Stress and Glucocorticoid Receptor Transcriptional Programming in Time and Space: Implications for the Brain–Gut Axis

Neurogastroenterology & Motility Neurogastroenterol Motil (2016) 28, 12–25 doi: 10.1111/nmo.12706

REVIEW ARTICLE

Stress and glucocorticoid receptor transcriptional programming in time and space: Implications for the brain–gut axis

J. W. WILEY,* G. A. HIGGINS†,‡ & B. D. ATHEY‡

*Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA †Department of Pharmacogenomic Science, Assurex Health, Inc., Mason, OH, USA ‡Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA

Key Messages • Chronic stress is associated with the exacerbation of common mental health and gastrointestinal disorders, including depression and irritable bowel syndrome. • Emerging evidence indicates that chronic stress alters physiological cortisol-mediated pulsatile (circadian and ultradian) gene transcription in a tissue-, cell-, and allele-speciﬁc manner. • Epigenetic studies indicate that chronic stress selectively alters both the DNA methylome and chromatin compaction state affecting gene transcription in central and peripheral pathways involved in a variety of important functions including mood and pain perception. • Chronic and early life stress-associated disruption in glucocorticoid receptor-regulated gene transcription has a potentially signiﬁcant impact on the brain–gut axis throughout the lifespan and across generations.

Abstract corticotropic hormone from the pituitary, ultimately Background Chronic psychological stress is associated leading to the release of cortisol (human) and corti- with enhanced abdominal pain and altered intestinal costerone (rodent) from the adrenal glands. Cortisol barrier function that may result from a perturbation in binds with the GR in the cytosol, translocates to the the hypothalamic–pituitary–adrenal (HPA) axis. The nucleus, and activates the NR3C1 (nuclear receptor glucocorticoid receptor (GR) exploits diverse mecha- subfamily 3, group C, member 1 [GR]) gene. This nisms to activate or suppress congeneric gene expres- review focuses on the rapidly developing observations sion, with regulatory variation associated with stress- that cortisol is responsible for driving circadian and related disorders in psychiatry and gastroenterology. ultradian bursts of transcriptional activity in the Purpose During acute and chronic stress, corti- CLOCK (clock circadian regulator) and PER (period cotropin-releasing hormone drives secretion of adreno- circadian clock 1) gene families, and this rhythm is disrupted in major depressive disorder, bipolar disorder, and stress-related gastrointestinal and immune Address for Correspondence John W. Wiley, MD, Professor, Internal Medicine, University disorders. Glucocorticoid receptor regulates different of Michigan Health System, 1150 West Medical Center Drive, sets of transcripts in a tissue-speciﬁc manner, through Ann Arbor, MI 48109, USA. pulsatile waves of gene expression that includes Tel: +1 (734) 615-6621; fax: +1 (734) 763-2535; e-mail: occupancy of glucocorticoid response elements located [email protected] within constitutively open spatial domains in chro- Received: 29 July 2015 Accepted for publication: 20 September 2015 matin. Emerging evidence supports a potentially piv-

otal role for epigenetic regulation of how GR interacts transcription.31 Epigenetics refers to external modifi- with other chromatin regulators to control the expres- cations to DNA and histones affecting gene transcrip- sion of its target genes. Dysregulation of the central tion that are independent of DNA sequence, but are and peripheral GR regulome has potentially signifi- often driven by DNA sequence variation. One example cant consequences for stress-related disorders affect- of an epigenetic change is DNA methylation, which ing the brain–gut axis. involves the addition of a methyl group to specific DNA sites, thereby preventing the expression of Keywords chronic stress, CLOCK genes, epigenomics, specific genes. Another example is histone modifica- intestinal barrier function, irritable bowel syndrome, tion. DNA wraps around histone proteins to form mental health disorders. compact DNA-histone complexes. Modifications that relax the DNA-histone compaction state, such as BACKGROUND acetylation at specific histone sites, allow accessibility to proteins that ‘read’ genes and, thereby, promote gene The human glucocorticoid receptor (GR) a isoform, transcription.32 herein referred to as GR, is responsible for the maintenance of physiological homeostasis. Glucocorticoid INSIGHTS FROM THE CENTRAL receptor has been implicated in the etiology of a wide NERVOUS SYSTEM AND PSYCHIATRY range of stress-related disorders. Molecular mechanisms underlying GR gene expression are widespread Expression quantitative trait loci (eQTLs) are genomic and diverse in the human genome. They include loci that regulate gene expression. The eQTLs may act opportunistic nucleosome occupancy, preprogramming in cis (locally) or trans (at a distance) to a gene. Cis- of chromatin to permit indiscriminate binding of eQTLs, methylation-quantitative trait loci, and QTLs steroid receptors, and distal enhancer–promoter loop- associated with the histone mark H3K27ac, which – ing to transcriptionally poised genes.1 9 The GR con- indicates enhancer and promoter sites of active gene trols target genes through long-distance looping to regulation, are significantly enriched in domains con- promoters within the three-dimensional milieu of taining the GR response element (GRE).33 These – euchromatin.10 12 A range of cooperative interactions domains are targeted by cis-regulatory, transcription with trophic factors, coactivators, corepressors, and factor-enriched sites within the human NR3C1 gene other nuclear receptors, including brain-derived neu- that contain individual SNPs, and elsewhere, depend- rotrophic factor (BDNF), are important in GR regula- ing on cell type.3,34 Chromatin remodeling, which can tion.13 Although greater than 1% of all transcripts in be observed near GR-regulated genes using live cell the human genome are regulated by the GR, circum- imaging immediately after treatment with the syn- scribed cell- and tissue-type expression is a prominent thetic glucocorticoid dexamethasone, occurs with a characteristic of glucocorticoid regulation. well-defined ultradian rhythm.6,9,35 Numerous studies Allele-specific bias is not uncommon among genes have shown that loss of data about the chromatin that are glucocorticoid responsive, including asymmet- environment of the human NR3C1 gene limits under- ric expression of targets resulting from allele-specific standing of the clinical consequences of genetic vari- methylation and parent-of-origin of monoallelic ation.36–38 In the context of genetic association studies, effects.14 In brain, studies in humans and in rodent one confounding problem has been the influence of models show that chronic stress and early-life adver- population structure, as the allele frequency of a given sity disrupts normal ultradian GR pulsatile chromatin variant, but also haplotype structure, varies widely interactions during critical periods of development in depending on ethnicity.39 an allele-specific manner. This stress-induced disrup- Most studies show that abuse, neglect, and/or lack of tion has been associated with psychiatric disorders parenting during early childhood are associated with which persists and may be transmitted through mater- blunted cortisol cycling in adolescence21,40,41; although – nal or paternal alleles across generations.15 26 Simi- the directionality of effect is still controversial as a larly, chronic stress and altered regulation of GR consequence of variability in study designs.42 Blunted expression and function have been implicated in cortisol and adrenocorticotropic hormone responses irritable bowel syndrome, intestinal barrier dysfunc- are associated with major depressive disorder, espe- tion, inflammatory bowel diseases, and alterations in cially in females.43 In humans, chronic stress of the – the gut microbiome.27 30 mother while the infant is in utero, and early life stress Epigenetic modifications are known to play an between birth and 5 years of age has been shown to important role in chronic stress-associated altered gene affect DNA methylation of specific sites within the

NR3C1 gene.25,26,44 It has been proposed that there is translocator-like protien (ARNTL; formally known as a critical period during child development when brain and muscle ARNT-like 1 [BMAL1]), which abuse and trauma can exert an irreversible lifelong program transcriptional dynamics of rhythmicity, and transgenerational impact.24 Hyper-suppression of including those of the period (PER1, PER2, and PER3) plasma cortisol is a feature of adult patients with and cryptochrome (CRY1 and CRY2) gene families. major depressive disorder and/or posttraumatic stress Maintenance of the circadian and ultradian rhythmic- disorder who have been abused as children. The ity of cortisol release appears to be regulated through flattening of the normal circadian cortisol curve in acetylation and deacetylation of the GR by the CLOCK women who were abused as children suggests a gene,46,54,55 and through CRY1 and CRY2 rhythmic mechanism that involves genomic variants in critical repression of GR expression in tissues, such as the genes that govern NR3C1 gene expression interacting liver.56 Disruption of the periodicity of GR expression with epigenetic modifications to disrupt regulated is associated with disease states characterized by GR periodicity. For example, data suggest that early glucocorticoid resistance or sensitivity.57,58 The peri- childhood trauma interacts with a risk allele in the odicity of GR function is important based on two FKBP5 (FK506 binding protein 5) gene that encodes a observations: (i) During acute physiological states, molecular chaperone of the GR. Early childhood such as the stress response, the need for rapid trans- stress caused by sexual or physical abuse or neglect, activation or transrepression of GR-regulated gene especially between the ages of 0–5 years, results in expression, mediated by the HPA axis, may explain demethylation of CpG sites within intron 7 of the the presence of pre-assembled, spatial chromatin FKBP5 gene, leading to the overexpression of FKBP5, domains where GR can bind in sec- GR resistance, and dysregulation of the central GR onds,6,9,12,33,37,38,57,59–61 and (ii) Cortisol release exhi- regulome.14 This may lead to flattening of the diu- bits both ultradian and circadian rhythmicity, and the rnal plasma cortisol curve in such individuals and temporal dynamics of GR binding to nuclear sites put them at greater risk of developing major depres- follows the circadian cycle as visualized using micro- sive disorder, posttraumatic stress, and functional scopy in living cells.6,9,61 Glucocorticoid receptor bowel disorders later in life. Further research is nee- chromatin immunoprecipitation sequencing (ChIP- ded to understand the molecular substrate of these Seq) studies confirm that the GR occupies existing phenomena. DNase I hypersensitive sites, allowing for rapid binding to GREs and inverted repeat negative GREs.62 DNase I hypersensitivity indicates open chromatin, RHYTHMIC PATTERNING OF GENE and is a primary indicator of sites of transcriptional EXPRESSION regulation. Although the GR initiates the opening of The GR is the major central nervous system determi- closed chromatin, along with the recruitment of other nant of metabolic rhythmicity in humans. Through the factors that can assist in ‘tethered’ binding, similar to hypothalamic–pituitary–adrenal (HPA) axis, the GR the androgen receptor,63 it also interacts with specific regulates peripheral glucocorticoids, such as corti- transcripts that are transcriptionally poised, acting sol.45,46 Mouse and human studies show that about through long-distance ‘looping’.11,12 10% of all genes exhibit diurnal rhythmicity. Neurons Recent studies show that chronic exposure to corti- of the suprachiasmatic nucleus of the hypothalamus sol in human cell lines or corticosterone in mouse are the only cell type known to exhibit endogenous mammary epithelial cells unmasks GR-binding sites circadian rhythmicity in the absence of all inputs,47 not evident after pulsatile steroid exposure.64 This may although when these neurons are dissociated in help explain why chronic stress in humans, which is mutant Cry1-/- knockout mice, their endogenous accompanied by continuous cortisol secretion, acts to rhythms become disrupted and asynchronous. In blunt healthy cortisol-mediated GR responses later in peripheral tissues, inputs such as light, temperature, life, compared to pulsatile increases in hormone levels and metabolic signaling reset various rhythms.48–53 in blood that are a normal component of the acute Peripheral tissue clocks regulate a variety of metabolic stress response.65–67 Thus, in a murine cell model processes, including lipogenesis, insulin secretion, involving a 60-min exposure to corticosterone, ChIP- glucose clearance, lipid accumulation, and food absorp- Seq demonstrated the presence of thousands of unique tion.49 Circadian clocks are based on feedback loops GR-binding sites, not found after 60-min pulses of that involve a heterodimer of the transcription factors hormone.64 Additionally, the Per1 transcription factor circadian locomotor output cycles kaput (CLOCK) and was more significantly associated with chronic expo- brain and muscle aryl hydrocarbon receptor nuclear sure than Hes1 (Hes family bHLH transcription factor

1), which was associated with pulsatile exposure.64 In specific transcription factors. Consequently, the humans, this may be related to the disruption of chronic nature of inflammatory conditions involving circadian, ultradian, and seasonal sleep habits associ- long-term glucocorticoid administration may lead to ated with a variety of diseases. constitutive repression of NR3C1 gene transcription, and thus to glucocorticoid resistance. This is one of several long-distance, epigenetic mechanisms used to THE COMPLEXITY OF NR3C1 GENE – stop expression of NR3C1 and its target genes.10 12 TRANSCRIPTIONAL REGULATION The NR3C1 promoter lacks a consensus TATA box CHROMATIN DYNAMICS and CCAAT motif, although it contains binding sites for transcription factors, such as activator protein 1 Upon entry into the nucleus, the GR diffuses through- (AP1), specificity protein 1, nuclear factor-jb (NFKB1), out chromatin and opportunistically finds poised spa- cAMP response element-binding protein (CREB), early tial domains of open chromatin, as defined by DNase I growth response 1/nerve growth factor 1, and others. hypersensitivity and chromatin conformation cap- The CLOCK gene has inherent histone acetyltrans- ture.5,8,12 The GR also modifies chromatin in these ferase activity, and acetylates lysine moieties located domains, rapidly cycling in and out of GREs, and within the hinge region of the GR, a lysine cluster recruiting other cofactors.59,60 Trans-repression can be containing a consensus sequence KXKK motif, and exerted by three mechanisms: (i) Through ‘negative’ represses NR3C1 transcriptional activity with a diur- palindromic GREs characterized by inverted repeats (IR nal rhythm.46,54,55 Neurotrophins, such as nerve nGREs), which act by direct binding of glucocorticoid growth factor and BDNF, also regulate NR3C1 tran- agonist-liganded GR, producing a silencing repressor scriptional activity, at least in animal models. For complex through the association of nuclear receptor example, rat cortical neurons treated with both BDNF corepressor 2 complex corepressors, and histone and dexamethasone produced a unique set of GR- deacetylases (HDACs),7 (ii) The GR forms complexes regulated genes associated with neuronal growth and with molecules, such as histone H3 lysine 9 methyl- differentiation.13 The BDNF receptor neurotrophic transferase G9a, which apparently can act as a molec- tyrosine kinase, receptor, type 2 increased GR induc- ular scaffold in modulating the periodicity of a subset tion of these genes, whereas BDNF has been shown to of GR-regulated genes,74 and (iii) Indirect suppression phosphorylate serine within the N-terminus of the using corepressors, such as GRIP1.75 Euchromatin is GR, increasing nucleosome occupancy and cofactor characterized by DNase 1 hypersensitivity and specific recruitment at glucocorticoid binding sites.13 Methy- combinations of histone marks that define active lation of the exon 1F promoter of the NR3C1 gene is genomic regulatory elements, such as promoters variable, but hyper-methylation is associated with: (i) H3K4me3 and H3K27ac, and enhancers H3K4me1 Blunted cortisol response in adults who have experi- and H3K27ac. An enhancer can either increase or enced childhood loss,68 (ii) Increased salivary cortisol decrease transcription. Recent research demonstrates response in the children of mothers who were that, in brain, the DNA sequence CAC is a common depressed during pregnancy, and (iii) Late gestational site of methylation, in contrast to other tissues, where age if this methylation occurs in the fetus.69 Hyper- CpG is most often methylated.76 Following activation methylation of NR3C1 has also been found in the by endogenous cortisol in humans or by exogenous postmortem brain tissue of suicide victims with a glucocorticoids, such as dexamethasone, GR protein is history of child abuse.70,71 Ultra-rapid negative feed- rapidly expressed and binds to molecular co-chaper- back of NR3C1 expression is mediated by FKBP5, ones, such as heat shock protein (hsp90), p23 (pros- which can lead to sustained repression of NR3C1 taglandin E synthase 3-cytosolic), and the expression through hyper-methylation of the 1F pro- immunophilin FKBP5, which are important for its moter, concomitant with abnormal disease states translocation to the nucleus. Glucocorticoid receptor associated with glucocorticoid resistance.72,73 A func- protein also participates in a process called assisted tional nGRE in exon 6 of the NR3C1 gene and a long- loading at some sites in chromatin,61 where it tran- range interaction occurring between this intragenic siently increases accessibility to closed nucleosomes, response element and the transcription start site are enabling other nuclear receptors, such as the estrogen instrumental in this repression. This auto-regulatory receptor, to bind at the same GRE, in what has been mechanism of repression shows that the GR concen- called a hit-and-run cycle.5,9 Research has shown that tration can coordinate repression with excess ligand, GR binding to open chromatin, as defined by DNase I regardless of the combinatorial associations of tissue- hypersensitivity, can be defined as: (i) Opportunistic

binding to spatial clusters of constitutively open show that these genes tend to be tissue-specific, at chromatin that can also be used by other transcription least in human and animal cell lines.87,88 Although factors, (ii) Glucocorticoid receptor binding to tran- some studies have attempted to define all of the GR- scriptionally poised genes and transcripts located regulated or glucocorticoid-sensitive genes or tran- within already DNase I–hypersensitive sites, and (iii) scripts in the whole genome, detailed comparison of Reprogramming of closed chromatin to open chro- GR-regulated genes from different tissues, species, and matin by activated GR bound to glucocorticoid, which cell types shows negligible overlap. However, there is probably occurs at the most conserved GRE motifs.62,77 considerable evidence from both rodent and human The transcription factor AP1 is present at all of these studies that CLOCK, PER, and CRY gene families are GRE-binding sites.77 highly regulated by nuclear receptors and constitute part of a master transcriptional system of peripheral circadian rhythmicity in humans. LONG-RANGE INTERACTIONS THAT The GR regulates the expression of restricted sets of CONTROL GR-REGULATED GENES BY tissue-specific transcripts in mouse and rat hippocam- THREE-DIMENSIONAL LOOPING pus, dorsal root ganglion (DRG) neurons, human brain, The architecture of the three-dimensional genome human liver, and various cell lines, and differences provides the basis for the regulation of gene expres- following maternal care in the mouse Nr3c1 gene in sion, bringing enhancers and promoters into close different tissues and cell types, as determined by proximity, although they may be far apart in linear assessing DNA methylation by sequencing. For exam- sequence.78 Thousands of significant long-range loop- ple, in liver, Grøntved et al.88 showed that for both ing interactions have been found to link gene pro- mouse and human, C/EBP (CCAAT/enhancer-binding moters and distal loci, reinforcing the notion that protein) maintains chromatin accessibility and enables many, if not all, gene promoters engage with distal GR recruitment to GREs and IR GREs in most GR- elements using a three-dimensional loop of chromatin binding sites. CCAAT/EBP is highly regulated by that confers spatial proximity, as detected by chro- glucocorticoids in liver, but not brain.87,88 The ubiqui- matin conformation capture.10 A common class of tous CTCF insulator, sometimes acting in concert long-range interactions mediated by the GR involves with cohesion, broadly defines the boundaries of the looping of promoters to sites bound by the specific gene bodies, and topologically associated insulator protein CTCF, the CCCTC-binding factor domains, which may be regulated by an interaction (zinc finger protein) whose gene (CTCF) is not only between the GR and transcriptionally poised genes, highly regulated by glucocorticoids,11,12 but exhibits similar to enhancer-regulated genes.11 In addition, significant allele-specific methylation in humans.79–86 certain genes, such as mouse mammary tumor virus One example of glucocorticoid-mediated looping is (MMTV) and FKBP5 are transcriptionally poised for trans-repression of the NR3C1 gene through intra- specific GR control, fitting into a model of tissue- genic auto-regulation.4 In some examples of tissue- specific gene expression.11,89–92 specific transcription induced by glucocorticoids, In the mammalian central nervous system, respon- there is evidence that looping between GREs and siveness to glucocorticoids appears to be mediated by their target genes takes place in a hormone-indepen- both the mineralocorticoid receptor and the GR in dent manner. For example, using associated chro- some cells of the hippocampus.93 However, although matin trapping, lipocalin 2 (LCN2) was found to be the mineralocorticoid receptor is expressed in many looped to a GRE upstream of the Cip1-interacting zinc tissues, such as the kidney, colon, heart, hippocampal finger protein (CIZ1) gene prior to treatment with formation, brown adipose tissue, and sweat glands, its dexamethasone, although both genes are regulated by expression is often protected from glucocorticoid acti- the GR.12 The loop structure is absent in pituitary vation through colocalization of HSD11K (11-beta- (AtT-20) cells, in which glucocorticoid induction of dehydrogenase isozyme 2), a GR-regulated gene, whose these genes does not occur.12 product inactivates cortisol to cortisone.94–98 In the context of inflammatory disease, GR acts in a reciprocal manner with NFKB1, a phenomenon that has been CELL AND TISSUE-SPECIFIC well-characterized; although a genome-wide coactiva- REGULATION OF TARGET GENES BY THE tion analysis shows that GRE and NFKB1 bind only in GR a fraction of GR- and p65-(NF-jb protein 65) binding There is no distinct set of GR-regulated genes in the sites.78 Studies of neuronal PC-12 cells suggest that human genome; instead, results from many studies greater than 40% of GR-binding sites are not GREs, as

defined by the canonical motif, and related motifs, negative feedback regulation, which is critical for consisting of A/G-G–ACA–T/A-GT-CTC.87 Of the limitation of the HPA-mediated stress response, is 1031 sites that bound the GR, only 25 were shared controlled by a composite element encompassing with 3857 GR-binding sites in A549 cells derived from high-affinity binding sites for AP1 and GR at adjacent human lung epithelium, and 73 shared with the 8265 elements within the nGRE. Corticotropin-releasing GR-binding sites found in the mouse adipocyte 3T3-L1 hormone trans-repression by glucocorticoids involves cell line.8,87 formation of a repressor complex consisting of GR, MECP2 (methyl CpG binding protein 2), and HDAC1, and recruitment of DNMT3b and associated changes COOPERATIVITY, CROSS TALK, PIONEER in proximal promoter methylation.102 Glucocorticoid MOLECULES, AND TRANSCRIPTION receptor trans-repression is known to act through FACTORS intermediary transcription factors that impact the As described above, pleiotropy and promiscuity are nervous and immune systems, including NFKB1 prominent characteristics of GR interactions with (NFKB p105 subunit), AP1, CREB1, nuclear factor of chromatin, and the subsequent regulation of gene activated T-cells, cytoplasmic, calcineurin-dependent expression. Other steroid receptors, such as the 1, signal transducer and activator of transcription 6, estrogen receptor, benefit from GR reprogramming interleukin-4 induced (STAT6), interferon regulatory of chromatin in a cell type-specific manner. For factor 3, STAT3, GATA binding protein 3 (GATA3), example, in a mouse mammary tumor cell line and T-box 21.103 Transcription factors are also designed to exclude progesterone receptor cross talk, involved in extensive remodeling of nucleosome ChiP-Seq analysis shows that priming with dexam- structure by the GR. ethasone treatment significantly increases the binding of estrogen receptor to sites within DNase-I– GR RHYTHMICITY, DYSREGULATION, accessible chromatin compared to treatment with AND THE BRAIN–GUT AXIS estradiol alone, suggesting that glucocorticoids provide accessibility to estrogen receptor–binding sites The CLOCK gene, whose translational product has a that are only accessible after GR programming.38 helix–loop–helix structure, has a primary self-oscilla- Specific cross talk between NFKB1 and GR involves tory component that drives circadian rhythm by significant reprogramming of gene expression under acting by acetylating the hinge region of the conditions when both tumor necrosis factor (TNF) GR.54,55 This mechanism normally represses GR- and glucocorticoids are present, lending support to regulated transcriptional activity, acting in a 180- the notion that GR and p65 are recruited by each degree phase shift relative to the HPA-mediated other, and probably by other factors, such as AP1, to oscillatory cycle.46 Changes in this inverse relation- binding sites gained on coactivation, in a mutually ship between HPA-mediated and CLOCK cycling dependent manner.78 At many genomic sites where have been implicated in shift work disorders,63,104 GR binding causes increased chromatin accessibility, cardiovascular diseases such as hypertension,104 obe- concurrent steady-state binding levels for another sity associated with sleep deprivation,105 and a range receptor are actually increased.61 Thus, although GR of inflammatory disorders,106 including irritable occupancy of a GRE may be short-lived, it acts bowel syndrome, gastroesophageal reflux disease, and cooperatively through assisted loading to enable GRE peptic ulcer disease.107 binding for the estrogen receptor at the same site.61 Dysregulation of the HPA-mediated stress response The mechanisms by which the GR enables other is critical to the decoupling of the phasing of GR- transcription factors to bind, resulting in either a regulated and CLOCK-regulated gene expression in positive or negative transcriptional outcome at com- many psychiatric disorders,108–115 although the molec- posite response elements, has been extensively stud- ular mechanisms that synchronize the periodicity of ied using the MMTV promoter as a model.99–101 In this relationship are not completely understood. the central nervous system, corticotropin-releasing Studies of the regulation of PER2, a clock-controlled hormone (CRH) expressed by neurons of the paraven- gene, show a unique overlapping of GRE and E-box tricular nucleus of the hypothalamus is critical for motifs in its proximal promoter. PER2 responds the release of adrenocorticotropic hormone from the rapidly to glucocorticoid-induction through a comp- anterior pituitary, and the subsequent release of lex including ARNTL, also known as BMAL1, cortisol from the adrenal glands. At the CRH pro- and CLOCK, and is involved in delaying circadian moter in these neurons, glucocorticoid-dependent rhythmicity mediated by the GR.116 However, many

GR-regulated genes are expressed in a variety of mice with a mutation of the clock gene Period 2 different phases relative to the circadian cycle in the (Per2; mPer2(m/m)). Occludin and claudin-1 mRNAs mouse.117 The circadian clock is regulated at the showed daily oscillations; Clock and Arntl (Bmal1) cellular level by a transcriptional feedback loop. bonded directly to the E-box elements of occludin CLOCK:ARNTL heterodimers activate the expression and claudin-1 promoters; and the overexpression of of the PER and CRY genes, acting as transcription Clock- and Arntl-activated occludin and claudin-1 factors directed to the PER and CRY promoters via promoters containing E-box elements. These observa- E-box elements. PER and CRY proteins form hetero- tions suggest that occludin and claudin-1 are CGs dimers and suppress the activity of CLOCK:ARNTL, with E-box elements in the promoter region directly completing the feedback loop. In addition, ARNTL regulated by Clock:Arntl heterodimers. The study did has been shown to directly mediate circadian control not exclude the possibility that posttranscriptional of protein translation.118 mechanisms may also underlie the circadian regula- It is largely unknown how chronic stress directly or tion of occludin and claudin-1 expression. Colonic indirectly affects GR expression and function in the GI permeability was also under circadian control, which tract. Chronic, intermittent water-avoidance stress was inversely associated with occludin and claudin-1 (WAS), 1 h/day for 10 days, significantly reduces the expression levels. Of interest, interleukin-1b (IL-1b) levels of the GR and epithelial tight junction protein and TNF-a mRNA levels did not show circadian levels in the colon, but not the jejunum.119 These oscillations in wild-type mice, and were comparable changes correlate with increased permeability to low between wild-type and mPer2m/m mice. Therefore, molecular weight molecules, and a significant increase Clock:Arntl heterodimers probably regulate colonic in visceral pain in response to colorectal distension. permeability in a circadian manner by timing the Glucocorticoid receptor appears to play a pivotal role in expression levels of claudin-1 and occludin, thereby these responses based on the observation that the GR affecting tight junction function. It remains to be antagonist RU-486 significantly reversed chronic determined whether the changes in occludin and stress-associated decrease in epithelial tight junction claudin-1 expression are primarily responsible for protein levels, increased paracellular permeability, and daily changes in tight junction permeability and visceral hyperalgesia. It is probably relevant that the susceptibility to DSS-induced colitis or, alternatively, baseline level of GR expression in healthy rats is the reduced susceptibility to DSS-induced colitis in markedly higher in the mucosa of the colon compared mPer2m/m mice may be due to some type of to the jejunum.119 immune cell dysfunction. Nevertheless, the chimeric Ontogenetic maturation of a circadian clock from mouse experiments suggest that the Per2 mutation in the fetal stage until weaning has been demonstrated in non-hematopoietic cells is responsible for the protec- the rat colon.120 This study suggests that the molec- tion against DSS-induced colitis. ular mechanism for entraining the circadian clock in the colon appears to involve maternal breast-feeding, ROLE OF THE GR IN CHRONIC STRESS- but subsequently, a suprachiasmatic nucleus-indepen- INDUCED VISCERAL HYPERALGESIA dent developmental signal switches the colonic clock from a maternal-dependent to maternal-independent Chronic stress is associated with downregulation of stage. Consistent with reports that cortisol is respon- the antinociceptive endocannabinoid CB1 receptor sible for driving circadian surges of transcriptional (CNR1 gene) and upregulation of the pro-nociceptive activity in the CLOCK and PER gene families in the endovanilloid TRPV1 receptor in the subpopulation of central nervous system, expression of the epithelial nociceptive DRG neurons innervating the pelvic tight junction proteins occludin and claudin-1 are organs, including the colon. The effect of chronic under circadian control in the mouse large intes- stress on both CB1 and TRPV1 receptor expression tine,121 which supports an earlier report that the and function are prevented by treatment with RU- circadian clock regulated intestinal permeability.122 486, supporting an important role for GR in this The expression of occludin and claudin-1 in the large pathway.123,124 Subsequent observations support a intestine epithelium demonstrated daily oscillations, potentially pivotal role for epigenetic regulatory path- which require normal Per2 activity. In addition, the ways in chronic stress-induced visceral hyperalge- temporal changes of the expression levels were sia.125 Male rats were subjected to WAS or inversely associated with colonic permeability and subcutaneous injections of corticosterone that repro- differential susceptibility to dextran sodium sulfate duced chronic stress levels. Lumbar 4–5 and lumbar (DSS)-induced colitis between wild-type mice and 6– lumbar 6 - sacral 2 DRGs sacral 2 DRGs were

collected and compared between stressed and sham- studies using cell cultures was observed.127 Epigenetic stressed control rats. L4–L5 DRG neurons contribute mechanisms appear to play a role in chronic stress- to the somatosensory innervation of the lower induced changes in NR3C1 receptor levels in the extremities by way of the sciatic nerve, whereas L6– amygdala.128 Trichostatin A, a potent HDAC inhibi- S2 DRG neurons innervate pelvic organs, including tor, significantly attenuated chronic stress-associated the colon. DNA methylation was measured at genes visceral hyperalgesia. Methylation of NR3C1 was encoding NR3C1 and CNR1. Protein levels of CNR1 increased following WAS, which was associated with and DNA (cytosine-5-)-methyltransferase 1 (DNMT1) decreased NR3C1 expression. In contrast, methylation were knocked down in DRG neurons in situ with of the corticotropin-releasing factor (CRF) promoter small interfering RNAs. Visceral pain was measured was decreased after WAS with a concomitant increase in response to colorectal distention. Chronic stress in CRF expression – an anticipated result. In a was associated with upregulation of DNMT1-medi- subsequent study, the authors examined the hypoth- ated methylation at CpG sites involving both NR3C1 esis that histone deacetylation contributes to the and CNR1 promoter regions, resulting in the down- maintenance of chronic anxiety and pain induced by regulation of NR3C1-induced expression of CNR1 in prolonged exposure of the CeA to corticosterone.129 L6–S2, but not L4–L5 DRGs. Gene silencing of Bilateral infusions of a HDAC inhibitor into the CeA DNMT1 in L6–S2 DRG neurons of rats reduced attenuated anxiety-like behavior, and somatic and DNA methylation and prevented chronic stress–in- visceral hypersensitivity resulting from elevated cor- duced increases in visceral pain. These studies indi- ticosterone exposure. Deacetylation of histone 3 cate that the NR3C1 receptor acts as a positive lysine 9 (H3K9), through the coordinated action of transcription factor on CNR1 expression. Chronic the NAD+-dependent protein deacetylase sirtuin-6 stress increases DNA methylation at upstream CpG (SIRT6) and NFKB1-sequestered NR3C1 expression, promoter sites for NR3C1 expression, resulting in leading to disinhibition of CRF. These observations decreased levels of the anti-nociceptive CNR1 recep- suggest that epigenetic programming in the amygdala, tor, culminating in enhanced visceral pain. involving both methylation and histone modification, Chronic, intermittent stress also alters the level of are important in the maintenance of chronic anxiety NR3C1 in regions of the central nervous system and pain. involved in visceral pain registration. In the WAS rodent model, exposing the central nucleus of the LIMITATIONS OF ANIMAL MODELS amygdala (CeA) to elevated glucocorticoid levels was associated with significant reduction in NR3C1 Much of what we know about how the GR regulates receptor expression, but no changes in mineralocorti- gene expression and behavior has been obtained from coid receptor expression,126 which is consistent with studies in rodents, with extension to human mecha- findings from experiments in the hippocampus and nisms of disease. However, recent studies show that

Figure 1 The glucocorticoid receptor, regulation of gene expression, and its clinical consequences. (A) The glucocorticoid receptor (GR) and regulation of the CLOCK gene family. This pathway contributes to regulation of circadian rhythmicity, and is driven by the hypothalamic–pituitary– adrenal axis and metabolic inputs from liver. Of all the thousands of genes whose expression is regulated by the GR, the most highly regulated include members of the CLOCK gene family such as PER1.33 Products of ARNTL and CLOCK form a heterodimer that acts as a transcription factor and binds to the E-box (E) in the promoter of other GR-regulated CLOCK and related genes, including CRY, PER, NR1D1, and RORA. In general, both CRY and PER genes are expressed in a circadian manner, acting either by repressing the CLOCK:ARNTL heterodimer, or blocking GR from binding to the GR response element (GRE) sequence. This latter mechanism is described in the section: GR rhythmicity, dysregulation, and the brain–gut axis. NR1D1 and RORA form a heterodimer that acts as a transcription factor that binds to the RORE sequence that is located in genes such as ARNTL. The ARNTL protein also acts independently of its role as a transcription factor to mediate translational control of specific proteins in a circadian manner.118 (B) Overview of results from the NIH Roadmap Epigenomics Mapping Consortium. Chromosomes are located in chromatin-bound territories in the nucleus. Euchromatin is characterized by DNase 1 hypersensitivity and specific combinations of histone marks that define active genomic regulatory elements, such as promoters H3K4me3 and H3K27ac, and enhancers H3K4me1 and H3K27ac. An enhancer can either increase or decrease transcription. Recent research demonstrates that, in brain, the DNA sequence CAC is a common site of methylation, in contrast to other tissues where CpG is most often methylated. Additionally, in brain, 5-hydroxymethylcytosine (5hmC), a reactive species, is common. In contrast, in the periphery, methylcytosine (hmC) is common.76 (C) Chronic and/or early life stress appear to lead to alteration of chromatin and allele-based gene expression, concomitant with psychiatric disorders and comorbid conditions including irritable bowel syndrome. In such cases, allele skewing of transcription 21,22 is most likely a consequence of epigenetic alterations arising from environmental adversity that contributes to a variety of stress- related disorders distinct from parental imprinting.29,30 POLR2A polymerase (RNA) II (DNA directed) polypeptide A, 220 kDa is a highly conserved, ubiquitously expressed gene that encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes and, therefore, plays an important role in gene transcription. Allelic imbalance refers to differential expression of the maternal vs paternal allele. POLR2A, DNA-dependent RNA polymerase II; H3K4me3, histone H3 trimethyl Lys4; H3K27ac, histone H3 Lys27 acetylation; H3Kme1, histone H3 monomethyl Lys4.

although the complexity of GR interactions with the eling factors for subsequent transient occupation by three-dimensional structure of programmed and un- the GR. Unprogrammed chromatin has not undergone programmed chromatin exhibits some degree of simi- this preparation for GR occupancy.88 In addition, larity between species, population differences in allele- rodents provide poor models of the human inflamma- specific and tissue-specific expression appear to be tory response,130 and do not adequately represent limited to humans.82 Programmed chromatin has been higher cognitive contributions to stress-related psychi- prepared by factors such as AP1, ER-alpha, or remod- atric disease and ameliorative drug therapy.131–133

A GR CRY NRIDI E PER E RORA

ARNTL CLOCK RORA NRIDI CRY PER

ARNTL

RORE

B Most tissue = cpG islands; mHC Brain = CpG, CAC; mostly 5mHC DNA methylation

Genes

DNase I hypersensitivity sites

Chromatin

Histone modifications

H3K27ac + H3K4me3 = active promoter RNA H3K27ac + H3K4me1 - active enhancer

C Chronic Altered Allelic Visceral stress chromatin imbalance pain

POLR2A

Studies from human cell lines underscore the inade- disorders, and the potential role of stress-induced quacy of the rodent to mirror the long-distance, alterations in the intestinal barrier function on visceral enhancer-based regulation of GR transcription that is hyperalgesia. It is probable that alterations in rhythmic a defining characteristic of glucocorticoid regulation of gene transcription, epigenetic regulation of gene tran- gene expression in humans.1,134–139 scription, and allele-specific gene expression play a significant role in the pathophysiology of functional gastrointestinal disorders that are characteristically SUMMARY inducible and reversible. These concepts are summa- Diurnal rhythms in the HPA axis and chronic inter- rized in Figure 1. mittent stress have differential, region-specific, and cell-specific effects involving the brain–gut axis, including intestinal barrier function, peripheral pain ACKNOWLEDGMENTS signaling mechanisms, and central nervous system The authors wish to thank Barbara Boughen for editorial support. modulation of pain perception. It appears that epigenetic regulation of NR3C1 expression plays a potentially key role in these actions. Epidemiological studies FUNDING suggest that chronic stress-associated functional gas- JWW receives grant support from NIH 1R01DK098205 and Takeda trointestinal disorders, such as irritable bowel syn- Pharmaceuticals. drome, are more prevalent in females and individuals 140 who have experienced significant stress early in life. CONFLICTS OF INTEREST Future studies will need to address the mechanistic basis for the distinct effects of gender and early life None. stress as predisposing risk factors in functional bowel

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