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Mol Cell Biol. Dec 2010; 30(23): 5531–5544.
Published online Sep 27, 2010. doi:  10.1128/MCB.00601-10
PMCID: PMC2976433

Unbiased, Genome-Wide In Vivo Mapping of Transcriptional Regulatory Elements Reveals Sex Differences in Chromatin Structure Associated with Sex-Specific Liver Gene Expression [down-pointing small open triangle]

Abstract

We have used a simple and efficient method to identify condition-specific transcriptional regulatory sites in vivo to help elucidate the molecular basis of sex-related differences in transcription, which are widespread in mammalian tissues and affect normal physiology, drug response, inflammation, and disease. To systematically uncover transcriptional regulators responsible for these differences, we used DNase hypersensitivity analysis coupled with high-throughput sequencing to produce condition-specific maps of regulatory sites in male and female mouse livers and in livers of male mice feminized by continuous infusion of growth hormone (GH). We identified 71,264 hypersensitive sites, with 1,284 showing robust sex-related differences. Continuous GH infusion suppressed the vast majority of male-specific sites and induced a subset of female-specific sites in male livers. We also identified broad genomic regions (up to ~100 kb) showing sex-dependent hypersensitivity and similar patterns of GH responses. We found a strong association of sex-specific sites with sex-specific transcription; however, a majority of sex-specific sites were >100 kb from sex-specific genes. By analyzing sequence motifs within regulatory regions, we identified two known regulators of liver sexual dimorphism and several new candidates for further investigation. This approach can readily be applied to mapping condition-specific regulatory sites in mammalian tissues under a wide variety of physiological conditions.

Sexual dimorphism in gene expression is common in mammalian somatic tissues (23) and has broad implications for human health. Sex differences in gene expression may contribute to differences between men and women in the prevalence, extent, and progression of disease, including autoimmune diseases (54), kidney disease (37), cardiovascular disease (45), and liver diseases, such as hepatocellular carcinoma (9, 58). In addition, sex-related differences in pharmacokinetics and pharmacodynamics are common and may affect drug response (52). Sex-related differences in gene expression have been widely studied in liver, where they affect >1,000 transcripts (5, 51, 57) and impact physiological and pathophysiological functions ranging from lipid and fatty acid metabolism to xenobiotic metabolism and disease susceptibility (52). In the liver, sex-related differences in gene expression are primarily determined by growth hormone (GH) signaling (3, 21), which shows important sex-related differences that reflect the sex-related differences in plasma GH profiles seen in many species, including rats, mice, and humans (53).

The underlying mechanisms of sexual dimorphism in mammalian tissues have been only partly elucidated at the molecular level. In the male rat liver, intermittent plasma GH pulses repeatedly activate the latent cytoplasmic transcription factor STAT5b, whose activity is essential for sex-related differences in the liver (5). The more continuous, female-like pattern of pituitary GH secretion can be mimicked by continuous GH infusion in male mice, which abolishes the normal male, pulsatile plasma GH profile and feminizes liver transcript patterns by suppressing many male-specific genes and inducing many female-specific genes (19). In spite of these findings, the molecular mechanisms whereby STAT5b and other transcription factors regulate sex specificity in the liver have remained elusive (26, 52).

DNase I hypersensitivity (DHS) analysis is a powerful tool to identify functional DNA elements involved in gene regulation. The temporal and spatial association of DHS sites with tissue-specific and developmentally regulated gene expression has long been established (14), and several instances of sex-related differences in DNase hypersensitivity characterizing genes that show sex-dependent transcription have been reported. Early studies identified a DHS site in the mouse liver upstream of C4a (the gene encoding sex-limited protein) that is more prominent in males, where an open chromatin structure correlated with a male-predominant pattern of gene expression (16), and examples of sex-regulated DHS sites have been reported for two sex-specific cytochrome P450 (Cyp) genes in rat liver (10, 47).

In order to identify sex-related differences in mouse liver chromatin structure on a global scale, we combined DHS analysis with ultrahigh-throughput sequencing (DNase-seq) to probe the open chromatin structure at single-base-pair resolution (1, 6, 40, 46). We show that DNase-seq, whose application until now has been limited to cultured cell lines, can readily be used to map DHS sites in mammalian liver, despite the added complexity of multiple cell types. We obtained high-resolution, genome-wide DHS maps for both male and female mouse livers under physiological conditions, and we demonstrate that these maps can be utilized to identify transcriptional regulators of sex-biased liver gene expression. We characterized more than 70,000 DHS sites and show that they encompass a large percentage (approximately 65 to 90%) of binding sites for six liver transcription factors identified earlier by chromatin immunoprecipitation (ChIP)-seq. We identified 1,284 DHS sites for which there are robust sex-related differences and that may contribute to sex-dependent gene expression; ~20% of these sites mapped within 100 kb of a sex-specific liver gene. In addition, a subset of the sex-dependent DHS sites was shown to respond to continuous GH treatment in male mice, likely representing functional DNA elements that mediate hormone-dependent, sex-dependent gene expression. Finally, analysis of the sex-dependent DHS sequences for enriched motifs identified binding sites for two transcription factors, STAT5b and HNF4α, known to be essential for sex-specific liver gene expression (5, 18, 20), as well as binding sites for other factors suggested to be involved (24) and several novel factors not previously implicated in liver sexual dimorphism. These findings highlight the utility of DNase-seq for elucidating condition-specific transcriptional regulatory sites associated with complex biological processes in mammalian tissue in vivo on a genome-wide scale.

MATERIALS AND METHODS

Mouse studies.

Adult male and female ICR mice (CD-1 mice) were purchased from Taconic Farms, Inc. (Germantown, NY), or Charles River Laboratories (Wilmington, MA) and were housed in the Boston University Laboratory Animal Care Facility in accordance with approved protocols. Livers were collected from 8-week-old mice euthanized by CO2 asphyxiation followed by cervical dislocation. Continuous GH treatment of 7-week-old male mice was achieved using Alzet model 1007D microosmotic pumps (Durect Corporation, Cupertino, CA) implanted subcutaneously (s.c.) under ketamine and xylazine anesthesia and delivering recombinant rat GH (obtained from Arieh Gertler, Protein Laboratories Rehovot, Ltd., Rehovot, Israel) at 20 ng rat GH per h per gram body weight for 7 days (19). RNA was extracted from individual livers by using Trizol reagent (Invitrogen, Carlsbad, CA) followed by reverse transcription using 1 μg total RNA and a high-capacity reverse transcription kit (Applied Biosystems, Carlsbad, CA). To verify feminization of liver gene expression by continuous GH infusion, real-time PCR analysis of a panel of established, continuous-GH-responsive genes (19) was performed using Power SYBR green PCR master mix and an ABS 7900HT sequence detection system (both from Applied Biosystems).

For in vivo transfection assays, genomic regions corresponding to six individual DHS sites were PCR amplified from ICR mouse genomic DNA and cloned into a modified pGL4.10 vector (Promega, Madison, WI), designated pAlbpmo, that includes a minimal mouse Alb promoter driving expression of a firefly luciferase gene. The DHS regions were cloned into pAlbpmo, upstream of the Alb promoter. A renilla luciferase reporter vector was constructed in a similar way by inserting an Alb enhancer into a modified pGL4.70 reporter vector containing the same mouse minimal Alb promoter. Twelve micrograms of firefly reporter vector and 3 μg of renilla reporter plasmids were delivered to adult male and female mouse livers by hydrodynamic injection by using the TransIT gene delivery system (Mirus, Madison, WI). Livers were harvested 7 days after injection, homogenized in 1× passive lysis buffer (Promega), and firefly luciferase activity normalized to renilla luciferase activity was assayed using a dual-luciferase assay kit (Promega).

Nucleus isolation.

Nuclei were isolated by using a high-sucrose-based protocol to minimize perturbation of chromatin structure during nucleus isolation (27). Livers were pooled from 2 to 5 mice, minced, and then homogenized in nuclear homogenization buffer (10 mM HEPES, pH 7.9, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 10 mM NaF, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM dithiothreitol [DTT]) supplemented with 1× protease inhibitor cocktail (Sigma P8340) by using a motor-driven Potter-Elvehjem homogenizer (5 ml homogenization buffer per g liver tissue). Twenty-five milliliters of homogenate was layered over a 10-ml cushion comprised of the same buffer and centrifuged at 25,000 rpm for 45 min at 4°C in an SW28 rotor. The pelleted nuclei were suspended in nucleus storage buffer (20 mM Tris-Cl, pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% [vol/vol] glycerol, 0.85 mM DTT, 0.125 mM PMSF) by using a Dounce homogenizer, counted by using a hemacytometer after ~100-fold dilution in phosphate-buffered saline, and snap-frozen in aliquots of approximately 5 × 107 to 10 × 107 nuclei/ml at −80°C.

DNase I digestion.

DNase I digestion was performed as described previously (40) with some modifications. Frozen nuclei were thawed on ice and washed twice with ice-cold buffer A (15 mM Tris-Cl, pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine). DNase digestion was initiated by incubating ~5 × 106 nuclei in 1 ml of buffer D (9 volumes of buffer A plus 1 volume of 60 mM CaCl2, 750 mM NaCl) for 2 min at 37°C with an optimized amount of RQ1 DNase I (Promega) (see below). Six tubes of nuclei were incubated in parallel. DNase digestion was halted by the addition of 1 ml stop buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 0.1% SDS, 100 mM EDTA) to each tube, followed by proteinase K digestion (100 μg/ml final concentration) overnight at 55°C. The next day, phenol-chloroform extraction was performed; after this, the aqueous phase (approximately 11 to 12 ml combined from the 6 parallel DNase digestion reactions) was removed and adjusted to 0.8 M NaCl by the addition of 5 M NaCl. Control samples were prepared in the same way by incubating ~5 μg purified genomic DNA in 0.1 ml digestion buffer with 0.0625, 0.125, 0.25, or 0.5 units of DNase I. The control DNase I digestion sample that yielded a smear of DNA fragments ranging from 100 bp to ~1.5 kb was selected and further purified by sucrose gradient ultracentrifugation.

Small DNA fragments (<1.5 kb) released during DNase I digestion were isolated by sucrose step gradient ultracentrifugation of the DNase-digested nuclei and also from the DNase-digested genomic DNA and the sonicated genomic DNA control samples. The small DNA fragments released from the digested nuclei correspond to DNase-sensitive regions containing multiple cut sites in close proximity, whereas larger DNA fragments present in the samples primarily result from random, nonspecific DNA degradation. The released fragments were size selected on a sucrose step gradient prepared by sequentially layering 3 ml of each sucrose concentration (20 mM Tris-Cl, pH 8.0, 5 mM EDTA, 1 M NaCl containing 40%, 35%, 30%, 25%, 20%, 17.5%, 15%, 12.5%, or 10% sucrose) in an SW28 tube. Half of each sample (~5.7 ml) was loaded on the top of each gradient, and the gradient was centrifuged for 24 h at 25,000 rpm at 25°C in an SW28 rotor. Fractions of 1.9 ml were collected from the top and assayed by agarose gel electrophoresis. Gels were stained with 1× SYBR green I nucleic acid gel staining solution (Invitrogen) in 1× TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.5) at room temperature for 30 min and visualized on a Typhoon imager (GE Healthcare, Piscataway, NJ). Fractions that primarily contained DNA fragments of <1.5 kb (typically fraction 7) were purified on Qiagen columns (catalog no. 28704) and assayed by quantitative PCR (qPCR) using primers designed to amplify known hypersensitive genomic regions (see Table S1 in the supplemental material). The typical yield was 1 to 2 ng released DNA fragments per 106 nuclei, as determined using the Quanti-IT kit (Invitrogen). Illumina sequencing (see below) was carried out for each of two independent pools of biological replicates (4 to 6 livers for each sex in each replicate). Each sequenced sample was comprised of ~30 ng DNA pooled from at least 3 independent batches of DNase-digested nuclei, to minimize the impact of intersample variability in DNase digestion.

Optimization of DNase digestion conditions.

qPCR primers used to optimize DNase digestion conditions and to assess the quality of DNase-released fragments are listed in Table S1 in the supplemental material: Intergenic-1 primers were used to amplify a genomic region distant (>100 kb) from any known genes, where no DHS regions were expected, and Rassf6 primers were selected to flank a strong DHS site near the Rassf6 promoter. For each batch of DNase I enzyme, initial experiments were carried out over a range of DNase I concentrations (5 to 120 units/ml) to identify a DNase concentration (typically ~40 units DNase I/ml) that resulted in <20% gene copy number loss in the Intergenic-1 region but >50% gene copy number loss in the Rassf6 promoter region. Purified, released DNase fragments were routinely tested for enrichment of Alb promoter sequences over intergenic region sequences to verify the quality of the DHS samples prior to Illumina sequencing analysis. This was done by qPCR using Alb primers, located within a strong DHS site within the Alb promoter, and Intergenic-2 primers, which are in a region near Intergenic-1 primers. Typically, the DNase-released Alb promoter fragments showed >16-fold enrichment compared to control genomic DNA. Sex-dependent release of genomic DNA fragments was also routinely verified for a male-specific DHS region near Ttc39c and a female-specific DHS region near Cyp2b9 (see Table S1).

Illumina sequencing.

Sequences of DNase I-released DNA fragments were sequenced using a Genome Analyzer II instrument (Illumina, Inc., San Diego, CA). Briefly, about 30 ng of DNase-released DNA fragments was subjected to end repair, adaptor ligation, and PCR enrichment using an Illumina sample preparation kit following the manufacturer's recommendations. A DNA library comprised of approximately 100- to 300-bp fragments was size purified by gel electrophoresis. The concentration of properly ligated samples was estimated by qPCR, and this was followed by cluster generation and sequencing. The sequencing reads were aligned to mouse genome mm9 by using Illumina's Eland extended software, with a maximum of 2 mismatches allowed in the first 25 bp. Totals of 36 million, 32 million, and 28 million reads were sequenced from the male, female, and GH-treated male samples, respectively, with ~82% mapped to unique genomic positions (see Table S2 in the supplemental material).

Identification of DHS sites.

PeakSeq (39) was modified as outlined below and used to identify DHS sites in male, female, and GH-treated male mouse liver nuclei in comparison to control samples; the latter consisted of sonicated mouse genomic DNA, as well as DNase I-digested mouse genomic DNAs, and were processed in parallel to the DNase I-digested nuclei. Sex-specific DHS sites and sites induced or suppressed in male mouse livers by continuous GH treatment were also identified. The PeakSeq algorithm was modified for identification of DHS peaks as follows. (i) Sequence read numbers from the two samples being compared were square-root normalized to improve the linearity of the data for calculation of the scaling factor by linear regression. (ii) When comparing sequence reads from male and female mouse livers, or from male and continuous GH-treated male mouse livers, only putative peak regions were included in linear regression due to the presence of differing amounts of background in each sample (see Table S3 in the supplemental material). (iii) Minimum thresholds of 7 sequence reads for autosomes and 5 sequence reads for sex chromosomes were applied to all putative DHS peaks to eliminate unusually long (≥10-kb) peaks with few sequence reads. (iv) Finally, putative peaks that were <100 bp in length were extended to 100 bp and then evaluated for statistical significance. Further details are provided in the supplementary methods and results in the supplemental material.

Association of DHS sites with genes, H3K4 methylation, and FoxA2 binding sites.

DHS sites were classified as intergenic, coding, or associated with promoter regions based on mapping to known genes, mRNAs, and spliced and unspliced expressed sequence tags (ESTs) in the University of California—Santa Cruz (UCSC) genome browser. To compare DHS sites with liver gene expression, the locations of sex-independent and sex-specific DHS sites were mapped to sex-specific and sex-independent genes expressed in mouse liver (50). ChIP-seq data for histone H3 lysine 4 monomethylation (H3K4-me1), histone H3 lysine 4 trimethylation (H3K4-me3), and FOXA2 binding in female mouse livers (38) were compared to locations of sex-independent and sex-specific DHS sites. The association of DHS sites with the two histone modifications listed above was calculated by determining the numbers of DHS sites that have a histone modification site within 150 bp of either side of the DHS site. To generate distribution plots, distances from the midpoints of DHS sites to sequence tags in peaks previously determined by ChIP-seq for H3K4-me1, H3K4-me3, and FOXA2 were computed.

Gene expression data.

Liver gene expression data were obtained from an earlier study (50), in which a total of 1,380 transcripts showed significant differences between expression levels in male and female mouse livers. After the removal of microarray probes that do not map to any known gene, probes that map to the same transcript as another probe, and probes mapping to chromosomes for which no DNase hypersensitivity data are present (chrY, chrUn, and chrN_random), a total of 1,209 genes showing >2-fold sex-related differences between expression levels at a P value of <0.005 (i.e., sex-specific genes) remained, as did 21,153 sex-independent genes. Of these, 343 sex-specific genes and 7,341 sex-independent genes met the additional criterion of a microarray signal intensity of ≥500 in the liver.

Identification of broad DHS regions.

SICER (59), an algorithm that uses a clustering approach to identify extended enriched domains from histone modification ChIP-seq data, was applied to detect broad regions of the genome that are enriched for DNase-seq reads in male or female liver samples compared to controls. Genomic regions that show sex-dependent DNase hypersensitivity were defined as those that were significantly enriched in DNase-digested liver nuclei of males compared to those of females or vice versa. Similarly, genomic regions that responded to continuous GH treatment were identified by comparison of the untreated male samples to the GH-treated male samples. A window size of 200 bp and a gap size of 1,200 bp were used, and significant regions that had a false-discovery rate (FDR) of <10−3 and ≥2-fold difference in expression levels of the pair of liver samples being compared were chosen.

Enriched transcription factor binding sites in sex-specific DHS sites.

THEME, a hypothesis-based algorithm that tests whether a given motif separates a foreground set of sequences from a background set (29), was used to identify enriched motifs in 18 sets of sex-specific DHS sites compared to sex-independent DHS sites, as follows: (i) male-specific sites either within 10 kb or up to 50 kb from the transcription start site (TSS) of a sex-specific gene, (ii) male-specific sites within 10 kb or 50 kb of a sex-independent gene, and (ii) male-specific sites distant from any gene. Each set of sex-specific DHS sites was divided into subgroups that respond and subgroups that do not respond to continuous GH treatment in males with a >2-fold difference between expression levels at a P value of <0.01, and similar criteria were used for female-specific DHS sites. Transcription factor binding profiles for 97 families of transcription factors were generated by clustering the vertebrate transcription factor position-specific scoring matrices (PSSMs) from the TRANSFAC and JASPAR databases (2, 33). The corresponding 97 motifs were considered enriched if they met the following conditions: a cross-validation error of <0.4, P value of <0.001, normalized log-likelihood ratio score of >0.4, and ≥2 enrichment compared to sex-independent sites. Motifs with a PSSM total information content (relative entropy) <8 bits were eliminated from further consideration. To identify the transcription factor(s) associated with each motif, the refined family binding profiles were matched back to the TRANSFAC and JASPAR databases by using STAMP (32), and the top factor(s) that matched with an E value of <10−8 was identified. One exception was motif 44, for which the best matches, to Fox factors, had an E value of <10−6. Discovered motifs were clustered by hierarchical clustering by Pearson correlation of fold enrichment over sex-independent sites in each of the 18 sets of sex-specific sites by using the hierarchical clustering module of the GenePattern suite of tools (36).

Microarray data accession numbers.

All high-throughput sequencing data are available in the GEO database (accession no. GSE-21777), in the Sequence Read Archive of NCBI (accession no. SRP002445), and in custom tracks submitted to the UCSC genome browser.

RESULTS

Generation of liver DHS maps.

DNase-seq was used to generate genome-wide DHS maps for liver tissue obtained from male and female mice and from male mice given a continuous infusion of GH for 7 days, which feminizes the pattern of liver gene expression (19). Mouse liver nuclei prepared from two independent pools of biological replicates were digested with DNase I under optimized conditions (see Materials and Methods), and fragments released from hypersensitive regions were separated from randomly cut DNA fragments, which tend to be much larger (40). DNase-released fragments ranging from approximately 100 to 300 bp were sequenced by using Illumina sequencing technology. The final combined data set is comprised of 29 million sequence reads mapped to unique locations in the mouse genome for male livers and 26 million reads for female livers; 23 million additional sequence reads were obtained for continuous (7-day)-GH-treated male livers (~82% uniquely mappable reads; see Table S2 in the supplemental material). The resultant DHS maps are of high quality, as seen in Fig. Fig.1A1A for the Alb gene region. Eight DHS regions were identified within ~47 kb of the Alb gene TSS, with very low background between peaks of hypersensitivity. In addition to identifying the DHS sites at kb −0.1, −3.5, −10.8, and −13.7 relative to the Alb TSS, previously identified by using classical Southern blotting methods (28), we identified DHS sites at four upstream locations, from kb −22 to −47 (Fig. (Fig.1A).1A). Closer examination of the kb −13.7 DHS site revealed a typical structure for a DHS peak, with a roughly symmetric distribution of positive- and negative-strand digestion sites that clearly define the DHS peak boundary (see Fig. S1B in the supplemental material). Using DNase I-digested genomic DNA as a control, we identified 71,264 DHS sites in male and female livers, covering 1.8% of the mouse genome. A total of 48,762 of the DHS sites were high-stringency sites, and the total number of DHS sites increased to 110,785 when the combined data sets were used (Table (Table1;1; see Table S4 in the supplemental material). There is a high degree of overlap between DHS sites and transcription factor binding sites identified by chromatin immunoprecipitation (ChIP)-chip or ChIP-seq analysis of mouse livers (Table (Table2),2), ranging from 67% for that corresponding to CEBP4A (42) to 93% for that corresponding to FXR/NR1H4 (49). Thus, the DHS sites identified here likely include a large fraction of the active regulatory elements in liver tissue.

FIG. 1.
DHS sites associated with the Alb gene (A), Cyp2d9, a male-specific gene (B), and Cux2, a female-specific gene (C). In panel A, DHS sites appear as sharp, narrow peaks and are marked by distance upstream of the Alb gene. The four sites marked in red are ...
TABLE 1.
DHS sites in male and female mouse liversa
TABLE 2.
Overlap between ChIP-chip/ChIP-seq transcription factor binding sites and DHS sites in the livera

DHS sites showing sex specificity and responsiveness to GH.

We hypothesized that liver chromatin is characterized by sex-associated differences between accessibilities to DNase and that these differences relate to the observed sex-associated differences in liver gene expression. We further anticipated that continuous GH treatment of male mice, which feminizes the overall pattern of liver gene expression (19), would alter the sex-dependent patterns of chromatin accessibility. By comparing the DHS profiles of male and female mouse livers, we identified genomic regions showing DNase I fragment release from male mouse liver nuclei that was significantly greater than that from female mouse liver nuclei, i.e., male-specific DHS sites. Correspondingly, female-specific DHS sites showed significantly greater DNase I cleavage in female mouse liver. Totals of 850 male-specific peaks and 434 female-specific peaks were identified as high-stringency sex-specific DHS sites based on their confirmation in each of two independent biological replicates (Table (Table1);1); examples are shown in Fig. Fig.1B1B (Cyp2d9 gene) and C (Cux2 intron 2) and in Fig. S4 in the supplemental material. A total of 4,182 sex-specific DHS sites were identified at lower stringencies (Table (Table1;1; see Tables S5B to E in the supplemental material). Continuous GH treatment of male mice suppressed 82% of the high-stringency male-specific DHS sites and induced 26% of the high-stringency female-specific DHS sites, whereas <3% of sex-independent DHS sites were GH responsive at the stringencies applied (Table (Table3).3). When weaker GH responses were included, 98% of male-specific DHS sites were suppressed and 44% of female-specific DHS sites were induced (see Table S5H).

TABLE 3.
Effect of continuous GH treatment on DHS sites in the male livera

Examination of the distribution of the 1,284 high-stringency sex-specific sites across chromosomes revealed significant enrichment of female-specific DHS sites on chromosomes 5 and X and enrichment of male-specific DHS sites on chromosomes 3 and 18 compared to the overall list of DHS sites (see Table S6 in the supplemental material). Overall, 65% of sex-specific DHS sites are in the coding region or within 5 kb of the TSS of a known transcript, compared to 78% of sex-independent DHS sites (see Fig. S5). The median lengths of sex-specific and sex-independent DHS were similar, 466 to 575 bp and 437 to 483 bp, respectively (see Table S7), corresponding to the depletion of ~2 nucleosomes.

Association of liver DNase hypersensitivity with liver gene expression.

In CD4+ T cells, the probability that a given gene harbors a 5′-end-proximal DHS site increases with the level of gene expression (1). We observed the same trend in mouse liver, where the proportion of sex-independent genes that have a DHS site within 200 bp of the TSS increased with increasing intensity of gene expression, leveling off at ~90% (Fig. (Fig.2A).2A). In contrast, the proportion of genes that show sex-specific expression (50) and have a 5′-end-proximal DHS site increased more gradually with increasing expression (P = 0.0006; Fig. Fig.2B).2B). The overall lower percentage of sex-specific genes with a 5′-end-proximal DHS site might indicate that these genes are more commonly regulated by distal elements. Alternatively, the sex-independent genes might simply be close to more nonfunctional DHS sites than are sex-specific genes.

FIG. 2.
Relationship between the level of gene expression and the presence of a 5′ DHS site. Shown is the fraction of sex-independent genes (A) or the fraction of sex-specific genes (B) that have at least one 5′ DHS site within 200 bp of the TSS, ...

Next, we tested the hypothesis that genes that show sex-specific expression in mouse liver are more likely to be associated with sex-specific DHS sites than are sex-independent genes. Supporting this hypothesis, we observed that sex-specific genes are 8.1-fold more likely than sex-independent genes to have a sex-specific DHS site in the coding region and 3.1-fold more likely to be within 100 kb; furthermore, the proportion of genes with a sex-specific DHS site rises with distance more steeply for sex-specific genes than for sex-independent genes (Fig. (Fig.3A,3A, left panel). Conversely, sex-specific DHS sites are more likely than sex-independent DHS sites to be located near a sex-specific gene (Fig. (Fig.3B,3B, left panel). Finally, the proportion of male-specific genes whose nearest DHS site is also male specific (20%) is ~10-fold greater than the proportion whose nearest DHS site is either female specific or sex independent (2% in both cases), and the case is similar for female-specific genes and female-specific DHS sites (Fig. (Fig.3C).3C). Thus, there is a strong association between sex-specific DHS sites and sex-specific gene expression. However, this association is seen for only a subset of sex-specific genes, insofar as only 20% of sex-specific genes have a high-stringency sex-specific DHS site in the coding region and only 43% have a sex-specific DHS site within 100 kb. This compares to 90% of liver-expressed sex-independent genes with a sex-independent DHS site in the coding region and 99% with at least one sex-independent DHS site within 100 kb (Fig. (Fig.3A,3A, right panel). Moreover, only 23% of sex-specific DHS sites are within 100 kb of a sex-specific gene, while 76% of sex-independent DHS sites are within 100 kb of a sex-independent gene (Fig. (Fig.3B,3B, right panel). Sex-specific DHS sites may therefore act as distant regulators. Alternatively, this finding may reflect more complex regulatory mechanisms of sex-specific genes, involving interactions between multiple regulatory sites and multiple genes (30), regulatory changes that have no effect on chromatin structure, or posttranscriptional regulation. The subsets of sex-specific genes that do and do not have a sex-specific DHS site within 100 kb include equal proportions of male-specific and female-specific genes; however, the female-specific genes that are within 100 kb of a sex-specific DHS site are 2.7-fold enriched (P < 10−4) for the subset of female-specific genes that are suppressed in the female liver upon ablation of pituitary and GH stimulation (class I female-specific genes [50]). The extensive loss of male-specific DHS sites and the induction of a substantial fraction of female-specific DHS sites in livers of continuous-GH-treated male mice (Table (Table3),3), in which the gene expression profile is feminized (19), support the conclusion that these sex-specific DHS sites play a functional role in the sex-specific expression of the genes associated with these sites. Indeed, the subset of female-specific DHS sites that respond to continuous GH is even more frequently associated with female-specific genes than the full set of female-specific DHS sites (Fig. (Fig.3C3C).

FIG. 3.
Proximity of sex-specific and sex-independent (indep) DHS sites to genes expressed in mouse liver. CI, confidence interval. (A) Cumulative distances from the gene body of sex-specific and sex-independent genes to the nearest sex-specific and sex-independent ...

Enhancer-like activity of sex-specific DHS sites.

Our finding described above that sex-specific DHS sites are more likely to be associated with genes of the same sex specificity suggests that sex-specific DHS sites serve as enhancers of sex-specific gene expression. This possibility is supported by a comparison of our DHS map with maps of histone H3 lysine 4 mono- and trimethylation (H3K4-me1 and H3K4-me3, respectively) reported for female mouse livers (38): 80% of high-stringency female-specific DHS sites are within 150 bp of nucleosomes marked by H3K4-me1 but not H3K4-me3, whereas only 15% are associated with a H3K4-me3 mark (Fig. (Fig.4A).4A). This pattern—the presence of H3K4-me1 in the absence of H3K4-me3—is indicative of an enhancer (15). A smaller proportion of sex-independent DHS sites exhibit an enhancer-like H3K4 methylation profile, with 32% of these DHS sites containing the H3K4-me3 mark and only 61% showing the H3K4-me1-only pattern (Fig. (Fig.4A).4A). The frequency of the H3K4-me1-H3K4-me3 double mark decreased dramatically with increasing distance from the promoter, as was seen for both female-specific and sex-independent DHS sites (Fig. (Fig.4A).4A). Peaks for both histone marks exhibited a trough at the midpoint of female-specific and sex-independent DHS sites, indicating nucleosome depletion (Fig. 4B and C).

FIG. 4.
Histone modifications at sex-independent and sex-specific DHS sites in female mouse livers. (A) Percentages of sex-independent and sex-specific DHS sites within 150 bp of an H3K4-me1 and/or H3K4-me3 mark in female mouse livers. These data are shown for ...

To assay for enhancer activity, we selected 6 sex-specific DHS sites, 5 of which were responsive to continuous GH treatment (see Table S8 in the supplemental material), and cloned them into a reporter vector containing a modified Alb promoter linked to a luciferase reporter (56). The 6 sex-dependent DHS sites were assayed for their intrinsic ability to enhance the Alb promoter following in vivo liver transfection by hydrodynamic injection. Five of the six sites exhibited enhancer activity when assayed 7 days after liver transfection (Fig. (Fig.5).5). This time point was selected to allow for decay of the transiently high activity of the Alb promoter by using this transfection method (56). The most active DHS fragment, from intron 2 of the highly female-specific Cux2 gene (see Table S8) (24), was >200-fold more active than the Alb promoter alone but showed similar activities in male and female mouse livers. Two male-specific DHS sites showed 8- to 17-fold higher activity than the Alb promoter, with the activity seen for a Cyp2d9 DHS site in male livers being 3-fold higher than that in female livers (Fig. (Fig.5).5). A female-specific DHS site adjacent to Acot4 showed female-enriched enhancer activity, albeit at a modest level. One of the six DHS sites (Cyp2c39) was inactive.

FIG. 5.
Enhancer activity of six sex-specific DHS sites assayed in mouse liver. Four high-stringency female DHS sites (Cux2, Acot4, Cyp3a16, and Cyp2c39) and two male DHS sites (Cyp7b1 and Cyp2d9), named after the closest gene to each site, were assayed for reporter ...

Broad genomic regions of DNase hypersensitivity.

While 87% of the above-identified DHS sites are <1 kb in length, we observed genomic regions with considerably longer sex-dependent hypersensitivity regions, some extending up to ~100 kb. To identify such extended DNase hypersensitivity regions, we used SICER (59), a clustering-based algorithm designed to identify diffuse domains of ChIP-enriched regions. We found 3,971 DHS regions >10 kb in length, 58 of which showed significant sex-related differences (Table (Table4).4). Continuous GH treatment suppressed 47% of the extended male-specific regions and induced 50% of the female-specific regions, compared to <0.2% of the sex-independent regions; the proportion of these >10-kb female-specific regions that are induced by GH is even higher than that for the short female-specific DHS peaks. Some of the extended DHS regions are comprised of clusters of the short DHS peaks identified above (Fig. (Fig.6A6A and B; see also the supplemental text and Fig. S6 in the supplemental material), while other extended DHS regions contain few sites identified as DHS peaks by PeakSeq, which is optimized for identification of short, well-defined discrete peaks (track marked “All DHS sites” in Fig. 6C and D). Additional examples, including GH responses, are shown in Fig. S6. The full list of SICER-identified regions is provided in Table S9.

FIG. 6.
Broad regions of DNase hypersensitivity. Tracks in each panel show (in order, from the top) RefSeq genes, DHS peaks identified using PeakSeq (all DHS sites), and DHS regions identified using SICER (broad DHS regions), with the length of the SICER-detected ...
TABLE 4.
Broad DHS regions and their response to continuous GH treatmenta

Transcription factor binding sites enriched in sex-specific DHS sites.

THEME, a hypothesis-based algorithm that tests for enrichment of predefined motifs (29), was used to examine sex-specific DHS sites for enrichment of transcription factor binding site motifs by using sex-independent DHS sites as a background. Given the expected heterogeneity of sex-specific DHS sites, we carried out these analyses by using subsets comprised of male- and female-specific DHS sites that are (i) within 10 kb or within 50 kb of the TSS of a sex-specific gene, (ii) within 10 kb or within 50 kb of a sex-independent gene, and (iii) distant (>50 kb) from any gene. Each set of DHS sites was further divided into sites that respond and sites that do not respond to continuous GH treatment in males with a >2-fold difference in intensity levels at a P value of <0.01 (see Table S5A in the supplemental material). Starting with motif families derived from the TRANSFAC and JASPAR databases, we identified 16 enriched motifs (see Table S10). The sets of sex-specific sites were then scanned for each of the 16 motifs, which were then clustered according to fold enrichment in each of the sets of DHS sites (Fig. (Fig.77).

FIG. 7.
Heat map of transcription factor binding motifs enriched in sex-specific DHS sites compared to sex-independent DHS sites. Motifs are clustered by fold enrichment in sets of male-specific (M) and female-specific (F) DHS sites that respond (GH) or do not ...

The discovered motifs include binding sites for two factors known to be required for sex-specific liver gene expression. Thus, a motif matching the binding site for STAT5b (motif 28) is enriched in male-specific GH-responsive sites, as is a motif matching HNF4α (motif 70), consistent with the earlier findings that these two transcription factors are essential for GH-regulated sex-specific gene expression in male mouse liver (5, 18, 20). While the HNF4α-like motif is most highly enriched in sites within 10 kb of a sex-specific gene, the STAT5 motif shows the highest enrichment in more distal sites, including sites proximal to sex-independent genes, consistent with other studies on STAT5 binding (8, 25, 34). The STAT5 motif clusters together with motifs that match 9 other transcription factors (or transcription factor families), all of which exhibit a common pattern of enrichment in male-specific, GH-responsive DHS sites (motif cluster A) (Fig. (Fig.7).7). Eight of these 10 motifs are underrepresented in female-specific GH-responsive sites (cluster A1). These 10 motifs include binding sites for CUX2, a highly female-specific, GH-regulated transcription factor (24), GFI1, a STAT-inducible transcriptional repressor (22, 60), OCT1 (POU2F1), which interacts with STAT5 in binding to the cyclin D1 promoter (31), PBX1, which interacts with OCT1 (35, 48) and may help penetrate repressive chromatin (41), and EVI1 (corresponding to the gene Mecom), a positive regulator of PBX1 (44) that interacts with the histone methyltransferase SUV39H1 (13). Two motifs, binding sites for MYC and MAX, were enriched in male-specific DHS sites not responsive to GH. Finally, motifs corresponding to binding sites for VDR, TCFAP2A, and TAL1 were most highly enriched at female-specific DHS sites. VDR activates the gene CYP3A4 (GH responsive and predominantly expressed in females) in human hepatocytes (4, 7, 55), and a female-specific DHS site containing the VDR motif is associated with a female-specific mouse homolog, Cyp3a41a (19).

DISCUSSION

We present a set of detailed, high-quality, genome-wide hypersensitivity maps comprised of more than 70,000 DHS sites, which encompass the transcriptional regulatory elements in the mouse liver in vivo. DHS maps were generated for both male and female mouse livers, from which we were able to identify 1,284 high-stringency sex-specific DHS sites, a subset of which was responsive to changes in plasma GH status, the major determinant of sex differences in liver gene expression. We demonstrate the utility of these maps for identifying binding sites for transcription factors previously shown to be essential for GH-regulated sex-specific gene expression (STAT5b and HNF4α [5, 18, 20]), as well as binding sites for several novel factors not previously implicated in this process. These DHS sites encompass 1.8% of the mappable mouse genome, which substantially narrows down the sequence space in searches for gene regulatory sequences, including binding site motifs important for liver gene expression. The fine structure of DHS sites with a high density of sequence reads (see Fig. S1B in the supplemental material) suggests that it might be possible to visualize transcription factor binding directly in the form of digital footprints within DHS sites (17). Further analysis of hypersensitivity data collected at greater sequencing depth will be required to establish the feasibility of this approach in mammalian tissues. DHS sites are expected to encompass key regulatory elements, including promoters, enhancers, silencers, and insulators associated with the expression of thousands of genes in their native chromatin structure under physiological conditions. The DHS maps presented here for mouse liver tissue, in combination with corresponding sets of genome-wide histone modification and transcription factor binding maps (11, 38, 42, 43, 49), can be expected to serve as a valuable resource for elucidation of transcriptional networks controlling a wide range of physiological and pathophysiological processes.

Most sex-dependent DHS sites were short and highly localized (median length, ~500 bp), but in several cases, sex-specific hypersensitivity extended over broad regions, up to ~100 kb in length (Fig. (Fig.6;6; see also Fig. S6 in the supplemental material). The accessibility of many of these sex-dependent DHS sites and regions was altered by continuous GH infusion in male mice, which both feminizes the overall pattern of liver gene expression (19) and rendered the vast majority of the male-specific DHS sites less accessible to DNase while increasing the hypersensitivity of a substantial subset of the female-specific DHS sites and extended regions. These findings support the proposal that these GH-responsive DHS sites play a functional role in liver sexual dimorphism and suggest that a common upstream pathway responsive to GH, such as the activation of STAT5b (26, 53), regulates their differential chromatin accessibility in male and female livers.

We also observed a strong association between sex-specific DHS sites and sex-specific gene expression, with sex-specific genes more likely than sex-independent genes to have a nearby sex-specific DHS site. Moreover, sex-specific DHS sites were more likely than sex-independent DHS sites to have a nearby sex-specific gene. In some cases, multiple DHS sites, or extended hypersensitivity regions (discussed above), were associated with sex-specific genes. These may act in concert to increase the magnitude of differences in gene expression between male and female livers. However, in other cases, we observed groups of sex-specific DHS sites not located near sex-specific genes: one striking example is a cluster of female-specific DHS sites on the X chromosome (see Fig. S6B in the supplemental material), and another is a cluster of male-specific sites on chr13, whose nearest sex-specific genes (the Cd180 and Sgtb genes) are weakly female specific and located 800 kb and 500 kb from the cluster, respectively (see Fig. S6A). Indeed, for a majority of sex-specific DHS sites, the closest gene was not a sex-specific gene, and only a subset of sex-specific genes have a sex-specific DHS site within 100 kb of the gene. These findings suggest the importance of long-range DNA interactions for sex-specific gene expression, as well as more complex interactions between multiple regulatory sites and multiple genes (30). While our results are consistent with regulation by distal sex-specific DHS sites, it is also possible that some sex-specific genes are regulated via sex-independent DHS sites whose cognate transcription factors are expressed or regulated in a sex-dependent manner. Other sex-specific genes may be regulated posttranscriptionally, i.e., by a mechanism that does not involve sex-related differences in chromatin accessibility.

Histone methylation marks, such as H3K4-me1 and H3K4-me3, are associated with active regions of chromatin, including enhancers and promoters, which are anticipated to coincide with DHS regions. Indeed, based on H3K4 methylation ChIP-seq maps for female mouse livers (38), we found that the fraction of H3K4-me1 marks associated with DHS sites was much higher than that of H3K4-me3 marks, particularly for female-specific DHS sites. As H3K4-me1 in the absence of H3K4-me3 is a characteristic of enhancers (15), we surmise that many liver DHS sites function as enhancers, some of which may exhibit sex-specific activities. This is supported by our in vivo reporter gene assays, in which 5 out of 6 DHS sites investigated demonstrated intrinsic enhancer activity when delivered to mouse livers by hydrodynamic injection. Moreover, several of the enhancer sequences tested showed sex-related differences in activity that match the sex specificity of the DHS site and the associated genes. The sex-related differences in in vivo enhancer activities seen here, were, however, considerably smaller than the sex-related differences in levels of expression of the genes themselves, suggesting that multiple DHS sites may be required to confer a high degree of sex specificity to gene expression. Indeed, multiple sex-dependent DHS sites are associated with the three genes whose enhancers showed sex-related differences in levels of activity (Acot4, Cyp7b1, and Cyp2d9). In the case of the Cux2 intron 2 DHS site tested, no sex-related difference between enhancer activities was observed, indicating that the cloned fragment does not recapitulate the strong sex-related difference in DNA accessibility seen in intact liver chromatin (female/male DHS site sequence read ratio, 7.3). Nevertheless, given the very strong enhancer activity of this genomic fragment (Fig. (Fig.5),5), coupled with its 7-fold-lower accessibility in the male liver, this DHS site could make a substantial contribution to the strong (~100-fold) female specificity that characterizes Cux2 gene expression (24). Together, these findings suggest that some sex-dependent DHS sites exhibit intrinsic sex-related differences in enhancer activity, e.g., due to the binding of transcription factors that are expressed or activated in a sex- and plasma GH pattern-dependent manner (e.g., STAT5b), while other sex-dependent DHS sites (e.g., the Cux2 intron 2 enhancer) impart strong sex-related differences to gene transcription by virtue of the large sex-related differences in their accessibility in intact liver chromatin per se, even though they might not directly bind sex-specific transcription factors. Further studies will be required to identify the factors and establish the underlying mechanisms that initiate and maintain these sex-related differences in chromatin structure.

Motif analysis identified 13 transcription factor binding motifs that are enriched in one or more subsets of male-specific DHS sites compared to sex-independent DHS sites (Fig. (Fig.7;7; see Table S10 in the supplemental material). Three other motifs were enriched in female-specific DHS sites, and one of these, the motif for TCFAP2A, was depleted in subsets of male-specific DHS sites. The male DHS site-enriched motifs include motifs that bind liver-expressed transcription factor families from the FOX and nuclear receptor families (e.g., HNF4α), as well as the binding site for STAT5b, which exhibits important sex-related differences in responsiveness to GH stimulation in vivo (52). Another male DHS-enriched motif, CDP, matches the binding site for CUX2 (12), a transcriptional repressor expressed in female livers at a level 100-fold higher than that in male livers (24), suggesting that CUX2 may enforce male specificity by binding to male-specific DHS sites in female livers, thereby suppressing the residual activity of enhancers that are partially accessible in females. A subcluster of 8 male DHS site-enriched motifs (subcluster A1) (Fig. (Fig.7),7), which includes motifs corresponding to binding sites for STAT5b and CUX2, was depleted in a subset of female-specific DHS sites responsive to GH. Given the high frequency of these 8 male-specific DHS site-enriched motifs, it is not surprising that many male-specific DHS sites contain matches for 6 or more of the 8 motifs (see Fig. S7). Further work will be needed to determine whether or not particular combinations of these 8 motifs have distinct functions and to identify the specific factors that actually bind to their cognate sequences at DHS sites in male and female livers.

Our finding that male-specific, GH-responsive DHS sites are enriched for both STAT5b-like and HNF4α-like (nuclear receptor) motifs is consistent with our earlier observation that these factors are both essential for sex-specific gene expression in mouse livers. STAT5b is one of the major direct effectors of GH signaling in liver, and its deletion downregulates ~90% of male-specific genes in the male mouse liver (5, 18). Similarly, knockout of the nuclear receptor HNF4α, enriched in the liver, abolishes sex-related differences in the liver (20). By carrying out the motif analysis separately for DHS sites that are near sex-specific genes, near sex-independent genes, and distant from genes, we showed that the HNF4α-like motif is most highly enriched at DHS sites within 10 kb of sex-specific genes, while the STAT5-like motif is highly enriched at distal sites, as well as at sites proximal to sex-independent genes. The latter finding is consistent with the occurrence of functional STAT5 binding sites at large distances from target genes (8).

In conclusion, the present investigation of sex-related differences in chromatin accessibility has identified condition-specific transcriptional regulatory sites in the mouse liver on a genome-wide scale. These differences are manifested as sex-specific DHS sites, which in some cases encompass extended chromatin regions. A subset of these sex-specific genomic sites and regions is associated with genes expressed in a sex-dependent manner, strongly suggesting that they play a functional role in liver sexual dimorphism; however, a majority of sex-specific DHS sites are distal to sex-specific genes, making it more difficult to establish their significance. Transcription factor binding motifs identified as enriched in these sites serve as candidates for further study of the molecular mechanisms that govern sex-specific liver gene transcription. Further study will be required to determine how sex-related differences in chromatin accessibility are established and maintained in response to sex-related differences in plasma GH patterns, which are programmed by early androgen exposure and first emerge at puberty.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported in part by NIH grant DK33765 (to D.J.W.). A.S. received Training Core support from the Superfund Research Program at Boston University (NIH grant 5 P42 ES07381).

We thank Minita Holloway of this laboratory for contributions to initial optimization of the DHS protocol and X. Shirley Liu (Dana-Farber Cancer Institute) for initial discussions about experimental design.

We have no financial interests to declare.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 September 2010.

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

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