William H. Light

DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery

Many genes in Saccharomyces cerevisiae are recruited to the nuclear periphery after transcriptional activation. We have identified two gene recruitment sequences (GRS I and II) from the promoter of the INO1 gene that target the gene to the nuclear periphery. These GRSs function as DNA zip codes and are sufficient to target a nucleoplasmic locus to the nuclear periphery. Targeting requires components of the nuclear pore complex (NPC) and a GRS is sufficient to confer a physical interaction with the NPC. GRS I elements are enriched in promoters of genes that interact with the NPC, and genes that are induced by protein folding stress. Full transcriptional activation of INO1 and another GRS-containing gene requires GRS-mediated targeting of the promoter to the nuclear periphery. Finally, GRS I also functions as a DNA zip code in Schizosaccharomyces pombe, suggesting that this mechanism of targeting to the nuclear periphery has been conserved over approximately one billion years of evolution.

A Conserved Role for Human Nup98 in Altering Chromatin Structure and Promoting Epigenetic Transcriptional Memory

In yeast and humans, interaction of a nuclear pore protein with promoters alters chromatin structure and allows RNA polymerase II to bind, poising them for faster reactivation for several generations.

Cohesinopathy mutations disrupt the subnuclear organization of chromatin

Nuclear morphology, chromosomal condensation, and transcriptional-mediated localization of genes to the nuclear periphery are disturbed by mutations in cohesin pathway genes.

Quantitative localization of chromosomal loci by immunofluorescence.

DNA within the yeast nucleus is spatially organized. Yeast telomeres cluster together at the nuclear periphery, centromeres cluster together near the spindle pole body, and both the rDNA repeats and tRNA genes cluster within the nucleolus. Furthermore, the localization of individual genes to subnuclear compartments can change with changes in transcriptional status. As such, yeast researchers interested in understanding nuclear events may need to determine the subnuclear localization of parts of the genome. This chapter describes a straightforward quantitative approach using immunofluorescence and confocal microscopy to localize chromosomal loci with respect to well characterized nuclear landmarks.

Nuclear pore proteins regulate chromatin structure and transcriptional memory by a conserved mechanism

Previous experience alters the rate of transcriptional induction of many genes in yeast, and this phenomenon persists through several cell division cycles. This phenomenon is called epigenetic transcriptional memory. For the yeast gene INO1, transcriptional memory requires a physical interaction with the nuclear pore complex (NPC) and changes in the chromatin structure of the promoter. These changes lead to binding of a preinitiation form of RNA Polymerase II (RNAPII) to the INO1 promoter, bypassing the need to recruit RNAPII to the promoter during reactivation. In our recent study, we found that in human cells, hundreds of interferon-γ responsive genes exhibit a mechanistically similar form of transcriptional memory. Transcriptional memory requires a homologous nuclear pore protein in yeast and humans, which interacts with the promoters of genes that exhibit transcriptional memory and promotes both alteration of chromatin structure and binding of RNAPII. Whereas the interaction of yeast genes with nuclear pore proteins occurs at the NPC, the interaction of human genes with nuclear pore proteins occurs in the nucleoplasm. Thus, the interaction of nuclear pore proteins with genes plays an important and conserved role in affecting long-term epigenetic changes in transcriptional regulation.

Approaches to studying subnuclear organization and gene-nuclear pore interactions

Many genes in budding yeast Saccharomyces cerevisiae associate with the nuclear pore complex (NPC), which impacts their location within the nucleus and their transcriptional regulation. To understand how eukaryotic genomes are spatially organized, we have used multiple approaches for analyzing the localization and transcription of genes. We have used these approaches to study the role of DNA elements in targeting genomic loci to the NPC and how these interactions regulate transcription, chromatin structure and the spatial organization of the yeast genome. These studies combine yeast molecular genetics with live-cell microscopy and biochemistry. Here, we present detailed protocols for these cytological and molecular approaches.

Erratum: DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery (Nature Cell Biol. (2010) 12 (111-118))

Nature Cell Biol. 12, 102–104 (2010); published online 17 January 2010; corrected after print, 21 January 2010 In the version of this News and Views initially published, cells are incorrectly referred to as “α7-integrin+”. They should actually be “α7-integrin–”. This error has been corrected in the HTML and PDF version of the article. DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery

Epigenetic Transcriptional Memory of GAL Genes Depends on Growth in Glucose and the Tup1 Transcription Factor in Saccharomyces cerevisiae

Previously expressed inducible genes can remain poised for faster reactivation for multiple cell divisions, a conserved phenomenon called epigenetic transcriptional memory. The GAL genes in Saccharomyces cerevisiae show faster reactivation for up to seven generations after being repressed. During memory, previously produced Gal1 protein enhances the rate of reactivation of GAL1, GAL10, GAL2, and GAL7. These genes also interact with the nuclear pore complex (NPC) and localize to the nuclear periphery both when active and during memory. Peripheral localization of GAL1 during memory requires the Gal1 protein, a memory-specific cis-acting element in the promoter, and the NPC protein Nup100. However, unlike other examples of transcriptional memory, the interaction with NPC is not required for faster GAL gene reactivation. Rather, downstream of Gal1, the Tup1 transcription factor and growth in glucose promote GAL transcriptional memory. Cells only show signs of memory and only benefit from memory when growing in glucose. Tup1 promotes memory-specific chromatin changes at the GAL1 promoter: incorporation of histone variant H2A.Z and dimethylation of histone H3, lysine 4. Tup1 and H2A.Z function downstream of Gal1 to promote binding of a preinitiation form of RNA Polymerase II at the GAL1 promoter, poising the gene for faster reactivation. This mechanism allows cells to integrate a previous experience (growth in galactose, reflected by Gal1 levels) with current conditions (growth in glucose, potentially through Tup1 function) to overcome repression and to poise critical GAL genes for future reactivation.