# Gene transcriptions/Boxes/CREs

CREB (cAMP response element-binding protein) is a cellular transcription factor that binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing the transcription of the genes. CREB was first described in 1987 as a cyclic adenosine monophosphate (cAMP)-responsive transcription factor regulating the somatostatin gene.
Genes whose transcription is regulated by CREB include: c-fos, BDNF, tyrosine hydroxylase, numerous neuropeptides (such as somatostatin, enkephalin, VGF, corticotropin-releasing hormone), and genes involved in the mammalian circadian clock (PER1, PER2).
CREB has a well-documented role in neuronal plasticity and long-term memory formation in the brain and has been shown to be integral in the formation of spatial memory. CREB downregulation is implicated in the pathology of Alzheimer's disease and increasing the expression of CREB is being considered as a possible therapeutic target for Alzheimer's disease.

## Consensus sequence

"Each species' promoter contains a cAMP/response element (CRE)$^1$ consensus sequence upstream of a TATA box. Two other members of this family of proteins, chromogranin B and secretogranin II (also known as chromogranin C), contain similar CRE and TATA homologies in their proximal gene promoters (8, 9)."
"Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP."

## cAMP response element

The cAMP response element (CRE) is the response element for CREB which contains the highly conserved nucleotide sequence, 5'-TGACGTCA-3’. CRE sites are typically found upstream of genes, within the promoter or enhancer regions. There are approximately 750,000 palindromic and half-site CREs in the human genome. However, the majority of these sites remain unbound due to cytosine methylation.

## Samplings of the A1BG promoters

For the Basic programs (starting with SuccessablesCRE.bas) written to compare nucleotide sequences with the sequences on either the template strand (-), or coding strand (+), of the DNA, in the negative direction (-), or the positive direction (+), including extension of nts from 958 to 4445, the programs are, are looking for, and found:
* negative strand in the negative direction (from ZSCAN22 to A1BG) is SuccessablesCRE--.bas, looking for 3'-TGACGTCA-5', 1, 3'-TGACGTCA-5', 4317,
* negative strand in the positive direction (from ZNF497 to A1BG) is SuccessablesCRE-+.bas, looking for 3'-TGACGTCA-5', 0,
* positive strand in the negative direction is SuccessablesCRE+-.bas, looking for 3'-TGACGTCA-5', 0,
* positive strand in the positive direction is SuccessablesCRE++.bas, looking for 3'-TGACGTCA-5', 0,
* complement, negative strand, negative direction is SuccessablesCREc--.bas, looking for 3'-ACTGCAGT-5', 0,
* complement, negative strand, positive direction is SuccessablesCREc-+.bas, looking for 3'-ACTGCAGT-5', 0,
* complement, positive strand, negative direction is SuccessablesCREc+-.bas, looking for 3'-ACTGCAGT-5', 1, 3'-ACTGCAGT-5', 4317,
* complement, positive strand, positive direction is SuccessablesCREc++.bas, looking for 3'-ACTGCAGT-5', 0,
* inverse complement, negative strand, negative direction is SuccessablesCREci--.bas, looking for 3'-TGACGTCA-5', 1, 3'-TGACGTCA-5', 4317,
* inverse complement, negative strand, positive direction is SuccessablesCREci-+.bas, looking for 3'-TGACGTCA-5', 0,
* inverse complement, positive strand, negative direction is SuccessablesCREci+-.bas, looking for 3'-TGACGTCA-5', 0,
* inverse complement, positive strand, positive direction is SuccessablesCREci++.bas, looking for 3'-TGACGTCA-5', 0,
* inverse, negative strand, negative direction, is SuccessablesCREi--.bas, looking for 3'-ACTGCAGT-5', 0,
* inverse, negative strand, positive direction, is SuccessablesCREi-+.bas, looking for 3'-ACTGCAGT-5', 0,
* inverse, positive strand, negative direction, is SuccessablesCREi+-.bas, looking for 3'-ACTGCAGT-5', 1, 3'-ACTGCAGT-5', 4317,
* inverse, positive strand, positive direction, is SuccessablesCREi++.bas, looking for 3'-ACTGCAGT-5', 0.

## Transcribed CRE boxes

GeneID: 627 BDNF brain derived neurotrophic factor. Also known as ANON2; BULN2. "This gene encodes a member of the nerve growth factor family of proteins. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed to generate the mature protein. Binding of this protein to its cognate receptor promotes neuronal survival in the adult brain. Expression of this gene is reduced in Alzheimer's, Parkinson's, and Huntington's disease patients. This gene may play a role in the regulation of the stress response and in the biology of mood disorders."
GeneID: 1113 CHGA chromogranin A. Also known as CGA. "The protein encoded by this gene is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. It is found in secretory vesicles of neurons and endocrine cells. This gene product is a precursor to three biologically active peptides; vasostatin, pancreastatin, and parastatin. These peptides act as autocrine or paracrine negative modulators of the neuroendocrine system. Two other peptides, catestatin and chromofungin, have antimicrobial activity and antifungal activity, respectively. Two transcript variants encoding different isoforms have been found for this gene."
Secretory "stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine."
"A CRE box containing somatostatin promoter (-71 bp to +55 bp)/luciferase reporter plasmid was obtained from Marc Montminy, Salk Institute, La Jolla, CA. Its CRE box (TGACGTCA) was at -41 to -48 bp."
"Since the proximal positive response region [...] contains a CRE homology ((-71 bp)-TGACGTAA-(-64 bp)), we tested the effect of a CRE box point-gap mutation (TGA-GTAA) in a transfected 77-bp chromogranin A promoter/luciferase reporter construct [...]. The nicotinic induction ratio (nicotine stimulation/mock stimulation) fell from 2.58-fold in the wild-type promoter down to 1.64-fold (i.e. a 60% fall, p < 0.05) in the promoter with the CRE box point-gap mutation."
"We then tested whether a CRE box alone could transfer nicotinic responsiveness to a heterologous (TK) promoter (in pTK-LUC). The chromogranin A CRE (TGACGTAA) insert conferred 3.97-fold nicotinic induction [...], while a consensus CRE (TGACGTCA) was induced 4.94-fold by nicotine; each of these nicotinic responses was significantly (p < 0.05) greater than that of the original TK promoter vector [...]. The nicotinic induction was blunted (down to 1.31-fold) by point-gap mutation of the CRE box (TGA-GTAA). Even a CRE box (TGACGTCA) in the transfected promoter of a gene (somatostatin) not ordinarily expressed in chromaffin cells responded 2.03-fold (p < 0.05) to nicotine (from 134 ± 10.1 to 272 ± 19.6 luciferase light units/mg protein; n = 4 transfected wells/condition)."
"In electrophoretic gel mobility shift assays, we tested the effect of PC12 nicotine exposure (at a dose (10$^{-3}$ M) and time (16 h) which stimulated the transfected promoter [...] on nuclear protein binding to four promoter regions: three that responded positively to nicotine during transfection (-432 to -312, -147 to -77, and -71 to -64 bp (the CRE box)), and one that seemed to respond negatively (-181 to -147 bp). None of these regions showed any significant change in band retardation pattern after nicotine [...]."
"Because CREB-bound CRE boxes are only transcriptionally transactivated when CREB itself has been activated by phosphorylation at serine 133 (33), we undertook antibody supershift assays [...], using not only an antibody directed against CREB (epitope: the CREB kinase-inducible domain), but also an antibody which specifically recognized CREB only in its activated, phosphorylated form (pS133-CREB). Cell exposure to nicotine did not alter the anti-CREB supershift by PC12 nuclear proteins, but did result in a qualitatively new anti-pS133-CREB supershift band. Competition experiments with a 100-fold molar excess of unlabeled CRE [...] indicated that both the anti-CREB and the anti-pS133-CREB supershifts represented CRE-binding complexes."
"The CRE box is at promoter position (-71 bp)TGACGTAA(-64 bp)."
"Each species' promoter contains a cAMP/response element (CRE)$^1$ consensus sequence upstream of a TATA box. Two other members of this family of proteins, chromogranin B and secretogranin II (also known as chromogranin C), contain similar CRE and TATA homologies in their proximal gene promoters (8, 9)."
"1. Abbreviations used: CMV, cytomegalovirus; CRE, cAMP responsive element; CREB, CRE-binding protein; CREM, CRE modulator protein; DBH, dopamine ,/-hydroxylase; LUC, luciferase; TK, thymidine kinase."
"Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP."
GeneID: 1114 CHGB chromogranin B. Also known as SCG1. "This gene encodes a tyrosine-sulfated secretory protein abundant in peptidergic endocrine cells and neurons. This protein may serve as a precursor for regulatory peptides."
The "isolated mouse chromogranin B promoter [specifically] the proximal chromogranin B promoter (from −216 to −91 bp); [...] contains an E box (at [−206 bp]CACCTG[−201 bp]), four G/C-rich regions (at[− 196 bp]CCCCGC[−191 bp], [−134 bp]CCGCCCGC[−127 bp],[− 125 bp]GGCGCCGCC[−117 bp], and [−115 bp]CGGGGC[−110 bp]), and a cAMP response element (CRE; at [−102 bp]TGACGTCA[−95 bp]). A 60-bp core promoter region, defined by an internal deletion from −134 to −74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from −134 to −74 bp). [...] CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene."
"Sequence analysis of 2908 bp of the 5′-flanking region of the mouse chromogranin B gene revealed several consensus matches for cis-acting transcriptional control elements [...] reported as base pairs upstream of the cap site (+1): 1) the TATA box at [−33 bp]TTCATAA[−27 bp]; 2) a cAMP response element (CRE) (24) at [−102 bp]TGACGTCA[−95 bp]; 3) eight G/C-rich elements of 6 consecutive bp or more (potential binding sites for such factors as Sp1, Ap2, or Egr1) at −8/−2 bp, −45/−39 bp, −62/−53 bp, −83/−64 bp, −115/−110 bp, −125/−117 bp, −134/−127 bp, and− 196/−191 bp [one of these G/C-rich sites contains a perfect (9/9 bp) consensus match for an Egr1 recognition site: −79/−71 bp (25); another G/C-rich site overlaps a perfect (10/10 bp) consensus match (GGGRNNYYCC; IUPAC nomenclature) for a nuclear factor-κB site: [−83 bp]GGGGCGCCCC[−74 bp] (25); several of these G/C-rich sites overlap partial matches for Ap2 motifs (e.g. GSSWGSCC; IUPAC nomenclature) (25)]; and 4) an E-box (CANNTG; IUPAC nomenclature) at [−206 bp]CACCTG[−201 bp] (25)."
"Mouse chromogranin B promoter sequence. Nucleotides are numbered from the transcription initiation site (+1) of the gene. Note the positions of the TATA box at [−33 bp]TTCATAA[−27 bp], the CRE at [−102 bp]TGACGTCA[−95 bp], and eight G/C-rich elements of 6 or more consecutive bp (potential binding sites for such factors as Sp1, Ap2, or Egr1) at [−8 bp]CCGCGCC[−2 bp], [−45 bp]CCCCGCC[−39 bp], [−62 bp]CGCCCCCGGG[−53 bp],[−83 bp]GGGGCGCCCCCGCCCGCCGC[−64 bp], [−115bp]CGGGGC[−110 bp], [−125 bp]GGCGCCGCC[−117 bp], [−134]CCGCCCGC[−127 bp], and [−196 bp]CCCCGC[−191 bp]. One of these G/C-rich sites is a perfect (9/9 bp) consensus match for an Egr1 recognition site: [−79 bp]CGCCCCCGC[−71 bp]. One of these G/C-rich sites overlaps a perfect (10/10 bp) consensus match (GGGRNNYYCC; IUPAC nomenclature) for a nuclear factor-κB site: [−83 bp]GGGGCGCCCC[−74 bp]. Several of these G/C-rich sites overlap partial matches for Ap2 motifs (e.g. GSSWGSCC; IUPAC nomenclature). There is an E box (CANNTG; IUPAC nomenclature) at [−206 bp]CACCTG[−201 bp]."
GeneID: 1392 CRH corticotropin releasing hormone. Also known as CRF; CRH1. "This gene encodes a member of the corticotropin-releasing factor family. The encoded preproprotein is proteolytically processed to generate the mature neuropeptide hormone. In response to stress, this hormone is secreted by the paraventricular nucleus (PVN) of the hypothalamus, binds to corticotropin releasing hormone receptors and stimulates the release of adrenocorticotropic hormone from the pituitary gland. Marked reduction in this protein has been observed in association with Alzheimer's disease. Autosomal recessive hypothalamic corticotropin deficiency has multiple and potentially fatal metabolic consequences including hypoglycemia and hepatitis. In addition to production in the hypothalamus, this protein is also synthesized in peripheral tissues, such as T lymphocytes, and is highly expressed in the placenta. In the placenta it is a marker that determines the length of gestation and the timing of parturition and delivery. A rapid increase in circulating levels of the hormone occurs at the onset of parturition, suggesting that, in addition to its metabolic functions, this protein may act as a trigger for parturition."
GeneID: 1843 DUSP1 dual specificity phosphatase 1. Also known as HVH1; MKP1; CL100; MKP-1; PTPN10. "The protein encoded by this gene is a phosphatase with dual specificity for tyrosine and threonine. The encoded protein can dephosphorylate MAP kinase MAPK1/ERK2, which results in its involvement in several cellular processes. This protein appears to play an important role in the human cellular response to environmental stress as well as in the negative regulation of cellular proliferation. Finally, the encoded protein can make some solid tumors resistant to both chemotherapy and radiotherapy, making it a target for cancer therapy."
Although "macrophage proliferation and activation induce MKP-1 with different kinetics, gene expression is mediated by the proximal promoter sequences localized between -380 and -180bp. Mutagenesis experiments of the proximal element determined that CRE/AP-1 is required for LPS- or M-CSF-induced activation of the MKP-1 gene. Moreover, the results from gel shift analysis and chromatin immunoprecipitation indicated that c-Jun and CREB bind to the CRE/AP-1 box."
The "same region, which contains a CREB/AP-1 box, is required for M-CSF- or LPS-dependent stimulation, although this stimulation is induced at different times after stimulation. [...] CREB and c-Jun are responsible for MKP-1 induction. The induction of c-Jun is correlated with the kinetics of MKP-1 induction by M-CSF or LPS."
"MKP-1 expression induced by M-CSF or LPS is regulated by a CREB/AP-1 box."
The "CRE/AP-1 box (TGACGTCT), which has been reported to be involved in the regulation of several genes [43], was critical."
"When we mutated either the CRE or the AP-1 box, M-CSF- or LPS- dependent inducibility was lost [...]. Surprisingly, although the kinetics of inductions differed, the same region controlled M-CSF- and LPS-stimulated MKP-1 expression."
"The DNA fragment spanned the promoter region from -193 to -169 bp containing the CRE/AP-1 box."
"AP-1 is not a single protein but a series of dimeric basic region-leucine zipper proteins that belong to the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1 and Fra2), and ATF (ATF2, LRF1/ATF3, B-ATF, JDP1, JDP2) subfamilies, which recognize either 12-otetradecanoylphorbol-13-acetate response elements (5'-TGAG/CTCA-3') or cAMP response elements (CRE, 5'-TGACGTCA-3') [44]."
GeneID: 2353 FOS Fos proto-oncogene, AP-1 transcription factor subunit. Also known as p55; AP-1; C-FOS. "The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death."
GeneID: 5179 PENK proenkephalin. Also known as PE; PENK-A. "This gene encodes a preproprotein that is proteolytically processed to generate multiple protein products. These products include the pentapeptide opioids Met-enkephalin and Leu-enkephalin, which are stored in synaptic vesicles, then released into the synapse where they bind to mu- and delta-opioid receptors to modulate the perception of pain. Other non-opioid cleavage products may function in distinct biological activities."
Two forms of enkephalin have been found, one containing leucine, and the other containing methionine, both are products of the proenkephalin gene.
The met-enkephalin peptide sequence is coded for by the enkephalin gene; the leu-enkephalin peptide sequence is coded for by both the enkephalin gene and the dynorphin gene.
During a stress response, several Met-enkephalin analogs had increased activity in the hippocampus, while Leu-enkephalin analogs as well as somatostatins were downregulated during stress; hence, stressors impact neuropeptides, and their action is localized to a specific brain region.
GeneID: 5187 PER1 period circadian regulator 1. Also known as PER; hPER; RIGUI. "This gene is a member of the Period family of genes and is expressed in a circadian pattern in the suprachiasmatic nucleus, the primary circadian pacemaker in the mammalian brain. Genes in this family encode components of the circadian rhythms of locomotor activity, metabolism, and behavior. This gene is upregulated by CLOCK/ARNTL heterodimers but then represses this upregulation in a feedback loop using PER/CRY heterodimers to interact with CLOCK/ARNTL. Polymorphisms in this gene may increase the risk of getting certain cancers. Alternative splicing has been observed in this gene; however, these variants have not been fully described."
GeneID: 6750 SST somatostatin. Also known as SMST. "The hormone somatostatin has active 14 aa and 28 aa forms that are produced by alternate cleavage of the single preproprotein encoded by this gene. Somatostatin is expressed throughout the body and inhibits the release of numerous secondary hormones by binding to high-affinity G-protein-coupled somatostatin receptors. This hormone is an important regulator of the endocrine system through its interactions with pituitary growth hormone, thyroid stimulating hormone, and most hormones of the gastrointestinal tract. Somatostatin also affects rates of neurotransmission in the central nervous system and proliferation of both normal and tumorigenic cells."
GeneID: 7054 TH tyrosine hydroxylase. Also known as TYH; DYT14; DYT5b. "The protein encoded by this gene is involved in the conversion of tyrosine to dopamine. It is the rate-limiting enzyme in the synthesis of catecholamines, hence plays a key role in the physiology of adrenergic neurons. Mutations in this gene have been associated with autosomal recessive Segawa syndrome. Alternatively spliced transcript variants encoding different isoforms have been noted for this gene."
GeneID: 7425 VGF VGF nerve growth factor inducible. Also known as SCG7; SgVII. "This gene is specifically expressed in a subpopulation of neuroendocrine cells, and is upregulated by nerve growth factor. The structural organization of this gene is similar to that of the rat gene, and both the translated and the untranslated regions show a high degree of sequence similarity to the rat gene. The encoded secretory protein also shares similarities with the secretogranin/chromogranin family, however, its exact function is not known."
GeneID: 8864 PER2 period circadian regulator 2. Also known as FASPS; FASPS1. "This gene is a member of the Period family of genes and is expressed in a circadian pattern in the suprachiasmatic nucleus, the primary circadian pacemaker in the mammalian brain. Genes in this family encode components of the circadian rhythms of locomotor activity, metabolism, and behavior. This gene is upregulated by CLOCK/ARNTL heterodimers but then represses this upregulation in a feedback loop using PER/CRY heterodimers to interact with CLOCK/ARNTL. Polymorphisms in this gene may increase the risk of getting certain cancers and have been linked to sleep disorders."