Faculty Bibliography

The mammalian SWI/SNF-related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. The recruitment of these complexes to promoters remains poorly understood and may involve histone modifications or direct interactions with site-specific transcription factors or other cofactors. Here we report the isolation of two related but distinct cDNA clones, hOsa1 and hOsa2, that encode the largest subunits of human SWI/SNF. hOsa1 is identical to previously reported BAF250, and hOsa2 shares a high degree of sequence similarity with hOsa1. Mass spectrometric analysis, and immunoblotting with antibodies specific to hOsa1 or hOsa2 demonstrate the presence of both proteins in SWI/SNF-A but not in the related polybromo-BRG1-associated factors complex purified from HeLa cells. Co-precipitation studies indicate that hOsa1 and hOsa2 associate with BRG1 and hBRM through the C-terminal domain of hOsa. We define multiple domains within hBRM and BRG1 that interact with the hOsa C terminus. In cultured mammalian cells, hOsa1 and hOsa2 stimulate transcription by the glucocorticoid, estrogen, and androgen receptors. The glucocorticoid receptor-mediated activation is not observed with the C-terminal domain or with the hOsa2 polypeptide lacking the ARID DNA binding domain. These results suggest that hOsa1 and hOsa2 participate in promoting transcriptional activation by the steroid hormone receptors

The general transcription factor TFIID facilitates recruitment of the transcription machinery to gene promoters and regulates initiation of transcription by RNA polymerase II. hTAF(II)130, a component of TFIID, interacts with and serves as a coactivator for multiple transcriptional regulatory proteins, including Sp1 and CREB. A yeast two-hybrid screen has identified an interaction between hTAF(II)130 and heterochromatin protein 1 (HP1), a chromatin-associated protein whose function has been implicated in gene silencing. We find that hTAF(II)130 associates with HP1 in an isoform-specific manner: HP1alpha and HP1gamma bind to hTAF(II)130, but not HP1beta. In addition, we show that endogenous hTAF(II)130 and components of TFIID in HeLa nuclear extracts associate with glutathione S-transferase-HP1alpha and -HP1gamma. hTAF(II)130 possesses a pentapeptide HP1-binding motif, and mutation of the hTAF(II)130 HP1 box compromises the interaction of hTAF(II)130 with HP1. We demonstrate that Gal4-HP1 proteins interfere with hTAF(II)130-mediated activation of transcription. Our results suggest that HP1alpha and HP1gamma associate with hTAF(II)130 to mediate repression of transcription, supporting a new model of transcriptional repression involving a specific interaction between a component of TFIID and chromatin

The human growth hormone (GH) gene family consists of five tandemly arranged and highly related genes, including the chorionic somatomammotropins (CSs), at a single locus on chromosome 17. Despite striking homologies in promoter and flanking DNA sequences, the genes within this locus have different tissue-specific patterns of expression: GH-N is expressed almost exclusively in the somatotrophs of the anterior pituitary; the remaining genes, including CS-A, are expressed in placental syncytiotrophoblast. Previously we proposed that active repression of the placental gene promoters in pituitary GC cells is mediated by upstream 'P' sequences and, specifically, a 263 bp region containing two 'P' sequence elements (PSE-A and PSE-B) and corresponding factors (PSF-A and PSF-B). We have now examined the possibility that PSF-A and PSF-B are members of the nuclear factor (NF)-1 family. Transcripts of NF-1A, NF-1C and NF-1X, but not of NF-1B, were readily detected in GC cells. High-affinity binding of NF-1 to PSE-B, but not to PSE-A, was confirmed by competition of DNA-protein interactions by using NF-1 DNA elements and antibodies. Functionally, a NF-1 element was able to substitute for PSE-B as a promoter-specific repressor in GC cells after gene transfer. However, there was a difference in the magnitude of repression exerted by the NF-1 and PSF-B elements on the CS-A promoter and, with the use of mutations, this difference was shown to be consistent with variations in NF-1-binding sequences. These results indicate that PSF-B, but not PSF-A, is a member of the NF-1 family, which participates in the PSF complex and in the repression of the CS-A promoter in pituitary GC cells

Numerous studies have demonstrated that the hepatitis B virus HBx protein stimulates signal transduction pathways and may bind to certain transcription factors, particularly the cyclic AMP response element binding protein, CREB. HBx has also been shown to promote early cell cycle progression, possibly by functionally replacing the TATA-binding protein-associated factor 250 (TAF(II)250), a transcriptional coactivator, and/or by stimulating cytoplasmic signal transduction pathways. To understand the basis for early cell cycle progression mediated by HBx, we characterized the molecular mechanism by which HBx promotes deregulation of the G(0) and G(1) cell cycle checkpoints in growth-arrested cells. We demonstrate that TAF(II)250 is absolutely required for HBx activation of the cyclin A promoter and for promotion of early cell cycle transit from G(0) through G(1). Thus, HBx does not functionally replace TAF(II)250 for transcriptional activity or for cell cycle progression, in contrast to a previous report. Instead, HBx is shown to activate the cyclin A promoter, induce cyclin A-cyclin-dependent kinase 2 complexes, and promote cycling of growth-arrested cells into G(1) through a pathway involving activation of Src tyrosine kinases. HBx stimulation of Src kinases and cyclin gene expression was found to force growth-arrested cells to transit through G(1) but to stall at the junction with S phase, which may be important for viral replication

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration

The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) compose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAF(II)s also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAF(II) subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAF(II)130 required for TAF(II)-TAF(II) interactions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II)250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAF(II)130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF(II)250 complex reconstituted with or without additional TAF(II)s was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAF(II)130 stabilized binding of hTBP to the TATA box, whereas hTAF(II)100 had little effect. Thus, our biochemical analysis supports the notion that TAF(II)s possess distinct functions to regulate the activity of TFIID

The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAF(II)s). The central domain of human TAF(II)130 contains four glutamine-rich regions Q1-Q4 that interact with transcriptional activators such as Sp1 and CREB and mediate activation. We screened in yeast random point mutations introduced into Q1-Q4 against the Sp1 activation domain and obtained a distinct set of hTAF(II)130s with alterations in TAF(II)-activator interaction. Here we characterize functionally an hTAF(II)130 mutant containing a phenylalanine to serine change at position 311 (F311S) that is compromised in its ability to associate with Sp1B and CREB-N activation domains. Substitution of phenylalanine with tyrosine but not with isoleucine or tryptophan also reduced hTAF(II)130 interaction, suggesting that the hydrophobic character rather than the specific amino acid at this position is a key determinant of interaction. Deletion of nine amino acids (Delta9) surrounding Phe(311) abolished the interaction of hTAF(II)130 with Sp1. Overexpression of hTAF(II)130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Delta9 only partially suppressed Sp1-mediated activation. Thus, a short hydrophobic sequence motif encompassing Phe(311) in hTAF(II)130 represents a critical surface with which Sp1B interacts to activate transcription

The yeast SWI/SNF complex and its Drosophila and mammalian homologs are thought to control gene expression by altering chromatin structure, but the mechanism and specificity of this process are not fully understood. The Drosophila osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. We present genetic and biochemical evidence that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. We show evidence that some Brahma-containing complexes do not contain Osa and that Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex