- Email: [email protected]
Expression of the transcriptional coactivators CBP and p300 in the hamster suprachiasmatic nucleus: possible molecular components of the mammalian circadian clock
Molecular Brain Research 111 (2003) 1–7 www.elsevier.com / locate / molbrainres
Research report
Expression of the transcriptional coactivators CBP and p300 in the hamster suprachiasmatic nucleus: possible molecular components of the mammalian circadian clock Paul Fiore, Robert L. Gannon* Department of Biology, Dowling College, Oakdale, NY 11769, USA Accepted 3 December 2002
Abstract Immediate early genes are expressed in the mammalian suprachiasmatic nucleus in response to photic information arriving from the retina at restricted times of the day, therefore their expression is regulated by the circadian biological clock. These light-induced genes are also activated by the phosphorylated form of CREB (pCREB) that binds to a cAMP response element upstream of the genes. The nuclear proteins CBP and p300 are known to be coactivators with pCREB in certain cell types, but their identification within the rodent SCN has not been reported. Therefore, in this study we examined the distribution of both CBP and p300 in the hamster suprachiasmatic nucleus. CBP and p300 immunoreactivity is detected in cells throughout the suprachiasmatic nucleus, and the pattern of staining within cells is indicative of a nuclear location for these proteins. The number of cells immunoreactive for both CBP and p300 significantly decreases at mid-night circadian times with respect to mid-day circadian times, although the reduction is less than 20%. Neither CBP nor p300 expression is affected by a circadian phase-resetting light pulse given late in the night. The ability of CBP and p300 to interact with pCREB as well as with the clock gene BMAL1 is discussed, and we propose that CBP and p300 may interact with, and link, both clock genes and clock-controlled genes in the generation of circadian rhythms in mammals. We further suggest that there will be a general importance for the role of transcriptional coactivators such as CBP and p300 in many of the molecular pathways related to the mammalian circadian clock. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Transcription factors; Biological rhythms; CREB; Hypothalamus
1. Introduction There has been considerable progress in the last decade in determining the molecular mechanisms that generate and regulate circadian rhythms in organisms. Arabidopsis, Neurospora, Drosophila and the mouse are all model systems for determining what genes comprise the ‘biological clock’ [2,9,15,26]. In mammals, circadian pacemakers may be located in diverse organ systems in the body [1,4], but the primary pacemaker for circadian activity rhythms is located in the suprachiasmatic nuclei *Corresponding author. Tel.: 11-631-244-3339; fax: 11-631-2441033. E-mail address: [email protected] (R.L. Gannon).
(SCN) of the hypothalamus [19]. Complex cycles of transcription and translation are believed to form a 24-h rhythm of gene expression in certain cells of the SCN that is then coupled by an unknown output signal to effector pathways that regulate activity. There are other molecular pathways in addition to the clock itself that are activated by photic input to the SCN, and these most likely entrain the time of the clock to the light:dark cycle of the environment in which the mammal resides [12]. Light pulses administered to a hamster kept in constant darkness during certain times of the subjective night were found to express protein encoded by the oncogene c-fos in the retino-recipient region of the SCN [20]. This was the first of a number of similar genes that were reported to be activated in a time-sensitive, or gated, manner. These
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0169-328X(02)00663-0
2
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
genes may also demonstrate a circadian rhythm in their expression in the absence of photic timing cues, and if so, they can thus be classified as clock-controlled genes [8,12]. Upstream of these genes is a region known as the cAMP response element (CRE) that may be involved in regulating the transcription of the clock-controlled genes. The CRE is in turn bound by the trans-acting factor cAMPresponse element-binding protein (CREB). CREB itself is activated by phosphorylation of serine residues in a region of the CREB activation domain [22]. CREB is also quickly phosphorylated in the hamster SCN in response to light pulses administered at the same circadian times (CT) used to induce immediate-early gene expression or phase shifts in activity rhythms [6]. These results indicate that phosphorylated CREB (pCREB) may be a critical component in the molecular pathway that mediates photic signaling in the hamster SCN. The activity of pCREB may be due to an interaction of this factor with coactivator proteins that is not possible until CREB is phosphorylated. Two coactivators of pCREB have been described in other systems. CREBbinding protein (CBP) and its homologue p300 have both been reported to bind to the phosphorylation box of pCREB [13,14]. CBP/ p300 binding to pCREB has been proposed to link pCREB bound to the CRE with the transcription factor TFIIB [13,14]. Therefore, our hypothesis is that a similar mechanism may exist in the hamster SCN whereby CBP and / or p300 link pCREB bound to the CRE with transcriptional factors regulating the transcription of clock controlled genes. As a first step in investigating our hypothesis, we have identified the presence of both CBP and p300 within the hamster SCN using immunohistochemical techniques. In addition, we evaluated the expression profile of these two proteins in the hamster SCN in response to different lighting conditions.
2. Materials and methods Adult male Syrian hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories (Kingston, NY) and were approximately 150 g at the time of use in this study. Hamsters were individually housed under either 14:10 h of light:dark (LD), or under conditions of constant darkness (DD). Each hamster had an exercise wheel in their cage along with access to food and water ad libitum. Room lighting was approximately 250 lux during the light period. Hamsters were maintained under either LD or DD conditions for 2 weeks prior to use in these experiments. CBP and p300 were not concurrently tested within the same hamster brain. For hamsters maintained under DD, activity level and thus CT times were determined using an activity monitoring system as described elsewhere [24]. To determine if light would affect expression of CBP or p300, hamsters were removed from LD during the dark phase
under dim red light (,1 lux) and exposed to 10 min of room light (250 lux) approximately 6 h after activity onset (CT 18). Hamsters were returned to darkness after the light pulse, and then terminally anesthetized with halothane at CT 19 under dim red light. To examine the expression profile of CBP and p300 at different circadian times without exposure to light, hamsters were removed from DD at mid-day and mid-night CT times and terminally anesthetized with halothane under dim red light (,1 lux). Terminally anesthetized hamsters were rapidly perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer for 5 min. Brains were removed and postfixed overnight in 4% paraformaldehyde before being cryopreserved in 30% sucrose. Hamster brains were cut into 40-mm sections using a cryostat and transferred to 24-well tissue plates for freefloating immunohistochemistry. Sections were incubated at room temperature in 1% hydrogen peroxide for 24 h. Sections were then washed with 0.1 M phosphate buffer, incubated with blocking serum for 1 h, washed again with 0.1 M phosphate buffer, and then incubated overnight with primary antibody (1:1000) at room temperature. The two primary antibodies used in this study were rabbit antihuman p300 (N-15) and rabbit anti-human CBP (C-20), obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody was removed and sections washed again with phosphate buffer before the addition of secondary antibody and ABC reagents according to kit instructions (Vectastain Elite ABC anti-rabbit peroxidase kit, Vector Laboratories, Burlingame, CA). Peroxidase staining was detected using the Vector SG Substrate kit. Finally, sections were washed with isotonic saline, mounted on Superfrost Plus slides, dehydrated and coverslipped. For controls, primary antibody was pre-absorbed with control peptides for 2 h at room temperature at a concentration of 20 mg / ml. Control peptides for p300 and CBP were purchased from Santa Cruz Biotechnology. Images of the SCN were digitally captured using a Pixera Professional CCD Camera (Pixera, Los Gatos, CA). The images were printed using a Hewlett-Packard PhotoSmart printer, the SCN region was outlined, and cell counts were then made using the printed images of the SCN. Two students using a blinded protocol performed the counting, and count totals for each SCN section were averaged.
3. Results
3.1. Immunoreactive patterns and controls Immunoreactivity for both CBP and p300 was widespread throughout the hamster brain, including dense labeling of both proteins in the SCN. Both CBP and p300 were identified throughout the entire SCN region, in both the rostral–caudal and dorsal–ventral axis (not shown).
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Immunoreactivity was confined to small circular regions within cells, indicative of a nuclear localization for both proteins. Preabsorption of CBP and p300 antisera with their respective control peptides completely blocked staining, demonstrating the specificity of these antisera for hamster proteins (Fig. 1).
3.2. Day versus night expression of CBP and p300 The number of cells in the SCN that were labeled by CBP and p300 at both mid-day and mid-night circadian times was determined using hamsters that were not exposed to light at either time. Mid-day circadian times for the CBP experiment ranged from CT 7–8, and mid-night time was at CT 16. Mid-day circadian times for p300 hamsters were CT 6–10, and mid-night circadian times were CT 15–19. There was an average of 1505658 (mean6S.E.M.) cells immunoreactive for CBP during midday times, and this number fell significantly to 1251632 cells during mid-night times (F(1,15)513.81, P,0.002, ANOVA; Figs. 2 and 4). Although there was on average fewer labeled cells for p300 in the SCN in this protocol, there was a similar difference in expression during the mid-day and mid-night times. The number of cells immunoreactive for p300 in the SCN during mid-day CT
3
times was 1156654, and this number fell significantly to 1005625 cells during the mid-night times (F(1,13)56.80, P,0.02, ANOVA; Figs. 2 and 4).
3.3. Effect of light on expression of CBP and p300 Hamsters were exposed to a 10-min light pulse at CT 18, 6 h into their dark phase, and then returned to darkness for 50 min prior to termination in order to allow time for any affect of light to alter expression levels of CBP or p300. The number of cells in the SCN immunoreactive to CBP or p300 was not significantly different than the levels observed during mid-night circadian times in the above study (Section 3.2). Hamsters not exposed to light and perfused at CT 19 were used as controls in this experiment. There was an average of 1215638 cells immunoreactive to CBP in the SCN of controls, and this level was not significantly altered by the exposure to light at CT 18 (1199659 cells; Figs. 3 and 5; ANOVA). Similarly, there was an average of 1067636 cells immunoreactive for p300 in the control SCN and 1036626 cells immunoreactive to p300 following light exposure at CT 18 (no significant difference; ANOVA). Therefore, a 10-min light pulse at CT 18 had no effect on the number of cells in the hamster
Fig. 1. Specificity of CBP and p300 antisera to hamster brain proteins. Dense cellular immunoreactivity to CBP (A) and p300 (C) in the rostral hippocampus is completely eliminated by preabsorption of the antisera with their respective control peptides (B,D). Hippocampus was used as the example since it demonstrated the most dense staining in the hamster brain. DG, dentate gyrus; CA3, CA3 cell layer.
4
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Fig. 2. SCN immunoreactivity to CBP/ p300 at mid-day and mid-night times. Left column, mid-day; right-column, mid-night sections. Cellular immunoreactivity to CBP (A,B) and p300 (C,D) The SCN region is outlined in (A). All sections are at the median region of the rostral–caudal SCN axis.
SCN immunoreactive for either CBP or p300 1 h after the beginning of the exposure to light.
4. Discussion The nuclear proteins CBP and p300 are localized within cells throughout the hamster SCN. Both CBP and p300 are found within the entire rostral–caudal axis of the SCN, with no shell region of the SCN being devoid of labeling for either protein. The number of cells immunoreactive to CBP and p300 decreases at mid-night circadian times as compared to mid-day times (Figs. 2 and 4), although the decrease in both instances is less than 20%. The potential significance, if any, of this decrease remains to be determined. In addition, with only two time points tested, it is not known if the expression profile of these proteins have a circadian rhythmicity. Finally, the number of cells in the SCN immunoreactive to either CPB or p300 is not affected by a light pulse at CT 18, suggesting that expression of neither protein is sensitive to light late in the dark phase in hamsters (Figs. 3 and 5). There are however,
some points that should be considered when interpreting these results: (1) immunohistochemistry techniques are not particularly good at detecting small changes of protein levels within labeled cells, (2) this study did not examine the phosphorylation state of either p300 or CBP, (3) there could be a significant change in protein levels if tests were conducted at either earlier or later time points following a light exposure, and (4) limiting cell counts to retinorecipient regions of the SCN (rather than the entire nucleus) following a light stimulus may have detected a change in cell numbers, although this is unlikely due to the density of labeling as indicated in Fig. 3. There are a number of immediate-early genes that are expressed in the hamster SCN in response to light pulses given at discrete times of the circadian night. The most studied of these genes are c-fos and c-jun [10,11]. Light induction of these genes is correlated to light-induced changes in circadian activity rhythms [8,10,20], so activation of these genes is considered to be involved with photic regulation of the circadian clock in some yet-undetermined manner. A cAMP response element can be found upstream of the immediate early genes [21], and this DNA region is
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
5
Fig. 3. SCN immunoreactivity to CBP/ p300 following a light pulse at CT 18. Control (no light) sections are in the left column, right column is 1 h following the beginning of a 10-min light pulse. CBP immunoreactivity is illustrated in (A,B); p300 examples are shown in (C,D). All sections are at the median region of the rostral–caudal SCN axis.
Fig. 4. Average number of cells per section of the median SCN immunoreactive to CBP/ p300 at mid-day and mid-night circadian times. The number of SCN sections used in each analysis is indicated in parentheses below the bars. *P,0.05 from respective mid-day group.
6
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Fig. 5. Average number of cells per section of the median SCN at CT 19 following no light (control) or a 10-min light pulse delivered at CT 18. The number of SCN sections used in each analysis is indicated in parentheses below the bars.
bound by the phosphorylated form of CREB [6]. CREB and pCREB immunoreactivity is dense in the hamster SCN [6], similar to that reported here for CBP and p300. In addition, both CBP and p300 are capable of binding to pCREB in other systems [13,14]. A recent report has also identified CREs in the promoter regions of the light-gated and clock-controlled genes mPer1 and mPer2 that bind CREB [25]. Therefore, it seems probable that CBP and / or p300 are interacting with pCREB in the hamster SCN as coactivators to drive transcription of clock-controlled genes. In addition to the light-induced and clock-controlled genes involved with circadian rhythms in the rodent SCN, there are the actual clock genes that form the time-keeping aspects of the biological clock through an intricate series of transcriptional / translational events [5,9]. In the rodent SCN, up to three period genes and two cryptochrome genes are regulated by the transcription factors BMAL1 and CLOCK acting as a heterodimer [5,9,16]. The BMAL1:CLOCK heterodimer drives transcription of the period and cryptochrome genes by interaction with the E-box upstream of the clock genes. The protein products of the period and cryptochrome genes then re-enter the nucleus and inhibit BMAL1:CLOCK activity, thereby forming a negative feedback loop and the core of the circadian clock. Recently, it has been reported that either CBP or p300 can interact via their CREB-binding sites with an activation domain at the c-terminal region of BMAL1 when it is heterodimerized with CLOCK [23]. Therefore, this raises the intriguing possibility that CBP and p300 may play important roles in the function of the BMAL1:CLOCK heterodimer regulation of clock genes in
the SCN. In fact, CBP and / or p300 may act as coactivators in several circadian transcriptional events. The precise nature of how the light-induced and clockcontrolled genes interact with the actual clock genes has not been resolved. However, we now suggest that CBP and p300 are worth investigating as a link between photically responsive immediate early genes and the time-keeping clock genes, perhaps through their interaction with pCREB. As indicated in the above paragraphs, CBP and p300 can interact with both pCREB and BMAL1. CBP and p300 have also been reported to link factors binding to an E-box (as found upstream of the period genes) and CREB activity in Sertoli cells [3]. Indeed, CBP and p300 appear to specialize in cross-coupling of diverse gene expression in cells [7]. CREB activation of transcription has recently been suggested to be a necessary component of the circadian clock in the SCN [18]. In addition, protein kinase activation of the human period genes has been reported to require pCREB [17], and mPer1 and mPer2 promoters contain CRE sites for CREB binding [25]. Therefore, as the role for pCREB in the activity of clock genes becomes more established, it will strengthen the link between this core time-keeping process and that of light-induced genes containing the cAMP response element in their upstream region. Likewise, when considering the well-known interaction between CBP and p300 with pCREB in other cell types, it seems likely that these two cofactors will also play a role in linking clock genes and gated light-induced genes. In conclusion, the pCREB coactivators CBP and p300 are substantially expressed within the hamster SCN. There is a decrease in the number of cells in the SCN immunoreactive for both CBP and p300 at night when compared to
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
day, although the expression level is still quite high even at night. Neither CBP nor p300 expression levels are sensitive to a phase-resetting light pulse administered in vivo at CT 18. Therefore, these preliminary studies suggest that both CBP and p300 are constitutively expressed at high levels in the SCN throughout the day. Finally, we propose that CBP and p300 are likely components of the molecular pathways mediating time keeping by the clock genes as well as the pathways mediating gated and clock-controlled gene expression in mammalian circadian rhythms. Indeed, CBP and or p300 are likely to be general coactivators involved in many of the transcriptional events comprising and modulating the mammalian circadian clock.
Acknowledgements Supported by NSF IBN-0116849. Paul Fiore is an undergraduate student in the biology program at Dowling College. Our thanks to Laura Mullally for her assistance with counting cells.
References [1] R.A. Akhtar, A.B. Reddy, E.S. Maywood, J.D. Clayton, V.M. King, A.G. Smith, T.W. Grant, M.H. Hastings, C.P. Kyriacou, Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus, Curr. Biol. 12 (2002) 540–550. [2] S. Barak, E.M. Tobin, C. Andronis, S. Sugano, R.M. Green, All in good time: the Arabidopsis circadian clock, Trends Plant Sci. 5 (2000) 517–522. [3] J. Chaudhary, M.K. Skinner, Role of the transcriptional coactivator CBP/ p300 in linking basic helix-loop-helix and CREB responses for follicle-stimulating hormone-mediated activation of the transferrin promoter in Sertoli cells, Biol. Reprod. 65 (2001) 568–574. [4] G.E. Duffield, J.D. Best, B.H. Meurers, A. Bittner, J.J. Loros, J.C. Dunlap, Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells, Curr. Biol. 12 (2002) 551–557. [5] J.C. Dunlap, Molecular bases for circadian clocks, Cell 96 (1999) 271–290. [6] D.D. Ginty, J.M. Kornhauser, M.A. Thompson, H. Bading, K.E. Mayo, J.S. Takahashi, M.E. Greenberg, Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock, Science 260 (1993) 238–241. [7] A. Giordano, M.L. Avantaggiati, p300 and CBP: partners for life and death, J. Cell. Physiol. 181 (1999) 218–230. [8] M.E. Guido, D. Goguuen, L. De Guido, H.A. Robertson, B. Rusak, Circadian and photic regulation of immediate-early gene expression in the hamster suprachiasmatic nucleus, Neuroscience 90 (1999) 555–571.
7
[9] M. Hastings, E.S. Maywood, Circadian clocks in the mammalian brain, BioEssays 22 (2000) 23–31. [10] J.M. Kornhauser, D.W. Nelson, K.E. Mayo, J.S. Takahashi, Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus, Neuron 5 (1990) 127–134. [11] J.M. Kornhauser, D.W. Nelson, K.E. Mayo, J.S. Takahashi, Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock, Science 255 (1992) 1581–1584. [12] J.M. Kornhauser, K.E. Mayo, J.S. Takahashi, Light, immediate-early genes, and circadian rhythms, Behav. Gen. 26 (1996) 221–240. [13] R.P. Kwok, J.R. Lundblad, J.C. Chrivia, J.P. Richards, H.P. Bachinger, R.G. Brennan, S.G. Roberts, M.R. Green, R.H. Goodman, Nuclear protein CBP is a coactivator for the transcription factor CRE, Nature 370 (1994) 223–226. [14] J.S. Lee, X. Zhang, Y. Shi, Differential interactions of the CREB / ATF family of transcription factors with p300 and adenovirus E1A, J. Biol. Chem. 27 (1996) 17666–17674. [15] J.J. Loros, J.C. Dunlap, Genetics and molecular analysis of circadian rhythms in Neurospora, Annu. Rev. Physiol. 63 (2001) 757–794. [16] P.L. Lowrey, J.S. Takahashi, Genetics of the mammalian circadian system: photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation, Annu. Rev. Genet. 34 (2000) 533–562. [17] D. Motzkus, E. Maronde, U. Grunenberg, C.C. Lee, W.-G. Forssmann, U. Albrecht, The human PER1 gene is transcriptionally regulated by multiple signaling pathways, FEBS Lett. 486 (2000) 315–319. [18] K. Obrietan, S. Impey, D. Smith, J. Athos, D.R. Storm, Circadian regulation of cAMP response element-mediated gene expression in the suprachiasmatic nuclei, J. Biol. Chem. 274 (1999) 17748– 17756. [19] M.R. Ralph, R.G. Foster, F.C. Davis, M. Menaker, Transplanted suprachiasmatic nucleus determines circadian period, Science 247 (1990) 975–978. [20] B. Rusak, H.A. Robertson, W. Wisden, S.P. Hunt, Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science 248 (1990) 1237–1240. [21] M. Sheng, M.E. Greenberg, The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron 4 (1990) 477–485. 21 [22] M. Sheng, M.A. Thompson, M.E. Greenberg, CREB: a Ca regulated transcription factor phosphorylated by calmodulin-dependent kinases, Science 252 (1991) 1427–1430. [23] S. Takahata, T. Ozaki, J. Mimura, Y. Kikuchi, K. Sogawa, Y. Fujii-Kuriyama, Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3, Genes Cells 5 (2000) 739– 747. [24] A. Tierno, P. Fiore, R.L. Gannon, Delta opioid inhibition of lightinduced phase advances in hamster circadian activity rhythms, Brain Res. 937 (2002) 66–73. [25] Z. Travnickova-Bendoza, N. Cermakian, S.M. Reppert, P. SassoneCorsi, Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK / BMAL1 activity, Proc. Natl. Acad. Sci. USA 99 (2002) 7728–7733. [26] J.A. Williams, A. Sehgal, Molecular components of the circadian system in Drosophila, Annu. Rev. Physiol. 63 (2001) 729–755.
Research report
Expression of the transcriptional coactivators CBP and p300 in the hamster suprachiasmatic nucleus: possible molecular components of the mammalian circadian clock Paul Fiore, Robert L. Gannon* Department of Biology, Dowling College, Oakdale, NY 11769, USA Accepted 3 December 2002
Abstract Immediate early genes are expressed in the mammalian suprachiasmatic nucleus in response to photic information arriving from the retina at restricted times of the day, therefore their expression is regulated by the circadian biological clock. These light-induced genes are also activated by the phosphorylated form of CREB (pCREB) that binds to a cAMP response element upstream of the genes. The nuclear proteins CBP and p300 are known to be coactivators with pCREB in certain cell types, but their identification within the rodent SCN has not been reported. Therefore, in this study we examined the distribution of both CBP and p300 in the hamster suprachiasmatic nucleus. CBP and p300 immunoreactivity is detected in cells throughout the suprachiasmatic nucleus, and the pattern of staining within cells is indicative of a nuclear location for these proteins. The number of cells immunoreactive for both CBP and p300 significantly decreases at mid-night circadian times with respect to mid-day circadian times, although the reduction is less than 20%. Neither CBP nor p300 expression is affected by a circadian phase-resetting light pulse given late in the night. The ability of CBP and p300 to interact with pCREB as well as with the clock gene BMAL1 is discussed, and we propose that CBP and p300 may interact with, and link, both clock genes and clock-controlled genes in the generation of circadian rhythms in mammals. We further suggest that there will be a general importance for the role of transcriptional coactivators such as CBP and p300 in many of the molecular pathways related to the mammalian circadian clock. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Transcription factors; Biological rhythms; CREB; Hypothalamus
1. Introduction There has been considerable progress in the last decade in determining the molecular mechanisms that generate and regulate circadian rhythms in organisms. Arabidopsis, Neurospora, Drosophila and the mouse are all model systems for determining what genes comprise the ‘biological clock’ [2,9,15,26]. In mammals, circadian pacemakers may be located in diverse organ systems in the body [1,4], but the primary pacemaker for circadian activity rhythms is located in the suprachiasmatic nuclei *Corresponding author. Tel.: 11-631-244-3339; fax: 11-631-2441033. E-mail address: [email protected] (R.L. Gannon).
(SCN) of the hypothalamus [19]. Complex cycles of transcription and translation are believed to form a 24-h rhythm of gene expression in certain cells of the SCN that is then coupled by an unknown output signal to effector pathways that regulate activity. There are other molecular pathways in addition to the clock itself that are activated by photic input to the SCN, and these most likely entrain the time of the clock to the light:dark cycle of the environment in which the mammal resides [12]. Light pulses administered to a hamster kept in constant darkness during certain times of the subjective night were found to express protein encoded by the oncogene c-fos in the retino-recipient region of the SCN [20]. This was the first of a number of similar genes that were reported to be activated in a time-sensitive, or gated, manner. These
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0169-328X(02)00663-0
2
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
genes may also demonstrate a circadian rhythm in their expression in the absence of photic timing cues, and if so, they can thus be classified as clock-controlled genes [8,12]. Upstream of these genes is a region known as the cAMP response element (CRE) that may be involved in regulating the transcription of the clock-controlled genes. The CRE is in turn bound by the trans-acting factor cAMPresponse element-binding protein (CREB). CREB itself is activated by phosphorylation of serine residues in a region of the CREB activation domain [22]. CREB is also quickly phosphorylated in the hamster SCN in response to light pulses administered at the same circadian times (CT) used to induce immediate-early gene expression or phase shifts in activity rhythms [6]. These results indicate that phosphorylated CREB (pCREB) may be a critical component in the molecular pathway that mediates photic signaling in the hamster SCN. The activity of pCREB may be due to an interaction of this factor with coactivator proteins that is not possible until CREB is phosphorylated. Two coactivators of pCREB have been described in other systems. CREBbinding protein (CBP) and its homologue p300 have both been reported to bind to the phosphorylation box of pCREB [13,14]. CBP/ p300 binding to pCREB has been proposed to link pCREB bound to the CRE with the transcription factor TFIIB [13,14]. Therefore, our hypothesis is that a similar mechanism may exist in the hamster SCN whereby CBP and / or p300 link pCREB bound to the CRE with transcriptional factors regulating the transcription of clock controlled genes. As a first step in investigating our hypothesis, we have identified the presence of both CBP and p300 within the hamster SCN using immunohistochemical techniques. In addition, we evaluated the expression profile of these two proteins in the hamster SCN in response to different lighting conditions.
2. Materials and methods Adult male Syrian hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories (Kingston, NY) and were approximately 150 g at the time of use in this study. Hamsters were individually housed under either 14:10 h of light:dark (LD), or under conditions of constant darkness (DD). Each hamster had an exercise wheel in their cage along with access to food and water ad libitum. Room lighting was approximately 250 lux during the light period. Hamsters were maintained under either LD or DD conditions for 2 weeks prior to use in these experiments. CBP and p300 were not concurrently tested within the same hamster brain. For hamsters maintained under DD, activity level and thus CT times were determined using an activity monitoring system as described elsewhere [24]. To determine if light would affect expression of CBP or p300, hamsters were removed from LD during the dark phase
under dim red light (,1 lux) and exposed to 10 min of room light (250 lux) approximately 6 h after activity onset (CT 18). Hamsters were returned to darkness after the light pulse, and then terminally anesthetized with halothane at CT 19 under dim red light. To examine the expression profile of CBP and p300 at different circadian times without exposure to light, hamsters were removed from DD at mid-day and mid-night CT times and terminally anesthetized with halothane under dim red light (,1 lux). Terminally anesthetized hamsters were rapidly perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer for 5 min. Brains were removed and postfixed overnight in 4% paraformaldehyde before being cryopreserved in 30% sucrose. Hamster brains were cut into 40-mm sections using a cryostat and transferred to 24-well tissue plates for freefloating immunohistochemistry. Sections were incubated at room temperature in 1% hydrogen peroxide for 24 h. Sections were then washed with 0.1 M phosphate buffer, incubated with blocking serum for 1 h, washed again with 0.1 M phosphate buffer, and then incubated overnight with primary antibody (1:1000) at room temperature. The two primary antibodies used in this study were rabbit antihuman p300 (N-15) and rabbit anti-human CBP (C-20), obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody was removed and sections washed again with phosphate buffer before the addition of secondary antibody and ABC reagents according to kit instructions (Vectastain Elite ABC anti-rabbit peroxidase kit, Vector Laboratories, Burlingame, CA). Peroxidase staining was detected using the Vector SG Substrate kit. Finally, sections were washed with isotonic saline, mounted on Superfrost Plus slides, dehydrated and coverslipped. For controls, primary antibody was pre-absorbed with control peptides for 2 h at room temperature at a concentration of 20 mg / ml. Control peptides for p300 and CBP were purchased from Santa Cruz Biotechnology. Images of the SCN were digitally captured using a Pixera Professional CCD Camera (Pixera, Los Gatos, CA). The images were printed using a Hewlett-Packard PhotoSmart printer, the SCN region was outlined, and cell counts were then made using the printed images of the SCN. Two students using a blinded protocol performed the counting, and count totals for each SCN section were averaged.
3. Results
3.1. Immunoreactive patterns and controls Immunoreactivity for both CBP and p300 was widespread throughout the hamster brain, including dense labeling of both proteins in the SCN. Both CBP and p300 were identified throughout the entire SCN region, in both the rostral–caudal and dorsal–ventral axis (not shown).
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Immunoreactivity was confined to small circular regions within cells, indicative of a nuclear localization for both proteins. Preabsorption of CBP and p300 antisera with their respective control peptides completely blocked staining, demonstrating the specificity of these antisera for hamster proteins (Fig. 1).
3.2. Day versus night expression of CBP and p300 The number of cells in the SCN that were labeled by CBP and p300 at both mid-day and mid-night circadian times was determined using hamsters that were not exposed to light at either time. Mid-day circadian times for the CBP experiment ranged from CT 7–8, and mid-night time was at CT 16. Mid-day circadian times for p300 hamsters were CT 6–10, and mid-night circadian times were CT 15–19. There was an average of 1505658 (mean6S.E.M.) cells immunoreactive for CBP during midday times, and this number fell significantly to 1251632 cells during mid-night times (F(1,15)513.81, P,0.002, ANOVA; Figs. 2 and 4). Although there was on average fewer labeled cells for p300 in the SCN in this protocol, there was a similar difference in expression during the mid-day and mid-night times. The number of cells immunoreactive for p300 in the SCN during mid-day CT
3
times was 1156654, and this number fell significantly to 1005625 cells during the mid-night times (F(1,13)56.80, P,0.02, ANOVA; Figs. 2 and 4).
3.3. Effect of light on expression of CBP and p300 Hamsters were exposed to a 10-min light pulse at CT 18, 6 h into their dark phase, and then returned to darkness for 50 min prior to termination in order to allow time for any affect of light to alter expression levels of CBP or p300. The number of cells in the SCN immunoreactive to CBP or p300 was not significantly different than the levels observed during mid-night circadian times in the above study (Section 3.2). Hamsters not exposed to light and perfused at CT 19 were used as controls in this experiment. There was an average of 1215638 cells immunoreactive to CBP in the SCN of controls, and this level was not significantly altered by the exposure to light at CT 18 (1199659 cells; Figs. 3 and 5; ANOVA). Similarly, there was an average of 1067636 cells immunoreactive for p300 in the control SCN and 1036626 cells immunoreactive to p300 following light exposure at CT 18 (no significant difference; ANOVA). Therefore, a 10-min light pulse at CT 18 had no effect on the number of cells in the hamster
Fig. 1. Specificity of CBP and p300 antisera to hamster brain proteins. Dense cellular immunoreactivity to CBP (A) and p300 (C) in the rostral hippocampus is completely eliminated by preabsorption of the antisera with their respective control peptides (B,D). Hippocampus was used as the example since it demonstrated the most dense staining in the hamster brain. DG, dentate gyrus; CA3, CA3 cell layer.
4
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Fig. 2. SCN immunoreactivity to CBP/ p300 at mid-day and mid-night times. Left column, mid-day; right-column, mid-night sections. Cellular immunoreactivity to CBP (A,B) and p300 (C,D) The SCN region is outlined in (A). All sections are at the median region of the rostral–caudal SCN axis.
SCN immunoreactive for either CBP or p300 1 h after the beginning of the exposure to light.
4. Discussion The nuclear proteins CBP and p300 are localized within cells throughout the hamster SCN. Both CBP and p300 are found within the entire rostral–caudal axis of the SCN, with no shell region of the SCN being devoid of labeling for either protein. The number of cells immunoreactive to CBP and p300 decreases at mid-night circadian times as compared to mid-day times (Figs. 2 and 4), although the decrease in both instances is less than 20%. The potential significance, if any, of this decrease remains to be determined. In addition, with only two time points tested, it is not known if the expression profile of these proteins have a circadian rhythmicity. Finally, the number of cells in the SCN immunoreactive to either CPB or p300 is not affected by a light pulse at CT 18, suggesting that expression of neither protein is sensitive to light late in the dark phase in hamsters (Figs. 3 and 5). There are however,
some points that should be considered when interpreting these results: (1) immunohistochemistry techniques are not particularly good at detecting small changes of protein levels within labeled cells, (2) this study did not examine the phosphorylation state of either p300 or CBP, (3) there could be a significant change in protein levels if tests were conducted at either earlier or later time points following a light exposure, and (4) limiting cell counts to retinorecipient regions of the SCN (rather than the entire nucleus) following a light stimulus may have detected a change in cell numbers, although this is unlikely due to the density of labeling as indicated in Fig. 3. There are a number of immediate-early genes that are expressed in the hamster SCN in response to light pulses given at discrete times of the circadian night. The most studied of these genes are c-fos and c-jun [10,11]. Light induction of these genes is correlated to light-induced changes in circadian activity rhythms [8,10,20], so activation of these genes is considered to be involved with photic regulation of the circadian clock in some yet-undetermined manner. A cAMP response element can be found upstream of the immediate early genes [21], and this DNA region is
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
5
Fig. 3. SCN immunoreactivity to CBP/ p300 following a light pulse at CT 18. Control (no light) sections are in the left column, right column is 1 h following the beginning of a 10-min light pulse. CBP immunoreactivity is illustrated in (A,B); p300 examples are shown in (C,D). All sections are at the median region of the rostral–caudal SCN axis.
Fig. 4. Average number of cells per section of the median SCN immunoreactive to CBP/ p300 at mid-day and mid-night circadian times. The number of SCN sections used in each analysis is indicated in parentheses below the bars. *P,0.05 from respective mid-day group.
6
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
Fig. 5. Average number of cells per section of the median SCN at CT 19 following no light (control) or a 10-min light pulse delivered at CT 18. The number of SCN sections used in each analysis is indicated in parentheses below the bars.
bound by the phosphorylated form of CREB [6]. CREB and pCREB immunoreactivity is dense in the hamster SCN [6], similar to that reported here for CBP and p300. In addition, both CBP and p300 are capable of binding to pCREB in other systems [13,14]. A recent report has also identified CREs in the promoter regions of the light-gated and clock-controlled genes mPer1 and mPer2 that bind CREB [25]. Therefore, it seems probable that CBP and / or p300 are interacting with pCREB in the hamster SCN as coactivators to drive transcription of clock-controlled genes. In addition to the light-induced and clock-controlled genes involved with circadian rhythms in the rodent SCN, there are the actual clock genes that form the time-keeping aspects of the biological clock through an intricate series of transcriptional / translational events [5,9]. In the rodent SCN, up to three period genes and two cryptochrome genes are regulated by the transcription factors BMAL1 and CLOCK acting as a heterodimer [5,9,16]. The BMAL1:CLOCK heterodimer drives transcription of the period and cryptochrome genes by interaction with the E-box upstream of the clock genes. The protein products of the period and cryptochrome genes then re-enter the nucleus and inhibit BMAL1:CLOCK activity, thereby forming a negative feedback loop and the core of the circadian clock. Recently, it has been reported that either CBP or p300 can interact via their CREB-binding sites with an activation domain at the c-terminal region of BMAL1 when it is heterodimerized with CLOCK [23]. Therefore, this raises the intriguing possibility that CBP and p300 may play important roles in the function of the BMAL1:CLOCK heterodimer regulation of clock genes in
the SCN. In fact, CBP and / or p300 may act as coactivators in several circadian transcriptional events. The precise nature of how the light-induced and clockcontrolled genes interact with the actual clock genes has not been resolved. However, we now suggest that CBP and p300 are worth investigating as a link between photically responsive immediate early genes and the time-keeping clock genes, perhaps through their interaction with pCREB. As indicated in the above paragraphs, CBP and p300 can interact with both pCREB and BMAL1. CBP and p300 have also been reported to link factors binding to an E-box (as found upstream of the period genes) and CREB activity in Sertoli cells [3]. Indeed, CBP and p300 appear to specialize in cross-coupling of diverse gene expression in cells [7]. CREB activation of transcription has recently been suggested to be a necessary component of the circadian clock in the SCN [18]. In addition, protein kinase activation of the human period genes has been reported to require pCREB [17], and mPer1 and mPer2 promoters contain CRE sites for CREB binding [25]. Therefore, as the role for pCREB in the activity of clock genes becomes more established, it will strengthen the link between this core time-keeping process and that of light-induced genes containing the cAMP response element in their upstream region. Likewise, when considering the well-known interaction between CBP and p300 with pCREB in other cell types, it seems likely that these two cofactors will also play a role in linking clock genes and gated light-induced genes. In conclusion, the pCREB coactivators CBP and p300 are substantially expressed within the hamster SCN. There is a decrease in the number of cells in the SCN immunoreactive for both CBP and p300 at night when compared to
P. Fiore, R.L. Gannon / Molecular Brain Research 111 (2003) 1–7
day, although the expression level is still quite high even at night. Neither CBP nor p300 expression levels are sensitive to a phase-resetting light pulse administered in vivo at CT 18. Therefore, these preliminary studies suggest that both CBP and p300 are constitutively expressed at high levels in the SCN throughout the day. Finally, we propose that CBP and p300 are likely components of the molecular pathways mediating time keeping by the clock genes as well as the pathways mediating gated and clock-controlled gene expression in mammalian circadian rhythms. Indeed, CBP and or p300 are likely to be general coactivators involved in many of the transcriptional events comprising and modulating the mammalian circadian clock.
Acknowledgements Supported by NSF IBN-0116849. Paul Fiore is an undergraduate student in the biology program at Dowling College. Our thanks to Laura Mullally for her assistance with counting cells.
References [1] R.A. Akhtar, A.B. Reddy, E.S. Maywood, J.D. Clayton, V.M. King, A.G. Smith, T.W. Grant, M.H. Hastings, C.P. Kyriacou, Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus, Curr. Biol. 12 (2002) 540–550. [2] S. Barak, E.M. Tobin, C. Andronis, S. Sugano, R.M. Green, All in good time: the Arabidopsis circadian clock, Trends Plant Sci. 5 (2000) 517–522. [3] J. Chaudhary, M.K. Skinner, Role of the transcriptional coactivator CBP/ p300 in linking basic helix-loop-helix and CREB responses for follicle-stimulating hormone-mediated activation of the transferrin promoter in Sertoli cells, Biol. Reprod. 65 (2001) 568–574. [4] G.E. Duffield, J.D. Best, B.H. Meurers, A. Bittner, J.J. Loros, J.C. Dunlap, Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells, Curr. Biol. 12 (2002) 551–557. [5] J.C. Dunlap, Molecular bases for circadian clocks, Cell 96 (1999) 271–290. [6] D.D. Ginty, J.M. Kornhauser, M.A. Thompson, H. Bading, K.E. Mayo, J.S. Takahashi, M.E. Greenberg, Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock, Science 260 (1993) 238–241. [7] A. Giordano, M.L. Avantaggiati, p300 and CBP: partners for life and death, J. Cell. Physiol. 181 (1999) 218–230. [8] M.E. Guido, D. Goguuen, L. De Guido, H.A. Robertson, B. Rusak, Circadian and photic regulation of immediate-early gene expression in the hamster suprachiasmatic nucleus, Neuroscience 90 (1999) 555–571.
7
[9] M. Hastings, E.S. Maywood, Circadian clocks in the mammalian brain, BioEssays 22 (2000) 23–31. [10] J.M. Kornhauser, D.W. Nelson, K.E. Mayo, J.S. Takahashi, Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus, Neuron 5 (1990) 127–134. [11] J.M. Kornhauser, D.W. Nelson, K.E. Mayo, J.S. Takahashi, Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock, Science 255 (1992) 1581–1584. [12] J.M. Kornhauser, K.E. Mayo, J.S. Takahashi, Light, immediate-early genes, and circadian rhythms, Behav. Gen. 26 (1996) 221–240. [13] R.P. Kwok, J.R. Lundblad, J.C. Chrivia, J.P. Richards, H.P. Bachinger, R.G. Brennan, S.G. Roberts, M.R. Green, R.H. Goodman, Nuclear protein CBP is a coactivator for the transcription factor CRE, Nature 370 (1994) 223–226. [14] J.S. Lee, X. Zhang, Y. Shi, Differential interactions of the CREB / ATF family of transcription factors with p300 and adenovirus E1A, J. Biol. Chem. 27 (1996) 17666–17674. [15] J.J. Loros, J.C. Dunlap, Genetics and molecular analysis of circadian rhythms in Neurospora, Annu. Rev. Physiol. 63 (2001) 757–794. [16] P.L. Lowrey, J.S. Takahashi, Genetics of the mammalian circadian system: photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation, Annu. Rev. Genet. 34 (2000) 533–562. [17] D. Motzkus, E. Maronde, U. Grunenberg, C.C. Lee, W.-G. Forssmann, U. Albrecht, The human PER1 gene is transcriptionally regulated by multiple signaling pathways, FEBS Lett. 486 (2000) 315–319. [18] K. Obrietan, S. Impey, D. Smith, J. Athos, D.R. Storm, Circadian regulation of cAMP response element-mediated gene expression in the suprachiasmatic nuclei, J. Biol. Chem. 274 (1999) 17748– 17756. [19] M.R. Ralph, R.G. Foster, F.C. Davis, M. Menaker, Transplanted suprachiasmatic nucleus determines circadian period, Science 247 (1990) 975–978. [20] B. Rusak, H.A. Robertson, W. Wisden, S.P. Hunt, Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science 248 (1990) 1237–1240. [21] M. Sheng, M.E. Greenberg, The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron 4 (1990) 477–485. 21 [22] M. Sheng, M.A. Thompson, M.E. Greenberg, CREB: a Ca regulated transcription factor phosphorylated by calmodulin-dependent kinases, Science 252 (1991) 1427–1430. [23] S. Takahata, T. Ozaki, J. Mimura, Y. Kikuchi, K. Sogawa, Y. Fujii-Kuriyama, Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3, Genes Cells 5 (2000) 739– 747. [24] A. Tierno, P. Fiore, R.L. Gannon, Delta opioid inhibition of lightinduced phase advances in hamster circadian activity rhythms, Brain Res. 937 (2002) 66–73. [25] Z. Travnickova-Bendoza, N. Cermakian, S.M. Reppert, P. SassoneCorsi, Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK / BMAL1 activity, Proc. Natl. Acad. Sci. USA 99 (2002) 7728–7733. [26] J.A. Williams, A. Sehgal, Molecular components of the circadian system in Drosophila, Annu. Rev. Physiol. 63 (2001) 729–755.