A transcriptional switch mediated by cofactor methylation.

We describe a molecular switch based on the controlled methylation of nucleosome and the transcriptional cofactors, the CREB-binding proteins (CBP)/p300. The CBP/p300 methylation site is localized to an arginine residue that is essential for stabilizing the structure of the KIX domain, which mediates CREB recruitment. Methylation of KIX by coactivator-associated arginine methyltransferase 1 (CARM1) blocks CREB activation by disabling the interaction between KIX and the kinase inducible domain (KID) of CREB. Thus, CARM1 functions as a corepressor in cyclic adenosine monophosphate signaling pathway via its methyltransferase activity while acting as a coactivator for nuclear hormones. These results provide strong in vivo and in vitro evidence that histone methylation plays a key role in hormone-induced gene activation and define cofactor methylation as a new regulatory mechanism in hormone signaling.

Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment.

The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. We show that unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites.

Site-specific acetylation by p300 or CREB binding protein regulates erythroid Krüppel-like factor transcriptional activity via its interaction with the SWI-SNF complex.

Recruitment of modifiers and remodelers to specific DNA sites within chromatin plays a critical role in controlling gene expression. The study of globin gene regulation provides a convergence point within which to address these issues in the context of tissue-specific and developmentally regulated expression. In this regard, erythroid Krüppel-like factor (EKLF) is critical. EKLF is a red cell-specific activator whose presence is crucial for establishment of the correct chromatin structure and high-level transcriptional induction of adult beta-globin. We now find, by metabolic labeling-immunoprecipitation experiments, that EKLF is acetylated in the erythroid cell. EKLF residues acetylated by CREB binding protein (CBP) in vitro map to Lys-288 in its transactivation domain and Lys-302 in its zinc finger domain. Although site-specific DNA binding by EKLF is unaffected by the acetylation status of either of these lysines, directed mutagenesis of Lys-288 (but not Lys-302) decreases the ability of EKLF to transactivate the beta-globin promoter in vivo and renders it unable to be superactivated by coexpressed p300 or CBP. In addition, the acetyltransferase function of CBP or p300 is required for superactivation of wild-type EKLF. Finally, acetylated EKLF has a higher affinity for the SWI-SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro. These results demonstrate that the acetylation status of EKLF is critical for its optimal activity and suggest a mechanism by which EKLF acts as an integrator of remodeling and transcriptional components to alter chromatin structure and induce adult beta-globin expression within the beta-like globin cluster.

Functional selectivity of recombinant mammalian SWI/SNF subunits.

The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood. We show that mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro. The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled beta-globin promoter. Interestingly, we find that two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain. In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB. We conclude that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling.

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology.

The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA.

LXIII Cold Spring Harbor Symposium on Quantitative Biology: Mechanisms of Transcription, 3-8 June 1998.

A new perspective is emerging in the transcription field towards understanding gene regulation not only at its most fundamental level but also in the context of chromatin, nuclear compartmentalization, and physiological processes. This direction is being fueled by several key observations. Among them is the discovery of multi-protein complexes whose components reveal a link between gene activity, nuclear structure, and cellular signaling pathways. This information will no doubt be extended by identifying expanded regulatory circuitry using the microchip oligonucleotide array technology. In addition to elucidating the regulatory consequences of these intricate connections, another frontier will be to analyze gene expression within chromosomes. This requires deciphering the mechanism of action of a variety of DNA elements that create a genetic domain such as locus control regions, distal enhancers, insulators, silencers, and matrix attachment regions. Hopefully, with the development of new assays these elements can be as rigorously defined as promoters have been. We can also look forward to capturing critical transcriptional processes by increasingly refined structural analyses. Thus, the scope of problems being addressed in gene regulation has been greatly expanded and the opportunity exists to answer very sophisticated questions in the future.

A SWI/SNF-related chromatin remodeling complex, E-RC1, is required for tissue-specific transcriptional regulation by EKLF in vitro.

Erythroid Krüppel-like factor (EKLF) is necessary for stage-specific expression of the human beta-globin gene. We show that EKLF requires a SWI/SNF-related chromatin remodeling complex, EKLF coactivator-remodeling complex 1 (E-RC1), to generate a DNase I hypersensitive, transcriptionally active beta-globin promoter on chromatin templates in vitro. E-RC1 contains BRG1, BAF170, BAF155, and INI1 (BAF47) homologs of yeast SWI/SNF subunits, as well as a subunit unique to higher eukaryotes, BAF57, which is critical for chromatin remodeling and transcription with EKLF. E-RC1 displays functional selectivity toward transcription factors, since it cannot activate expression of chromatin-assembled HIV-1 templates with the E box-binding protein TFE-3. Thus, a member of the SWI/SNF family acts directly in transcriptional activation and may regulate subsets of genes by selectively interacting with specific DNA-binding proteins.

Transcription of chromatin: these are complex times.

Transcription of chromatin-packaged genes involves highly regulated changes in nucleosomal structure that control DNA accessibility. Two systems that facilitate these changes are ATP-dependent chromatin remodeling complexes and enzymatic complexes which control histone acetylation and deacetylation. Recent studies provide insight on the role of these remodeling machines and specific transcription factors in the expression of viral, inducible, and tissue-restricted genes.

Pre-operative fasting for paediatric anaesthesia. A survey of current practice.

There has recently been much debate about pre-operative fasting for paediatric anaesthesia. There is no consensus about the optimum fasting times for children undergoing elective surgery. In order to establish a standard for paediatric pre-operative fasting times, we undertook a postal survey, targeting members of the Association of Paediatric Anaesthetists resident in the United Kingdom and Ireland in 1995. One hundred and sixty-three questionnaires were dispatched, 131 (80%) were returned and 110 (67%) were complete. The results show that the following guidelines for duration of fast are acceptable to the majority of respondents-neonates: 2 h for clear fluids, 4 h for breast and formula milk; infants: 2 h for clear fluids, 4 h for breast milk, 6 h for formula milk and solids; children: 2 h for clear fluids, 6 h for milk and solids. We suggest that these times be used as guidelines and audited for pre-operative fasting in paediatric anaesthesia.

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro.

Long-range promoter-enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken betaA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein-protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

An HMG I/Y-containing repressor complex and supercoiled DNA topology are critical for long-range enhancer-dependent transcription in vitro.

The 3' enhancer of the T cell receptor alpha-chain (TCR alpha) gene directs the tissue- and stage-specific expression and V(D)J recombination of this gene locus. Using an in vitro system that reproduces TCR alpha enhancer activity efficiently, we show that long-range promoter-enhancer regulation requires a T cell-specific repressor complex and is sensitive to DNA topology. In this system, the enhancer functions to derepress the promoter on supercoiled, but not relaxed, templates. We find that the TCR alpha promoter is inactivated by a repressor complex that contains the architectural protein HMG I/Y. In the absence of this repressor complex, expression of the TCR alpha gene is completely independent of the 3' enhancer and DNA topology. The interaction of the T cell-restricted protein LEF-1 with the TCR alpha enhancer is required for promoter derepression. In this system, the TCR alpha enhancer increases the number of active promoters rather than the rate of transcription. Thus, long-range enhancers function in a distinct manner from promoters and provide the regulatory link between repressors, DNA topology, and gene activity.

NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.

The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

Regulated expression of the beta-globin gene locus in synthetic nuclei.

Regulated gene expression within a complex chromosomal locus requires multiple nuclear processes. We have analyzed the transcriptional properties of the cloned chick beta-globin gene family when assembled into synthetic nuclei made by use of Xenopus egg extracts. Assembly in an erythroid protein environment correctly recapitulates tissue-specific chromatin structure and long-range promoter-enhancer interaction within the chromosomal locus, resulting in beta-globin gene activation. Nucleosome-repressed beta-globin templates can be transcriptionally activated by double-stranded DNA replication in the presence of staged erythroid proteins by remodeling of the chromatin structure within the promoter region and establishment of distal promoter-enhancer communication. The programmed transcriptional state of a gene, as encoded by its chromatin structure and long-range promoter-enhancer interactions, is stable to nuclear decondensation and DNA replication unless active remodeling occurs in the presence of specific DNA-binding proteins.

The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes.

The chick beta-globin gene is regulated developmentally within erythroid cells by the interaction of multiple proteins with the promoter and the 3' enhancer. These interactions are correlated with changes in chromatin structure, which are characteristic of the actively expressed gene. Using in vitro chromatin assembly and transcription with staged erythroid extracts, we have determined the critical proteins required to activate expression of nucleosome-reconstituted beta-globin genes. These genes contain a specialized TATA box at -30 (GATA) through which the erythroid-restricted protein cGATA-1 and TFIID both function to regulate different steps in beta-globin expression. We find that TBP (TATA-binding protein) alone can activate transcription of beta-globin chromatin templates from promoters mutated to a canonical TATA box but is ineffective on those containing the normal -30 GATA site. The occupancy of this site by cGATA-1 also fails to generate efficient expression of beta-globin chromatin unless combined with a stage-specific protein, NF-E4, that binds to an adjacent site. However, NF-E4 does not function with TBP to derepress nucleosome-assembled beta-globin genes. We propose that the developmental regulation of beta-globin expression is achieved, in part, by the requirement of an erythroid protein and a stage-specific factor, rather than TBP, to activate chromatin through a specialized TATA box.

The erythroid-specific protein cGATA-1 mediates distal enhancer activity through a specialized beta-globin TATA box.

The erythroid-specific protein cGATA-1 regulates the chick beta-globin gene through GATA sequences present at the canonical TATA location in the promoter as well as the distal 3' enhancer. We have analyzed beta-globin transcription in transfected erythroid cells and in erythroid extracts to determine whether cGATA-1 binding at -30 regulates promoter or enhancer activity. The interaction of both cGATA-1 and TFIID at different times with the -30 GATA site is required for efficient beta-globin expression in vivo, and the GATA enhancer site can functionally replace the TATA element in the beta-globin promoter. TFIID initiates transcription in vitro by complexing with adaptor proteins and displacing cGATA-1 from the -30 GATA site. Mutations that abolish TFIID binding to the -30 GATA box inactivate the promoter, whereas elimination of cGATA-1 binding to this site selectively diminishes enhancer-dependent transcription. We propose that interaction of cGATA-1 with the distal 3' enhancer and the specialized TATA box confers erythroid specificity to the initiation complex by mediating promoter-enhancer communication. Thus, one mechanism of action for tissue-specific proteins that recognizes noncanonical TATA motifs is to enable TFIID to be regulated by distal control elements. In this way, the initiation complex can be responsive to specific regulators that may not recognize a canonical TFIID-TATA structure.

Site-directed, recombination-mediated mutagenesis of a complex gene locus.

We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae. This gene cluster contains four beta-type globin genes which share a high degree of sequence homology. In this procedure, a small fragment of beta A-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integrating vector (YIp). This mutated globin subclone is introduced into cells that carry the 38 kb globin cluster clone on a single-copy, circular vector derived from a yeast artificial chromosome (YAC). Insertion of the 17 bp oligomer is achieved by targeted integration of the Ylp subclone. The recombinant contains the normal beta A-globin gene, the mutant gene and Ylp vector sequences between the two copies. Excision of the vector sequences and one copy of the duplicated globin sequences by homologous recombination is required for cell survival when exposed to the selective agent 5-fluoroorotic acid, which is toxic to ura+ yeast cells. Depending upon the point of the cross-over, a ura- yeast cell bearing either a wild-type globin gene or a 17 bp insertion mutation will result. By restriction mapping and in vitro transcription analysis, the beta A-globin gene containing the 17 bp insert has no nonspecific mutations generated during the recombination and selection procedures. Specific mutations of regulatory regions, including protein-DNA binding sites, can be accurately targeted within extensive DNA clones by this method.

Erythroid-specific activation and derepression of the chick beta-globin promoter in vitro.

Transcriptionally active extracts were prepared from chick red cells isolated at different stages of development. The template activity of cloned beta-globin genes is highest in extracts from definitive red cells, where the endogenous gene is normally expressed, and lowest in extracts from primitive red cells or nonerythroid tissues. This system has been used to identify regulatory elements and to assign functions to the proteins that bind within the beta-globin promoter. Regulation of expression is achieved, in part, by factors whose composition changes during red cell development. Two proteins, PAL and CON, bind at adjacent sites but have opposite effects on transcription in vitro. Levels of PAL, a potent repressor, are highest in mature red cells while those of CON, an activator, are highest in actively transcribing red cells. The effect of PAL can be overcome by blocking its binding site with a protein having a similar recognition sequence but a dissimilar function.

Analysis of the tissue-specific enhancer at the 3' end of the chicken adult beta-globin gene.

In an earlier paper we identified a tissue-specific enhancer in the 3' flanking region of the chicken adult beta-globin gene. In this paper we analyze the properties of this enhancer. Deletion analysis and transient expression assays show that the domain responsible for activation of transcription is at most 136 base pairs long. Specific factors that bind to discrete sequences within the enhancer DNA are found in extracts of embryonic and adult erythrocytes and in brain. These factors are specific for the tissue or the erythrocyte developmental stage and protect at least five discrete regions in or near the enhancer against DNase I digestion in "footprinting" experiments. Four of these regions reside wholly within the 136-base-pair functional enhancer domain, which also comprises a site in chromatin that is hypersensitive to nucleases. The nature of the binding sites and the program of appearance of the factors during development suggest that a subset of these interactions may be responsible for the developmental specificity of the enhancer.