The glucocorticoid receptor is pivotal to the physiological response to stress and peripheral circadian rhythms. The GR is broadly expressed, and nearly all cells have ready access to cortisol provided by the bloodstream. Therefore, how the GR separates stress and circadian effects remains an enigma. Here we have shown that, in A549 cells, the GR can drive expression of many glucocorticoid-responsive genes in a dose-dependent manner. Of the genes responding to <5 nM DEX, PER1
is uniquely sensitive in A549 cells, exhibiting 2-fold overexpression at 0.5 nM DEX, a dose at which no other genes have a significant response. To the best of our knowledge, our study is the first to show that in some tissue or cell types, low doses of glucocorticoids can directly regulate the expression of one and only one gene. A 274-bp enhancer upstream of PER1
is sufficient for a hypersensitive response to DEX or to cortisol, and the endogenous promoter or proximity to additional GR binding sites nearby does not affect the response. The region does not contain an instance of a previously reported glucocorticoid modulatory element (62
). Instead, our results suggest that a complex recognition motif likely coordinates the binding of numerous additional factors, or perhaps even additional GR molecules through noncanonical binding motifs (60
), that tune the glucocorticoid response. Furthermore, we have found that those prebound factors are associated with open chromatin. However, the identity of those factors, and their specific roles in remodeling chromatin or otherwise modulating the glucocorticoid responses, remains unclear.
Current models of GR binding suggest that chromatin remodeling is an integral component of GR activity (e.g., references 27
). Pioneer factors, such as the FOXA family of TFs, actively open regions of chromatin and in turn promote GR occupancy (5
). The GR itself can also act to remodel chromatin at some sites, and in doing so it appears to assist in the loading of additional factors into the same region (67
). Our data revealed that increased chromatin availability prior to GR occupancy is strongly associated with GR occupancy at low doses of glucocorticoids and significantly more so than for medium sensitive GR binding sites (P
= 1 × 10−12
, Fisher's exact test). In contrast, sites where the GR directs chromatin remodeling in response to DEX are uniformly bound at higher doses of DEX. The distinction is not absolute, and open chromatin is generally associated with GR binding regardless of sensitivity, suggesting that open chromatin only partially contributes to tuning glucocorticoid responses and that genetic mechanisms are also likely to be important. For example, mutation mapping of the hypersensitive PER1
enhancer revealed that a number of DNA sequences outside the core DNA binding motif contribute to the sensitivity of the corticosteroid response. These sites may contribute both to the occupancy of pioneer factors as well as to the prior recruitment of additional interacting transcription factors and cofactors that would in turn decrease the Kd
of the GR for DNA upstream of PER1
. In this model, low concentrations of DEX would result in a low concentration of active GR in the nucleus, which would only be able to productively bind the hypersensitive sites. Fully confirming the role of open chromatin will require additional experiments to determine if disrupting the chromatin state near hypersensitive GR binding sites affects the sensitivity of those sites.
Given the potential importance of open chromatin for hypersensitive GR binding, it is interesting that we see similar expression responses originating from plasmid reporters. Transiently transfected plasmids are known to have incomplete nucleosome structure and often to not faithfully model chromosomal DNA structure (49
). It may be that the minimal enhancer element contains enough information to establish an open chromatin state on the plasmid, or that incomplete nucleosome structure on transfected plasmids may be permissive to GR binding. Our observed associations with open chromatin in the genome may therefore reflect prebound TFs that help to recruit the GR rather than a strict prerequisite for gene expression responses to low glucocorticoid concentrations. Alternatively, while our motif analysis did not indicate specific GR binding sequences associated with increased or decreased sensitivity, we cannot exclude the possibility that some GR binding motifs may limit remodeling and influence sensitivity. Resolution of whether indeed plasmid chromatin structure matches that seen on the genome at the PER1
response elements may therefore provide further insights into if and how chromatin is established near hypersensitive GR binding sites and the extent to which that chromatin may tune glucocorticoid responses. One possible approach is to determine how stably integrated versions of hypersensitive GR binding sites respond relative to transfected plasmids. Alternatively, it is now possible to directly modify genomic GR binding regions, allowing study of the genetics and epigenetics of glucocorticoid responses in the native chromatin context (11
As described recently, monomeric GR interacting with DNA (even at a high-affinity site) is expected to follow a first-order dose response (47
). In that model, increased affinity of the GR for DNA at hypersensitive sites would shift the dose-response curve toward lower concentrations of DEX but would not cause an increase in the steepness of the dose-response curve (i.e., the Hill coefficient). The model further predicts, as we see in many of our nonhypersensitive response curves, that a Hill coefficient of 1 is characteristic of diffusion-mediated interactions of the GR with DNA. In our results, however, the PER1
enhancer elements that respond to a low concentration of DEX follow a steeper dose-response curve in A549 cells. That change in the shape of the dose-response curve suggests that cooperative binding of the GR with other molecules also contributes to the sensitivity of the response (47
). It may be that a fraction of nuclear GR binds effector molecules prior to binding chromatin and that that complex then binds specific regions of the genome at a composite DNA binding motif.
Consistent with the model showing that both increased affinity to DNA and cooperative interactions with other molecules contribute to the hypersensitive gene expression response, deletion constructs that respond to higher DEX concentrations in A549 cells also lack the higher-order response. Meanwhile, in ECC-1 cells, the higher-order dose response is not evident despite increased sensitivity, indicating that the two mechanisms may be distinct and tissue specific. Together, the results point to a model where a combination of chromatin state, occupancy of additional transcription factors and cofactors, and cooperative interactions with other molecules work together to tune GR binding at specific genomic loci, including a locus responsible for the regulation of PER1.
The level of cortisol required for expression from the GR binding site upstream of PER1
was 21 nM, below the normal range of plasma cortisol. However, a substantial fraction of plasma cortisol is bound by proteins, such as corticosteroid binding globulin (32
), and is unavailable to activate the GR. Estimates of free cortisol levels are as low as 5 nM in blood (3
) and in tissues (57
), similar to the range that we expect would be required to dynamically regulate PER1
expression through the day.
Cortisol released from the adrenal gland plays an important role in the regulation of PER1
in some but not all mouse peripheral tissues and is an important messenger in peripheral circadian rhythms (36
). Some peripheral clocks can be entrained independent of neuronal signals by restricted feeding times, suggesting additional clock mechanisms (9
). Researchers have shown that either a serum shock or a DEX shock can induce circadian patterns of gene expression in tissue culture cells (3
) and that glucocorticoids can also influence circadian rhythms in peripheral tissues (3
). Our work suggests that hypersensitive PER1
expression may contribute to peripheral circadian timing by controlling the time of day when PER1
is first expressed. Supporting our hypothesis, a recent study of adrenalectomized rats showed that glucocorticoids are essential for oscillation in rPer1
expression and that daily injection of cortisol into the adrenalectomized rodents entrained circadian rhythms (58
). These results suggest that cortisol-mediated regulation of PER1
is an important component of circadian gene expression in peripheral tissues. Our time course study also showed that triggering of PER1
by low-dose glucocorticoids is sufficient to regulate expression of other circadian rhythm genes many hours later. That regulation reveals coordinated expression of PER1
, as well as of CRY1
. In our study, PER1/2
expression gave way to CRY1/2
gene expression, which was followed by expression of ARNTL
. While we did not observe clear circadian oscillation after specific induction of PER1
expression, the order of expression matched that of in vivo
studies of circadian gene expression (3
). Together, these studies suggest that GR regulation of PER1
plays an important role in maintaining circadian rhythms in peripheral tissues and that specific induction of PER1
may help better dissect the regulatory network controlling circadian gene expression.
The ability to regulate circadian rhythms via a highly targeted PER1
response may ultimately be pharmacologically useful. Abnormal circadian rhythms, including abnormal cortisol levels, are involved in many diseases, including depression (29
), schizophrenia (15
), and metabolic syndrome (41
). Correcting the circadian oscillation of cortisol presents a treatment option in some patients (18
). In such cases, the use of low doses of corticosteroids to target expression of PER1
and to renormalize circadian rhythms and related metabolic oscillations may provide a novel treatment option that is free of typical and serious side effects of high-dose exogenous glucocorticoids (16
). It may be that different cortisol levels regulate different physiological functions. Testing that hypothesis will require studies in diverse primary tissues to understand if the genes sensitive to low doses of glucocorticoids coordinately regulate specific pathways or functions.