Jonathan D Turner

Simone R Alt

Lei Cao

Sara Vernocchi

Nadia Battello

Claude P Muller
email: claude.muller@lns.etat.lu20

Transcriptional control of the glucocorticoid receptor: CpG islands, epigenetics and more. 1 2

The unique variability in the 5' region of the GR gene, with 9 alternative first exons and 2 13 splice variants plays a critical role in transcriptional control maintaining homeostasis 3 of the glucocorticoid receptor (GR). This 5'mRNA heterogeneity, common to all species 4 investigated, remains untranslated since the alternative first exons are spliced to exon 2 5 immediately upstream of the translation initiation codon. These alternative first exons are 6 located either immediately upstream of the coding exons in the CpG island (exons B-H 7 and J), or further upstream (exons 1A and 1I). The mechanisms regulating the differential 8 usage of these first exons in different tissues and individuals, and the role of the 5' UTR 9 in the splicing of the coding exons are still poorly understood. Here we review some of 10 the mechanisms that have so far been identified. Data from our laboratory and others 11 have shown that the multiple first exons represent only a first layer of complexity 12 orchestrated probably by tissue-specific transcription factors. Modulation of alternative 13 first exon activity by epigenetic methylation of their promoters represents a second layer 14 of complexity at least partially controlled by perinatal programming. The alternative [START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF] promoter usage also appears to affect the 3' splicing generating the different GR coding 16 variants, GR, GRβ, and GR-P. Aberrant GR levels are associated with stress-related 17 disorders such as depression, and affect social behaviour, mood, learning and memory. [START_REF] Geng | Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter[END_REF] Dissecting how tissue-specific GR levels are regulated, in particular in the brain, is a first 19 step to understand the significance of aberrant GR levels in disease and behaviour. 20 Stress causes an increasing disease burden at least in Western societies. Economic losses 2 due to stress may be as high as 3-4% of the European gross national product. In addition, 3 stress has detrimental effects on behaviour, mood, learning and memory that are difficult 4 to quantify. Glucocorticoids (GCs), the key stress mediators, exert profound effects on a 5 wide range of physiological and developmental processes that are crucial for the 6 adaptation to stress. Psychosocial stress has also been implicated in the development of 7 mental disorders, including schizophrenia, anxiety disorders, and depression. 8

GCs act via binding to two types of intracellular nuclear receptors, i.e. the glucocorticoid 9 receptor (GR) and the mineralocorticoid receptor (MR). Upon binding of GCs, these 10 receptors translocate to the nucleus where they regulate the activity of specific target 11 genes, in a cell-type specific manner, as transcription factors. This review focuses on the 12 classical GR, NR3C1. Numerous factors have been demonstrated to affect the 13 responsiveness to GC by regulating GR activity, such as GR co-activators and co-14 repressors [START_REF] Kumar | Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions[END_REF], GR splice-variants [START_REF] Derijk | Very low levels of the glucocorticoid receptor beta isoform in the human hippocampus as shown by Taqman RT-PCR and immunocytochemistry[END_REF][START_REF] Derijk | A human glucocorticoid receptor gene variant that increases the stability of the glucocorticoid receptor beta-isoform mRNA is associated with rheumatoid arthritis[END_REF][START_REF] Schaaf | Molecular mechanisms of glucocorticoid action and resistance[END_REF], and GR isoforms [START_REF] Lu | Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor[END_REF][START_REF] Lu | Glucocorticoid receptor isoforms generate transcription specificity[END_REF][START_REF] Lu | Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes[END_REF]. In addition, and perhaps 15 most important for GC responsiveness is the expression level of GR protein [START_REF] Bamberger | Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids[END_REF][START_REF] Reichardt | Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock[END_REF][START_REF] Ridder | Mice with genetically altered glucocorticoid receptor expression show altered sensitivity for stress-induced depressive reactions[END_REF][START_REF] Van Der Laan | Nuclear receptor coregulators differentially modulate induction and glucocorticoid receptor-mediated repression of the corticotropin-releasing hormone gene[END_REF], [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF] determined by the mRNA level. 17 The human GR gene (OMIM + 138040; NR3C1) is located on chromosome 5q31-q32 5 [START_REF] Hollenberg | Primary structure and expression of a functional human glucocorticoid receptor cDNA[END_REF] and contains 8 translated exons (2-9) and 9 untranslated alternative first exons. We 6 and others have shown that GR levels are under the transcriptional control of a complex 7 5' structure of the gene, containing the untranslated first exons important for differential 8 expression of the GR. All of the alternative first exons identified are located in one of the 9 two promoter regions: the proximal or the distal promoter region, located approximately 10 5kb and 30kb upstream of the translation start site, respectively [START_REF] Barrett | Coordinate regulation of glucocorticoid receptor and c-jun gene expression is cell type-specific and exhibits differential hormonal sensitivity for down-and up-regulation[END_REF][START_REF] Wei | Regulation of the glucocorticoid receptor gene by the AP-1 transcription factor[END_REF][START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF][START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF][START_REF] Nunez | Characterization of promoter 1B in the human glucocorticoid receptor gene[END_REF][START_REF] Geng | Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter[END_REF]. Alternative first 11 exons 1A, and 1I are under the control of promoters in the distal promoter region, 12 whereas the promoters of exons 1D, 1J, 1E, 1B, 1F, 1C (1C1-3), 1H (Fig. 1A) are located 13 in the proximal promoter region [START_REF] Turner | Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1[END_REF][START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF]. Exons 1D to 1H are found in an upstream CpG 14 island with a high sequence homology between rats and humans. 15

The region-or tissue-specific usage of alternative first exons leading to different GR 16 mRNA transcripts [START_REF] Turner | Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1[END_REF][START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF] (Fig. 1 B) provides a mechanism for the local fine-tuning of 17 GR levels. Since the ATG start codon lies only in the common exon 2, this 5'mRNA 18 heterogeneity remains untranslated, but is important for translational regulation [START_REF] Pickering | The implications of structured 5' untranslated regions on translation and disease[END_REF]. [START_REF] Turner | Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1[END_REF] Alternative mRNA transcript variants are generated by splicing of these alternative first 20 exons to a common acceptor site in the second exon of the GR. Exon 2 contains an in-21 frame stop codon immediately upstream of the ATG start codon to ensure that this 5 ' 22 A c c e p t e d M a n u s c r i p t GR Transcription 5 heterogeneity remains untranslated, and that the sequence and structure of the GR is not 1

affected. 2

The GR also has a variable 3' region. Unlike the 5' region, the 3' variability encodes 3 splice variants with different functions. The 3 main 3' splice variants of the GR are GRα, 4

GRβ, and GR-P (Fig. 1B). GRα and GRβ are generated by two alternatively spliced 3 ' 5 exons, 9α and 9β. GR-P lacks both exons 8 and 9 and is translated into a protein with a 6 truncated ligand binding domain (LBD) which is thought to enhance GR activity. GRα 7 is by far the most active form of the receptor, GRβ is thought to be a dominant negative 8 regulator of the receptor, and little is known about the function of GR-P. 9 10

Alternative first exon usage and 3' splice variants 11

The recent observation that transcription factors binding to pol II transcribed promoters 12 modulate alternative splicing, supports a physical and functional link between 13 transcription and splicing [START_REF] Kornblihtt | Promoter usage and alternative splicing[END_REF]. Several factors were identified that were critical for the 14 recruitment of a specific set of co-regulators to pol II transcribed gene promoters and the 15 production of a specific splice variants. The splice variant produced depends on the 16 structural organisation of the gene and the nature of the co-regulators involved [START_REF] Auboeuf | Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes[END_REF]. A 17 link between transcription initiation sites and the resulting splice variant was suggested 18 since it was shown that promoters controlled alternative splicing also via the regulation of 19 pol II elongation rates or processivity. Slow pol II elongation paired with internal 20 elongation pauses favoured the inclusion of alternative exons governed by an exon 21 skipping mechanism, whereas high elongation rates of pol II, without internal pauses 22 favoured the exclusion of such exons. However, the GR with its variable 5' UTR, and alternative splicing in the 3' coding 3 region is unique. Little is known about the association between the promoter usage and 4 the resulting GR protein isoform. The 5´ UTR has tight control over local GR expression 5 levels. There seems to be also a poorly understood statistical link between the 5'UTR and 6 3' splice variants produced. One of the first studies to address this question showed that 7 exon 1A3, and to a lesser extend 1B and 1C contribute most to the expression of GR-α 8 isoform [START_REF] Pedersen | Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines[END_REF]. By comparing the most abundant exon 1 containing transcripts (1A, 1B 1C) 9

with GRα, GRβ, and GR-P containing transcripts in different tissues and cell lines, 10

Russcher et al. found a correlation between promoter usage and alternative splicing of the 11 GR gene [START_REF] Russcher | Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene[END_REF]. More specifically they found that the expression of GRα is preferentially 12 regulated by promoter 1C, whereas 1B usage favours the expression of GR-P isoform. No 13 association was found with transcripts including exon 9β or with those transcribed from 14 1A, suggesting that GRβ splicing may be associated with one of the recently identified 15 exon 1 variants such as 1D to 1F and 1H that were not included in the above study [START_REF] Russcher | Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene[END_REF]. [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF] We also confirmed that in post-mortem brain tissues of patients with major depressive 17 disorder (MDD) altered promoter usage influenced the resulting 3'GR isoform, with a 18 negative correlation between GR-P expression and promoter 1B usage in all brain areas 19 of MDD patients but not in normal control brains. A negative correlation was also found 20 between the 1C promoter usage and GR-P expression in MDD brains. These results 21 suggest that the promoters 1B or 1C do not play a significant role in GR-P expression in 22 MDD, and that they were rather linked to other forms with lower expression [START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF]. Thus, The hGR was initially described as a housekeeping or constitutively expressed gene with 5 promoters that contain multiple GC boxes and no TATA or TATA-like box [START_REF] Zong | The promoter and first, untranslated exon of the human glucocorticoid receptor gene are GC rich but lack consensus glucocorticoid receptor element sites[END_REF]. A wide 6 variety of transcription factors have been identified that bind in the CpG island upstream 7 of the gene. The description of the transcription factors active within this region is 8 complicated by their tissue-specific usage. These transcription factors were not assigned 9 to the different exon 1 promoters since most of this work was performed before our 10 detailed description of the first exons in this region. The transcription factors so far 11 identified are summarised (Table 1) and their location within the CpG island shown in 12 Figure 1C. Initially, 11 DNAse 1 footprints representing unique transcription factor 13 binding sites were found in the 1C to 1F region of the CpG island (-3259 to -2522 from 14 the ATG start codon) including, one AP-2 and 5 Sp-1 binding sites were identified [START_REF] Nobukuni | Characterization of the human glucocorticoid receptor promoter[END_REF]. [START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF] It was initially thought that the latter transcription factors played an essential role in the 16 basal expression of the hGR, although this is now less clear. Further studies identified 17 one of the footprints in promoter 1C as a binding site for the transcription factor Yin 18

Yang 1 [START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF]. YY1, expressed in a wide variety of mammalian cell types, is a zinc-finger 19 transcription factor that can act as an activator, a repressor, or an initiator of transcription 20 [START_REF] Nunez | Characterization of promoter 1B in the human glucocorticoid receptor gene[END_REF][START_REF] Shrivastava | An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1[END_REF]. The same authors also revealed three other YY1 sites and another Sp1 site, 21 initially assigned to promoter 1B. The later identification of promoter 1D suggested that 22

A c c e p t e d M a n u s c r i p t GR Transcription 8 these YY1 sites are probably associated with this promoter [START_REF] Turner | Tissue specific glucocorticoid receptor expression, a role for alternative first exon usage?[END_REF]. Similarly, the Sp1 sites 1 correspond to a region that was later identified as promoter 1J [START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF]. 2

Similarly, several transcription factors initially assigned to promoter 1C should be 3 reassigned to promoter 1F. AP-1, a transcription complex whose components are encoded 4 by c-fos and c-jun proto-oncogenes binds to the AP-1 site within the hGR promoter 1F 5 [START_REF] Wei | Regulation of the glucocorticoid receptor gene by the AP-1 transcription factor[END_REF][START_REF] Breslin | The Glucocorticoid Receptor and c-jun Promoters Contain AP-1 Sites That Bind Different AP-1 Transcription factors[END_REF]. This same region was also shown to bind Ku70 and Ku 80 in a tissue-specific 6 manner [START_REF] Warriar | Expression of human glucocorticoid receptor gene and interaction of nuclear proteins with the transcriptional control element[END_REF]. Whilst most of the transcription factors identified upregulate GR 7 expression, GRF-1 (glucocorticoid receptor DNA binding factor 1) has been identified as 8 a repressor of GR transcription [START_REF] Leclerc | Molecular cloning and characterization of a factor that binds the human glucocorticoid receptor gene and represses its expression[END_REF][START_REF] Leclerc | Purification of a human glucocorticoid receptor gene promoter-binding protein. Production of polyclonal antibodies against the purified factor[END_REF]. At the 3' end of the rat 1 7 promoter a NGFI-A 9 binding site was identified only 2 bp upstream of the transcription initiation site of this 10 exon [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF]. Recently, the homologous human NGFI-A binding site, together with 11 numerous non-canonical NGFI-A sites were identified in promoter 1F of the hGR [START_REF] Mcgowan | Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse[END_REF]. 12 As a transcription factor, GR also auto-regulates its own CpG island promoters. Several 13 glucocorticoid response element (GRE) half-sites, acting in concert with c-Myb, and c-14 Ets protein members have been identified in promoter 1D, 1E, 1F and 1C [START_REF] Geng | A conserved molecular mechanism is responsible for the auto-up-regulation of glucocorticoid receptor gene promoters[END_REF]. [START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF] The currently known transcription factors provide only an incomplete picture of the 16 complex regulatory mechanisms. For instance, little is known about the proximal 17 elements in promoters 1B and 1H. Using an in silico phylogenetic footprinting technique 18 we were able to find the majority of the experimentally identified transcription factors, [START_REF] Turner | Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1[END_REF] and predicted a wide variety of factors that are conserved between many species [START_REF] Turner | Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms[END_REF]. 20

These are interesting candidate regulators of GR expression that warrant further 21 investigations. Whilst the majority of the GR first exons and their promoters are located within the CpG 9 island, exons 1A and 1I map 25 Kb upstream of the CpG island and 32 Kb upstream of 10 the main GR ORF in the distal promoter region [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF][START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF]. Exon 1A has also been 11 identified in the mouse, and three possible homologues 1 1 , 1 2 , and 1 3 have been found in 12 the rat [START_REF] Strahle | At least three promoters direct expression of the mouse glucocorticoid receptor gene[END_REF][START_REF] Mccormick | 5'heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events[END_REF]. The human promoter 1A generates 3 alternatively spliced transcripts, 13 1A1, 1A2 and 1A3 [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF]. Expression of the 1A transcripts appears to be limited to the 14 immune system in both humans and rodents. The human 1A3 transcript is widely 15 expressed in both acute lymphoblastic leukemia (ALL) cell lines and in children with this 16 malignancy [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF][START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF][START_REF] Russcher | Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene[END_REF][START_REF] Chen | Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells[END_REF][START_REF] Tissing | Glucocorticoid-induced glucocorticoid receptor expression and promoter-usage is not linked to glucocorticoid resistance in childhood ALL[END_REF][START_REF] Chen | Multiple glucocorticoid receptor transcripts in membrane glucocorticoid receptor-enriched S-49 mouse lymphoma cells[END_REF]. Similarly, exon 1I is used predominantly in T cells, 17 although it is also present in HeLa cells [START_REF] Presul | Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor[END_REF]. [START_REF] Geng | Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter[END_REF] Promoter 1A is regulated by the GR itself. The human 1A promoter contains a GC-19 responsive cassette containing a non-canonical GRE adjacent to overlapping binding sites 20 for c-Myb and c-Ets protein family members. In the presence of c-Myb the ligand-bound 21 GR is recruited to the promoter and up-regulates 1A transcripts, while the interaction 22 with c-Ets family members leads to a repression of 1A promoter activity [START_REF] Geng | Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter[END_REF][START_REF] Geng | Human glucocorticoid receptor alpha transcript splice variants with exon 2 deletions: evidence for tissueand cell type-specific functions[END_REF]. This decreases GR expression, but in and certain T cells GCs increase GR expression. The 2 synergy between c-Ets and the GR has been shown to be responsible for the down 3 regulation of 1A3 in the B lymphoblastoid cell line IM-9 after GC exposure. In contrast, 4 the presence of c-Myb in T cells increased the activity of promoter 1A in human cell lines 5 such as the ALL T cell line CEM-C7. As a result of the higher level of total GR T cells 6 are more sensitive to GC induced apoptosis, and 1A3-transcripts were shown to be the 7 most GC responsive of all first exons investigated [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF][START_REF] Pedersen | Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines[END_REF]. Although 1A containing 8 transcripts correspond to only about 10% of all GR transcripts [START_REF] Tissing | Glucocorticoid-induced glucocorticoid receptor expression and promoter-usage is not linked to glucocorticoid resistance in childhood ALL[END_REF], their contribution to 9 the tissue-specific response to GC treatment was considered essential [START_REF] Breslin | Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids[END_REF][START_REF] Pedersen | Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines[END_REF]. The 10 human promoter 1A also has a functional binding site for Interferon Regulatory Factors 11 (IRF-1 and IRF-2), however IFNγ stimulation of CEM-C7 cells did not increase 1A 12 transcript levels, nor did it alter their susceptibility to GC-mediated apoptosis [START_REF] Nunez | Interaction between the Interferon Signaling Pathway and the Human Glucocorticoid Receptor Gene 1A Promoter[END_REF]. [START_REF] Barrett | Coordinate regulation of glucocorticoid receptor and c-jun gene expression is cell type-specific and exhibits differential hormonal sensitivity for down-and up-regulation[END_REF] Thus the evidence suggests that 1A transcript levels are critically involved in mechanisms 14 of therapeutic induction of apoptosis in ALL T cells. However, GC resistance in pediatric [START_REF] Breslin | The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1[END_REF] T-and B-ALL cells obtained directly from patients did not correlate with either the basal 16 or the stimulated expression of the 1A-, 1B-or 1C-transcripts. The relation between GC 17 sensitivity and expression of GR transcripts may be complicated by the overlapping 18 effects such as multi drug resistance genes that prevent GC concentrations from building 19 up in the cells. In addition, ALL cells were shown to have an up-regulated GR expression 20 upon prednisolone treatment regardless of their phenotype or sensitivity to GC-induced 21 apoptosis [START_REF] Tissing | Glucocorticoid-induced glucocorticoid receptor expression and promoter-usage is not linked to glucocorticoid resistance in childhood ALL[END_REF]. In mice the presence of the membrane-bound GR was a better correlate of GC-induced 1 apoptosis than the intracellular GR level [START_REF] Gametchu | Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines[END_REF][START_REF] Gametchu | Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells[END_REF]. Exon 1A was found to be highly 2 expressed in a T lymphoma cell line with elevated levels of membrane-bound GR and 3 enhanced sensitivity to GC-dependent cytotoxicity. 1A transcripts appear to contain all 4 the necessary information for both the synthesis and the subcellular trafficking of the 5 membrane GR, although the exact mechanism remains unknown [START_REF] Chen | Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells[END_REF]. It is interesting to 6 hypothesise that the initially presumed link between GC induced apoptosis and 1A 7 transcript levels is indirect, with 1A transcripts producing membrane GR the true 8 correlate of GC sensitivity. Recently a similar membrane GR has been detected in human 9 cells, but its sequence is not known [START_REF] Bartholome | Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis[END_REF][START_REF] Spies | Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway[END_REF]. The membrane GR may eventually explain 10 the discrepancy between the in vivo and in vitro observations in ALL. It is interesting to 11 speculate that, as in the mouse, the different first exons determine the cellular fate of the 12 human GR. [START_REF] Barrett | Coordinate regulation of glucocorticoid receptor and c-jun gene expression is cell type-specific and exhibits differential hormonal sensitivity for down-and up-regulation[END_REF] Further sequence analysis of mouse exon 1A found 5 small upstream-ORFs preceding the 14 major ORF of the GR. It has been shown that GR expression from the 1A transcript 15 requires translation of the peptide encoded by uORF-2. The peptide was detectable in 16 both cells with naturally 1A transcripts and cells transfected with 1A. The role of this 17 peptide in the regulation of the membrane GR is still unclear [START_REF] Diba | 5'UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor[END_REF]. imprinting and inactivation of one X-chromosome [START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF]. The epigenetic chromatin status 1 is sensitive to the host environment. Thus, epigenetic methylation represents a link 2 between the environment and gene activity. In particular, early life events can have a 3 long-lasting effect on epigenetic programming [START_REF] Meaney | Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health[END_REF][START_REF] Szyf | The early life environment and the epigenome[END_REF]. In many instances, minor 4 changes in GR levels can have a significant impact, for example on feedback regulation 5 of the HPA axis, where hippocampal or pituitary GR levels determine the HPA axis set-6 points and the response to stress. 7

Experimentally, maternal care such as licking-grooming (LG) and arched-back nursing 8 (ABN) has been shown to translate into epigenetic methylation of the GR promoter 1 7 9

with profound and lasting effects on the stress response of the off-springs. [START_REF] Weaver | Epigenetic programming by maternal behavior and pharmacological intervention. Nature versus nurture: let's call the whole thing off[END_REF]. The 10 NGFI-A binding site in the GR promoter 1 7 (Fig. 3), homologous to the human 1F, was 11 highly methylated (>80%) in the offspring of low caring mothers whereas it was rarely 12 methylated in the offspring of high caring rats [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF]. As a result, binding of NGFI-A to the 13 GR 1 7 promoter was inhibited in the hippocampus of offspring of low caring mothers and 14 GR 1 7 expression was reduced [START_REF] Weaver | The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: altering epigenetic marks by immediate-early genes[END_REF]. Interestingly, these effects were reversed by cross-15 fostering indicating a direct effect of maternal care on the epigenome of the offspring 16 [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF]. Infusion of L-methionine reversed these effects on methylation and NGFI-A 17 binding to the exon 1 7 promoter in the rat brain [START_REF] Weaver | Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life[END_REF]. [START_REF] Geng | Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter[END_REF] In Lewis and Fisher rats that naturally differ in their stress response and hippocampal GR 19 levels, the 1 7 promoter was shown to be un-or poorly methylated throughout (mostly 20 below 10% and never exceeding 30%), with no difference between the two strains. 21

Feeding these rats a methyl-supplemented diet had no significant effect on the GR 22 promoter 1 7 methylation levels [START_REF] Herbeck | Conserved methylation of the glucocorticoid receptor gene exon 1(7) promoter in rats subjected to a maternal methyl-supplemented diet[END_REF]. Using the maternal separation model to change the stress response in rat pups (Table 2), 1 Daniels et al. observed elevated NGFI-A levels and significant behavioural changes. In 2 this model, the 1 7 promoter, including both CpG sites within the NGFI-A binding site, 3 was uniformly un-methylated even after applying the maternal separation stressor [START_REF] Daniels | Maternal separation alters nerve growth factor and corticosterone levels but not the DNA methylation status of the exon 1(7) glucocorticoid receptor promoter region[END_REF]. [START_REF] Schaaf | Molecular mechanisms of glucocorticoid action and resistance[END_REF] Epigenetic programming of the GR is not limited to central tissues such as the 5 hippocampus. It has also been proposed that dietary restriction could lead to changes in 6 DNA methyl content, affecting epigenetic programming of the GR promoter both 7 centrally and peripherally [START_REF] Mcgowan | Diet and the epigenetic (re)programming of phenotypic differences in behavior[END_REF]. Feeding a protein-restricted diet to pregnant dams lead to 8 a hypomethylation of the major GR promoter 1 10 and to an increased expression of GR in 9 the liver of these rat pups [START_REF] Lillycrop | Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during A c c e p t e d M a n u s c r i p t GR Transcription 23 pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications[END_REF]. 10

These animal models showing changes in GR promoter methylation are not easily 11 transferred to humans. Nevertheless maternal adversities like depression or protein 12 restriction and their effects on the epigenome of offspring have been investigated (Table 13 2). Oberlander et al. showed that prenatal exposure to maternal depression leads to 14 increased methylation levels of the GR promoter 1F at the NGFI-A binding site in cord 15 blood of newborns [START_REF] Oberlander | Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses[END_REF]. Like in most rat experiments, methylation levels were uniformly 16 low (5-10 %) with small but significant differences between children of depressed and 17 healthy mothers. 18

Several studies also investigated alterations in the human GR 1F promoter in specific 19 disease populations based on the rat 1 7 data of Weaver et al. [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF]. In neurological 20 disorders such as Parkinson's disease, Alzheimer's or dementia no hypermethylation of 21 the 1F promoter and the NGFI-A binding site could be found [START_REF] Moser | The glucocorticoid receptor gene exon 1-F promoter is not methylated at the NGFI-A binding site in human hippocampus[END_REF]. We showed that in 22 major depressive disorder there was no methylation of the NGFI-A binding site of the 1F patterns compared to suicide victims without abuse [START_REF] Mcgowan | Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse[END_REF]. In this study, methylation of 3 another putative NGFI-A binding site within promoter 1F resulted in decreased 4 expression levels of 1F transcripts and overall GR levels. The known NGFI-A binding 5 site was completely unmethylated in all of the suicide victims. Thus, it is possible that 6 other transcription factor binding sites are important for the transcriptional regulation of 7 the GR 1F promoter and that these are more sensitive to epigenetic modifications. 8

Interestingly, in all four of the above studies [START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF][START_REF] Mcgowan | Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse[END_REF][START_REF] Oberlander | Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses[END_REF][START_REF] Moser | The glucocorticoid receptor gene exon 1-F promoter is not methylated at the NGFI-A binding site in human hippocampus[END_REF], levels of methylation were 9 always very low in comparison to those in the LG-ABN rats [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF]. Investigating the 10 complete GR CpG island, we were able to show highly variable methylation patterns 11 among different GR promoters in PBMC's of healthy donors, suggesting that epigenetic 12 programming may not be restricted to the 1F promoter, but operates throughout the CpG 13 island [START_REF] Leclerc | Molecular cloning and characterization of a factor that binds the human glucocorticoid receptor gene and represses its expression[END_REF]. It remains unclear, however, what triggers changes in methylation, and when 14 are the different tissues most susceptible to epigenetic programming. Despite some 15 contradictions it seems that levels of methylation are consistently low in the brain, and 16 somewhat higher and more variable at least in the blood mononuclear cells and liver. 17 The future -GR post-transcriptional regulation by miRNAs ? 19 MicroRNAs (miRNAs) were discovered in 2001 [START_REF] Lim | The microRNAs of Caenorhabditis elegans[END_REF][START_REF] Lee | An extensive class of small RNAs in Caenorhabditis elegans[END_REF]. In mammalian, miRNAs were 20 predicted to regulate up to 30% of all genes [START_REF] Filipowicz | Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight?[END_REF]. So far they have been shown to be 21 involved in almost every cellular process investigated [START_REF] Bushati | microRNA functions[END_REF][START_REF] Kloosterman | The diverse functions of microRNAs in animal development and disease[END_REF]. It is now recognised that 22 miRNAs account for about 1% of the human genome and that they play a key role in that bind through imperfect base pairing to their target mRNAs, interfering with 3 translational output [START_REF] Bagga | Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation[END_REF]. miRNAs can either be encoded within an intronic locus [71, 72] 4 or they cluster within intergenic regions [START_REF] Lee | MicroRNA maturation: stepwise processing and subcellular localization[END_REF]. miRNA gene transcription gives a primary 5 miRNA transcript (pri-miRNA) that after subsequent processing (reviewed in [START_REF] Kim | MicroRNA biogenesis: coordinated cropping and dicing[END_REF]) is 6 incorporated into the RNA Induced Silencing Complex (RISC). The seed region, a 6-8 7 nucleotide long sequence situated at the 5' end of the miRNA, nucleates the binding of 8 miRNAs to their mRNA targets, requiring several contiguous and perfect by matching 9 base pairs [START_REF] Knight | A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans[END_REF]. With a few exceptions, miRNA-binding sites in metazoan mRNA lie in 10 the 3'UTR of the target gene and are usually present in multiple copies [START_REF] Brennecke | Principles of microRNA-target recognition[END_REF][START_REF] Doench | Specificity of microRNA target selection in translational repression[END_REF][START_REF] Grimson | MicroRNA targeting specificity in mammals: determinants beyond seed pairing[END_REF][START_REF] Lewis | Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[END_REF][START_REF] Nielsen | Determinants of targeting by endogenous and exogenous microRNAs and siRNAs[END_REF]. The 11 exact mechanisms by which miRNAs repress translation remain unclear. However, it is 12 known that miRNAs not only induce mRNA deadenylation and degradation but they also 13 seem to interfere with protein synthesis [START_REF] Behm-Ansmant | mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes[END_REF]. 14

There is now evidence that miRNAs are involved in the regulation of hippocampal GR 15 levels modulating the HPA axis responsiveness [START_REF] De Kloet | Glucocorticoid signaling and stress-related limbic susceptibility pathway: about receptors, transcription machinery and microRNA[END_REF]. The 3' UTR of the GR was 16 predicted to contain numerous seed regions recognized by a variety of miRNAs two of 17 which were miR-18 and miR-124a [START_REF] Uchida | Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNA-mediated down-regulation of the glucocorticoid receptor[END_REF]. Both reduced GR protein levels and the overall 18 GR activities measured by target gene expression levels. miR-18 inhibited GR mRNA 19 translation in cultured neuronal cells and was shown to be up-regulated in the 20 paraventricular nucleus of Fischer 344 rats, a strain hypersensitive to stress. Interestingly, 21 it has been known for some time that Fischer 344 rats have low GR levels in the 22 hippocampus compared to Lewis rats. Together, these results suggest a role of miR-18 in GR down-regulation, increasing the vulnerability to repeated stress [START_REF] Lytle | Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5' UTR as in the 3' UTR[END_REF]. In contrast to 1 miR-18, which was expressed in numerous tissues, miR-124a expression was restricted to 2 certain brain regions and changed during the stress neonatal hyporesponsive period [START_REF] Vreugdenhil | MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: implications for glucocorticoid responsiveness in the brain[END_REF]. 3

Little is known about miRNA targeting 5'UTRs, although target sites for endogenous 4 miRNAs can also be identified in these upstream gene regions. A recent study reported 5 mRNAs to be repressed as efficiently by miRNA binding in the 5' as in the 3' UTR 6 although previously 5' miRNAs were shown to be less effective than those binding in the 7 3'UTRs [START_REF] Farh | The widespread impact of mammalian MicroRNAs on mRNA repression and evolution[END_REF]. Most surprisingly, by binding to the 5'UTR, miRNA family member miR-8 10 enhanced the translation of its target mRNA [START_REF] Orom | MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation[END_REF]. 9

Nothing is known so far about miRNAs targeting the 5' UTR of GR mRNAs. 10

Considering the high variability of the GR 5'UTRs and tissue-dependent first exon usage 11 [START_REF] Turner | Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1[END_REF][START_REF] Turner | Tissue specific glucocorticoid receptor expression, a role for alternative first exon usage?[END_REF], it is attractive to hypothesise that miRNAs may add yet another layer of 12 transcriptional complexity to the GR. Using the online version of PITA (Probability of 13

Interaction by Target Accessibility) [START_REF] Kertesz | The role of site accessibility in microRNA target recognition[END_REF], we predicted a number of highly significant 14 miRNA binding sites within the GR first exon Figure [START_REF] Schaaf | Molecular mechanisms of glucocorticoid action and resistance[END_REF]. To determine if and how these 15 miRNAs are involved in GR posttranscriptional regulation remains a rewarding 16 challenge. 17 The abundance of the alternative exons is modulated by epigenetic methylation of their 1 promoters. Finally, we suggest that the mature mRNA will be susceptible to miRNA 2 effects that are transcript specific. Whilst the amount of the GR is thought to be the main 3 determinant of the GC response, the third layer of complexity, beyond the scope of this 4 review is the interaction of the mature GR with its numerous co-regulators and co-5 repressor proteins, as well as their interactions with the other steroid receptors upon 6 ligation. 7 8 Amongst the many open questions is the effect of the alternative 5' transcripts on 9 translation. It is likely that the 5'UTR sequence will affect the recruitment of the 10 translational machinery, translation rates, as well as mRNA stability. Together these will 11 further regulate the amount of GR generated, the ultimate determinant of GC sensitivity. 12 Determining how these different mechanisms converge to produce an adequate stress 13 response in the healthy and the sick is one of the next major challenges. 14 15 When we published the structure of the GR 5'UTR its complexity was unique. However, 16 over the last few years an unexpected variability in the 5' UTR of genes throughout the 17 complete genome has unfolded. In 2001 there were only 2 human genes known to have 18 >10 first exons [START_REF] Zhang | Multiple variable first exons: a mechanism for cell-and tissue-specific gene regulation[END_REF], in 2006 this had expanded to at least 43 [START_REF] Kimura | Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes[END_REF]. Although there is no 19 more recent data, comprehensive analysis of the ENCODE project data suggests that 20 >20% of all human genes have multiple alternative promoters [START_REF] Cooper | Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome[END_REF]. Data from the 21 Database of Transcription Start sites (DBTSS) even suggests that >50% of all genes have 22 alternative first exons and associated promoters [START_REF] Tsuritani | Distinct class of putative "non-conserved" promoters in humans: comparative studies of alternative promoters of human and mouse genes[END_REF]. The discrepancy between these two estimates is most probably due to the greater variety of tissue and cell types available in 1 the DBTSS, underscoring tissue specificity of these first exons. Thus, genes with 2 alternative first exons, translated or not, seem to be the rule rather than the exception as 3 initially thought for the GR. Epigenetic modifications can lead to methylation of the transcription factor binding site in the GR promoter (full ovals). Therefore binding to the promoter is inhibited and expression of the GR is reduced. Figure from [START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF] with permission.

Figure 3: Alignment of the rat GR promoter 1 7 and the human GR promoter 1F. Solid-lined boxes represent known canonical NGFI-A binding sites either in the rat [START_REF] Weaver | Epigenetic programming by maternal behavior[END_REF][START_REF] Herbeck | Conserved methylation of the glucocorticoid receptor gene exon 1(7) promoter in rats subjected to a maternal methyl-supplemented diet[END_REF][START_REF] Daniels | Maternal separation alters nerve growth factor and corticosterone levels but not the DNA methylation status of the exon 1(7) glucocorticoid receptor promoter region[END_REF] or the human [START_REF] Turner | Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms[END_REF][START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF][START_REF] Oberlander | Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses[END_REF][START_REF] Moser | The glucocorticoid receptor gene exon 1-F promoter is not methylated at the NGFI-A binding site in human hippocampus[END_REF] promoter. The broken-lined box in the human 1F promoter represents a hypothetical non-canonical NGFI-A binding site [START_REF] Mcgowan | Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse[END_REF].

Figure 4: The GR first exon 1C is rich in potential microRNA binding sites. MicroRNA binding was predicted based on the probability of interaction by target accessibility (PITA) [START_REF] Kertesz | The role of site accessibility in microRNA target recognition[END_REF]. Individual binding scores are from the colour scale. Values lower then -11 are normally considered highly relevant.

21 Keywords:

 21 Glucocorticoid receptor; Promoter; Methylation; Epigenetic variability; 22 alternative exon splicing 23 Page 3 of 32 A c c e p t e d M a n u s c r i p t Introduction 1

2 3

 2 In eukaryotic cells, gene expression is controlled by a variety of mechanisms, both at 18 transcriptional and translational levels, including chromatin condensation, transcription 19 initiation, DNA methylation, alternative RNA splicing, mRNA stability and others. The 20 GR is an ubiquitously expressed nuclear hormone receptor, however, levels of both 21 mRNA and protein vary widely between cell and tissue types. Over the last few years we 22 and others have contributed to the significant progress that has been made to unravel the Page 4 of 32 A c c e p t e d M a n u s c r i p t GR Transcription 4 transcriptional mechanisms determining the tissue specific control of GR levels, that will 1 be reviewed here. Structure of the NR3C1 gene 4

  contain multiple promoters that are alternatively used for the 1 production of different protein isoforms, with important physiological consequences. 2

2 3

 2 a link between the two ends of the mRNA transcript, but there is no 1 consensus as to the nature of this link.Transcription factors and transcriptional control within the CpG island 4

22

 22

6 7

 6 A c c e p t e d M a n u s c r i p t GR Transcription 9 It has not yet been shown whether the transcription factors that bind immediately 1 upstream of exons D, E, F, H, and I, activate the expression of these exons. Only site 13, 2 one of 6 in the region immediately upstream of exon 1F (Fig. 1C), has been shown to 3 activate transcription of the downstream exon. Furthermore, the link between the 4 transcription factors previously identified, or predicted, and the transcription of the new 5 CpG island first exons must be established. Transcriptional control upstream of the CpG island: Exon 1A and 1I 8

Page 10 of 32 A

 32 c c e p t e d M a n u s c r i p t GR Transcription 10 explains in part the opposite effects observed in different tissues: in most tissues GC 1

22

 22

18 19

 18 Epigenetic programming of GR promoters 20Epigenetic methylation of the 5'-cytosine of a CpG dinucleotide is associated with gene 21 silencing either by inhibition of transcription factor binding (Fig.2) or by chromatin 22 inactivation [51-53]. For instance, prenatal epigenetic methylation governs genomic Page 12 of 32 A c c e p t e d M a n u s c r i p t GR Transcription 12

  regions of human post-mortem brains[START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF]. However, in suicide 1 victims with a history of child abuse, McGowan et al. found increased methylation 2

18

 18

  such as development timing, cell differentiation and apoptosis 1[START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]. MicroRNAs are single-stranded RNA molecules of about 21 nucleotides in length 2

18 Summary 19 The

 1819 studies presented in this review demonstrate the multiple layers of complexity 20 involved in the maintenance of the homeostasis of the ubiquitously expressed GR. Data 21 from our laboratory and others have shown that the multiple first exons represent a first 22 layer of complexity playing a particular role in tissue-specific transcriptional regulation. Page 17 of 32 A c c e p t e d M a n u s c r i p t

4

 4

Figure 1 :

 1 Figure 1: Structure of the GR gene (NR3C1; OMIM + 138040; NR3C1; 5q31-q32), the potential mRNA transcripts and the binding sites within the CpG island. Panel A The genomic structure of the GR. 5' untranslated distal exons; 5'untranslated CpG island exons; Common exons; 3' alternatively spliced exons. Panel B shows the potential mRNA transcripts encoding the three GR isoforms: GR, GR and GR-P. Panel B shows the location of the known transcription factor binding sites. IRF 1 and IRF2 (position 1); c-Myb, c-Ets ½ and PU1 (position 4); Ying Yand 1 (positions 5,6,7 and 25); Glucocorticoid response elements (GRE, positions 2, 3, 8, 21, and 22); Sp1 binding sites (positions 9, 10, 12, 13, 16, 19, 20, 21, and 24); NGFIA binding site (position 17); Glucocorticoid response factor-1 (GRF1, position 18); Ap-1 (position 15); and Ap-2 (position 23).

Figure 2 :

 2 Figure 2: DNA methylation inhibits transcriptional regulation of genes. A complex of transcription factors (TF), the DNA polymerase (Pol) and co-activators (open circles) normally binds to the GR promoter and thus regulates gene expression.Epigenetic modifications can lead to methylation of the transcription factor binding site in the GR promoter (full ovals). Therefore binding to the promoter is inhibited and expression of the GR is reduced. Figure from[START_REF] Alt | Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed[END_REF] with permission.

A c c e p t e d M a n u s c r i p

A c c e p t e d M a n u s c r i p Figure 1 Figure 3 Page 31 of 32 A c c e p t e d M a n u s c r i p

 1332 Figure 1

Figure 4 Page

 4 Figure 4

Table 1 . Transcription factor binding sites in the hGR proximal promoter region

 1 RG: reporter gene. FP: DNAse protection / DNA footprinting. ChIP: chromation precipitation. D: deletion analysis. EMSA: electrophoretic mobility shift assay c Locationswith repect to the ATG start codon in exon 2.

	Promoter	TF a	N°Sequences	Cell lines / Tissues	Technique b	Location	c	reference
							start	end
	Distal Promoter
		IRF-1		GTAGAGGCGAATCACTTTCACTTCTGCTGGG	CEM-C7	FP, EMSA, RG	-34574 -34544	[16, 45]
		IRF-2		GTAGAGGCGAATCACTTTCACTTCTGCTGGG	CEM-C7, Jurkat	FP, EMSA, RG	-34574 -34544	[16, 45]
		GR-α		TCTGATACCAAATCACTGGACCTTA	CEM-C7	FP, EMSA	-34490 -34466	[18]
		GR-α		GACCGTAAAATGCGCATG	CEM-C7, IM-9	FP, EMSA, ChIP	-34436 -34419	[18, 37, 44]
		GR-β		GAGAAGGAGAAAACTTAGATCTTCTGATACCAA	CEM-C7	FP, EMSA	-34512 -34480	[16]
		c-Myb		ATGTGTCCAACGGAAGCACT	CEM-C7	FP, EMSA, ChIP	-34421 -34402	[18, 44]
	c-Ets 1/2		ATGTGTCCAACGGAAGCACT	CEM-C7	FP, EMSA	-34421 -34402	[18]
		PU.1		ATGTGTCCAACGGAAGCACT	IM-9	EMSA, ChIP	-34421 -34402	[37, 44]
	Proximal promoter
	1D	dYY1	1	CCAAGATGG	NIH 3T3, Hela	FP, D, E	-4807	-4799	[15]
		mYY1	2	CCAAGATGG	NIH 3T3, Hela	FP, D, E	-4635	-4627	[15]
		pYY1	3	CCAAGATGG	NIH 3T3, Hela	FP, D, E	-4591	-4583	[15]
		GRE	4	GGCTTCCGGGACGCGCTTCCCCAATCGTCTTCAAG	Jurkat, IM-9, CEM-C7	ChIP, E	-4574	-4540	[37]
	1J	Sp1	5	GCTGGGGCGGGGGCTT	NIH 3T3, Hela	FP, E	-4250	-4235	[15]
		Sp1	6	TTCGGGGGTGGGG	Jurkat, HepG2, Hela	RG, FP, E	-4011	-3999	[17]
	1E	GRE	7	GTGGAAGAAGAGGTCAGGAGTTTC	Jurkat, IM-9, CEM-C7	ChIP, E	-3962	-3939	[37]
	1B	Sp1	8	CACATTGGGCGGGAGGGG	Jurkat, HepG2, Hela	RG, FP, E	-3774	-3757	[17]
		Sp1	9	TTGAACTTGGCAGGCGGCGCC	Jurkat, HepG2, Hela	RG, FP, E	-3750	-3730	[17]
	1F	GRE	10 GCACCGTTTCCGTGCAACCCCGTAGCCCCTTTCGAAGTGACACACT	Jurkat, IM-9, CEM-C7	ChIP, E	-3438	-3393	[37]
		AP-1	11	TGACACA (consensus TGAC/GTCA)	AtT-20,NIH3T3	EMSA	-3401	-3395	[31][14]
		Sp1	12	TGGGCGGGGGCGGGAA	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-3228	-3213	[28]
		NGFIA 13	GGGCGGGGGCGG	Rat Hippocampi / HEK293	ChIP	-3227	-3216	[35, 36]
		GRF-1 14	GAAGGAGGTAGCGAGAAAAGAAACTGGAGAAACTCGGTGG	MCF7, CV-1	EMSA	-3215	-3176	[33, 34]
		Sp1	15	TCTTAACGCCGCCCCAGAGA	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-3172	-3153	[28]
	1C	Sp1	16	GGAGTTGGGGGCGGGGGGCG	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-3107	-3088	[28]
		Sp1	17	GCGCACCGGGCGGGGCGGCC	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-3080	-3061	[28]
		GRE	18	CTGCAGTTGCCAAGCGTCACCAACAGGTTGCATCGTTCCCC	Jurkat, IM-9, CEM-C7	ChIP, E	-2971	-2931	[37]
		AP-2	19	CCGCGCGGCCCCTCGGGCGGGGA	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-2923	-2901	[28]
		Sp1	20	CGCCGTGGCGCCGCCTCCA	Hela, NIH3T3, CV1, HepG2	RG, FP, EMSA	-2856	-2838	[28]
		iYY1	21	CTCCTCCATTTTG	NIH 3T3, Hela	FP, D, E	-2755	-2743	[28]

a TF: Transcription Factor b

Table 1

 1 Page 27 of 32

Table 2 : Methylation analyses of GR promoter regions

 2 Abbreviations: r. nor, rattus norwegicus; h. sap, homo sapiens; LG-ABN, licking-grooming and arched-back nursing; met sup, methy-supplemented diet;

	Overall

maternal sep, maternal seperation; PR, protein-restricted; HV, healthy volunteer; Dep, depression; Al, Alzheimer's disease; Park, Parkinson's disease; Dem, dementia; MDD, major depressive disorder; MS-PCR, methylation-specific PCR

Table 2

 2 Page 28 of 32


Dirk Zahn
email: dirk.zahn@chemie.uni-erlangen.de

Molecular dynamics simulation of ionic conductors: perspectives and limitations

Keywords:

Concepts of modeling ion conductors by means of molecular dynamics simulations are outlined and exemplified by a small series of applications. Computational studies offer atomic level of detail insights into ionic mobility and thus help to rationalize experimental findings in particular for complex ion conduction mechanisms. Standard techniques are discussed within their thermodynamic context i.e. chemical potentials, conductivity and activation energies.

The limitations of molecular dynamics simulations are defined by statistics and the accuracy in evaluating atomic interactions.

Introduction

The phenomenon of ion conductivity in solids is of tremendous technical importance. To support the experimentalists search for compounds of high conductivity and their characterization, atomistic simulation models are becoming increasingly popular. The profound understanding of transport phenomena in ionic devices requires in-situ investigations at the atomistic level of detail. While hard to achieve from experiment, molecular dynamics (MD) simulations may provide such a tool of investigation.

So far, MD simulations related to ion conductivity were mainly focused on either singlecrystalline or amorphous systems. The accessible insights include the mechanisms of Frenkel defect formation, interstitial and/or vacancy migration and recombination. Inspired by experimental work on nanoscale ionic conductors [1][2][3], recent MD simulations also addressed nanoionics systems [4][5][6]. For such systems, also structural insights may be derived from simulation [4].

While MD simulations offer understanding at atomic level of detail and the calculation of experimentally assessable properties such as conductivity, chemical potentials and activation energies, there is a number of technical complications that still limit the elsewise quite impressive potential of MD simulations. Here, we outline standard and more specific techniques to model ion conductors.

Molecular dynamics simulations

Based on ab initio or empirically derived forces acting between the atoms, MD simulations allow the time propagation of atomic model systems [START_REF] Haile | Molecular dynamics simulation[END_REF]. As the latter are typically large, numerical approaches to solve the Newton's equations of motions are employed. The key idea is to divide a trajectory into many small time intervals. Within such an interval, the forces are assumed to be constant, hence allowing the prediction of atomic positions r at time t + Δt on the basis of positions, velocities and acceleration at time t. The Verlet algorithm reads [8]:

i i i i i i i i i (1)
where the index i runs over all atoms of the model system. To keep the error O(Δt 4 ) negligible, Δt has to be chosen rather small, typically around 1 fs. Longer trajectories are produced from using Eq. 1 iteratively, hence positions, velocities and forces are calculated in time intervals using small time steps of Δt = 0.1 -5 fs, depending on atomic masses and the applied temperature.

To mimic realistic simulation conditions, MD simulations may be coupled to algorithms that scale the simulation cell and the atomic velocities to induce specific values of pressure and temperature, respectively [START_REF] Haile | Molecular dynamics simulation[END_REF].

Modeling of atomic interactions

The atomic interaction forces may be derived from quantum mechanical calculations or from less accurate, but less expensive empirical interaction models (force fields). While quantum calculations are accurate in evaluating the forces acting on a specific configuration, this does not imply that ab initio MD simulations would automatically provide the most reliable results. Indeed, with the currently accessible hardware many properties may be predicted more accurately when using empirical force-fields. This apparent contradiction is due to statistical errors. The large computational costs related to ab initio MD simulations typically lead to small model systems and short simulation times. Both give rise to limited configuration sampling which may have severe effects on the statistical significance of the simulation data.

Depending on the properties to be explored, the modeler should hence perform a reasonable balance between two diametrically aspects: accurate forces and statistical quality. Initial criteria to assess the quality of empirical force fields for studying ionic mobility may be the lattice constants and elastic moduli of single crystalline models and the energy required Frenkel for defect incorporation. These properties may be used for comparing force field based simulations with ab initio data. For the sake of computational efficiency, both simulations are typically related to zero Kelvin, i.e. the model systems are subjected to simple energy minimization before assessing the propensities described above. More sophisticated benchmarks are given by the evaluation of ionic conductivity and the related activation energy from MD simulation and comparison with impedance spectroscopy experiments. This implies MD trajectories computed at temperature and pressure conditions close to the experiment and reflects a further, highly desired if not indispensable benchmark of the force-fields.

Thermodynamics

In a crystalline ion conductor, ionic mobility is associated with the spontaneous formation of a Frenkel defect, hence by displacement of an ion to an interstitial position i and formation of a vacancy ν. i 0

(

The chemical potential of the Frenkel defects are thus given by the sum of vacancy and interstitial chemical potentials, i.e.

i Frenkel (3) To assess chemical potentials from MD simulation data, it is convenient to employ Boltzmann statistics. Differences in free energy (free enthalpy) may be evaluated on the basis of the relative occurrences within a constant temperature (constant temperature, constant pressure) MD simulation run. Thus, by calculating the average density of Frenkel defects and considering the density of the mobile ionic species the difference in chemical potential for reaction (Eq. 2) is given by:

ion F B Frenkel T k MD 0 ln ( 4
)
where the average <ρ F > MD is taken from a sufficiently long MD trajectory to ensure accurate sampling of ρ F .

A simple way to assess the appropriate sampling is given by dividing an MD trajectory in several parts and comparing the corresponding averages separately. This also allows the estimation of error margins for the chemical potential difference as derived from Eq. 4.

In particular for compounds of low ionic conductivity, the required sampling times often turn out to be extremely long compared to the typical ns-μs scope of an MD simulation (using force-fields, ab initio MD often reach a few ps, only). Thus, to improve the statistical quality of sampling <ρ F > MD a common trick is to apply high temperature. In principle the same procedure is applied in the experiments. However, the temperature of the MD simulation runs may be (and often have to be) chosen even larger than experiments would allow. Within the short time scale of an MD simulation, the system may remain in a metastable state and the sampling of solid state ionic mobility may often be performed at temperatures above the actual melting point. Moreover, typical simulation models do not include bond breaking or formation and thus (artificially) prevent decomposition reactions.

Kinetics

To actually calculate ionic conductivity, it is in principle possible to discriminate interstitial mobility and vacancy mobility. While this requires geometric criteria for identifying the corresponding positions, it is much simpler to directly calculate the overall conductivity from the diffusion constant of all ions.

T R F D z c T R F D z c T R F D z c ion ion ion i i i i 2 2 2 (5)
Here c i , z i , D i and c v , z v , D v refer to the concentration, charge and diffusion constant of the interstitials and vacancies, respectively. Depending on the way how the interstitial and vacancy diffusion coefficients are sampled from molecular simulation, it may become necessary to consider a correction factor k corr to account for the interactions between the charge carriers [9]. This particularly holds for simulations in which the migration of a single carrier is explored.

T R F D z c T R F D z c k i i i corr i 2 2 (5a)
On the other hand, on may directly calculate the average diffusion constant D ion of all ions of the mobile ionic species. If this is based on sufficiently large simulation models, the interplay between nearby and remote charge carriers may be observed directly (see also section 6 and figs. 1-2). Moreover, no correction terms are required as the effective diffusion constant already accounts for the charge carrier interactions:

Complex simulation systems

Single-crystalline models typically require the setup of a supercell model to ensure a reasonably large simulation system. When applying periodic boundary conditions, selfinteraction of the vacancy and interstitial defects must be avoided. This can typically be provided by choosing box dimensions of several nm or more. Because of ionic migration, the self-interaction of Frenkel defects cannot be avoided. However, sufficiently large model systems may accommodate several Frenkel defects at the same time and thus allow recombination of interstitials and vacancies originating to different Frenkel defects. Amorphous models may be prepared from a single-crystalline supercell by means of hightemperature MD simulation to induce melting, followed by quenching to reach a glassy state.

A similar strategy was recently demonstrated for polycrystalline models [5,6]. To promote the formation of crystalline domains, here quenching is replaced by somewhat slower cooling combined with the application of high-pressure. Moreover, the preparation of defined nanostructures was recently outlined for CaF 2 / BaF 2 nanolayer systems [4]. In this study, we first prepared sandwiches of single-crystalline CaF 2 and BaF 2 layers and then promoted structural relaxation by repeated heating and annealing circles until convergence was reached. The resulting system exhibited a pattern of dislocations to reduce the stress induced by the mismatching lattice constants of the heterosystem.

Mechanistic investigations

The elaboration of the conductivity mechanisms clearly depends on the model system under investigation. For crystalline compounds, nearest neighborhood analyses offer the possibility to calculate coordination numbers for individual atoms as a function of time. Thus, simple geometric criteria may help to identify interstitials and vacancies. On the other hand, amorphous systems may be characterized by a statistic of ion-ion contacts which may help to discriminate particularly stable constellations from more mobile regions. Fig. 1 illustrates an nm-sized simulation model (with periodic boundaries) mimicking a CaF 2 crystal [10]. The ionic pathways within a time-sketch of 10 ps are illustrated by solid lines.

From this the calcium ions may be seen to undergo vibrations only, whilst the fluoride ions display a complex pattern of multiple Frenkel defect formation/recombination and vacancy transport events. The latter is also shown in Fig. 2, highlighting an individual Frenkel defect formation event, followed by subsequent vacancy migration whilst the interstitial is pinned [10].

While ionic conductivity in fluorite is achieved via a single interstitial type, more complex compounds may provide a variety of different interstitial sites. By the example of Li-Argyrodites we recently demonstrated the application of Boltzmann statistics to assign free energy levels to different interstitial types [11]. The corresponding energy levels were then associated to NMR and impedance spectroscopy. This allowed to discriminate local mobility and real ionic conductivity, i.e. locally confined vibrations and occupation of low energy interstitial sites and ion migration throughout the whole argyrodite unit cell [12].

Conclusions

While ab initio MD is still far from computationally feasible to provide sufficient statistics, the increasing number of high-quality force-fields allows the study ionic conductivity for a steadily increasing variety of compounds. This is documented by a large number of successful applications of molecular simulation techniques. While detailed references to such studies are beyond the scope of the present concept paper, we outlined the MD simulation methodology and connected approaches to explore ionic mobility. MD simulations offer atomic level of detail insights into ionic mobility and may help to rationalize experimental findings in particular for complex ion conduction mechanisms. The model simulations may be confirmed from thermodynamic and kinetic properties, namely chemical potentials, conductivity and activation energies. Comparison with experimental data is needed, as limited statistics and limited accuracy in evaluating atomic interactions may produce severe artifacts [13].

Figure captions

  plots of σ·T, σ i ·T and σ ν ·T yields: interstitial or vacancy migration individually, both the migration barrier and the formation energy must be considered. On the other hand, the activation energy of the average diffusion constant of all ions already contains the formation energy of Frenkel defects according to Eq. 3. (Note that c i and c ν , but not c ion change exponentially with temperature)

Fig. 1 Fig. 2

 12 Fig. 1Visualization of a molecular dynamics simulation mimicking a CaF 2 crystal modeled at 1800 K and ambient pressure. To illustrate ion transport pathways, solid lines mark the ion pathways for a time sketch of 10 ps. While the calcium ions exhibit vibrations only, i.e. remain at their lattice positions, the fluoride ions display a complex pattern of multiple Frenkel defect formation/recombination and vacancy transport events

Acknowledgments

The author wishes to thank Joachim Maier and Paul Heitjans for stimulating discussions.