Selective gene regulation by SWI/SNF-related chromatin remodeling factors.

Chromatin is a highly dynamic structure that plays a key role in the orchestration of gene expression patterns during cellular differentiation and development. The packaging of DNA into chromatin generates a barrier to the transcription machinery. The two main strategies by which cells alleviate chromatin-mediated repression are through the action of ATP-dependent chromatin remodeling complexes and enzymes that covalently modify the histones. Various signaling pathways impinge upon the targeting and activity of these enzymes, thereby controlling gene expression in response to physiological and developmental cues. Chromatin structure also underlies many so-called epigenetic phenomena, leading to the mitotically stable propagation of differential expression of genetic information. Here, we will focus on the role of SWI/SNF-related ATP-dependent chromatin remodeling complexes in developmental gene regulation. First, we compare different models for how remodelers can act in a gene-selective manner, and either cooperate or antagonize other chromatin-modulating systems in the cell. Next, we discuss their functioning during the control of developmental gene expression programs.

A homeotic mutation in the trithorax SET domain impedes histone binding.

Trithorax (TRX) is a Drosophila SET domain protein that is required for the correct expression of homeotic genes. Here, we show that the TRX SET domain efficiently binds to core histones and nucleosomes. The primary target for the SET domain is histone H3 and binding requires the N-terminal histone tails. The previously described trx(Z11) mutation changes a strictly conserved glycine in the SET domain to serine and causes homeotic transformations in the fly. We found that this mutation selectively interferes with histone binding, suggesting that histones represent a critical target during developmental gene regulation by TRX.

Chromatin silencing and activation by Polycomb and trithorax group proteins.

The Polycomb group (PcG) of repressors and the trithorax group (trxG) of activators maintain the correct expression of several key developmental regulators, including the homeotic genes. PcG and trxG proteins function in distinct multiprotein complexes that are believed to control transcription by changing the structure of chromatin, organizing it into either a 'closed' or an 'open' conformation. The hallmark of gene regulation by PcG/trxG proteins is that it can lead to a mitotically stable pattern of gene expression, often referred to as epigenetic regulation. Although much remains to be learned, recent studies have provided insights into how this epigenetic switch is set, how PcG/trxG proteins might be linked to cis-acting DNA elements and what potential mechanisms underlie stable inheritance of gene expression status over multiple cell divisions. Finally, the study of the evolutionarily conserved PcG/trxG factors has recently gained additional urgency with the realization that they play a pertinent role in certain human cancers.

Biochemical analyses of the AF10 protein: the extended LAP/PHD-finger mediates oligomerisation.

Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region.

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste.

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator.

Basal transcription factor TFIID comprises the TATA-box-binding protein, TBP, and associated factors, the TAF(II)s. Previous studies have implicated TAF(II)250 and TAF(II)150 in core promoter selectivity of RNA polymerase II. Here, we have used a random DNA binding site selection procedure to identify target sequences for these TAFs. Individually, neither TAF(II)250 nor TAF(II)150 singles out a clearly constrained DNA sequence. However, a TAF(II)250-TAF(II)150 complex selects sequences that match the Initiator (Inr) consensus. When in a trimeric complex with TBP, these TAFs select Inr sequences at the appropriate distance from the TATA-box. Point mutations that inhibit binding of the TAF(II)250-TAF(II)150 complex also impair Inr function in reconstituted basal transcription reactions, underscoring the functional relevance of Inr recognition by TAFs. Surprisingly, the precise DNA sequence at the start site of transcription influences transcriptional regulation by the upstream activator Sp1. Finally, we found that TAF(II)150 specifically binds to four-way junction DNA, suggesting that promoter binding by TFIID may involve recognition of DNA structure as well as primary sequence. Taken together, our results establish that TAF(II)250 and TAF(II)150 bind the Inr directly and that Inr recognition can determine the responsiveness of a promoter to an activator.

Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology.

The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodelling and human cancers. Here, we study the role of the POZ domain of the GAGA transcription factor in promoter recognition. Natural target promoters for GAGA typically contain multiple GAGA-binding elements. Our results show that the POZ domain mediates strong co-operative binding to multiple sites but inhibits binding to single sites. Protein cross-linking and gel filtration chromatography experiments established that the POZ domain is required for GAGA oligomerization into higher order complexes. Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy revealed that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. Our results indicate a novel mode of DNA binding by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. We suggest a model in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation.

A conserved motif in goosecoid mediates groucho-dependent repression in Drosophila embryos.

Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, which mediates recruitment of the Groucho (Gro) corepressor to target promoters. We recently showed that Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. Here, we characterize a repressor domain from the Goosecoid (Gsc) developmental regulator that includes an eh1/GEH-like motif. We demonstrate that this domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and that repression by GscR requires Gro function. GscR and Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, we investigate whether Gro is part of a stable multiprotein complex in the nucleus. Our results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes.

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster.

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

TAFs mediate transcriptional activation and promoter selectivity.

The TATA-binding protein (TBP)-associated factors (TAFs) of TFIID play a central role in RNA polymerase II transcriptional regulation. Some TAFs can function as co-activators that mediate the activation signal from enhancer-bound regulators. In addition, interactions between selected TAFs and core elements direct promoter selectivity by RNA polymerase II.

CIF, an essential cofactor for TFIID-dependent initiator function.

The core promoters for mammalian protein-coding genes often contain a TATA box, an initiator (Inr) element, or both of these control elements. The TFIID complex is essential both for TATA activity and for the activity of a common class of Inr elements characterized by an approximate consensus sequence PyPyA+1NT/APyPy. Although the complete set of proteins required for basal TATA-mediated transcription has been established, the requirements for TFIID-dependent Inr activity remain undefined. In this study we set out to reconstitute Inr activity with purified and recombinant general transcription factors. For this analysis, Inr activity was measured as the ability of an Inr to enhance the strength of a core promoter containing an upstream TATA box. Inr activity was not detected in reactions containing TFIIB, RAP30, RAP74, RNA polymerase II, and either TBP or TFIID, even though these factors were sufficient for TATA-mediated transcription from supercoiled templates. By use of a complementation assay, a factor that imparts Inr activity was identified. This factor, named CIF, stimulated Inr activity in reactions containing the TFIID complex, but activity was not detected with TBP. Further characterization of CIF suggested that it contains multiple components. Functional and immunological experiments demonstrated that one of the CIF components is the mammalian homolog of Drosophila TAF(II)150, which is not tightly associated with mammalian TFIID. These results reveal significant differences in the factor requirements for basal TATA and Inr activity. Further elucidation of these differences is likely to explain the need for the core promoter heterogeneity found within protein-coding genes.

Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II.

The mechanisms that govern core promoter recognition and basal transcription efficiency remain poorly understood. Here, we have assessed the potential role of TAFs and the TFIID complex in directing basal promoter function. Reconstituted transcription reactions revealed the ability of TFIID versus TBP to discriminate between distinct core promoters. A comparison of different partial TBP-TAF assemblages established that a trimeric TBP-TAFII250-TAFII150 complex is minimally required for efficient utilization of the initiator and downstream promoter elements. Depending on the promoter structure, TAFs can increase or decrease the stability of TFIID-promoter interactions. These findings suggest that TAFs play a critical role in promoter selectivity and transcription regulation through direct contacts with core promoter elements.

Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation.

Drosophila transcription factor IIA (TFIIA) is composed of three subunits (30, 20, and 14 kD) that function during initiation of transcription. We reported previously the characterization of cDNAs that encode a precursor (dTFIIA-L) of the Drosophila TFIIA 30- and 20-kD subunits. In the absence of the smallest subunit, dTFIIA-S (14 kD), the unprocessed large subunit failed to exhibit any detectable promoter binding or transcriptional activity. Here, we report the molecular cloning and expression of dTFIIA-S, which has allowed the assembly of holo-dTFIIA (dTFIIA-L/S). Subunit interaction studies indicate that dTFIIA-S binds to an amino-terminal domain of dTFIIA-L, which likely corresponds to the endogenous 30-kD processed species. In addition, both dTFIIA-S and the carboxy-terminal domain of dTFIIA-L, which corresponds to the 20-kD species, independently interact weakly with the TATA-binding protein (TBP). In contrast, the holo-dTFIIA (L/S) binds TBP with high affinity. The dTFIIA-L/S complex also binds cooperatively with TBP to TATA box DNA sequences, generating an extended DNase footprint pattern. The reconstituted holo-dTFIIA is able to stimulate basal transcription of several core promoter templates. Interestingly, dTFIIA-L/S is also able to significantly enhance transcriptional activation by upstream transcription factors including Sp1, VP16, and NTF-1. These results suggest that dTFIIA is a multifunctional transcription factor capable of influencing DNA binding as well as interactions with the basal machinery, thereby enhancing activator-dependent transcription.

Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators.

We previously reported that transcriptional regulators can bind selected TAF subunits of the TFIID complex. However, the specificity and function of individual TAFs in mediating transcriptional activation remained unknown. Here we report the in vitro assembly and transcriptional properties of TBP-TAF complexes reconstituted from the nine recombinant subunits of Drosophila TFIID. A minimal complex containing TBP and TAFII250 directs basal but not activator-responsive transcription. By contrast, reconstituted holo-TFIID supports activation by an assortment of activators. The activator NTF-1, which binds TAFII150, stimulates transcription with a complex containing only TBP, TAFII250, and TAFII150, whereas Sp1 binds and additionally requires TAFII110 for activation. Interestingly, TAFII150 enhances Sp1 activation even though this subunit does not bind directly to Sp1. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggest that TAFs perform multiple functions during activation.

Two independent pathways for transcription from the MMTV promoter.

The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.

Drosophila TAFII150: similarity to yeast gene TSM-1 and specific binding to core promoter DNA.

In Drosophila and human cells, the TATA binding protein (TBP) of the transcription factor IID (TFIID) complex is tightly associated with multiple subunits termed TBP-associated factors (TAFs) that are essential for mediating regulation of RNA polymerase II transcription. The Drosophila TAFII150 has now been molecularly cloned and biochemically characterized. The deduced primary amino acid sequence of dTAFII150 reveals a striking similarity to the essential yeast gene, TSM-1. Furthermore, like dTAFII150, the TSM-1 protein is found associated with the TBP in vivo, thus identifying the first yeast homolog of a TAF associated with TFIID. Both the product of TSM-1 and dTAFII150 bind directly to TBP and dTAFII250, demonstrating a functional similarity between human and yeast TAFs. Surprisingly, DNA binding studies indicate that purified recombinant dTAFII150 binds specifically to DNA sequences overlapping the start site of transcription. The data demonstrate that at least one of the TAFs is a sequence-specific DNA binding protein and that dTAFII150 together with TBP are responsible for TFIID interactions with an extended region of the core promoter.

NMR studies of the POU-specific DNA-binding domain of Oct-1: sequential 1H and 15N assignments and secondary structure.

The 1H and 15N resonances of the POU-specific DNA-binding domain of transcription factor Oct-1 have been assigned sequentially using two-dimensional homo- and heteronuclear NMR techniques, as well as three-dimensional heteronuclear NMR techniques, including TOCSY, 2D NOE, and NOESY-HMQC experiments. A number of typical short- and medium-range NOE contacts, as well as amide proton exchange data, gave evidence for the presence of four alpha-helices, in the peptide segments 1-19, 23-34, 40-49, and 54-71, which are connected by short loops of irregular structure. Interestingly, the second helix contains three glycine residues and the fourth helix a proline in the middle of the helix. Although the regular pattern of hydrogen bonds in the fourth helix is interrupted, due to the absence of an amide proton in proline, the helix is remarkably stable. All four helices are amphipathic, which suggests a packing of the apolar sides of the helices in the folded structure of the protein.