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Molecular and Cellular Biology, November 2007, p. 7641-7648, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00742-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Regulation of Gene Transcription by the Histone H2A N-Terminal Domain{triangledown} ,{dagger}

Michael A. Parra and John J. Wyrick*

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660

Received 27 April 2007/ Returned for modification 19 June 2007/ Accepted 13 August 2007


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ABSTRACT
 
Histone N-terminal domains play critical roles in regulating chromatin structure and gene transcription. Relatively little is known, however, about the role of the histone H2A N-terminal domain in transcription regulation. We have used DNA microarrays to characterize the changes in genome-wide expression caused by mutations in the N-terminal domain of histone H2A. Our results indicate that the N-terminal domain of histone H2A functions primarily to repress the transcription of a large subset of the Saccharomyces cerevisiae genome and that most of the H2A-repressed genes are also repressed by the histone H2B N-terminal domain. Using the histone H2A microarray data, we selected three reporter genes (BNA1, BNA2, and GCY1), which we subsequently used to map regions in the H2A N-terminal domain responsible for this transcriptional repression. These studies revealed that a small subdomain in the H2A N-terminal tail, comprised of residues 16 to 20, is required for the transcriptional repression of these reporter genes. Deletion of either the entire histone H2A N-terminal domain or just this small subdomain imparts sensitivity to UV irradiation. Finally, we show that two residues in this H2A subdomain, serine-17 and arginine-18, are specifically required for the transcriptional repression of the BNA2 reporter gene.


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INTRODUCTION
 
The packaging of DNA into chromatin has profound consequences on the regulation of gene transcription. The principal packaging unit of chromatin is the nucleosome core particle, consisting of two copies each of histones H2A, H2B, H3, and H4 and 147 bp of DNA (16). The arrangement and organization of genomic DNA into nucleosomes are not random processes, but instead appear to play an important regulatory function in transcription (14). Regions of genomic DNA are also distinguished by distinct patterns of posttranslational modifications that decorate the associated histone proteins (11). A large body of scientific literature has shown that histone posttranslational modifications are important regulators of transcription (2).

The N-terminal domains of histone proteins play critical roles in both the organization and posttranslational modification of chromatin. Previous studies have shown that the histone N-terminal domains function to control the translational positioning of nucleosomes in vitro (24). Histone N-terminal domains can mediate internucleosome interactions that are required for the formation of higher-order chromatin structures (4, 25). Histone N-terminal domains are also primary sites of posttranslational modifications. These modifications, such as lysine acetylation, lysine and arginine methylation, and serine phosphorylation, can alter the stability of the nucleosome and regulate the association of transcriptional regulatory proteins (2, 6, 21).

In Saccharomyces cerevisiae, a single histone H2A N-terminal modification has been verified, i.e., acetylation of H2A K7 (20, 22). An additional acetylation site at H2A K4 is suspected (3, 18); however, this modification has yet to be verified in vivo. Both H2A K4 and K7 are acetylated in vitro by the Esa1 histone acetyltransferase (3, 18, 20). Mutants in which H2A K4 and K7 have been changed to arginine show defects in transcriptional silencing at yeast telomeres (23). Beyond this phenotype, little is known about the functional roles of H2A K4 and K7 acetylation in gene regulation.

Deletion of the entire histone H2A N-terminal domain (residues 1 to 20) leads to a defect in the repression of basal transcription from the GAL1 and HTA1 promoters (7, 15). Deletions or mutations in the H2A N-terminal domain result in a defect in SUC2 transcription (5) and cause growth defects on various carbon sources, including raffinose and galactose (5).

To define the function of the histone H2A N-terminal domain in transcription regulation, we used genome-wide expression profiling to identify genes whose expression is altered by histone H2A N-terminal mutations or deletions. Our results indicate that H2A acetylated lysine residues K4 and K7 are required for the transcriptional repression of a small set of genes involved in NAD biosynthesis and vitamin metabolism. In contrast, we found that the H2A N-terminal domain is required for the transcriptional repression of a much larger set of yeast genes (~4% of the yeast genome) and that most of these genes are also repressed by the histone H2B N-terminal domain. We have mapped functional regions in the H2A N-terminal domain and found that a small subdomain comprised of H2A residues 16 to 20 is required for this repression. We show that deletion of this subdomain or of the entire H2A N-terminal domain confers sensitivity to UV irradiation. Finally, we identify two functional residues in this H2A subdomain that are specifically required for transcriptional repression.


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MATERIALS AND METHODS
 
Yeast strains and growth conditions. The yeast strains used in this study are listed in Table 1. Each was derived from strain PY013, which has a W303 genetic background (see reference 12 for more details). For the genome-wide expression experiments, three mutant and three wild-type yeast cultures were grown in yeast extract-peptone-dextrose medium to a final optical density at 600 nm of 0.5 to 0.7 and then harvested as described previously (10).


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  		TABLE 1. Yeast strains used in microarray experiments

Plasmid construction. All histone H2A mutants were generated by site-directed mutagenesis (QuikChange kit; Stratagene), using plasmid pMP002 as a template. Plasmid pMP002 contains wild-type histone H2A (HTA1) and H2B (HTB1) genes (12). Mutagenic primer sequences are listed at http://wyrick.sbs.wsu.edu/histoneH2A/. Each histone H2A mutation was confirmed by DNA sequencing.

Genome-wide expression profiling. Total RNA was isolated from each yeast culture and used to prepare cDNA and biotin-cRNA, as described elsewhere (10). The biotin-labeled cRNA was hybridized to a single S98 genome microarray (Affymetrix) and scanned following standard protocols (Affymetrix). Probe fluorescence intensities were captured using GeneChip software (Affymetrix), and a single raw expression level was determined for each gene. Array data sets are available at http://wyrick.sbs.wsu.edu/histoneH2A/.

Data analysis. The data from each chip were normalized using GeneChip software (version 5; Affymetrix), as no global changes in mRNA levels were detected. A modified version of the error model analysis method was used to identify differentially expressed genes, as previously described (12). A P value cutoff of 0.001 was used to identify differentially expressed genes. Lists of differentially expressed genes in the H2A {Delta}4-20 and H2A K4,7G mutant strains are provided in Tables S1 and S2, respectively, in the supplemental material.

Statistical analysis of array data. A hypergeometric probability model was used to calculate the statistical significance of the overlap between the lists of up- and down-regulated genes in the various histone mutants. The resulting P values were calculated numerically, using the Mathematica software package (Wolfram Research).

Significant functional enrichments were identified in the lists of up- and down-regulated genes by using the FunSpec tool (17). Searches were conducted at http://funspec.med.utoronto.ca/, using a P value cutoff of 0.05 and a Bonferroni correction for multiple hypothesis testing.

Reverse transcription-PCR (RT-PCR). Total RNA was isolated from histone H2A mutant or wild-type yeast following the methods described above. DNase I-treated RNA was used as a template for cDNA synthesis, using a cDNA synthesis kit (Roche) according to the manufacturer's instructions. The resulting cDNA was used as a template for multiplex PCR. Primers were selected to amplify BNA1, BNA2, or GCY1 in combination with ACT1 (as an internal loading control). Primer sequences are provided at http://wyrick.sbs.wsu.edu/histoneH2A.

The following PCR cycling conditions were used for RT-PCR analysis: for BNA1, 30 s at 95°C, 1 min at 52.6°C, and 1 min at 72°C, with a 10-min extension at 72°C after 33 cycles; for BNA2, 30 s at 95°C, 1 min at 57.2°C, and 1 min at 72°C, with a 10-min extension at 72°C after 32 cycles; and for GCY1, 30 s at 95°C, 1 min at 47°C, and 1 min at 72°C, with a 10-min extension at 72°C after 30 cycles. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Gel images were taken using a GelDoc EQ imager (Bio-Rad), and band intensities were quantified using Quantity One software (Bio-Rad). BNA1, BNA2, and GCY1 expression levels were normalized using the ACT1 intensity as a loading control, since ACT1 expression was not significantly altered in any of the histone H2A mutants.

UV sensitivity assay. Triplicate cultures of strains containing mutant or wild-type histone H2A were grown in yeast extract-peptone-dextrose medium to mid-log phase (optical density at 600 nm of 0.5 to 0.7) and then serially diluted and plated (from 1 x 105 to 1 x 102 cells per plate). Plates were treated with 0, 50, or 100 J/m2 of UV light (primarily at 254 nm). Following treatment, strains were allowed to grow for 2 days at 30°C in the dark. Colonies were counted and compared to untreated (0 J/m2) controls.

Microarray accession numbers. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession numbers GSE7337 and GSE7338.


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RESULTS
 
The histone H2A N-terminal domain is required for the repression of a large set of yeast genes. We investigated the importance of the histone H2A N-terminal domain in regulating yeast transcription. To this end, we deleted the H2A N-terminal domain (H2A {Delta}4-20) and characterized the consequent changes in yeast gene expression by using whole-genome oligonucleotide microarrays (Affymetrix). Each microarray experiment (mutant and wild type) was performed on RNA samples isolated from three independent biological replicates. To analyze the microarray data, we employed a triple-array error model as described previously (12). The error model estimates the significance (or P value) of the observed changes in mRNA levels. In these experiments, we deemed genes with P values of <0.001 to be expressed differentially in the mutant strain compared to the wild type. When we compared the array data for independent sets of wild-type samples by using this method, only 6 of 6,063 genes were deemed differentially expressed (Fig. 1A).


Figure 1
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  		FIG. 1. Genome-wide expression analysis of histone H2A mutants. (A and B) Average fluorescence intensities from triplicate array data sets for all yeast genes. In panel A, two independent wild-type data sets are compared (data are from reference 12); in panel B, wild-type and histone H2A mutant ({Delta}4-20) data sets are compared. Each point represents the expression level of a single gene. Genes whose mRNA levels are significantly altered (according to the error model analysis method) are shown in red, and those whose mRNA levels are unchanged are shown in blue. (C) Numbers of up- and down-regulated genes in each of the histone H2A mutant strains.

Analysis of the array data for the histone H2A {Delta}4-20 mutant strain revealed that the mRNA levels of 248 genes were up-regulated and the mRNA levels of 44 genes were down-regulated in the mutant compared to the wild type (Fig. 1). The list of up-regulated genes showed significant enrichment of genes involved in vitamin metabolism (P = 1.5 x 10–6), NAD biosynthesis (P = 4.6 x 10–6), and arginine biosynthesis (P = 3.3 x 10–5). These results indicate that under standard growth conditions, the histone H2A N-terminal domain regulates the transcription of 4.8% of the yeast genome and functions primarily to repress transcription.

Transcriptional repression by the histone H2A N-terminal domain is largely independent of H2A K4 and K7. Previous studies have shown that histone H2A can be acetylated at K4 and K7 in its N-terminal domain (20, 22). While the role of these acetylated lysine residues in transcription regulation is largely unknown, we hypothesized that deletion of these functional residues was responsible for the changes in genome expression observed in the H2A {Delta}4-20 mutant strain. To test this hypothesis, we profiled the changes in gene expression in a histone H2A mutant in which K4 and K7 were mutated to glycine. To our surprise, we found that the mRNA levels of only 28 genes were up-regulated and the mRNA levels of 6 genes were down-regulated in the H2A K4,7G mutant relative to those in the wild type (Fig. 1C). A large fraction of the genes up-regulated in the H2A K4,7G mutant overlap with genes up-regulated in the H2A {Delta}4-20 mutant (P = 6.1 x 10–13), particularly genes that function in NAD or arginine biosynthesis (e.g., ARG1, ARG3, ARG4, ARG6, BNA2, and HIS4). It is clear, however, that H2A K4 and K7 were not responsible for the majority of the gene expression changes observed in the H2A {Delta}4-20 mutant. Taken together, these data indicate that lysine-4 and lysine-7 are responsible for the repression of only a small subset of genes repressed by the entire H2A N-terminal domain, indicating that other regions in the H2A N-terminal domain must play important roles in gene repression.

It is possible that the mutations in the H2A N-terminal domain might affect gene expression indirectly by destabilizing the histone H2A protein. A reduction in histone H2A protein levels could presumably lead to the induction of gene transcription. We tested this model by performing Western blot analysis of chromatin extracts isolated from the histone H2A mutants profiled above. The results indicated that the levels of the histone H2A protein were not significantly altered in the H2A {Delta}4-20 and H2A K4,7G mutants compared to that in the wild type (see Fig. S1 in the supplemental material).

The histone H2A and H2B N-terminal domains have similar effects on gene transcription. Because histone H2A and H2B are present as heterodimers in the nucleosome core particle and because the H2A and H2B N-terminal domains are in close proximity to one another in the nucleosome structure (8, 9), we hypothesized that the H2A and H2B N-terminal domains might act jointly to repress gene transcription. To test this hypothesis, we compared the gene expression effects of the H2A N-terminal deletion to effects of the H2B N-terminal deletion mutant (12). Figure 2A shows that 79% of the genes up-regulated in the H2A {Delta}4-20 mutant were also up-regulated in the H2B {Delta}3-37 mutant, which is a highly significant overlap (P = 2.1 x 10–151). In contrast, only 14% of the genes up-regulated in the histone H4 N-terminal deletion mutant (H4 {Delta}2-26) (19) were also up-regulated in the H2A {Delta}4-20 mutant (Fig. 2B). While the degree of overlap between the genes up-regulated in the H4 {Delta}2-26 and H2A {Delta}4-20 mutants is statistically significant (P = 1.5 x 10–10), the magnitude of the overlap is clearly smaller than that observed between the H2A and H2B N-terminal deletion mutants.


Figure 2
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  		FIG. 2. Histone H2A and H2B N-terminal deletion mutants have similar effects on the expression of a core set of yeast genes. (A) Overlap in the set of genes up-regulated in the H2A {Delta}4-20 and H2B {Delta}3-37 mutants, depicted using a Venn diagram. The size of the circle is proportional to the number of genes up-regulated in each mutant; the degree of overlap is proportional to the number of genes shared between data sets. (B) As a control, the overlap between the sets of genes up-regulated in the histone H4 {Delta}2-26 (data are from reference 19) and H2A {Delta}4-20 mutants is shown. The histone H4 data were processed prior to this comparison to filter out redundant gene entries in the data set. (C) Comparison of changes in mRNA levels for all yeast genes in the H2A {Delta}4-20 and H2B {Delta}3-37 mutants. The log2 ratio of the change in mRNA level (mutant/wild type) was plotted for each gene. (D) Changes in mRNA levels in the H2A {Delta}4-20 and H4 {Delta}2-26 mutants.

We also directly compared the changes in mRNA levels for all yeast genes in the histone H2A and H2B mutant strains (Fig. 2C). Examination of the plot in Fig. 2C indicates that most genes showed similar changes in mRNA levels in the H2A {Delta}4-20 and H2B {Delta}3-37 mutants, although the magnitudes of the changes were generally smaller for the H2A {Delta}4-20 mutant. Indeed, the expression changes in the H2A {Delta}4-20 and H2B {Delta}3-37 mutant strains are fairly well correlated (r = 0.78). As a control, we compared the expression changes of the H2A and H4 N-terminal deletion strains, as these mutants have a relatively small degree of overlap based on our analysis criteria (Fig. 2B). Inspection of Fig. 2D indicates that the changes in mRNA levels in the H2A {Delta}4-20 and H4 {Delta}2-26 mutant strains are not well correlated (r = 0.36). Hence, the H2A and H2B N-terminal domains repress a shared core set of genes, although the effect of the H2A N-terminal domain on gene transcription is generally smaller in magnitude than that of the H2B N-terminal domain.

Deletion of the H2A N-terminal domain confers UV sensitivity to yeast cells. Previous studies have shown that chromatin can play an important role in the repair of DNA lesions (1). We previously found that the histone H2B N-terminal domain is involved in the DNA damage response, as deletion of this domain confers a strong UV sensitivity phenotype (12). We therefore determined if H2A N-terminal deletion mutants exhibited a similar sensitivity to UV-induced DNA damage. The histone H2A mutant and wild-type strains were treated with various doses of UV light (0, 50, and 100 J/m2) and scored for the number of surviving cells. As shown in Fig. 3, the H2A {Delta}4-20 mutant exhibited an ~10-fold lower survival rate than the wild-type control when irradiated with a dose of 50 J/m2 and an ~100-fold lower survival rate when irradiated with a dose of 100 J/m2. While the H2A {Delta}4-20 mutant was less sensitive to UV damage than the H2B {Delta}30-37 mutant, it was considerably more sensitive than the wild type. In contrast, the H2A K4,7G mutant strain exhibited only a slight UV sensitivity phenotype compared to the wild type.


Figure 3
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  		FIG. 3. Yeast strains lacking the histone H2A N-terminal domain are sensitive to UV irradiation. The percentage of viable cells for each histone H2A strain was plotted as a function of UV dose. The data for the histone H2B {Delta}30-37 mutant (12) are included for comparison.

The histone H2A repression (HAR) domain is required for transcriptional repression and UV sensitivity. To identify residues in the histone H2A N-terminal domain that function to repress transcription, we tested the effects of H2A N-terminal deletions on the expression of two reporter genes, BNA1 and BNA2. These genes were chosen because they encode enzymes in the NAD biosynthesis pathway (an enriched functional category) and because their mRNA levels are significantly up-regulated in the H2A {Delta}4-20 mutant. In the array data, the mRNA level of BNA1 is up-regulated 3.0-fold (P = 1.5 x 10–6) and the mRNA level of BNA2 is up-regulated 6.0-fold (P = 4.4 x 10–14).

RT-PCR analysis of BNA1 and BNA2 expression confirmed the results obtained with the array data. As shown in Fig. 4, in the H2A {Delta}4-20 mutant the mRNA level of BNA1 increased 4.1-fold relative to the wild-type level, and the mRNA level of BNA2 increased 6.1-fold relative to the wild-type level. In the H2A K4,7G mutant strain (Fig. 4), both BNA1 and BNA2 showed small increases in mRNA level (1.7- and 1.5-fold, respectively). These results are in agreement with our array data, in which BNA1 is up-regulated 1.6-fold (P = 8.3 x 10–3) and BNA2 is up-regulated 1.8-fold (P = 3.8 x 10–5) in the H2A K4,7G mutant. In summary, the results of the RT-PCR experiments confirm the array data and indicate that the transcriptional repression mediated by the H2A N-terminal domain is largely independent of H2A K4 and K7.


Figure 4
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  		FIG. 4. RT-PCR analysis of BNA1 and BNA2 mRNA levels in histone H2A N-terminal mutants. The calculated changes in mRNA levels are relative to wild-type levels. The signal obtained from ACT1 mRNA was used as a loading control for normalization.

To identify other residues in the H2A N-terminal domain required for the transcriptional repression of BNA1 and BNA2, we constructed a series of overlapping deletions of the H2A N-terminal domain (Fig. 5D). Each deletion mutant was tested for its effects on the mRNA levels of BNA1 and BNA2 by RT-PCR. The results are shown in Fig. 5A and B. We observed the following simple pattern in these data: all deletion strains that eliminated residues 16 to 20 in histone H2A (e.g., H2A {Delta}8-20, {Delta}12-20, and {Delta}16-20) displayed increases in BNA1 and BNA2 mRNA levels that were indistinguishable from those in the H2A {Delta}4-20 mutant. In contrast, H2A deletions that did not eliminate residues 16 to 20 (e.g., H2A {Delta}4-16, {Delta}4-12, and {Delta}4-8) did not increase the expression of BNA1 or BNA2 to the extent observed in the H2A {Delta}4-20 mutant. The same trend was observed in the RT-PCR analysis of a third reporter gene, GCY1, which is involved in carbohydrate metabolism (see Fig. S2 in the supplemental material). In summary, we concluded that residues 16 to 20 comprise a minimal subdomain necessary for the repression of BNA1, BNA2, and GCY1. We refer to this subdomain as the HAR domain.


Figure 5
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  		FIG. 5. Dissecting the functional residues in the histone H2A N-terminal domain. RT-PCR analysis was used to measure the changes in mRNA level for BNA1 (A) and BNA2 (B) in a series of histone H2A N-terminal deletion mutants. Data are represented as described in the legend to Fig. 4. (C) UV sensitivities of histone H2A {Delta}16-20 and {Delta}4-16 mutants. (D) Amino acid sequences of H2A N-terminal deletion mutants.

We next tested whether deletion of the HAR subdomain was responsible for the enhanced sensitivity of the H2A {Delta}4-20 mutant to UV-induced DNA lesions. As shown in Fig. 5C, the H2A {Delta}16-20 mutant displayed a UV sensitivity phenotype very similar to that of the H2A {Delta}4-20 mutant. In contrast, the H2A {Delta}4-16 mutant displayed only slight sensitivity to UV damage. Based on these data, we concluded that deletion of the HAR domain in histone H2A leads to enhanced sensitivity to UV-induced DNA lesions.

Dissecting the functional residues in the HAR domain required for transcriptional repression. To identify which residues in the HAR domain were required for transcriptional repression, we systematically mutated H2A residues 16 to 20 to alanine (Fig. 6D). Residue 20 was not mutated in this case, as it is an alanine in wild-type histone H2A. We tested the effects of these mutations on the expression of the BNA2 reporter gene by RT-PCR. As shown in Fig. 6A, simultaneous replacement of residues 16 to 20 with alanine led to a 5.5-fold increase in the BNA2 mRNA level relative to that in the wild type. This increase in BNA2 mRNA level was similar to that observed for the H2A {Delta}16-20 mutant (Fig. 6A), indicating that the alanine mutations mimicked the effects of the deletion mutant.


Figure 6
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  		FIG. 6. Alanine scanning mutagenesis of the HAR domain reveals residues required for the transcriptional repression of BNA2. (A to C) RT-PCR analysis was used to measure the change in mRNA level for BNA2 in a series of histone H2A alanine substitution mutants. Data are represented as described in the legend to Fig. 4. (D) Amino acid sequences of H2A alanine substitution mutants. The HAR domain is indicated.

Next, we constructed a single alanine substitution for each residue in the HAR domain (except for H2A A20). The effects of these mutants on the BNA2 mRNA level are shown in Fig. 6B. Inspection of these data indicated that mutating either S17 or R18 to alanine led to a significant increase in the BNA2 mRNA level. We therefore constructed an H2A S17A;R18A double mutant and tested its effects on BNA2 expression. Our results indicate that the effect of the H2A S17A;R18A double mutant on the BNA2 mRNA level is indistinguishable from that of the H2A 16-20 alanine mutant (Fig. 6C). These results indicate that histone H2A S17 and R18 are specifically required for the transcriptional repression of BNA2.


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DISCUSSION
 
In this study, we have determined the role of the histone H2A N-terminal domain in regulating genome-wide transcription in S. cerevisiae. Our results indicate that the H2A N-terminal domain is responsible for repressing the transcription of a large set of yeast genes under standard growth conditions. Surprisingly, we found that transcriptional repression by the histone H2A N-terminal domain is largely independent of the acetylated lysine residues H2A K4 and K7. Our microarray analysis indicates that these residues are required for the transcriptional repression of 28 genes, many of which function in NAD and arginine biosynthesis. In contrast, the H2A N-terminal domain as a whole represses the transcription of 248 genes, indicating that there must be other functional residues in the H2A N-terminal domain required for transcriptional repression.

We systematically mapped which residues in the histone H2A N-terminal domain are responsible for this repression. These experiments identified a small subdomain, comprised of residues 16 to 20, that is required for the repression of the three reporter genes that we selected (BNA1, BNA2, and GCY1). Based on these results, we have tentatively labeled this subdomain the HAR domain. These results are in accordance with a previous study, which showed that residues 17 to 20 in histone H2A were required for the repression of the GAL1 promoter (7). It is important to note, however, that our data do not distinguish whether this subdomain is required for all genes repressed by the histone H2A N-terminal domain.

We subsequently performed alanine scanning mutagenesis of the HAR domain. These studies revealed that two residues in the HAR domain—S17 and R18—are specifically required for transcriptional repression of BNA2. Simultaneous mutation of these two residues to alanine recapitulated the effects of deletion or mutation of the entire HAR domain. It is tempting to speculate that H2A S17 and R18 may be the key residues in the histone H2A N-terminal domain required for transcriptional repression, but this conclusion has yet to be tested rigorously.

We also show that deletion of the HAR subdomain confers a UV sensitivity phenotype. Deletion of the H2A N-terminal domain does not affect the expression of any genes known to be involved in the DNA repair pathway. We also tested whether deletion of the HAR domain affected the induction of DNA repair genes (i.e., RAD51) in cells exposed to UV irradiation. RT-PCR analysis of RAD51 mRNA levels following UV irradiation did not reveal any significant difference between the wild-type and H2A {Delta}4-20 mutant strains (see Fig. S3 in the supplemental material). These results suggest that the UV sensitivity phenotype may be a direct consequence of the histone H2A N-terminal mutations. However, the mechanism by which deletion of the HAR domain compromises the repair or recovery of UV-induced DNA lesions is unknown.

Sequence alignment of histone H2A N-terminal sequences indicates that a number of the residues in the HAR subdomain are well conserved, particularly S17, R18, and S19 (Fig. 7A). This sequence conservation is in contrast to the rest of the H2A N-terminal tail (i.e., residues 1 to 15), which shows relatively little sequence similarity from yeast to humans. Analysis of the nucleosome structure indicates that HAR domain residues, such as R18, contact the minor groove of the DNA on the outside of the superhelix (Fig. 7B) (8). The previously identified histone H2B repression (HBR) domain (12) sits in a minor groove channel between the DNA strands of the superhelix, in close proximity to the HAR domain residues (Fig. 7B). This juxtaposition of the H2A and H2B subdomains was previously noted by Luger and Richmond, who postulated that these subdomains might act to "tether the H2A-H2B dimer to the nucleosome" (9).


Figure 7
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  		FIG. 7. Sequence and structural analyses of the HAR domain. (A) Alignment of histone H2A N-terminal domain sequences. The locations of the HAR domain (residues 16 to 20 in yeast) and the first alpha helix ({alpha}N) are indicated. (B) Locations of the HAR and HBR subdomains in the nucleosome structure. The HAR domain (residues 15 to 19 in Xenopus H2A) is colored red, and the HBR domain (residues 24 to 31 in Xenopus H2B) is colored blue. The rest of the histone residues are shown as cartoons in white. The DNA backbone is colored gray. The structural data (PDB code 1AOI; www.rcsb.org) are from reference 8. The figure was generated using POLYVIEW-3D (13).

This structural connection is intriguing, as analysis of our microarray data indicates that a shared set of genes are repressed by the histone H2A and H2B N-terminal tails, though to a lesser extent by the H2A N-terminal domain. Taken together, these observations suggest that the HAR and HBR domains may regulate gene transcription through a similar mechanism, perhaps by regulating the association and dynamics of the H2A-H2B dimer with nucleosomal DNA.


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ACKNOWLEDGMENTS
 
We are grateful to Deirdre Fahy, Lisa Gloss, Yi Jin, McKenna Kyriss, Ray Reeves, and Julie Stanton for helpful discussions and comments on the manuscript.

This work was supported by American Cancer Society grant RSG-03-181-01-GMC.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Molecular Biosciences, Washington State University, Fulmer Hall 675, Pullman, WA 99164-4660. Phone: (509) 335-8785. Fax: (509) 335-9688. E-mail: jwyrick{at}wsu.edu Back

{triangledown} Published ahead of print on 27 August 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, November 2007, p. 7641-7648, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00742-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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