Circadian Regulation of cAMP Response Element-mediated Gene Expression in the Suprachiasmatic Nuclei

Abstract

A program of stringently-regulated gene expression is thought to be a fundamental component of the circadian clock. Although recent work has implicated a role for E-box-dependent transcription in circadian rhythmicity, the contribution of other enhancer elements has yet to be assessed. Here, we report that cells of the suprachiasmatic nuclei (SCN) exhibit a prominent circadian oscillation in cAMP response element (CRE)-mediated gene expression. Maximal reporter gene expression occurred from late-subjective night to mid-subjective day. Cycling of CRE-dependent transcription was not observed in other brain regions, including the supraoptic nucleus and piriform cortex. Levels of the phospho-active form of the transcription factor CREB (P-CREB) varied as a function of circadian time. Peak P-CREB levels occurred during the mid- to late-subjective night. Furthermore, photic stimulation during the subjective night, but not during the subjective day, triggered a marked increase in CRE-mediated gene expression in the SCN. Reporter gene experiments showed that activation of the p44/42 mitogen-activated protein kinase signaling cascade is required for Ca2+-dependent stimulation of CRE-mediated transcription in the SCN. These findings reveal the CREB/CRE transcriptional pathway to be circadian-regulated within the SCN, and raise the possibility that this pathway provides signaling information essential for normal clock function. A program of stringently-regulated gene expression is thought to be a fundamental component of the circadian clock. Although recent work has implicated a role for E-box-dependent transcription in circadian rhythmicity, the contribution of other enhancer elements has yet to be assessed. Here, we report that cells of the suprachiasmatic nuclei (SCN) exhibit a prominent circadian oscillation in cAMP response element (CRE)-mediated gene expression. Maximal reporter gene expression occurred from late-subjective night to mid-subjective day. Cycling of CRE-dependent transcription was not observed in other brain regions, including the supraoptic nucleus and piriform cortex. Levels of the phospho-active form of the transcription factor CREB (P-CREB) varied as a function of circadian time. Peak P-CREB levels occurred during the mid- to late-subjective night. Furthermore, photic stimulation during the subjective night, but not during the subjective day, triggered a marked increase in CRE-mediated gene expression in the SCN. Reporter gene experiments showed that activation of the p44/42 mitogen-activated protein kinase signaling cascade is required for Ca2+-dependent stimulation of CRE-mediated transcription in the SCN. These findings reveal the CREB/CRE transcriptional pathway to be circadian-regulated within the SCN, and raise the possibility that this pathway provides signaling information essential for normal clock function. In mammals, the suprachiasmatic nuclei (SCN) 1The abbreviations used are: SCN, suprachiasmatic nuclei; CRE, cAMP response element; CREB, CRE-binding protein; P-CREB, phospho-active CREB; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; L, light; D, dark; CT, circadian time; PBS, phosphate-buffered saline; PBST, PBS with Triton X-100; RHT, retinohypothalamic tract; ERK, extracellular signal-regulated kinase; D/N, dominant negative; DM, dissociation medium; MEK, MAPK kinase. 1The abbreviations used are: SCN, suprachiasmatic nuclei; CRE, cAMP response element; CREB, CRE-binding protein; P-CREB, phospho-active CREB; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; L, light; D, dark; CT, circadian time; PBS, phosphate-buffered saline; PBST, PBS with Triton X-100; RHT, retinohypothalamic tract; ERK, extracellular signal-regulated kinase; D/N, dominant negative; DM, dissociation medium; MEK, MAPK kinase. of the hypothalamus contain a circadian oscillator that functions as the major biological clock (1Hastings M.H. Best the to and to the is a of and circadian of the clock is light; clock is to the photic from the to the the retinohypothalamic a during the of the the circadian is from the of from the the of the clock is thought to in gene expression. In of that gene is triggered work has information and transcriptional essential for circadian and to form a transcription factor that the enhancer in gene expression In transcription a of of circadian M.H. that this transcriptional is essential for normal clock function. is that the enhancer element the expression of other within the SCN. the transcriptional activation of circadian-regulated of of enhancer elements the of transcription in circadian gene within the SCN. of the of transcriptional circadian transcriptional that to the to signaling information from a of as as that for is the CREB/CRE transcriptional pathway has to be including protein kinase A and mitogen-activated protein kinase CREB/CRE transcriptional pathway has the to the activation of signaling and the of in gene transcription Furthermore, CRE-mediated transcription be a of including cAMP as a of CREB with transcription These to this transcriptional pathway a role in circadian this we used a reporter to CRE-mediated transcription in that the exhibit a prominent circadian oscillation in CRE-mediated gene expression in Furthermore, photic stimulation during the subjective night triggered CRE-dependent during the subjective was not that the pathway is essential for Ca2+-dependent stimulation of CRE-mediated transcription in the the CREB/CRE transcriptional pathway to of CRE-mediated is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. In mammals, the suprachiasmatic nuclei (SCN) 1The abbreviations used are: SCN, suprachiasmatic nuclei; CRE, cAMP response element; CREB, CRE-binding protein; P-CREB, phospho-active CREB; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; L, light; D, dark; CT, circadian time; PBS, phosphate-buffered saline; PBST, PBS with Triton X-100; RHT, retinohypothalamic tract; ERK, extracellular signal-regulated kinase; D/N, dominant negative; DM, dissociation medium; MEK, MAPK kinase. 1The abbreviations used are: SCN, suprachiasmatic nuclei; CRE, cAMP response element; CREB, CRE-binding protein; P-CREB, phospho-active CREB; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; L, light; D, dark; CT, circadian time; PBS, phosphate-buffered saline; PBST, PBS with Triton X-100; RHT, retinohypothalamic tract; ERK, extracellular signal-regulated kinase; D/N, dominant negative; DM, dissociation medium; MEK, MAPK kinase. of the hypothalamus contain a circadian oscillator that functions as the major biological clock (1Hastings M.H. Best the to and to the is a of and circadian of the clock is light; clock is to the photic from the to the the retinohypothalamic a during the of the the circadian is from the of from the the of the clock is thought to in gene expression. In of that gene is triggered work has information and transcriptional essential for circadian and to form a transcription factor that the enhancer in gene expression In transcription a of of circadian M.H. that this transcriptional is essential for normal clock function. is that the enhancer element the expression of other within the SCN. the transcriptional activation of circadian-regulated of of enhancer elements the of transcription in circadian gene within the SCN. of the of transcriptional circadian transcriptional that to the to signaling information from a of as as that for is the CREB/CRE transcriptional pathway has to be including protein kinase A and mitogen-activated protein kinase CREB/CRE transcriptional pathway has the to the activation of signaling and the of in gene transcription Furthermore, CRE-mediated transcription be a of including cAMP as a of CREB with transcription These to this transcriptional pathway a role in circadian this we used a reporter to CRE-mediated transcription in that the exhibit a prominent circadian oscillation in CRE-mediated gene expression in Furthermore, photic stimulation during the subjective night triggered CRE-dependent during the subjective was not that the pathway is essential for Ca2+-dependent stimulation of CRE-mediated transcription in the the CREB/CRE transcriptional pathway to of CRE-mediated is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. that the exhibit a prominent circadian oscillation in CRE-mediated gene expression in Furthermore, photic stimulation during the subjective night triggered CRE-dependent during the subjective was not that the pathway is essential for Ca2+-dependent stimulation of CRE-mediated transcription in the the CREB/CRE transcriptional pathway to of CRE-mediated is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. of CRE-mediated is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. of CRE-mediated is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of is a of that the of from protein the enhancer elements transcriptional activation not that the a role in the of to gene expression in the clock. In of we that CRE-mediated gene expression. the that a a of CRE-mediated gene expression with a of to of CRE-mediated gene and the of thought to be in the clock that the for and contain is to that the a role in the of to gene that gene expression in a with work that CREB is to the subjective night These that of the pathway during the be a element that the of role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent A role for in photic of the clock has the that activation the a transcriptional component to we the signaling that to gene expression in the SCN. In of we that in CRE-dependent In the of and cAMP in a activation of CRE-dependent a of cAMP of of and cAMP be for activation of the CREB/CRE transcriptional pathway in the SCN. we observed that stimulation of cAMP signaling was to increase CRE-mediated Ca2+-dependent CRE-mediated transcription in the SCN. and CRE-mediated gene expression is a of a of kinase work that stimulation of CRE-mediated transcription was activation of the MAPK signaling with a form of with the gene expression. of to activation of the MAPK cascade has to be of Ca2+-dependent activation is triggered a of signaling including and the factor MAPK pathway to the nucleus the of has to triggered CREB activation of and of CREB A for MAPK was the that CREB was the In CREB was that the signaling pathway in activation of the MAPK cAMP has to the MAPK cascade in and the SCN, has to CREB a the of Ca2+-dependent activation was to in a pathway MAPK Although other transcription be is to that and a clock and the activation of a transcriptional pathway in CRE-mediated gene expression. the a stimulation to CRE-dependent transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. transcription to be to circadian a form of the transcription factor circadian M.H. that CRE-mediated gene expression is in a circadian Levels of reporter protein to during the subjective night and during mid-subjective day. the transcription and reporter that of CRE-mediated gene expression is to the subjective night, and the subjective that the of CRE-mediated gene with the of CRE-mediated gene that a signaling is the stimulation of CRE-mediated gene expression was the in reporter expression from activation of signaling signaling that and the clock. we that MAPK activation in the SCN, and that MAPK is in a circadian in the work in has a circadian oscillation in CRE-mediated gene expression and CRE-dependent transcription and that the transcriptional pathway is a component of the circadian clock work in reveal a the transcriptional pathway and in the of the transcription factor CREB in the P-CREB the reporter gene for transcriptional activation and protein expression. circadian P-CREB not from CREB levels of CREB in the subjective night that circadian in P-CREB from circadian in the activation of CREB is CRE-mediated gene expression is that extracellular signaling CRE-mediated gene to circadian in the of extracellular of CRE-mediated gene expression. In of this circadian in the of observed within the of the and in in CRE-dependent transcription be a of transcriptional program of possibility is that the of CREB within the the circadian In of this not circadian circadian expression of a of within the from circadian CRE-dependent the for that circadian the protein in the SCN, including during contain the to CRE-dependent and to to the the circadian expression of be that CRE-mediated the we that the transcriptional pathway role in the of transcriptional essential for clock and the of to the clock. for the for the for the and and for during and was in the for of