Monoclonal Antibodies Directed against the Amino-Terminal Domain of Human TBP Cross-React with TBP from Other Species

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of FBI-1, a Cellular Factor That Binds to the Human Immunodeficiency Virus Type 1 Inducer of Short Transcripts

Peßler

Pendergrast

Hernandez

1997

“…Anti-SNAP43 (CS48) and anti-TBP (SL2) antibodies have been described previously (16,24). The anti-Bdp1 (CS913), anti-Brf1/Brf2 (CS1043), and anti-RNA polymerase III (CS377) antibodies were gifts from Nouria Hernandez (Cold Spring Harbor Laboratory).…”

Section: Methodsmentioning

confidence: 99%

Distinct Mechanisms for Repression of RNA Polymerase III Transcription by the Retinoblastoma Tumor Suppressor Protein

Hirsch

Jawdekar²,

Lee

et al. 2004

The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAP C and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.The retinoblastoma (RB) protein controls the cell cycle, differentiation, apoptosis, and general growth. Typically, RB functions to repress RNA polymerase II transcription of genes that act at control points in these key cellular processes (10,11,14). The classical understanding of RB function is exemplified by its ability to control the cell cycle (reviewed in reference 45). For example, RB can target genes that are required to enter into the S phase of the cell cycle and whose promoters contain binding sites for the E2F family of transcriptional activator proteins (4,17,31,46). RB can directly block the transactivation function of E2F (34) or alter chromatin structure to repress transcription by recruiting coregulatory proteins, such as histone deacetylases (HDACs) (2, 25, 26), ATP-dependent chromatin remodeling complexes (39,43,53), and histone methyltransferases (32).RB is related to two other pocket domain proteins, p107 and p130, that have also been shown to repress E2F target genes and inhibit cell growth when overexpressed (reviewed in references 12 and 29). The different pocket domain proteins are utilized at distinct phases of the cell cycle to control transcription of E2F target genes (27, 42), suggesting that RB, p107, and p130 have specialized nonredundant functions for controlling gene expression. Interestingly, the minimal regions of RB required for RNA polymerase II transcriptional repression are different from those essential for tumor suppression. RB contains 928 amino acids and can be functionally divided into at least three regions: the N-terminal region between amino acids 1 and 378, ...

“…All rabbit polyclonal sera were produced by CoCalico Biologicals (Reamstown, Pa.). The simian virus 40 T-antigenspecific antibody 419 (22) and the TBP-specific monoclonal antibody SL8 (42) were provided by Ed Harlow and Nouria Hernandez, respectively. The BRG1-and INI-1-specific polyclonal antibodies (50) used for Fig.…”

Section: Methodsmentioning

confidence: 99%

“…NM1 (lane 4) is a monoclonal antibody raised against human p300 that cross-reacts with CBP and p270 (13). SL8 (lane 5) is a monoclonal antibody directed against human TBP (42). The specifities of the antibodies used to probe the nitrocellulose blot are indicated at the right.…”

Section: Figmentioning

confidence: 99%

p300/CREB Binding Protein-Related Protein p270 Is a Component of Mammalian SWI/SNF Complexes

Dallas

Cheney

Liao

et al. 1998

p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex.The cellular protein p300 is a direct target of the transforming functions of the adenovirus E1A gene (19, 51) and as such is implicated in the regulation of both cell cycle-specific and tissue-specific gene expression (18,27,38,43,46,52,54). p300 is highly homologous (about 64% identical) to the cyclic AMP response element binding protein (CREB) coactivator, CBP (CREB binding protein) (5,11,17,30,34). Both p300 and CBP are present in intracellular complexes with the TATA binding protein (TBP) (1, 13). Both act as cofactors for p53 (6,21,33,44) and nuclear hormone receptors (9,24,28). Both also contain intrinsic and associated histone acetyltransferase (HAT) activity (39, 53), suggesting that chromatin modification is an essential part of their role in regulating transcription.A recent detailed characterization of a panel of antibodies raised against a mixture of native p300 and CBP revealed the existence of a 270-kDa cellular protein, distinct from p300 and CBP but sharing at least two independent antigenic determinants with p300 (13). Four of the eleven antibodies in the panel recognize p270. The subset of p300/CBP-derived antibodies that recognizes p270 consistently coprecipitates a series of cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Typical of these is the antibody designated NM1, whose immunoprecipitation pattern is shown in Fig. 1. TBPspecific antibodies coprecipitate a subset of these proteins including p300, CBP, and the phosphoprotein species indicated in Fig. 1 as p64 and p59 (1, 13). Because the TBP-specific antibodies do not coprecipitate all of the p300 family-associated proteins, it is likely that the array of proteins seen in Fig. 1 represents more than one intracellular complex.We have now identified four of the remaining p300/CBP/ p270-associated proteins as members of another important cellular complex: the mammalian SWI/SNF complex. The 190-kDa protein visible in the p300-related complex is BRG1, the human homolog of the yeast transcriptional activator, SWI2/ SNF2. The 170-and 155-k...