RÉSUMÉ
ING2 (inhibitor of growth family member 2) is a component of a chromatin-regulatory complex that represses gene expression and is implicated in cellular processes that promote tumor suppression. However, few direct genomic targets of ING2 have been identified and the mechanism(s) by which ING2 selectively regulates genes remains unknown. Here we provide evidence that direct association of ING2 with the nuclear phosphoinositide phosphatidylinositol-5-phosphate (PtdIns(5)P) regulates a subset of ING2 targets in response to DNA damage. At these target genes, the binding event between ING2 and PtdIns(5)P is required for ING2 promoter occupancy and ING2-associated gene repression. Moreover, depletion of PtdIns(5)P attenuates ING2-mediated regulation of these targets in the presence of DNA damage. Taken together, these findings support a model in which PtdIns(5)P functions as a sub-nuclear trafficking factor that stabilizes ING2 at discrete genomic sites.
Sujet(s)
Noyau de la cellule/métabolisme , Chromatine/métabolisme , Altération de l'ADN , Protéines à homéodomaine/métabolisme , Phosphates phosphatidylinositol/métabolisme , Récepteurs cytoplasmiques et nucléaires/métabolisme , Protéines suppresseurs de tumeurs/métabolisme , Lignée cellulaire , Altération de l'ADN/génétique , Protéines à homéodomaine/génétique , Humains , Régions promotrices (génétique) , Récepteurs cytoplasmiques et nucléaires/génétique , Protéines suppresseurs de tumeurs/génétiqueRÉSUMÉ
In Saccharomyces cerevisiae, the repressive chromatin environment at telomeres gives rise to telomere position effect (TPE), the epigenetic silencing of telomere-proximal genes. Chromatin-modifying factors that control TPE in yeast have been extensively studied, and, among these, the lifespan regulator and silencing protein Sir2 has a pivotal role. In contrast, the factors that generate and maintain silent telomeric chromatin in human cells remain largely unknown. Here we show that the Sir2 family member SIRT6 is required for maintenance of TPE in human cells. RNAi-mediated depletion of SIRT6 abrogates silencing of both an integrated telomeric transgene and an endogenous telomere-proximal gene. Moreover, enhanced telomeric silencing in response to telomere elongation is associated with increased repressive chromatin marks, and this heterochromatic milieu is lost in SIRT6-deficient cells. Together, these findings establish a new role for SIRT6 in regulating an ageing-associated epigenetic silencing process and provide new mechanistic insight into chromatin silencing at telomeres.
Sujet(s)
Sirtuines/métabolisme , Télomère/métabolisme , Chromatine/génétique , Chromatine/métabolisme , Extinction de l'expression des gènes , Cellules HeLa , Humains , Sirtuines/génétique , Télomère/génétiqueRÉSUMÉ
Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4(me3)) while heterochromatin contains the H3K9(me3) marks. The H3K9(me3) modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4(me3). In addition, we show that SETDB1 fails to methylate substrates containing the H3K4(me3) mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4(me3) substrates. Accordingly, we provide in vivo evidence that H3K9(me2)-enriched histones are devoid of H3K4(me2/3) and that histones depleted of H3K4(me2/3) have elevated H3K9(me2/3). The correlation between the loss of interaction of these KMTs with H3K4 (me3) and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4(me3) and H3K9(me3). Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.
Sujet(s)
Hétérochromatine/métabolisme , Histone/métabolisme , Protein Methyltransferases/métabolisme , Maturation post-traductionnelle des protéines/physiologie , Homologue-5 de la protéine chromobox , Cellules HeLa , Hétérochromatine/génétique , Histone-lysine N-methyltransferase , Histone/génétique , Humains , Lysine/génétique , Lysine/métabolisme , Méthylation , Protein Methyltransferases/génétiqueRÉSUMÉ
Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.
Sujet(s)
Chromatine/métabolisme , Épigenèse génétique , Analyse par réseau de protéines/méthodes , Protéome/analyse , Transduction du signal , Anticorps/analyse , Génome humain , Histone/métabolisme , Humains , Liaison aux protéinesRÉSUMÉ
Since their discovery, the members of the ING (inhibitor of growth) family of tumor suppressors have emerged as essential and core components of chromatin modifying complexes. Recent work has identified the ING family as histone mark sensors that orchestrate cellular responses to genotoxic insults and regulate chromatin homeostasis. Dysregulation of chromatin homeostasis is implicated in tumorigenesis through mechanisms such as silencing of tumor suppressor genes, inappropriate activation of oncogenes, and genomic instability due to failure to repair DNA damage. This review will concentrate on the chromatin signaling aspects of the ING proteins, focusing on how recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the PHD (plant homeodomain) finger of ING proteins is critical for regulating cellular functions such as gene expression. We will also discuss how H3K4me3-recognition by ING proteins plays a critical role in their tumor suppressive functions. Finally, we will discuss the relevance of the association between one ING protein (ING2) and the nuclear phosphoinositide, phosphatidylinositol-5-phosphate (PtdIns(5)P). Interestingly, the ING2-PtdIns(5)P interaction involves the PHD finger and an adjacent polybasic region. The level of nuclear PtdIns(5)P sharply increases upon genotoxic stress, and this increase positively regulates ING2-mediated responses. Thus, the PHD finger of ING2 integrates phosphoinositide and chromatin signaling networks to prevent unchecked cell growth.