Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation.

Frank Uhlmann,Yasutaka Kakui

doi:10.1007/978-1-4939-9520-2_3

Abstract

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in controlling chromatin organization. Condensin is an essential SMC complex that compacts chromatin to form condensed chromosomes in mitosis. Complete condensin inactivation is necessary to reveal how condensin converts interphase chromatin into mitotic chromosomes. Here, we have developed a condensin depletion system in fission yeast that combines transcriptional repression with auxin-inducible protein degradation. This achieves efficient condensin depletion without need for a temperature shift. Our system is useful when studying how condensin contributes to chromosome architecture and is applicable to the study of other SMC complexes.

Highlights

Spatial chromatin organization by Structural maintenance of chromosomes (SMC) complexes is at the heart of genome stability and faithful chromosome segregation
SMC complexes are evolutionary conserved, large proteinaceous rings that topologically entrap one or more DNAs to engage in higher order chromatin architecture [1]
Condensin consists of two SMC coiled-coil subunits, SMC2/Cut14 and SMC4/Cut3, and three non-SMC accessory subunits, CAP-D2/Cnd1, CAP-H/ Cnd2, and CAP-G/Cnd3 (Fig. 1a)

Summary

Introduction

Spatial chromatin organization by SMC complexes is at the heart of genome stability and faithful chromosome segregation. The SMC family member, condensin, plays a crucial role in the compaction of interphase chromatin to form condensed chromosomes in mitosis [2]. It plays roles in genome maintenance during interphase. In case of budding yeast condensin, cytoplasmic sequestration using the anchor-away approach successfully abolishes nuclear condensin function [5–7]. Condensin depletion in vertebrates has been achieved using RNA interference or promoter shut-off [8–10]. In these cases, depletion progresses slowly, typically over the duration of several cell divisions. An alternative approach is the use of TEV protease to target and inactivate an engineered condensin complex more quickly [11]. Efficient depletion of chicken DT40 cell condensin was reported using an auxin-inducible degron (aid) [12]

Cell Culture

Reagents for Western Blotting

Confirmation of Condensin Depletion by Western Blotting

26. Blocking

Anti-Tat1 antibody