Role of SIRT6 in Chromatin

Abstract

Chromatin compaction is regulated by different factors, among them histone posttranslational modifications. There are different histone modifications, and among them, acetylation and methylation of lysine residues. Acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), enzymes that catalyze addition and removal, respectively, of acetyl groups from histone lysine residues. Among the four classes of HDACs, the class III which correspond to the members of the Sir2 family or Sirtuins are quite unique. They participate in the response to a wide variety of stress stimuli through their requirement of NAD+ as a cofactor in their enzymatic activity. Mammals harbor seven Sirtuins (SirT1-SirT7). Among them, SirT6 is a nuclear protein involved in both genomic stability and metabolic homeostasis. Interestingly, SirT6 regulates the majority of these processes through an effect on chromatin, based on deacetylation of a histone mark, H3K9ac. However, the mechanism involved has not been fully characterized. In this work, we aim to study the consequences of SirT6 function in chromatin organization. We show that SirT6 overexpression induces gene silencing, which fits with the role of SirT6 as a repressor, shown by previous reports. Our studies show that SirT6 interacts with proteins or multi-protein complexes also involved in gene silencing, such as components of NuRD complex, or the HMTs EZH2, Suv39h1. However, we have focused the project in understanding the functional relationship between SirT6 and a H3K9-specific methyltransferases that we have identified as Suv39h1 and G9a. Suv39h1 trimethylates H3K9 and is essential in the establishment and maintenance of pericentromeric and telomeric constitutive heterochromatin. Interestingly, Suv39h1 was previously found to interact with SirT1 in the context of constitutive and facultative heterochromatin formation. Our work shows that the functional relationship between Suv39h1 and SirT6 is quite different from the already described between SirT1 and Suv39h1. SirT6 mediates a non canonical monoubiquitination in Suv39h1 in three conserved cysteines of the pre-SET domain. We have also identified SKP2 as the E3 ubiquitin ligase responsible of this monoubiquitination. SKP2 has a well-known role in promoting poliubiquitination and degradation of protein involved in G1/S checkpoint such as p21 and p27 and its levels are regulated during cell cycle progression. Our data show that SKP2 levels are also regulated by SirT6 through deacetylation, which in turn induce a double phosphorylation that prevents its degradation. Suv39h1 monoubiquitination is induced by double thymidine block and nocodazole treatments that arrest cells in G1/S phase and early mitosis, respectively. Furthermore, Suv39h1 monoubiquitination is induced by NF-kB pathway activation such as TNFa treatment, the overexpression of the NF-kB transcription factor RelA, or of the activator IKKa. Moreover, the promoter of the NF-kB global repressor, IkBa, is regulated by the interplay between Suv39h1, SKP2 and SirT6. Thus, upon activation of the pathway by TNFa treatment, SirT6 induces the Suv39h1 monoubiquitination through SKP2 activation and monoubiquitinated Suv39h1 is removed from the promoter allowing the transcription activation of IkBa by RelA. This novel model provides not only new insights in the regulation of NFkB pathway but also unveils new roles for SirT6, SKP2 and Suv39h1.

La compactación de la cromatina es regulada por diferentes factores entre los cuales destacan las modificaciones post-traduccionales de las histonas. Hay diferentes modificaciones de histonas entre ellas las desacetilationes y las metilaciones. Los niveles de acetilación están regulados por las histonas acetiltransferasas (HATs) y las histonas deacetilasas (HDACs), proteínas que añaden o quitan grupos acetil a las lisinas de las histonas, respectivamente. Los miembros de la familia Sir2 o de las sirtuínas constituyen la clase III de las HDACs y participan en respuesta a muchas formas de estrés. Entre ellas, está SirT6 que desacetila H3K9 acetilado para promover silenciamiento. En este proyecto mostramos que la sobreexpresión de SirT6 produce silenciamiento génico tal y como se había descrito previamente. Además, SirT6 interacciona con proteínas involucradas en silenciamiento génico como es el complejo NuRD y EZH2 y la metiltransferases de H3K9, G9a y Suv39h1. Suv39h1 trimetila H3K9 necesario para la estructura de la heterocromatina. Está caracterizada la interacción entre Suv39h1 y SirT1 en el contexto de formación de la formación de heterocromatina. La relación entre Suv39h1 y SirT6 es bastante diferente a la de Suv39h1 y SirT1. SirT6 media una monoubiquitinación no canónica en tres cisteínas conservadas del dominio pre-SET de Suv39h1. Entre las E3 ubiquitina ligasas que interaccionan con Suv39h1 y SirT6 como CHIP y CHFR, encontramos que la responsable de la monoubiquitinación de Suv39h1 es SKP2. Mostramos que los niveles de SKP2 están regulados también por SirT6 la cual desacetila SKP2 e induce su fosforilación evitando así su degradación. La monoubiquitinación de Suv39h1 es inducida con tratamiento de doble bloqueo y timidina, que paran las células en G1/S y mitosis temprana, respectivamente. Además, la activación de la vía de NFkB induce la monoubiquitinación de Suv39h1. La expresión del regulador negativo de la vía de NFkB, IkBa, está regulada por la interacción entre Suv39h1, SirT6 y SKP2 y bajo tratamiento con TNFa. SirT6 induce la monoubiquitinación de Suv39h1 a través de SKP2 y Suv39h1 monoubiquitinizado es desplazado del promotor permitiendo la activación de la transcripción de IkBa por RelA. Este modelo proporciona nuevas perspectivas de la regulación de la vía NFkB.