SIR proteins create compact heterochromatin fibers

Abstract

2 SummaryHeterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins --Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to previous studies, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which two molecules of Sir3 bridge and stabilize two adjacent nucleosomes, while a single Sir2/4 heterodimer binds the intervening linker DNA, driving fiber compaction. IntroductionEukaryotic cells regulate the accessibility of their genome to enzymatic processes by organizing it into two functionally distinct compartments, known as euchromatin and heterochromatin. Euchromatin consists of actively transcribed gene regions, whereas heterochromatin is refractory to external processes such as transcription and recombination 1 .Heterochromatin organizes and protects centromeres and telomeres, guards against the spreading of transposons, and prevents aberrant homologous recombination within repetitive regions that can lead to chromosomal abnormalities such as deletions, inversions, and translocations. [2][3][4] Additionally, heterochromatin formation is an essential developmental process All rights reserved. No reuse allowed without permission.

The Sir4 H‐ BRCT domain interacts with phospho‐proteins to sequester and repress yeast heterochromatin

“…The SIR complex, composed of the NAD + -dependent histone deacetylase Sir2, the histone binding protein Sir3, and the 152 kDa scaffold protein Sir4, serves as a model system to study epigenetically silenced heterochromatin in higher eukaryotes (reviewed in refs Gartenberg & Smith, 2016;Oppikofer et al, 2013). Binding of these factors initiates chromatin repression by the recruitment of Sir1 and/or a heterotrimeric SIR complex that can spread along the chromatin fiber by oligomerization (Rudner et al, 2005;Cubizolles et al, 2006;Swygert et al, 2018). Binding of these factors initiates chromatin repression by the recruitment of Sir1 and/or a heterotrimeric SIR complex that can spread along the chromatin fiber by oligomerization (Rudner et al, 2005;Cubizolles et al, 2006;Swygert et al, 2018).…”

“…Repression of genes in S. cerevisiae is initiated by DNAbinding proteins repressor activator protein 1 (Rap1) (Shore et al, 1987;Moretti et al, 1994), ARS-binding factor 1 (Abf1) (Sussel & Shore, 1991), and origin recognition complex subunit 1 (Orc1) , which recognize specific DNA sequences located in silencer elements or telomeric repeats. Binding of these factors initiates chromatin repression by the recruitment of Sir1 and/or a heterotrimeric SIR complex that can spread along the chromatin fiber by oligomerization (Rudner et al, 2005;Cubizolles et al, 2006;Swygert et al, 2018). Central to nucleation is Rap1, which binds with high affinity to silencer elements and telomeric DNA (Buchman et al, 1988); it then interacts with Sir4 to recruit Sir2 to nucleosomes, allowing Sir2 to deacetylate the N-terminal histone H4 tail (Moretti et al, 1994;Hecht et al, 1995;Imai et al, 2000;Tanner et al, 2000).…”

Sir3 heterochromatin protein promotes non‐homologous end joining by direct inhibition of Sae2

“…The competition between Sae2 and Sir4 for Sir3 binding questions the relevance of Sae2-Sir3 interaction in subtelomeric heterochromatin. Recent in vitro data support a stoichiometry of two Sir3 molecules and one Sir2-4 dimer per nucleosome (Swygert et al, 2018). This suggests that one Sir3 molecule per nucleosome might not be interacting with Sir4 on chromatin, leaving room for the binding and inhibition of Sae2 on heterochromatin-bound Sir3.…”

Physical principles and functional consequences of nuclear compartmentalization in budding yeast

“…These foci concentrate the silent regulatory factors Sir2, Sir3 and Sir4 that form the SIR complex [43]. In vitro, this complex associates with nucleosome with 1:2:1 stoechiometry and compacts chromatin [44]. Once recruited at the telomeres through interaction with the telomeric protein Rap1, this complex has the ability to spread on chromatin, and repress underlying genes [45], possibly by forming a compact chromatin structure [44].…”