ABSTRACT
Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1ß is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1ß bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1ß genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.
Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Amino Acid Sequence , Blotting, Western , Cell Line, Tumor , Chromatin/genetics , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Crystallography, X-Ray , Heterochromatin/genetics , Histones/chemistry , Humans , Kinetics , Lysine/chemistry , Methylation , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Binding , Protein Multimerization , Sequence Homology, Amino Acid , Static ElectricityABSTRACT
Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.
Subject(s)
Chromatin/chemistry , Chromatin/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Histones/chemistry , Histones/metabolism , Animals , Animals, Genetically Modified , Chromatin/genetics , Chromatin Assembly and Disassembly , DNA/chemistry , DNA/genetics , DNA/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Histones/genetics , Male , Mutagenesis, Site-Directed , Protamines/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spermatozoa/metabolism , Static ElectricityABSTRACT
Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that 'read' the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1-ETO resides in and functions through a stable AML1-ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.
Subject(s)
Cell Transformation, Neoplastic , Core Binding Factor Alpha 2 Subunit/metabolism , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Multiprotein Complexes/metabolism , Oncogene Proteins, Fusion/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cell Division , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Core Binding Factor Alpha 2 Subunit/chemistry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Oncogene Proteins, Fusion/chemistry , Point Mutation , Protein Binding , Protein Multimerization , Protein Stability , Protein Structure, Tertiary , RUNX1 Translocation Partner 1 ProteinABSTRACT
Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1ß to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1ß with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1ß-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.
