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P B Becker

Publications and source records attributed to P B Becker.

At least 55 records · Page 3Linked to original sources
PubMed1998

Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA.

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo, yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A Drosophila embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperature shift pointing to profound alterations of DNA/histone interactions. This successful reconstitution of accessible and flexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.

Acetylation↗
PubMed1998

Structural and functional analysis of chromatin assembled from defined histones.

In this review we describe how the extract-mediated chromatin assembly system derived from preblastoderm Drosophila embryos can be modified to assemble chromatin from defined histones. This approach combines the advantages of assembling (i) chromatin templates from homogeneous histones with (ii) an assembly system that generates chromatin with physiological nucleosome spacing and density and that contains the biological complexity of in vivo chromatin. We have used this technique to assemble nonacetylated and hyperacetylated histones into chromatin (K. P. Nightingale, R. Wellinger, J. Sogo, and P. B. Becker, 1998, EMBO J. 17, 2865-2876; W. A. Krajewski and P. B. Becker, 1998, Proc. Natl. Acad. Sci. USA 95, 1540-1545), and use this as an example to detail the structural and transcriptional assays used to compare and characterize these chromatin templates. The application of this procedure to assemble chromatin from recombinant histones should facilitate a wide variety of studies on the role(s) of histone mutants and variants.

Animals↗
PubMed1998

Chromatin-remodeling factors: machines that regulate?

Chromatin has shifted into the focus of attention as a key to understanding the regulation of nuclear processes such as transcription. Protein machines have been described that use the energy of ATP to render chromatin dynamic and hence active, but which may also be involved in chromatin assembly. The discovery of three different Drosophila nucleosome remodeling complexes that contain imitation switch (ISWI), an ATPase with a high degree of sequence conservation from yeast to human, points to a central function of this ATPase in chromatin dynamics.

Adenosine Triphosphatases↗
PubMed1998

Heat shock factor increases the reinitiation rate from potentiated chromatin templates.

Transcription by RNA polymerase II is highly regulated at the level of initiation and elongation. Well-documented transcription activation mechanisms, such as the recruitment of TFIID and TFIIB, control the early phases of preinitiation complex formation. The heat shock genes provide an example for transcriptional regulation at a later step: in nuclei TFIID can be detected at the TATA box prior to heat induction. Using cell-free systems for chromatin reconstitution and transcription, we have analyzed the mechanisms by which heat shock factor (HSF) increases transcription of heat shock genes in chromatin. HSF affected transcription of naked DNA templates in multiple ways: (i) by speeding up the rate of preinitiation complex formation, (ii) by increasing the number of productive templates, and (iii) by increasing the reinitiation rate. Under the more physiological conditions of potentiated chromatin templates, HSF affected only the reinitiation rate. Activator-dependent reinitiation of transcription, obviating the slow assembly of the TFIID-TFIIA complex on a promoter, may be especially crucial for genes requiring a fast response to inducers.

Animals↗
PubMed1997

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.

Animals↗
PubMed1997

Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II.

Repressive chromatin structures need to be unravelled to allow DNA-binding proteins access to their target sequences. This de-repression constitutes an important point at which transcription and presumably other nuclear processes can be regulated. Energy-consuming enzyme complexes that facilitate the interaction of transcription factors with chromatin by modifying nucleosome structure are involved in this regulation. One such factor, nucleosome-remodelling factor (NURF), has been isolated from Drosophila embryo extracts. We have now identified a chromatin-accessibility complex (CHRAC) which uses energy to increase the general accessibility of DNA in chromatin. However, unlike other known chromatin remodelling factors, CHRAC can also function during chromatin assembly: it uses ATP to convert irregular chromatin into a regular array of nucleosomes with even spacing. CHRAC combines enzymes that modulate nucleosome structure and DNA topology. Using mass spectrometry, we identified two of the five CHRAC subunits as the ATPase ISWI, which is also part of NURF, and topoisomerase II. The presence of ISWI in different contexts suggests that chromatin remodelling machines have a modular nature and that ISWI has a central role in different chromatin remodelling reactions.

Adenosine Triphosphatases↗
PubMed1997

RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression.

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.

Animals↗
PubMed1997

Solid phase technology improves coupled gel shift/footprinting analysis.

For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now describe an improved experimental strategy that employs transient coupling of DNA fragments to a solid support in order to extract DNA of high purity quantitatively, rapidly and reliably. As an example, we describe the application of our protocol to the 'in-gel footprinting' by copper phenanthroline. The method should also find application to the chemical interference assays.

Bacterial Proteins↗
PubMed1997

The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids.

The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer. HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding. Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homotetramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids. Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human alpha1-antitrypsin TATA box region. Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH. Interestingly, DCoH retains its enzymatic activity while complexed with HNF1. Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

Animals↗
PubMed1997

Genomic footprinting of Drosophila embryo nuclei by linker tag selection LM-PCR.

The unmatched power of Drosophila genetics revealed the complex regulatory network of gene activities that governs the development of higher eukaryotes. An understanding of gene control at the level of transcription requires insight into the protein/DNA interactions that regulate transcription in the developing embryo. Genomic footprinting allows the direct visualization of these protein/DNA interactions within intact nuclei or cells. In combination with other in vivo assays such as protein/DNA crosslinking and classical biochemistry, genomic footprinting can give valuable insight into the architecture of promoters in various states of activity. In this article we summarize our experience in analyzing Drosophila embryos by genomic footprinting and describe modifications of the ligation-mediated PCR procedure that have improved this analysis. Applications of genomic footprinting to embryos are currently limited by the fact that all target nuclei must be uniform with respect to the protein/DNA interactions at the chosen site. We discuss strategies that should allow the analysis of small numbers of cells derived from heterogeneous populations and tissues.

Animals↗
PubMed1997

Biochemical analysis of chromatin structure and function using Drosophila embryo extracts.

The biochemical analysis of chromatin structure and function is greatly facilitated by the availability of cell-free systems that assemble chromatin under physiological conditions. One such system that has shown great potential is derived from extracts of early Drosophila embryos. These embryos contain large maternal stocks of chromatin constituents, such as histones and assembly factors. Chromatin assembled in these extracts resembles native chromatin in many respects: it displays physiological nucleosome repeat lengths, it is complex, containing a wealth of nonhistone proteins as well as enzymatic activities, and it has dynamic properties that allow the interaction of DNA-binding proteins that regulate important cellular processes. Most importantly, chromatin with variant properties, e.g., with respect to the basic geometry of the nucleosomal array, histone modifications, and its content of linker histones or nonhistone proteins, can be obtained by manipulating the reconstitution conditions. The synthesis of uniform chromatin with specific characteristics should allow the analysis of the functional significance of the structural and biochemical heterogeneity observed in vivo.

Animals↗
PubMed1996

The effect of nucleosome phasing sequences and DNA topology on nucleosome spacing.

The distances between the nucleosomes in eukaryotic chromatin that define the nucleosome repeat length are not universally constant, but vary between different cell types and activity states. We have previously established in a cell-free system that nucleosome spacing is essentially governed by electrostatic principles, most likely through charge neutralisation of linker DNA by cations either free in solution or on flexible histone domains. On the basis of the tight correlation between the parameters that affect nucleosome spacing and those that influence the folding of the nucleosomal fiber into higher order structures, we suggested that there is an intimate relationship between nucleosome spacing and chromatin folding. Here we describe DNA topology as a new parameter that influences nucleosome spacing in a predictable way. The effects of topology and cation concentrations integrate to define the final repeat length. The phenomenon of "nucleosome phasing" describes nucleosomal arrays that are generated through positioning of nucleosomes by the underlying DNA sequence. To determine the relative contribution of DNA sequence and the parameters intrinsic to physiological chromatin for nucleosomal positions, we created situations where these two principles were in conflict. We found that nucleosome repeats directed by a strong positioning sequence are dominated by the cation-induced spacing as well as by the effects of topology. We conclude that the DNA sequence effects nucleosome spacing only by "fine tuning" of nucleosome positions within the framework of a repeat pattern that is established by other principles.

Animals↗
PubMed1996

The architecture of the heat-inducible Drosophila hsp27 promoter in nuclei.

Transcriptional activation of the Drosophila hsp27 gene in response to heat shock critically relies on binding sites for heat shock factor (HSF) about 300 bp upstream of the transcription start site. In contrast to the well-characterised heat-inducible hsp70 and hsp26 promoters, no other transcription factor binding sites have been identified closer to the TATA box. In order to understand the structural requirements for activation from a distance we studied the protein-DNA interactions at the hsp27 promoter in Drosophila embryos and tissue culture cells before and after heat induction. Genomic footprinting with nucleases and a chemical probe, the 1,10-phenanthroline cuprous complex (OP-Cu), suggests that the DNA between the TATA box and the heat shock elements (HSEs) is constitutively organised by a positioned nucleosome, effectively shortening the distance between the distal HSEs and the TATA box. Protection of the TATA element from nuclease attack and the OP-Cu reactivity pattern around the start site of transcription is consistent with the constitutive presence of TFIID and the "poised polymerase", a transcription machinery blocked in an early phase of elongation. The general transcription factors at the TATA box and the positioned nucleosome are separated by a stable structure, presumably a protein bound to a palindromic sequence. These constitutive features define the "preset" architecture of the promoter within which the induced binding of HSF in vivo is observed. Our study highlights the importance of positioned nucleosomes as architectural elements within promoters and identifies a new regulatory sequence that may function either to direct a nucleosome boundary or to mediate signals of distant activator proteins.

Animals↗
PubMed1995

Electrostatic mechanism of nucleosome spacing.

Native bulk chromatin is characterized by regular arrays of nucleosomes with defined internucleosomal distances. The nucleosome repeat length is not a constant but varies between species and cell-types, during differentiation and during gene activation. Previous studies have highlighted the importance of linker histones as a major determinant of nucleosome repeat length in vivo. We used a physiological reconstitution system derived from Drosophila embryos to study nucleosome spacing. In these extracts, histone H1 incorporation increases the apparent linker length in a gradual way. Manipulation of the chromatin assembly conditions in vitro allowed us to define additional parameters that modulate nucleosomal distances, such as protein phosphorylation events and the precise ionic conditions during the reconstitution. Interestingly, moderate changes in the concentrations of mono-, di-, and multivalent cations affect the precise distances between nucleosome cores remarkably. These changes in the ionic environment are unlikely to affect the association of linker proteins but are known to influence the folding of the nucleosomal fiber by modulation of electrostatic forces. Our results suggest electrostatic interactions in chromatin units as major determinants of nucleosome spacing. Nucleosome spacing and the folding of the nucleosomal fiber can therefore be explained by common principles, most notably the neutralization of charges in linker DNA.

Animals↗
PubMed1995

Transcription factor-mediated chromatin remodelling: mechanisms and models.

The association of DNA with nucleosomes in chromatin severely restricts the access of the regulatory factors that bring about transcription. In vivo active promoters are characterised by altered, almost transparent chromatin structures that allow the interaction of the transcriptional machinery. Recently, enzymatic activities have been discovered that facilitate the binding of transcription factors to chromatin by modifying nucleosomal structures in a process that requires energy. The mechanisms by which chromatin is remodelled may involve nucleosome movements, their transient unfolding, their partial or even complete disassembly. The dynamic properties of chromatin that underlie these structural changes are fundamental to the process of regulated gene expression.

Chromatin↗
PubMed1995

Dual regulation of the Drosophila hsp26 promoter in vitro.

Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). We have evaluated the contribution of hsp26 promoter sequences to transcriptional activity in extracts from either heat shocked or unstressed fly embryos. Efficient transcription in either extract was governed by distinct regulatory principles. Transcription in extracts from unstressed embryos relied solely on GAGA elements which efficiently counteracted repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depended only a little on GAGA elements, relying mainly on functional HSEs. Constitutively active recombinant HSF or native factor in an extract from heat shocked embryos was able to truly activate transcription essentially via proximal HSEs, but not when bound to distal sites. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo.

Animals↗
PubMed1995

Energy-dependent chromatin accessibility and nucleosome mobility in a cell-free system.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.

Adenosine Triphosphate↗