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J L Workman

Publications and source records attributed to J L Workman.

At least 73 records · Page 4Linked to original sources
PubMed1996

Remodeling chromatin structures for transcription: what happens to the histones?

Activation of gene transcription in vivo is accompanied by an alteration of chromatin structure. The specific binding of transcriptional activators disrupts nucleosomal arrays, suggesting that the primary steps leading to transcriptional initiation involve interactions between activators and chromatin. The affinity of transcription factors for nucleosomal DNA is determined by the location of recognition sequences within nucleosomes, and by the cooperative interactions of multiple proteins targeting binding sites contained within the same nucleosomes. In addition, two distinct types of enzymatic complexes facilitate binding of transcription factors to nucleosomal DNA. These include type A histone acetyltransferases (e.g. GCN5/ADA transcriptional adaptor complex) and ATP-driven molecular machines that disrupt histone-DNA interactions (e.g. SWI/SNF and NURF complexes). These observations raise the important question of what happens to the histones during chromatin remodeling. We discuss evidence supporting the retention of histones at transcription factor-bound sequences as well as two alternative pathways of histone loss from gene control elements upon transcription factor binding: histone octamer sliding and histone dissociation.

Acetyltransferases↗
PubMed1996

Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning.

To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells.

Abscisic Acid↗
PubMed1995

Stimulation of transcription factor binding and histone displacement by nucleosome assembly protein 1 and nucleoplasmin requires disruption of the histone octamer.

To investigate the mechanisms by which transcription factors invade nucleosomal DNA and replace histones at control elements, we have examined the response of the histone octamer to transcription factor binding in the presence of histone-binding proteins (i.e., nucleosome assembly factors). We found that yeast nucleosome assembly protein 1 (NAP-1) stimulated transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. In addition, disruption of the histone octamer was required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP-1. While NAP-1 and nucleoplasmin stimulated the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation was lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP-1 and nucleoplasmin were able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity was also forfeited when the histone octamers were cross-linked. These data indicate that octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP-1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA did not require disruption of the histone octamer. Thus, histone displacement in this instance occurred by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP-1.

Base Sequence↗
PubMed1995

Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

Base Sequence↗
PubMed1994

Differential repression of transcription factor binding by histone H1 is regulated by the core histone amino termini.

In order to investigate the interrelated roles of nucleosome cores and histone H1 in transcription repression, we have employed a purified system to analyze the function of H1 in the repression of transcription factor binding to nucleosomes. H1 binding to nucleosome cores resulted in the repression of USF binding to nucleosomes. By contrast, H1 only slightly inhibited the binding of GAL4-AH, indicating that H1 differentially represses the binding of factors with different DNA-binding domains. H1-mediated repression of factor binding was dependent on the core histone amino-terminal tails. Removal of these domains alleviated H1-mediated repression and increased acetylation of these domains partly alleviated repression by H1. H1 binding assays suggest a less stable interaction of histone H1 with the core particle in the absence of the amino termini.

Acetylation↗
PubMed1994

Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex.

The SWI/SNF protein complex is required for the enhancement of transcription by many transcriptional activators in yeast. Here it is shown that the purified SWI/SNF complex is composed of 10 subunits and includes the SWI1, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products. The complex exhibited DNA-stimulated adenosine triphosphatase (ATPase) activity, but lacked helicase activity. The SWI/SNF complex caused a 10- to 30-fold stimulation in the binding of GAL4 derivatives to nucleosomal DNA in a reaction that required adenosine triphosphate (ATP) hydrolysis but was activation domain-independent. Stimulation of GAL4 binding by the complex was abolished by a mutant SWI2 subunit, and was increased by the presence of a histone-binding protein, nucleoplasmin. A direct ATP-dependent interaction between the SWI/SNF complex and nucleosomal DNA was detected. These observations suggest that a primary role of the SWI/SNF complex is to promote activator binding to nucleosomal DNA.

Adenosine Triphosphatases↗
PubMed1994

Nucleosome binding by the constitutive transcription factor Sp1.

The constitutive transcription factor Sp1 plays a role in the transcription of numerous viral and cellular genes including constitutive "housekeeping" genes and inducible genes. Sp1 has also been implicated in the formation of a nucleosome-free region in Simian virus 40 (SV40) minichromosomes. To investigate the potential functions of Sp1 in remodeling chromatin structures, Sp1 was analyzed for the ability to bind its recognition sites (GC boxes) in DNA fragments reconstituted into nucleosome cores. Sp1 was found to bind to the GC boxes of the SV40 early promoter when this element was reconstituted into nucleosome cores. The affinity of Sp1 for the nucleosomal SV40 early promoter DNA was reduced approximately 10-20-fold relative to naked DNA. A peptide containing only the zinc fingers of Sp1 was also capable of binding nucleosomal DNA, indicating that the glutamine-rich and serine/threonine-rich domains of Sp1 are not required for nucleosome binding. The binding of Sp1 to nucleosome cores resulted in the formation of a ternary Sp1-nucleosome complex.

Animals↗
PubMed1994

A histone-binding protein, nucleoplasmin, stimulates transcription factor binding to nucleosomes and factor-induced nucleosome disassembly.

The binding of a GAL4-AH, USF or Sp1 to nucleosome cores was stimulated by the presence of the histone-binding protein, nucleoplasmin. Stimulation of factor binding by nucleoplasmin was specific for nucleosome reconstituted DNA and was not mimicked by non-specific histone sinks (i.e. polyglutamate or RNA). Upon GAL4-AH binding, nucleoplasmin specifically removed histones H2A and H2B from the nucleosome which enhanced the subsequent loss of the H3/H4 tetramers onto competing DNA. Thus, nucleoplasmin participated in the complete conversion of nucleosome cores to transcription factor-DNA complexes. These data indicate that proteins which bind histones can increase transcription factor binding to nucleosomal DNA and that transcription factor binding can initiate nucleosome disassembly. Similar activities of histone-binding proteins may participate in the displacement of nucleosomes at enhancers and promoters in vivo.

Base Sequence↗
PubMed1994

Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores.

Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied. However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA. Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end. When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions. These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini. This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin.

Base Sequence↗
PubMed1994

Experimental analysis of chromatin function in transcription control.

Chromatin structure plays a crucial role in the regulation of eukaryotic gene transcription. Nucleosomes and higher orders of chromatin structure repress promiscuous gene expression by increasing its dependence on the function of activator proteins that regulate transcription in eukaryotic cells. Here we review several parameters governing the dynamic interactions between transcription factors and chromatin structures. These include functions of the core histones and their modification by acetylation, histone H1, HMG proteins, nucleosome positioning, DNA replication, cooperative nucleosome-binding by transcription factors, histone chaperones and nucleosome displacement, the SWI/SNF protein complex, and higher-order domains of chromatin structure. All of these impact on the interactions of transcription factors with chromatin templates. Experimental analysis of these parameters provides new insights into mechanisms of eukaryotic transcription regulation.

Animals↗
PubMed1993

Multiple functions of nucleosomes and regulatory factors in transcription.

The in vivo packaging of DNA with histone proteins to form chromatin makes its transcription a difficult process. Biochemical and genetic studies are beginning to reveal mechanistic details of how transcriptional regulatory factors confront at least two hurdles created by nucleosomes, the primary structural unit of chromatin. Regulatory factors must gain access to their respective binding sites and activate the formation of transcription complexes at core promoter elements. Distinct regulatory factors may be specialized to perform these functions.

Animals↗
PubMed1992

Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex.

In order to function, transcription factors must compete for DNA binding with structural components of chromatin, including nucleosomes. Mechanisms that could be used in this competition have been characterized with the use of the DNA binding domain of the yeast GAL4 protein. The binding of GAL4 to a nucleosome core resulted in a ternary complex containing GAL4, the core histone proteins, and DNA. This ternary complex was unstable; upon the addition of nonspecific competitor DNA, it dissociated into either the original nucleosome core particle or GAL4 bound to naked DNA. Nucleosome core destabilization by GAL4 did not require a transcriptional activation domain. These data demonstrate the displacement of nucleosome cores as a direct result of binding by a regulatory factor. Similar mechanisms might affect the establishment of factor occupancy of promoters and enhancers in vivo.

Base Sequence↗
PubMed1992

Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16.

Studies of DNA viruses have provided evidence that eukaryotic transcriptional activator proteins can enhance the efficiency of DNA replication as well as transcription. The mechanism of this effect was studied in vitro using the chimeric transcription factor GAL4-VP16 and a DNA template containing GAL4 binding sites adjacent to the simian virus 40 origin of DNA replication. The binding of GAL4-VP16 prevented the repression of DNA replication which otherwise occurred when the template was assembled into chromatin. Relief of repression by GAL4-VP16 required both its DNA-binding and transcriptional activation domains but did not require RNA synthesis. The results are consistent with a general model in which transcriptional activators stimulate eukaryotic DNA replication by modifying the outcome of the competition between initiation factors and histones for occupancy of the origin.

DNA Replication↗
PubMed1991

Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes.

GAL4 derivatives containing an activation domain alleviated repression of a promoter during nucleosome assembly. A GAL4 derivative lacking an activation domain stably bound the promoter during nucleosome assembly but was not sufficient to preserve promoter function. The activation domain of GAL4 derivatives was essential for preserving promoter function, and thus the transcriptional stimulatory activity attributable to these activation domains increased dramatically during nucleosome assembly. Furthermore, promoter-bound activation domains allowed the formation of preinitiation complexes after nucleosome assembly. Finally, GAL4 derivatives containing activation domains significantly stimulated transcription through bacterially produced yeast TFIID only from nucleosome-assembled templates. These data indicate that acidic activation domains stimulate transcription by enhancing the ability of basal transcription factors to compete with nucleosomes for occupancy of the promoter.

Animals↗