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2.
Nat Struct Mol Biol ; 30(11): 1628-1639, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37770717

ABSTRACT

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.


Subject(s)
Chromatin , Nucleosomes , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Promoter Regions, Genetic , Enhancer Elements, Genetic
3.
J Mol Biol ; 428(14): 2931-42, 2016 07 17.
Article in English | MEDLINE | ID: mdl-27117189

ABSTRACT

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.


Subject(s)
Histone Deacetylases/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Nucleosomes/metabolism , Animals , Cell Nucleus/metabolism , Chromatin/metabolism , Chromatin Assembly and Disassembly/physiology , Cytosol/metabolism , Drosophila/metabolism , Mice , Protein Subunits/metabolism , Stem Cells/metabolism
4.
Trends Biochem Sci ; 26(10): 577-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11589999

ABSTRACT

Currently, the combination of library selection and directed evolution is the most powerful approach for finding proteins with novel folds or functions. In the past, most studies concentrated either on protein scaffolds with a given fold or on short peptides. With the recent development of potent in vitro selection and evolution techniques, the screening of much larger sequence space is possible, allowing for the de novo generation of proteins.


Subject(s)
Protein Folding , Adenosine Triphosphate/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Directed Molecular Evolution , In Vitro Techniques , Peptide Library
5.
Proc Natl Acad Sci U S A ; 98(15): 8572-7, 2001 Jul 17.
Article in English | MEDLINE | ID: mdl-11438689

ABSTRACT

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.


Subject(s)
Antibodies, Protozoan/immunology , DNA, Protozoan/immunology , DNA/immunology , Hypotrichida/genetics , Immunoglobulin Fragments/immunology , Immunoglobulin Variable Region/immunology , Telomere , Animals , Antibodies, Protozoan/biosynthesis , G-Quadruplexes , Guanine , Humans , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Variable Region/biosynthesis , Repetitive Sequences, Nucleic Acid
6.
Nat Biotechnol ; 18(12): 1287-92, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11101809

ABSTRACT

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.


Subject(s)
Antibody Affinity , Directed Molecular Evolution , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Peptide Library , RNA, Messenger/genetics , Ribosomes/metabolism , Amino Acid Sequence , Animals , Cattle , Codon , Frameshifting, Ribosomal , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , Insulin/immunology , Insulin/metabolism , Molecular Sequence Data , Mutation , RNA, Messenger/metabolism , Radioimmunoprecipitation Assay
8.
FEBS Lett ; 450(1-2): 105-10, 1999 Apr 30.
Article in English | MEDLINE | ID: mdl-10350066

ABSTRACT

Ribosome display is a technology for library selection and simultaneous molecular evolution in vitro. We present here a comparison between an optimized Escherichia coli system and different rabbit reticulocyte ribosome display systems, optimized in a number of parameters, as a coupled eukaryotic system had been suggested to result in high enrichment factors [He and Taussig (1997) Nucleic Acids Res. 25, 5132-5134]. With all systems, antibody scFv fragments, complexed to the ribosomes and the corresponding mRNA, were enriched by binding to their cognate antigen and enrichment was always dependent on the absence of a stop codon and the presence of cognate antigen. However, the efficiency of the E. coli ribosome display system was 100-fold higher than an optimized uncoupled rabbit reticulocyte ribosome display system, with separate in vitro transcription and translation, which was in turn several-fold more efficient than the reported coupled system. Neither the E. coli nor the rabbit reticulocyte ribosome display system was dependent on the orientation of the domains of an antibody scFv fragment or on the spacer sequence. In summary, we could not detect any intrinsic advantage of using a eukaryotic translation system for ribosome display.


Subject(s)
Escherichia coli/genetics , Reticulocytes/metabolism , Ribosomes/metabolism , Amino Acid Sequence , Animals , Antigens/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Molecular Sequence Data , Peptide Fragments/immunology , Protein Biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins/analysis , RNA-Binding Proteins/immunology , Rabbits , Transcription, Genetic
9.
J Immunol Methods ; 231(1-2): 119-35, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10648932

ABSTRACT

Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment.


Subject(s)
Immunoglobulin Fragments/biosynthesis , Peptide Library , Ribosomes , Animals , Humans , Immunoglobulin Variable Region/biosynthesis
10.
J Bacteriol ; 180(10): 2689-93, 1998 May.
Article in English | MEDLINE | ID: mdl-9573154

ABSTRACT

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na+ dependent, and imposition of a DeltapNa+ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate- is the transported malonate species. The stimulation of malonate uptake by Na+ ions showed Michaelis-Menten kinetics, and a Km for Na+ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na+/Hmalonate- symporter and that it is dependent on two separate genes.


Subject(s)
Genes, Bacterial/genetics , Gram-Negative Anaerobic Bacteria/genetics , Gram-Negative Anaerobic Bacteria/metabolism , Malonates/metabolism , Biological Transport , Potassium/metabolism
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