Elsevier

Methods

Volume 40, Issue 3, November 2006, Pages 272-278
Methods

Genome-wide location analysis of the stress-activated MAP kinase Hog1 in yeast

https://doi.org/10.1016/j.ymeth.2006.06.007Get rights and content

Abstract

MAP kinase signal transduction pathways play a critical role in eukaryotic cells to unleash complex transcriptional programs to properly adapt to changing environments. The MAP kinase Hog1 upon activation is physically recruited to the chromatin of osmostress responsive genes. This allowed us to use in vivo chromatin immunoprecipitation in combination with microarrays (ChIP–Chip) to identify the transcriptional targets of Hog1 at the genomic scale. The ChIP–Chip method described here revealed that the stress-activated MAP kinase gets recruited to most of the osmoinducible genes. Interestingly Hog1 associates with both the 5′ upstream and the 3′ downstream sequences of stress genes. We confirmed by targeted ChIP at several stress genes that the MAP kinase crosslinks all over the transcribed regions in all cases tested. Taken together the genome-wide location analysis reported here is a powerful approach to determine the genomic binding patterns of an activated MAP kinase and will be of great interest to analyze other SAPKs under different environmental conditions.

Introduction

Mitogen-activated protein (MAP) kinases are key signaling molecules of eukaryotic cells to orchestrate adaptive responses to a wide variety of stress conditions. MAP kinase signaling pathways are evolutionarily conserved from yeast to humans [1]. One of the most important physiological functions of MAP kinases is the transcriptional activation of a large number of stress-regulated genes in the nucleus. The role of MAP kinases in stimulating gene expression goes far beyond the simple modulation of specific transcription factor activity. It has become clear, mainly from the work in the yeast model, that MAP kinases regulate transcription directly at the chromosome forming part of transcription complexes [2]. Their functions involve physical recruitment to the chromatin, phosphorylation of specific transcription factors, RNA polymerase II recruitment, chromatin modification, and transcript elongation. Whether MAP kinases of higher eukaryotes play similar roles as structural adaptors and essential subunits of transcriptional activator complexes remains to be confirmed experimentally. However, the stable interaction of mammalian MAP kinases with many specific transcription factors (reviewed in [2]) and the conservation of MAP kinase functions during evolution suggest that direct transcriptional activation in the chromatin context might be a general feature of MAP kinases.

Yeast cells have served as a powerful model to decipher the molecular mechanisms of MAP kinase cascade signaling and the functions of activated MAP kinases. One of the best studied signal transduction pathways in Saccharomyces cerevisiae is the high osmolarity glycerol (HOG) MAP kinase pathway [3]. Hyperosmotic stress leads to the rapid activation of its terminal MAP kinase Hog1 which is directly involved in an unexpectedly great number of adaptive processes. In a pre-transcriptional fast response Hog1 counteracts acute ion imbalances by directly phosphorylating the Na+/H+ antiporter Nha1 and the K+ channel Tok1, thus enabling the cell to respond at the level of transcription [4]. Hog1 also inhibits cell cycle progression during stress by directly targeting the CDK inhibitor protein Sic1 [5] and may regulate translation efficiency by phosphorylating the Rck2 kinase [6], [7]. Once activated, Hog1 is rapidly translocated to the nucleus [8] where it activates the complex transcriptional program upon hyperosmotic stress. Genomic expression profiling experiments revealed that Hog1 is necessary for the proper induction of the vast majority of >150 stress defense genes [9], [10].

The mechanisms by which Hog1 stimulates transcription are surprisingly complex. The MAP kinase interacts with and phosphorylates at least three structurally unrelated specific transcription factors: Sko1, Hot1, and Smp1 [11], [12], [13]. These DNA binding molecules have been shown to be required for the targeted recruitment of Hog1 to the chromatin at some osmoresponsive promoters [11], [14]. Once present at the chromatin structure of inducible genes, Hog1 can stimulate transcription by distinct mechanisms. The kinase modulates the transcription factor output by stimulating the entry of chromatin modifying complexes. This has been described for the histone acetyl transferase (HAT) complex SAGA or the chromatin remodeling complex SWI/SNF in the case of the Sko1 repressor/activator switch upon stress [14]. Hog1 recruits directly the histone deacetylase Rpd3 to osmostress regulated promoters [15]. Finally the MAP kinase seems to recruit the RNA polymerase II machinery directly to Hot1 regulated stress genes [16]. The events leading to stimulated transcription upon activation of Hog1 are depicted in Fig. 1. Taken together, the activated MAP kinase Hog1 forms an intimate complex of the chromatin at various stress regulated promoter regions.

Here, we describe a general methodology for analyzing the transcriptional targets of Hog1 that can be applied to other MAP kinase pathways. We determined the genomewide binding pattern of Hog1 upon hyperosmotic stress by the combination of chromatin immunoprecipitation and microarray hybridization (ChIP–Chip). The ChIP–Chip method is a powerful approach to identify the physical target sequences on a genomic scale for DNA-binding proteins and proteins that get recruited indirectly to chromatin. We used formaldehyde crosslink, chromatin immunoprecipitation, and hybridization of the co-precipitated DNA fragments to intergenic regions microarrays to identify the sequences bound by Hog1. The method reported here confirms the targeted recruitment of a MAP kinase to stress responsive promoters and reveals new findings about Hog1. We show that Hog1 recruitment strongly correlates with osmoinducibility of the gene. Furthermore we found that in many cases Hog1 is present at the 3′ downstream region of a stress regulated gene. Targeted ChIP analysis of some osmoresponsive genes confirmed Hog1 occupancy at the whole transcribed region and therefore a role of the MAP kinase in the process of transcript elongation [17]. Therefore the ChIP–Chip method described here successfully identified the genes directly up-regulated by a stimulated MAP kinase at the genomic scale and gave invaluable insights into the mechanisms of transcriptional activation.

Section snippets

Method

Here we describe the experimental steps to analyze the in vivo association of the Hog1 MAP kinase with promoter sequences in the whole yeast genome by ChIP–Chip. They essentially include: (1) formaldehyde-induced protein–DNA and protein–protein crosslinking of osmotically stressed yeast cultures; (2) ChIP: preparation of sheared chromatin, immunoprecipitation with specific antibodies, reversal of the crosslinks, protease treatment, and purification of co-precipitated genomic DNA fragments; (3)

Conclusions

The methodology described here illustrates how the ChIP analysis can be used to identify the genome wide location of a stress-activated MAP kinase. We successfully applied this approach to the stress-activated MAP kinase Hog1. The genomic ChIP–Chip data identify the genes which are directly targeted and activated by the MAP kinase. Moreover, the Hog1 occupancy all over the transcribed regions of stress genes characterizes the MAP kinase as a transcription elongation factor. The technology

Acknowledgments

This work was supported by an EMBO Long Term Fellowship and a grant (BFU2005-01714, partially founded by FEDER) from Ministerio de Educación y Ciencia, Spain to M.P. The authors thank to Drs. Stefan Hohmann and Francesc Posas for making available their expression profile data.

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