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Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.
In a eukaryotic nucleus, DNA is wrapped around an octamer of basic histone proteins (H2A, H2B, H3 and H4) to form nucleosomes, the building blocks of chromatin. An important regulatory mechanism that governs overall chromatin structure for factor access is the covalent posttranslational modifications of histones. Histones are extensively adorned with a wide variety of modifications, including acetylation, phosphorylation, methylation, ubiquitination and sumoylation (1–2). Methylation of lysine residues (K) in histones can be present as a mono- (me1), di- (me2) or trimethylated (me3) form, further diversifying the function and complexity of this modification. While histone modifications serve as “marks” for cellular processes (2–3) and are recognized by factors with specific interaction modules (1, 4), their regulation is highly dynamic with the presence of different types and families of enzymes that can either add or remove them (5). Additionally, these enzymes, which might exist as components of multi-protein complexes, are themselves regulated by both endogenous and exogenous cues. Another interesting facet to the regulation of histone modifications is that one modification can regulate another within the same histone (in cis) or on different histones (in trans), a phenomenon termed the “histone crosstalk” (6–8). Regulation of H3K4 and -K79 methylation by H2BK123 monoubiquitination (H2Bub1) during transcription is a well-studied example of a trans-histone modification crosstalk.
This trans-histone regulatory circuit is evolutionarily conserved, as H2Bub1 is important for H3K4 and -K79 methylation in most eukaryotes (9). However, several valuable characteristics, such as the short life cycle, powerful genetics, ease of genome manipulation and being easily amenable to biochemical and high-throughput genome-wide studies, have made budding yeast (Saccharomyces cerevisiae) as the model system of choice to identify and extensively investigate the regulatory pathways, factors and enzymes involved in mediating this trans-histone crosstalk. Rad6, the E2 ubiquitin-conjugating enzyme and Bre1, the E3 ubiquitin ligase are required to conjugate ubiquitin to K123 present in the H2B C-terminal helix (10–11). Ubp8 and Ubp10 are the deubiquitinases involved in the removal of this conjugated ubiquitin to maintain the total H2Bub1 levels in the cell (9). Set1-COMPASS, a multi-protein complex consisting of the methyltransferase (Set1) and seven subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1), catalyzes all forms of H3K4 methylation (H3K4me). Dot1, a non-SET domain methyltransferase, catalyzes all forms of H3K79 methylation (H3K79me) (1, 11). A role for H2Bub1 in modulating the enzymatic functions of these methyltransferases by affecting their ability to catalyze the different forms of H3K4 and K79 methylation is now well established (9, 11): H2Bub1 is required for H3K4me2, H3K4me3 and H3K79me3. Additionally, H3K4me1 and H3K79me2 are severely reduced in the absence of H2Bub1.
To explain the mechanism of trans-histone crosstalk, it was proposed that the ubiquitin conjugated onto H2B might act as a “bridge” to directly recruit the methyltransferases (12). Since Set1 and Dot1 associate with chromatin even in the absence of H2Bub1 (13–14), their recruitment does not appear to be the basic mechanism by which H2Bub1 participates in the crosstalk. Two studies have alluded to Swd2, a Set1-COMPASS subunit, as a key link in this crosstalk (15–16), but their conflicting findings and conclusions have left the regulation of methyltransferase functions by H2Bub1 as an open question. H2Bub1 has also been proposed to act as a “wedge” to open-up the chromatin and allow access to the enzymes (12, 17). However, contrary to its supposed role in opening up chromatin, using chromatin immunoprecipitation assays (ChIP) and salt-dependent nucleosome disruption assays, we recently showed that H2Bub1 stabilizes the nucleosome by preventing the constant H2A-H2B eviction (18). This finding addresses a longstanding question in chromatin biology as to whether conjugation of bulky ubiquitin moiety onto histones affects nucleosome structure. Further, it has provided a new working model for the trans-histone crosstalk: addition of ubiquitin onto H2B acts as a “glue” to hold the nucleosome together and provides a stable platform for the prolonged chromatin association of Set1-COMPASS and Dot1 to promote their processive methylation. However, understanding this trans-histone crosstalk is an ongoing saga that is far from completion.
Several questions pertaining to the mechanism of nucleosome stabilization by H2Bub1 and the chromatin association of Set1-COMPASS and Dot1 remain to be explored. The basic patch in H4 N-terminal plays a role in a novel trans-histone pathway by controlling the chromatin binding and functions of Dot1 (19). However, the question as to how H2Bub1 controls Dot1 function remains unanswered. While a “docking site” for Dot1 on chromatin via the H4 tail region is now known, how Set1-COMPASS associates with chromatin and how this multi-subunit protein complex is assembled on chromatin remain unknown. We recently found that residues R119 and T122 in the H2B C-terminal helix interact with Spp1, a Set1-COMPASS subunit, and they modulate the chromatin association, integrity and overall stability of Set1-COMPASS independent of H2Bub1 (20). Importantly, we have uncovered a “docking” surface for only Set1-COMPASS, since mutations in R119 and T122 do not affect the functions of Dot1; thereby, revealing an uncoupling of the H2Bub1-mediated co-regulation of H3K4 and -K79 methylation. Therefore, a simple model that can be proposed for the trans-histone crosstalk between H2Bub1 and H3K4 methylation is as follows: H2Bub1 stabilizes the nucleosome by preventing H2A-H2B eviction. In turn, this leads to the retention of a “docking site” for Set1-COMPASS present in H2B on chromatin, culminating in increased complex integrity and stability of Set1-COMPASS needed for high levels of processive H3K4 methylation. While considerable effort has been invested in understanding how the methyltransferases associate with chromatin, the binding of Rad6/Bre1 and Ubp8/Ubp10 to chromatin remains poorly understood and needs further investigation.
In this report, we provide detailed procedures used in our previous studies to address some of the questions mentioned above. An improved method is provided to assess the steady state levels of histones and any other proteins in total cell extracts or nuclear extracts isolated under native or denaturing condition (sections 1.1 and 1.2). A quick and easy method to isolate and detect H2Bub1, a highly labile modification, employing a simple boiling procedure is described in section 1.3. To measure changes in the chromatin association of histones, histone modifying enzymes and their regulatory factors, two different assays are described in section 2. The chromatin association assay measures global changes in protein levels on chromatin obtained from isolated nuclei (section 2.1). On the other hand, local changes in the occupancy and distribution of histones, histone modifications and factors on genes or in different regions of a gene can be assessed employing the ChIP assay (section 2.2.1). A procedure to evaluate changes in the distribution and occupancy of H2Bub1 using chromatin-double immunoprecipitation is provided in section 2.3.
Many histone-modifying enzymes (HME) and chromatin-remodeling factors (CRF) exist as multi-subunit protein complexes. Their intermolecular interactions and chromatin association are likely subjected to highly dynamic regulation in vivo with high turnover or on/off rates. Therefore, it is conceivable that the integrity and overall stability of a given complex and its biochemical activity purified from total cell populations might not necessarily reflect the events that occur on chromatin in vivo. The fractionation procedure described below in section 2.1, which is derived from previous studies (26–28), allows direct assessment of the global chromatin-bound levels of the components of a specific protein complex containing HME/CRF under a given experimental condition. Additionally, a refined protocol for ChIP assay to examine local, gene and region-specific changes in the chromatin binding of components present in HME/CRF-containing complexes is provided in section 2.2.
Antibodies specific to mammalian (human, mouse and rat) and budding yeast H2Bub1 have been generated and used to perform ChIP assays (31–32). However, only the mammalian-specific H2Bub1 antibody is commercially available. Therefore, two sequential immunoprecipitations are needed to measure H2Bub1 occupancy in yeast and in any other species for which an H2Bub1-specific antibody is not commercially available. As described in this section, N-terminally Flag epitope-tagged H2B is isolated using α-Flag in the first IP followed by Flag peptide elution. The eluate is then used in a second round of IP using an antibody that recognizes mono- or poly-ubiquitinated proteins. For IP using α-Flag, chromatin from a yeast strain harboring H2B without the Flag tag (no tag control) should included as a control to confirm enrichment of Flag-H2B. Chromatin from a yeast strain harboring a mutation in the site of ubiquitination (Flag-H2B-K123R) should also be included as a control to measure and confirm enrichment of H2Bub1.
We have adopted and refined many of the standard and fundamental procedures used in studying the regulation and functions of histone modifications. Although the procedures described here have been tailored to investigate the crosstalk between H2Bub1 and H3K4/-K79 methylation in budding yeast, they are in general applicable to any histone or non-histone proteins and their modifications that might occur in yeast or in other model organisms. Advantages of the improved methodologies are as listed below.
We thank Vincent Geli for the Set1 epitope-tagging plasmids; Ethan Lee for the HA and Myc mouse monoclonal antibodies; and Mary Ann Osley, Brian Strahl and Tony Weil for some of the yeast strains used in this study. We also thank Ya-Ting Chung and Yi-Chun Chen for their valuable contributions in the standardization of the experimental procedures. This work was supported by funds from The Vanderbilt-Ingram Cancer Center, The Robert J. and Helen C. Kleberg Foundation, and National Institutes of Heath (RO1CA109355).
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