multiple stages, which are designated preinitiation, initiation, elongation, and termination and which have come to be referred to collectively as the transcription cycle (Fig. 1). The past decade was a watershed for biochemical studies of eukaryotic messenger RNA synthesis. A diverse collection of transcription factors and other nuclear proteins that govern the activity of RNA polymerase II during messenger RNA synthesis was identified and characterized, and unprecedented progress in several key research areas has provided a deeper understanding of the biochemical mechanisms underlying many aspects of eukaryotic transcriptional regulation. First, major breakthroughs in investigations of the structures of eukaryotic protein-coding genes and the role of chromatin in the regulation of their expression were achieved. Chromatin proteins, such as histones and HMG proteins, were found to play crucial roles in gene regulation by packaging genes into inactive or transcriptionally repressed configurations (4–9). Second, many DNA binding transactivators that interact specifically with upstream promoter elements and enhancer sequences located in the promoter-regulatory regions of genes were isolated, classified according to their structures, and found to regulate the expression of specific genes or gene families by controlling the rate of initiation (10) and, as shown more recently, the efficiency of elongation by RNA polymerase II (11–13). Third, chromatin remodeling proteins, such as the multisubunit SWI/SNF (14, 15) and NURF (16, 17) complexes, were discovered and found to play key roles in transcriptional activation by promoting conversion of regions of inactive chromatin into transcriptionally active, open chromatin, thereby allowing DNA binding transactivators and RNA polymerase II access to the promoter-regulatory regions of genes (4, 18–23). Fourth, coactivators, such as the SRB-containing mediator complex (2, 3, 24, 25), CREB binding protein (CBP) 1

A TATA sequence-dependent transcriptional repressor activity associated with mammalian transcription factor IIA.

In the process of characterizing cellular proteins that modulate basal transcription by RNA polymerase II, we identified a novel repressor activity specific for promoters containing consensus TATA boxes. This activity strongly represses TATA‐binding protein (TBP)‐dependent transcription initiation from core promoter elements containing a consensus TATA sequence, but activates TBP‐dependent transcription from core promoter elements lacking a consensus TATA sequence. Purification of this activity to near homogeneity from rat liver nuclear extracts led to the surprising discovery that it co‐purifies closely with mammalian transcription factor IIA (TFIIA). The close association of TATA sequence‐dependent transcriptional repressor activity with TFIIA adds a new and unexpected dimension to the already complex picture of this factors function in transcription by RNA polymerase II.