RESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Secuencia de Aminoácidos , Western Blotting , Línea Celular Tumoral , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Cristalografía por Rayos X , Heterocromatina/genética , Histonas/química , Humanos , Cinética , Lisina/química , Metilación , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/química , Nucleosomas/metabolismo , Unión Proteica , Multimerización de Proteína , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/química , Histonas/química , Fosfatos de Fosfatidilinositol/química , Regulación Alostérica , Sitios de Unión/fisiología , Línea Celular Tumoral , Metilación de ADN , Células HeLa , Heterocromatina/fisiología , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Ubiquitina-Proteína LigasasRESUMEN
Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Mitosis , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Adenosina Trifosfatasas/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Lisina/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismoRESUMEN
The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.