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
]), 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
), 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. ; 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
), 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
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
, 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. ), 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. ; 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
). 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. ), 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
). 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.