ABSTRACT
The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.
Subject(s)
Adenosine Triphosphatases/metabolism , Histones/chemistry , Histones/metabolism , Nucleosomes/metabolism , Transcription Factors/metabolism , Animals , DNA/metabolism , In Vitro Techniques , Macromolecular Substances , Multiprotein Complexes , Protein Structure, Quaternary , Substrate Specificity , Xenopus laevisABSTRACT
Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.
Subject(s)
Adenosine Triphosphatases/metabolism , DNA Polymerase III , DNA-Binding Proteins/metabolism , Drosophila Proteins , Histones/metabolism , Nucleoproteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Animals , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/chemistry , Drosophila , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nucleoproteins/chemistry , Nucleosomes/metabolism , Protein Binding , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Transcription Factors/chemistryABSTRACT
The ISWI ATPase of Drosophila is a molecular engine that can drive a range of nucleosome remodelling reactions in vitro. ISWI is important for cell viability, developmental gene expression and chromosome structure. It interacts with other proteins to form several distinct nucleosome remodelling machines. The chromatin accessibility complex (CHRAC) is a biochemical entity containing ISWI in association with several other proteins. Here we report on the identification of the two smallest CHRAC subunits, CHRAC-14 and CHRAC-16. They contain histone fold domains most closely related to those found in sequence-specific transcription factors NF-YB and NF-YC, respectively. CHRAC-14 and CHRAC-16 interact directly with each other as well as with ISWI, and are associated with functionally active CHRAC. The developmental expression profiles of both subunits suggest specialized roles in chromatin remodelling reactions in the early embryo for both histone fold subunits.
Subject(s)
Adenosine Triphosphatases/metabolism , CCAAT-Binding Factor , Chromatin/metabolism , Drosophila Proteins , Drosophila/genetics , Nucleoproteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Expression Regulation , Histones/chemistry , Molecular Sequence Data , Nucleoproteins/chemistry , Nucleoproteins/genetics , Nucleosomes/metabolism , Protein Binding , Protein Conformation , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.
Subject(s)
Dosage Compensation, Genetic , Drosophila Proteins , Drosophila/embryology , Nuclear Proteins/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Transcription Factors/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Animals , Binding Sites/genetics , DNA-Binding Proteins , Gene Expression Regulation , Poly A/genetics , RNA Caps/geneticsABSTRACT
In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.
Subject(s)
CCAAT-Enhancer-Binding Proteins , Cell Cycle Proteins , Chromosomes/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins , Pyrophosphatases , Receptors, Progesterone/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Animals , Chromatin/metabolism , DNA Footprinting , Drosophila/embryology , Humans , Insect Proteins/metabolism , Mammary Tumor Virus, Mouse/genetics , NFI Transcription Factors , Nuclear Proteins , Nucleic Acid Conformation , Nucleosomes/genetics , Promoter Regions, Genetic , Recombinant Proteins/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription, Genetic , Y-Box-Binding Protein 1ABSTRACT
The chromatin accessibility complex (CHRAC) belongs to the class of nucleosome remodeling factors that increase the accessibility of nucleosomal DNA in an ATP-dependent manner. We found that CHRAC induces movements of intact histone octamers to neighboring DNA segments without facilitating their displacement to competing DNA or histone chaperones in trans. CHRAC-induced energy-dependent nucleosome sliding may, in principle, explain nucleosome remodeling, nucleosome positioning, and nucleosome spacing reactions known to be catalyzed by CHRAC. The catalytic core of CHRAC, the ATPase ISWI, also mobilized nucleosomes at the expense of energy. However, the directionality of the CHRAC- and ISWI-induced nucleosome movements differed drastically, indicating that the geometry of the native complex modulates the activity of its catalytic core.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin/metabolism , Histones/metabolism , Nucleosomes/metabolism , Transcription Factors/metabolism , Animals , DNA/metabolism , Drosophila , Polyglutamic Acid/metabolismABSTRACT
The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.
Subject(s)
Adenosine Triphosphatases/physiology , Nucleosomes/ultrastructure , Transcription Factors/physiology , Adenosine Triphosphatases/genetics , Animals , Binding Sites , Chromatin/metabolism , Chromatin/ultrastructure , DNA/pharmacology , Drosophila melanogaster/genetics , Escherichia coli , Gene Expression Regulation , Macromolecular Substances , Mutagenesis, Site-Directed , Nucleosomes/metabolism , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The human TRIP-1 (transforming growth factor-beta (TGBbeta)-receptor interacting protein-1) cDNA encodes a protein able to associate specifically with the type II TGFbeta receptor. It is phosphorylated on serine and threonine by this receptor kinase which makes it a strong candidate as part of the TGFbeta signal transduction pathway. We have isolated the genomic sequence of TRIP-1 and found that the complete coding region is organised into 11 exons ranging from 39 to 397 bp and spanning approximately 9 kb of genomic DNA. The 5' flanking region lacks a TATA box but is GC-rich, suggesting that it is a constitutively expressed gene which is in agreement with its wide pattern of expression. Fluorescence in situ hybridisation mapped the TRIP-1 gene to chromosome 1p34.1 whereas a pseudogene is located on chromosome 7q32.
Subject(s)
Chromosomes, Human, Pair 1 , Proteins/genetics , Chromosomes, Human, Pair 7 , Eukaryotic Initiation Factor-3 , Exons , Genes , Humans , In Situ Hybridization, Fluorescence , Introns , Pseudogenes , Restriction MappingABSTRACT
In this descriptive-exploratory study, we sought to describe and compare the similarities and differences between Filipino and Mexican-American primigravidas in social support networks, type of support received, and expectations regarding care from health care providers. Convenience sampling was used, and face-to-face interviews using the Norbeck Social Support Questionnaire (Norbeck, Lindsey, & Carrieri, 1983) were conducted in Filipino and Spanish by trained data collectors. The profiles obtained for both groups were similar in sociodemographic characteristics; possession of an adequate social support network, predominantly composed of the spouse and family members; and expectations of more personalized care and decreased waiting time than transpired. The groups differed slightly with regard to ability to discuss pregnancy with persons reported in the network. Overall findings reflect the central role of the family as a source of support during pregnancy. Recommendations are made to conduct research addressing populations of varied ethnic groups and with different educational backgrounds, populations of single and teenage pregnant women, and spousal support.
