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.

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.

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.

A conserved role for human Nup98 in altering chromatin structure and promoting epigenetic transcriptional memory.

The interaction of nuclear pore proteins (Nups) with active genes can promote their transcription. In yeast, some inducible genes interact with the nuclear pore complex both when active and for several generations after being repressed, a phenomenon called epigenetic transcriptional memory. This interaction promotes future reactivation and requires Nup100, a homologue of human Nup98. A similar phenomenon occurs in human cells; for at least four generations after treatment with interferon gamma (IFN-γ), many IFN-γ-inducible genes are induced more rapidly and more strongly than in cells that have not previously been exposed to IFN-γ. In both yeast and human cells, the recently expressed promoters of genes with memory exhibit persistent dimethylation of histone H3 lysine 4 (H3K4me2) and physically interact with Nups and a poised form of RNA polymerase II. However, in human cells, unlike yeast, these interactions occur in the nucleoplasm. In human cells transiently depleted of Nup98 or yeast cells lacking Nup100, transcriptional memory is lost; RNA polymerase II does not remain associated with promoters, H3K4me2 is lost, and the rate of transcriptional reactivation is reduced. These results suggest that Nup100/Nup98 binding to recently expressed promoters plays a conserved role in promoting epigenetic transcriptional memory.

Interaction of a DNA zip code with the nuclear pore complex promotes H2A.Z incorporation and INO1 transcriptional memory.

DNA "zip codes" in the promoters of yeast genes confer interaction with the NPC and localization at the nuclear periphery upon activation. Some of these genes exhibit transcriptional memory: after being repressed, they remain at the nuclear periphery for several generations, primed for reactivation. Transcriptional memory requires the histone variant H2A.Z. We find that targeting of active INO1 and recently repressed INO1 to the nuclear periphery is controlled by two distinct and independent mechanisms involving different zip codes and different interactions with the NPC. An 11 base pair memory recruitment sequence (MRS) in the INO1 promoter controls both peripheral targeting and H2A.Z incorporation after repression. In cells lacking either the MRS or the NPC protein Nup100, INO1 transcriptional memory is lost, leading to nucleoplasmic localization after repression and slower reactivation of the gene. Thus, interaction of recently repressed INO1 with the NPC alters its chromatin structure and rate of reactivation.

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.

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.

Cohesinopathy mutations disrupt the subnuclear organization of chromatin.

In Saccharomyces cerevisiae, chromatin is spatially organized within the nucleus with centromeres clustering near the spindle pole body, telomeres clustering into foci at the nuclear periphery, ribosomal DNA repeats localizing within a single nucleolus, and transfer RNA (tRNA) genes present in an adjacent cluster. [corrected] Furthermore, certain genes relocalize from the nuclear interior to the periphery upon transcriptional activation. The molecular mechanisms responsible for the organization of the genome are not well understood. We find that evolutionarily conserved proteins in the cohesin network play an important role in the subnuclear organization of chromatin. Mutations that cause human cohesinopathies had little effect on chromosome cohesion, centromere clustering, or viability when expressed in yeast. However, two mutations in particular lead to defects in (a) GAL2 transcription and recruitment to the nuclear periphery, (b) condensation of mitotic chromosomes, (c) nucleolar morphology, and (d) tRNA gene-mediated silencing and clustering of tRNA genes. We propose that the cohesin network affects gene regulation by facilitating the subnuclear organization of chromatin.

Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells.

Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. Here, we show that the small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population.