Gic1 is a novel heterochromatin boundary protein in vivo

Abstract

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

Spt3 and Spt8 Are Involved in the Formation of a Silencing Boundary by Interacting with TATA-Binding Protein

“…ChIP was performed to determine the boundary position of the Chromatin III right telomere region. ChIP assays were performed as described previously [ 45 , 46 ]. Yeast cells were grown until reaching OD600 = 1.5 at 30 °C in YPD medium and collected by centrifugation at 4 °C for 5 min.…”

<i>IMD2</i>, located near the boundary of heterochromatin regions, is regulated by multiple HAT-related factors

“…Boundary-forming factors have been identified by screening at the HMR region (Hatanaka et al 2011;Mitsumori et al 2016b;Oki et al 2004) and include histone modifiers other than Sas2, such as components of NuA4 (a histone H4 acetylation complex) and Dot1 (involved in histone methylation) (Feng et al 2002;Lacoste et al 2002;van Leeuwen et al 2002). Many of the identified genes are components of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex, which activates transcription via histone acetylation and deubiquitination of histone H2B.…”