Casein kinase 2 mediated phosphorylation of Spt6 modulates histone dynamics and regulates spurious transcription

CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we showed that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production are associated with a defective adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription.

The Pygo2-H3K4me2/3 interaction is dispensable for mouse development and Wnt signaling-dependent transcription

Michel Aguet

Pygopus has been discovered as a fundamental Wnt signaling component in Drosophila. The mouse genome encodes two Pygopus homologs, Pygo1 and Pygo2. They serve as context-dependent beta-catenin coactivators, with Pygo2 playing the more important role. All Pygo proteins share a highly conserved plant homology domain (PHD) that allows them to bind di- and trimethylated lysine 4 of histone H3 (H3K4me2/3). Despite the structural conservation of this domain, the relevance of histone binding for the role of Pygo2 as a Wnt signaling component and as a reader of chromatin modifications remains speculative. Here we generate a knock-in mouse line, homozygous for a Pygo2 mutant defective in chromatin binding. We show that even in the absence of the potentially redundant Pygo1, Pygo2 does not require the H3K4me2/3 binding activity to sustain its function during mouse development. Indeed, during tissue homeostasis, Wnt/beta-catenin-dependent transcription is largely unaffected. However, the Pygo2-chromatin interaction is relevant in testes, where, importantly, Pygo2 binds in vivo to the chromatin in a PHD-dependent manner. Its presence on regulatory regions does not affect the transcription of nearby genes; rather, it is important for the recruitment of the histone acetyltransferase Gcn5 to chromatin, consistent with a testis-specific and Wnt-unrelated role for Pygo2 as a chromatin remodeler.

Company Of Biologists Ltd2013

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates

Mathias Fournier

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.

2010

Toward a mechanistic understanding of variable chromatin modules

Gerard Llimos Aubach

Our genome is a long sequence of DNA that contains all the information to be able to constitute a living organism like us, similarly to what the letters in a book do to create a story. This sequence, which is a stretch of molecules called nucleotides, is almost identical between organisms of the same species (>99.9% in humans), however it varies in a small percentage due to some nucleotides being different at specific positions of the genome. This variation in the DNA will therefore influence our traits and affect our susceptibility to a certain condition. Thus, their study is highly relevant in genetics and biomedicine since they allow not only to predict the predisposition to a disease, such as cancer, but also to understand the molecular and cellular mechanism behind a certain phenotype. The effect of a genetic variant on a phenotype is given by their capacity to modulate the expression of a gene, either by directly impacting its sequence and thus changing the protein, or by regulating its expression levels. Interestingly, more than 88% of the disease-associated genetic variants present in humans are located outside genes, on the so-called non-coding part of the genome. This poses a challenge to identify what gene the genetic variant affects and to understand how it does so. Genetic variants do not only influence gene expression but they also affect the epigenetics state of the DNA (i.e. DNA methylation, histone marks, transcription factor (TF) binding...) and its 3D conformation, consequently modifying the activity of gene regulatory elements like promoters and enhancers. Previously, it has been shown that some of these regulatory regions located in the same locus exhibit a high level of molecular coordination and interindividual variation, mostly affected by genetic variation or environmental effects. These regions were termed variable chromatin modules (VCMs) or cis-regulatory domains (CRDs) and are thought to be the manifestation of fine-grained regulatory units of the genome. Understanding how a VCM is formed and the molecular basis behind the cooperativity between regulatory elements is an outstanding question in the field. In this work, we aim at resolving part of this puzzle by mechanistically dissecting one VCM that is influenced by a non-coding germline variant in B cells. Using a set of tools such as genetic engineering, TF binding assays and chromosome conformation studies, we demonstrate that this variant creates a de novo binding site for the TF MEF2, which acts as a pioneering factor for dozens of other collaborating TFs. In turn, this actuates a cluster of enhancers spanning a region over 150 kilobases (kb) that regulates AXIN2 gene expression, and is associated with a global compaction of the chromatin. In addition, in line with the known tumor-suppressor properties of AXIN2, we found that this variant influences chronic lymphocytic leukemia (CLL) progression and predisposition. Together, the characterization of the AXIN2 VCM provides an unique example to the community of how a germline variant is able to switch on an entire genomic locus with high degree of cooperativity between regulatory elements, since it captures both the formation and transition behavior of a VCM. Furthermore, given the link with CLL, this focal variant could have translational potential by serving as a patient-stratifying or treatment diagnostic.

EPFL2021

Toward a mechanistic understanding of variable chromatin modules

Gerard Llimos Aubach

EPFL2021