Groupthink: chromosomal clustering during transcriptional memory.

Gene expression is regulated by the dynamic nature of DNA modification, histone modification, and recruitment of activating and repressing protein complexes at specific loci. Chromatin itself is further organized structurally into regions of varying accessibility, an aspect of which includes transient or sustained redistribution of portions of the genome from the nucleoplasm to the nuclear periphery. Targeting of genes to the nuclear membrane, frequently via association with components of the nuclear pore complex (NPC), has both positive and negative effects on the timing and magnitude of gene expression [1]. Moreover, chromatin localization to the nuclear membrane endows certain genes with a form of multigenerational transcriptional memory after inactivation, allowing faster reactivation in the progeny upon return to inducing conditions [2]. The mechanisms underlying these various phenomena are beginning to be understood at the molecular level, perhaps best exemplified by studies in the baker’s yeast Saccharomyces cerevisiae, where precise manipulation of environmental conditions can be coupled with state of the art single-cell imaging and genetics. For example, genes have been found to localize to the nuclear periphery in response to nutrient shift (GAL1-10, INO1, SUC2, HXK1) and heat shock (HSP104, TSA2) (summarized in ref [3]). This dynamic association requires NPC proteins and, in the case of INO1, two sequence elements (gene recruitment sequence I and II; GRS) in the promoter [4]. While the role of GRSII remains unclear, GRSI recruits the Put3 transcription factor as an intermediary for NPC targeting [5]. After shift to repressing conditions, the INO1 locus remains associated with the nuclear periphery for multiple generations, conferring transcriptional memory via stabilization of a poised RNA polymerase II complex [6, 7]. This phenomenon requires a different promoter element termed the memory recruitment sequence (MRS) and a different nuclear pore protein (Nup100) than those involved in GRS recruitment [6]. Therefore, unique “DNA zip codes” dictate targeting of a gene to the nuclear periphery via distinct protein interactions. Importantly, these targeting events are independent of each other – GRSI, II and Put3 are not required during memory and the MRS is apparently dispensable for localization during gene activation [6]. The Brickner laboratory has paved the way in our understanding of these events, and elucidated much of the molecular detail. Central to their discoveries has been the development of methods to quantitatively assess subnuclear chromatin positioning. These approaches utilize DNA arrays consisting of Lac (LacO) and Tet (TetO) Operator elements integrated at loci of interest paired with fluorescent protein-DNA binding protein fusions. Cells bearing these endogenous and/or ectopic constructs can then be visualized with confocal microscopy and intra-nuclear distances measured (i.e., gene-gene or gene-nuclear periphery) for individual cells within a population. A key observation derived from these analyses was that two copies of the same locus, for example INO1/INO1 in a diploid cell, identified with different color fluorophores, were found to cluster together in three-dimensional space [8]. Moreover, two independent loci, each bearing a GRSI element, likewise cluster during activation [8]. In the article published in this issue of Microbial Cell, Brickner and co-workers reveal that clustering also occurs during transcriptional memory [9]. Multiple aspects of INO1 memory clustering differentiate this phenomenon from that observed upon gene activation. Transcriptional memory at the GAL1-10 locus requires the nuclear basket protein Mlp1, in contrast to INO1 which targets to the NPC through Nup100 [6, 10]. Consistently, GAL1 and INO1, when differentially labeled, were found not to cluster after shift to repressing conditions, indicating specificity [9]. The MRS zip code was also shown to be required for clustering, functionally linking this feature with targeting to the nuclear periphery during memory. Fascinatingly, the MRS was seen in this study to be insufficient for memory-based clustering – both GRSI and GRSII elements are also required. The same held true for the role of the GRSI binding protein Put3 and the gene activation nuclear periphery recruitment protein Nup100 [9]. Therefore, clustering during transcriptional memory requires previous clustering during gene activation, and each process relies on its own previ-