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J Biol Chem. 2009 March 20; 284(12): 7606–7622. doi: 10.1074/jbc.M808394200. | PMCID: PMC2658055 |
Copyright © 2009, The American Society for Biochemistry and
Molecular Biology, Inc. Night/Day Changes in Pineal Expression of >600 Genes CENTRAL
ROLE OF ADRENERGIC/cAMP
SIGNALING*![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Michael J. Bailey, ‡1 Steven L. Coon, ‡ David A. Carter, §2 Ann Humphries, §2 Jong-so Kim, ‡3 Qiong Shi, ‡4 Pascaline Gaildrat, ‡5 Fabrice Morin, ‡6 Surajit Ganguly, ‡7 John B. Hogenesch, ¶8 Joan L. Weller, ‡ Martin F. Rath,  9 Morten Møller, 9 Ruben Baler, **10 David Sugden, ‡‡11 Zoila G. Rangel, §§ Peter J. Munson, §§ and David C. Klein ‡12‡Section on Neuroendocrinology, Program on Developmental Endocrinology and Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, §School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom, ¶Genomics Institute, Novartis Research Foundation, San Diego, California 92121, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvei 3, DK-2200 Copenhagen, Denmark, **Unit on Temporal Gene Expression, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892, ‡‡Division of Reproduction and Endocrinology, School of Biomedical and Health Sciences, King's College London, London SE1 1UL, United Kingdom, and §§Mathematical and Statistical Computing Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 Received November 3, 2008; Revised December 12, 2008. The pineal gland plays an essential role in vertebrate chronobiology by
converting time into a hormonal signal, melatonin, which is always elevated at
night. Here we have analyzed the rodent pineal transcriptome using Affymetrix
GeneChip® technology to obtain a more complete description of pineal cell
biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez
Gene identifiers are differentially expressed greater than 2-fold between
midnight and mid-day (false discovery rate <0.20). Expression is greater at
night in ~70%. These findings were supported by the results of
radiochemical in situ hybridization histology and quantitative real
time-PCR studies. We also found that the regulatory mechanism controlling the
night/day changes in the expression of most genes involves
norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression
profile with that in other tissues identified 334 genes (496 probe sets) that
are expressed greater than 8-fold higher in the pineal gland relative to other
tissues. Of these genes, 17% are expressed at similar levels in the retina,
consistent with a common evolutionary origin of these tissues. Functional
categorization of the highly expressed and/or night/day differentially
expressed genes identified clusters that are markers of specialized functions,
including the immune/inflammation response, melatonin synthesis,
photodetection, thyroid hormone signaling, and diverse aspects of cellular
signaling and cell biology. These studies produce a paradigm shift in our
understanding of the 24-h dynamics of the pineal gland from one focused on
melatonin synthesis to one including many cellular processes. A defining feature of the pineal gland is a 24-h rhythm in melatonin
synthesis. Melatonin provides vertebrates with a circulating signal of time
and is essential for optimal integration of physiological functions with
environmental lighting on a daily and seasonal basis
( 1– 4). The melatonin rhythm in mammals is driven by a circadian clock located in
the suprachiasmatic nucleus
(SCN), 13 which is
hard-wired to the pineal gland by a polysynaptic pathway that courses through
central and peripheral neuronal structures. The pineal gland is innervated by
projections from the superior cervical ganglia (SCG) in the form of a dense
network of catecholamine-containing sympathetic fibers. Activation of the SCN
→ pineal pathway occurs at night and results in the release of
norepinephrine (NE) from the sympathetic fibers into the pineal perivascular
space ( 5). NE activates the
pinealocyte through adrenergic receptors
( 5,
6). The best studied mechanism
involves coincident “AND” gate activation of α 1b-
and β 1-adrenergic receptors, which maximally stimulates
adenylate cyclase, thereby elevating cAMP
( 7– 13).
Activation of α 1b-adrenergic receptors alone elevates
intracellular calcium and phospholipid signaling
( 1,
14– 16).
cAMP is believed to mediate the effects of NE on melatonin production to a
large part by activating cAMP-dependent protein kinase. In rodents, this
induces expression of Aanat, the penultimate enzyme in melatonin
synthesis ( 17). Induction
occurs through phosphorylation of cAMP-response element-binding protein (CREB)
bound to cAMP-response elements (CREs) in the Aanat gene. A similar
NE/cAMP mechanism also controls expression of Adra1b, Atp7b,
Crem, 14
Dio2, Fosl2, Id1, Dusp1, Mat2a, Nr4a1, Slc15a1, Pde4b2, Ptch1, and
Rorb
( 18– 27).
In addition, a NE/cAMP mechanism decreases expression of Hs3st2
( 28). Although it is likely
that some of the effects of cAMP involve CREs, it is also likely that cAMP
influences pineal gene expression through epigenetic mechanisms that alter
chromatin structure, e.g. histone phosphorylation
( 29,
30), thereby having the
potential of altering the expression of many genes and broadly promoting
transcription by factors other than CREB. Whereas there is abundant evidence
that the SCN/SCG/NE/cAMP system controls rhythmic gene expression in the
pineal gland, it is also possible that other regulatory mechanisms exist,
involving release of other transmitters, and additional second messengers
( e.g. cGMP, Ca 2+, and phospholipids). The increased abundance of some of these night/day differentially expressed
genes and of other genes in the pineal gland is determined in part by members
of the OTX2/CRX family of homeodomain proteins, which play a similar role in
the retina
( 31– 34).
These factors bind to photoreceptor conserved elements and closely related
sequences. In addition, Pax6 and Otx2 are essential for
development of both tissues
( 35– 37).
This developmental similarity is consistent with the common evolutionary
origin of the pineal gland and retina from a primitive photodetector
( 38). Examples of
OTX2/CRX-controlled genes expressed in both tissues include Aanat, Asmt,
Sag, and Grk1
( 20,
39– 46).
The first two encode proteins dedicated to melatonin synthesis; the latter two
encode proteins associated with phototransduction in the retina. It is not
clear whether the proteins encoded by these phototransduction genes play
parallel roles in NE/cAMP signal transduction in the pinealocyte or if they
are functionally vestigial in the context of the pinealocyte. Although OTX2
and CRX are of central importance in these tissues, it appears that other
transcription factors and regulatory cascades are involved. For example, the
importance of E-boxes in determining tissue-specific expression of
Aanat is evident from several studies
( 42,
47), and NeuroD1 may also play
a role in determining pineal gland-specific expression patterns
( 48). Whereas in both the pineal gland and retina, photoreceptor conserved
elements control developmental expression of the same gene, different
mechanisms can operate in each tissue to control rhythmicity. For example, in
the case of Aanat, CREs mediate cAMP control of 24-h rhythms in the
pineal gland ( 49,
50). In the retina, however,
E-box elements mediate circadian clock control of the 24-h rhythm in
Aanat expression
( 51). In addition to the accepted SCN/SCG/NE/cAMP pathway, reports in the
literature have claimed that a circadian clock regulates daily changes in the
expression of some genes in the mammalian pineal gland
( 52), as in the submammalian
pineal gland ( 53,
54). The physiological impact
of this remains unknown. Here we have expanded our understanding of the transcriptional regulation
and physiology of the pineal gland by employing Affymetrix GeneChip®
technology, including a microarray that interrogates more than 13,663 genes
that have been assigned Entrez Gene
identifiers. 15
Previous studies of this nature in the rat have identified 39 night/day
differentially expressed genes
( 26); a more recent study
identified 35 such genes with Entrez Gene identifiers (59 probe sets)
( 55). Our study had three
specific goals. The first goal was to produce a comprehensive listing of genes
that are differentially expressed on a night/day basis. The second goal was to
identify the highly enriched genes that define pineal function, independent of
whether they are tonically or night/day differentially expressed; this was
done by comparing gene expression in the pineal gland to median expression
among other tissues. The third goal was to determine the scope of the NE/cAMP
regulatory cascade; this approach utilized an in vitro organ culture
system. In addition to establishing the importance of this cascade, the organ
culture studies identified sets of genes that were spontaneously up- or
down-regulated more than 10-fold during culture in defined medium, providing
evidence of the existence of unknown regulatory mechanisms. An unexpected
discovery was that the pineal transcriptome includes a large number of
immune/inflammation response-associated genes. The findings of this study are of value to investigators interested in the
pineal gland, chronobiology, neuroendocrinology, and immunology and to those
who study specific genes that are night/day differentially and/or highly
expressed in the pineal gland. Animals Three microarray experiments were done (experiments A, B, and C). For
microarray experiments A and B (Cardiff University), Sprague-Dawley rats
(2–3 months old) were maintained in standard laboratory conditions in a
14:10 light-dark (LD) cycle (lights on, 05:00 h). Animals were killed at
mid-day (ZT7) or midnight (ZT19) by cervical dislocation, and pineal glands
were rapidly dissected, placed in tubes on solid CO2, and stored at
–80 °C. For microarray experiment C (NICHD, National Institutes of
Health), for the time series analysis of gene expression by qRT-PCR
(), and for organ culture
experiments, Sprague-Dawley rats (2–3 months old, female) were housed
for 2 weeks in LD 14:10 lighting cycles, killed by CO2
asphyxiation, and decapitated; pineal glands were rapidly dissected and either
placed in tubes on solid CO2 and stored at –80 °C or were
prepared for organ culture. Other tissues were also removed, and 10-mg samples
were frozen and stored in a similar manner. For the qRT-PCR experiment
(), tissues were
collected at ZT1, -7, -13.5, -15, -16, -17.5, -19, and -22, placed in tubes on
solid CO2, and stored at –80 °C. Glands for organ culture
experiments were obtained at ZT4–6 and placed in culture within 60 min.
For radiochemical in situ hybridization histology studies (University
of Copenhagen), Sprague-Dawley and Wistar rats (Charles River, Germany) were
housed for 2 weeks in a controlled lighting environment (LD 12:12). Animals
were killed by decapitation at ZT6 and ZT18; their brains were removed,
immediately placed in solid CO2, and stored at –80 °C
until sectioned. Animal use and care protocols were approved by local ethical review, and
they were in accordance with National Institutes of Health guidelines, United
Kingdom Home Office Regulations, and Health Sciences Animal Policy European
Union Directive 86/609/EEC (approved by the Danish Council for Animal
Experiments). For organ culture, rat pineal glands were cultured in BGJ b
medium as described previously
( 56) and detailed in the
supplemental material. Glands were incubated (1 gland/well) with fresh media
containing NE (1 μ m), dibutyryl cAMP (Bt 2cAMP; 0.5 or
1 m m), or forskolin (10 μ m) (Sigma). Following a 6-h
treatment, glands were placed in microtubes on solid CO 2. To confirm that the glands were activated by the drugs, melatonin
production in the culture media was measured by tandem mass spectroscopy as
described ( 57), with an
internal d4-melatonin standard. The amount of melatonin
produced (nanomoles/gland/6 h; means ± S.E.) for the control,
NE-treated, Bt 2cAMP-treated, and forskolin-treated groups was
(number of samples) 1.4 ± 0.1
( 9); 20.3 ± 1.1
( 9); 9.9 ± 0.9
( 9); and 15.0 ± 1.2
( 9), respectively. Microarray For the analysis of pineal glands in experiments A and B, two sets of six
pooled samples of four rat pineal glands each were prepared (three night and
three day). In experiment C, four pools, each containing three glands, were
prepared for each time point; as part of this experiment, single retinas and
10-mg samples of the cerebellum, neocortex, hypothalamus, liver, and heart
were also obtained. Glands were also obtained from organ culture experiments
in which each treatment group was comprised of three pools, each containing
four glands. Total RNA was isolated, labeled and used to interrogate
Affymetrix GeneChips® as detailed in the supplemental material. Microarray Data Sets The microarray data presented here are derived from the experiments
described below (A, B, and C) in conjunction with a published tissue profiling
effort (Genomics Institute of the Novartis Research Foundation (GNF), Entrez
Gene Expression Omnibus (GEO), dataset GDS589
( 58)). Microarray experiment A (Cardiff University) used the Affymetrix RG_U34A
microarray (8,799 probe sets, 4,996 genes). Results from microarray experiment
A were compared with data from the GNF data base, which had been generated
using the same microarray. Expression data for the following 23 Sprague-Dawley
tissues and isolated cells were used (number of samples per tissue is in
parentheses): neocortex (39), cerebellum (17), striatum (13), hippocampus (3),
hypothalamus (2), pituitary (2), amygdala (10), nucleus accumbens (6), locus
ceruleus (2), dorsal raphe (2), ventral tegmental area (2), pineal gland (2),
dorsal root ganglion (2), cornea (2), heart (2), intestine (4), kidney (2),
spleen (2), thymus (2), bone marrow (2), muscle (2), Sertoli cells (10), and
endothelial cells (2). Microarray experiment B (Cardiff University) used the RAE230A microarray
(15,923 probe sets, 10,174 genes). Microarray experiment C (NICHD, National
Institutes of Health) used the Rat230_2 microarray (31,099 probe sets, 13,663
genes); this experiment included pineal glands and other tissues (retina,
neocortex, cerebellum, hypothalamus, heart, and liver) obtained at mid-day and
midnight, and glands obtained from organ culture. Analysis of Microarray Results Night/Day Differences in Gene Expression—Affymetrix MAS5
Signal and Present Call values were stored in the NIH-LIMS, a data base for
storage and retrieval of microarray data. The microarray data are available at
the Entrez Gene Expression Omnibus, National Center for Biotechnology
Information ( 59), and are
accessible through GEO series accession number GSE12344
( ncbi.nlm.nih.gov),
and at
sne.nichd.nih.gov.
Data were statistically analyzed using the MSCL Analyst Toolbox (P. J. Munson,
J. J. Barb,
abs.cit.nih.gov)
and the JMP statistical software package (SAS, Inc., Cary, NC). Affymetrix
signal values were incremented by a value of 0.1× microarray median
value, then normalized to 1.1× microarray median values, and finally
decimal log-transformed. This transformation is termed “Lmed” and
has the desirable effect of reducing the influence of very small expression
values. One-way, two-level analysis of variance testing differences between
night and day were performed on the transformed data, and significance
( p values, or false discovery rate (FDR)
( 60)) was reported (see
supplemental Table S3). Night-day log fold changes were computed as the
difference between the night and day Lmed values; in experiment C, NE/control
and Bt 2cAMP/control log fold change values were calculated
similarly. Expression ratios are reported as linear values; values less than
one are reported using the 1/ X convention in which X = the
night/day ratio, i.e. a night/day ratio of 0.01 is displayed as
1/100. Table 1 details the
expression ratios (night/day, NE/control, Bt 2cAMP/control) of all
genes with a Entrez Gene identifier and with a night/day ratio greater than 4
or less than ¼. The supplemental Table S3 presents the expression
ratios of all probe sets with a night/day ratio greater than 2 or less than
½. | TABLE 1Differential expression of genes in the pineal gland |
Expression of Genes in the Pineal Gland Relative to Other
Tissues—Gene expression in one tissue relative to expression in
other tissues was defined as the relative tissue expression (rEx) value, which
was calculated as the ratio of maximum expression (the highest of day or
night) to the median expression of that gene in other tissues. In experiment
A, median values were calculated from 23 Sprague-Dawley tissues in the GNF
data base (see above) plus the day and night pineal gland values generated in
experiment A. In experiment C, the median values were calculated from the
average expression levels in each of seven tissues (see above). These averages
were based on single mid-day and midnight values, except in the case of the
pineal gland for which four mid-day and four midnight values were used. The
larger of two rEx values obtained using the two experiments is presented in
Table 3, which contains genes
with rEx values greater than 8. The supplemental Table S4 includes rEx values
from both experiments for probe sets with rEx values greater than 2. | TABLE 3Genes highly expressed in the pineal gland relative to other
tissues |
Comparison of Results across Different Microarray
Platforms—Results from the three platforms were compared using the
following mapping algorithm. The RG_U34A probe sets were first mapped to the
Rat230_2 microarray using the Affymetrix ortholog map (available online).
Exact matches for probe sets on the RAE230A microarray are found on the
Rat230_2 microarray. In cases where a probe set maps to several probe sets on
another microarray, each mapped probe set pair is considered as an independent
pair, resulting in multiple pairs, each sharing a common probe set. Probe sets
from the different microarrays were annotated using Affymetrix annotation
files (available online) dated November 5, 2007. Each probe set was mapped to
an Entrez Gene identifier. In cases of discrepant gene identification for the
probe set pair, the annotations from the most recent microarray type were
used. In cases where probe sets identify two or more genes, the gene symbol of
the first gene on the annotation file is listed, unless otherwise indicated.
Summaries over multiple probe sets mapping to the same gene were calculated by
taking the maximum observed ratios for night/day. The rEx values were
calculated in a similar manner. Radiochemical in Situ Hybridization Histology Sagittal sections of frozen rat brains were analyzed by in situ
hybridization histology as described previously
( 34,
61) and detailed in the
supplemental material. Sections were hybridized with 35S-labeled
38-mer oligonucleotide probes (supplemental Table S1). The sections were
exposed to x-ray film or dipped into an LM-1® emulsion (Amersham
Biosciences). The in situ hybridization images presented in
are available at high
resolution on line at
sne.nichd.nih.gov. qRT-PCR For data in and
Table 6, pools of glands were
used for each time point or treatment group. cDNA was synthesized from
DNase-treated total RNA, and qRT-PCR results were quantitated and normalized
( 62) as detailed in the
supplemental material. The primers used are described in supplemental Table
S2. | TABLE 6Effects of NE, Bt2cAMP, or forskolin on transcript abundance
in cultured pineal glands |
Functional Analysis The programs used to identify clusters of genes associated with discrete
functions were DAVID Bioinformatics Resources 2007
( david.abcc.ncifcrf.gov/),
ModuleMiner ( 63) and
Affymetrix NetAffx. The results of these analyses and common knowledge were
used to generate Table 7. | TABLE 7Functional grouping of genes that are night/day differentially expressed
(N/D >2 or <1/2) or at high levels (rEx >4) in the pineal
gland |
Detection of cis-Regulatory Elements Computational detection of enriched cis-regulatory elements
(position weight matrices (PWMs)) within microarray-derived gene sets was
conducted using ModuleMiner
( 63). Microarray Analysis A Large Number of Genes Exhibit Night/Day Differences in Expression in
the Pineal Gland—The results of analysis of night/day differences
in gene expression using the RG_U34A, the RAE230A, and Rat230_2 microarrays
are presented in Table 1 and
supplemental Table S3. To examine the degree of agreement of data obtained by
these microarrays, we compared results from the RG_U34A and Rat230_2
microarray; the latter and the RAE230A microarray contain the same probe sets,
representing 10,156 Entrez Gene identifiers, and did not require comparative
analysis. Comparison of the expression levels of the 4,459 genes (6,392 probe
sets) present on both the RG_U34A and Rat230_2 microarrays revealed the
results were in excellent agreement ( r = 0.44) (supplemental Fig.
S1). Expression of 604 genes (1,268 probe sets) exhibits a significantly greater
than 2-fold change on a night/day basis (FDR <0.20;
Table 1 and supplemental Table
S3). Approximately 2,000 additional genes exhibit a smaller but significant
night/day change in expression (FDR < 0.20). These findings increase by
more than 50-fold the known number of genes differentially expressed in the
pineal gland. Among the 604 genes with a greater than 2-fold difference in
expression, 72% increase in expression at night and 28% decrease. A scatter
plot of the night versus day expression (supplemental Fig.
S2 A) provides an indication of the range of night/day differences;
this plot used the largest night/day difference observed with any microarray
type. The amplitude of these changes varied from a downward 20-fold to an
upward ~100-fold change. A set of 142 genes (209 probe sets) changes
greater than 4-fold ( Table 1
and supplemental Table S3). Among the genes listed in
Table 1 are those previously
reported to be night/day differentially expressed (see Introduction). Although
the results obtained with the different microarrays are in excellent overall
agreement, there are differences in the absolute magnitude of the night/day
changes, which may reflect different probe set design, biological variation,
technical differences, or a combination. NE/cAMP Signaling Plays a Dominant Role in the Control of Night/Day
Changes in Gene Expression—As noted in the Introduction, 13 genes
were previously known to be differentially expressed on a night/day basis in
the pineal gland and to be controlled by NE/cAMP signaling
( 1,
4). To determine whether
additional genes exhibiting night/day differences in expression are also
controlled by NE/cAMP signaling, we used a well established organ culture
method in which glands are incubated for 48 h, during which time nerve endings
disintegrate. After 48 h glands are treated with NE. A 6-h treatment period
was selected to approximate the time period between lights off and midnight
sampling in the in vivo experiments; a dose of 1 μ m NE
was selected because it is known to selectively activate α-adrenergic
and β-adrenergic receptors in this system
( 6,
10,
12,
13,
56). Gene expression was
studied using the Rat230_2 microarray (experiment C). Approximately 98% of the probe sets that exhibited increased expression at
night also exhibited increased expression following NE treatment, and 85% of
the probe sets that exhibited decreased expression at night also exhibited
decreased expression following NE treatment (supplemental Fig. S2 B,
Table 1, and supplemental Table
S3). This finding supports the conclusion that night/day differences in gene
expression in the pineal gland are due to a large degree to the release of NE
from nerve terminals in the pineal gland
( 4). NE activates adenylate cyclase and elevates intracellular cAMP levels in
the pineal gland. Here it was found that most effects of NE were mimicked by
treatment with 0.5 m m Bt 2cAMP (supplemental Fig.
S2 C, Table 1, and
supplemental Table S3). 95% of the probe sets that exhibited decreased
expression following NE treatment also exhibited decreased expression
following Bt 2cAMP treatment. The finding that Bt 2cAMP
treatment broadly mimics the effects of NE on gene expression provides
evidence that cAMP is the primary second messenger mediating NE control of
gene expression in this tissue. Although it is apparent that NE or Bt 2cAMP treatments change
gene expression in a pattern similar to the changes seen on a night/day basis,
there are striking exceptions, i.e. genes that exhibit marked
night/day changes in expression that exhibit 10-fold lower response to NE
treatment. These include Ccl9, Cd8a, Cyp1a1, Drd4, Mfrp, Per2, Prlr,
Slco1a5, and several genes that do not have gene symbols
( Table 1). Organ Culture Has Marked Effects on Expression of a Minor Component of
Genes—To determine whether changes in gene expression are induced
by organ culture itself, we compared the day values from the in vivo
study to control values from the organ culture study (experiment C). The
normalized expression levels of more than 95% of the genes were unchanged
after organ culture. However, marked changes occurred in 5% of the genes, and
most notable were the greater than 10-fold decreases in gene expression that
occurred in 51 genes (102 probe sets; Table
2), including 11 genes (24 probe sets) that decrease greater than
30-fold, and the greater than 10-fold increase in expression of 13 genes (20
probes sets; Table 2). Some of
the genes that exhibit a greater than 30-fold decrease in expression are
hemoglobin genes, suggesting that in some cases expression of a gene is low
because blood cells that express these genes are present in the pineal gland
when removed for in vivo experiments but are lost from the pineal
gland during culture. | TABLE 2Genes expressed 10-fold higher or lower following organ culture |
Among the nine non-hemoglobin genes that exhibited the largest decrease
(>30-fold) in expression during culture in control glands, six were also
highly rhythmic, suggesting day levels seen in vivo may reflect
physiological regulation by NE and/or another factor. The decrease in
expression during culture may reflect the absence of a factor that is
necessary for NE stimulation of these genes. Highly Expressed Genes That Characterize the Pineal
Gland—Highly expressed genes were identified by determining the
ratio of expression in the pineal gland relative to the median expression
among other tissues (see “Experimental Procedures”), yielding rEx
values. This was done using data obtained in experiment A (RG_U34A microarray)
and in experiment C (Rat230_2 microarray); the median expression values were
based on 23 ( 58) and 7
tissues, respectively. In both cases, brain tissues comprise approximately
half of the tissues sampled. The calculated tissue medians are given in
supplemental Table S4. This effort identified 996 genes (1,654 probe sets) with rEx values of 4 to
~300. One hundred fifty six genes (255 probe sets) had rEx values greater
than 16 ( Table 3; supplemental
Table S4). The rEx values for the pineal gland were compared with those of six other
tissues in an effort to identify other tissues strongly expressing the same
genes. Only in the case of the retina was there a striking similarity in the
genes with high rEx values, consistent with evidence that both tissues evolved
from a common ancestral photodetector
( 64). Approximately 17% of the
highly expressed genes (rEx > 8) in the pineal gland are also expressed in
the retina at similar levels (; Table 4, and
supplemental Table S5). Among these highly expressed genes are those that
encode signal transduction proteins ( e.g. Sag, Pdc, and
Grk1) and genes encoding developmental and regulatory transcription
factors ( e.g. Otx2, Crx, Pax6, and Neurod1). | TABLE 4Genes highly expressed in the pineal gland and/or the retina |
Genes with high rEx values in the pineal gland but not the retina include
genes encoding enzymes required for melatonin synthesis (e.g. Tph1, Gch1,
Ddc, Aanat, Asmt, and Mat2a). Among those with high rEx values
in the retina but not the pineal gland are Rho and Opn1mw,
which encode photosensitive G-protein-coupled receptors. A surprising
observation was that expression of Opn1sw, which encodes another
G-protein-coupled light receptor, is 4-fold greater in the pineal gland; this
is surprising because the mammalian pineal gland is not generally regarded as
being directly photosensitive. Radiochemical in Situ Hybridization Histological Analysis Confirms
Gene Profiling Results Radiochemical in situ hybridization analysis of sagittal brain
sections () was used to
confirm night/day differences in gene expression and to obtain a detailed
anatomical analysis of areas of expression of the genes with a high rEx value
from the microarray analysis. Quantitation of the radiochemical labeling in
the pineal gland revealed the results of histological studies were in
excellent agreement with the microarray results, as regards both the degree of
night/day differences in expression and rEx values
( Table 5). | TABLE 5Quantitative analysis of the in situ hybridization signal in
the pineal gland |
The rEx values provided by microarray analysis were also confirmed by the
results of in situ hybridization; genes with high rEx values were
found by in situ hybridization to be expressed highly or exclusively
in the pineal gland relative to other brain regions on the section
(). In some cases,
however, genes with high pineal rEx values are also strongly and selectively
expressed in other brain regions. For example, Acly is highly
expressed in the habenular nucleus and in the mesencephalic raphe complex;
Dusp1 is highly expressed in the cortex, cerebellum, and thalamus;
Gnb3 is expressed at moderate levels in many brain regions;
Nptx1 is expressed in the cerebellum and dentate gyrus; and
Ttr is very strongly expressed in the choroid plexus
(). In addition, the
results of in situ hybridization provide examples of nocturnally
elevated extra pineal expression of specific genes, including Per2 in
the cerebellum and cortex and Fosl2 in the cortex, and this was
consistently observed upon repeated examination of brain sections from all
animals. Although these findings require further in-depth investigation, this
is beyond the scope of this study. In sections that contained the pineal stalk or deep pineal gland, both
structures were labeled with the same density and pattern as the superficial
pineal gland. An example of this is found in the Crx panel (see
). However, because the
deep pineal gland was absent from most sections because of its small size and
to differences in the plane of section, this was not seen on a regular
basis. It is highly likely that most of these genes are expressed in the dominant
cell type in the pineal gland, the pinealocyte
( 5), as occurs with
Aanat (). However, it is also clear that some of the expressed
genes with high rEx values may be expressed in non-pinealocytes, as
demonstrated by the endothelial cell marker Esm1, which has a high
rEx value (supplemental Table S4) and is expressed in a pattern consistent
with localization in cells confined to the perivascular spaces of the gland,
where endothelial cells occur (). qRT-PCR Analysis Confirms Results of Gene Profiling qRT-PCR was used to confirm and extend in vitro and in
vivo results obtained from microarrays. This effort included genes known
to exhibit large night/day differences in the pineal gland, which serve as
references ( e.g. Aanat, Fosl2, Crem, and Dio2), in addition
to genes that did not exhibit a night/day difference; 44 genes were examined.
qRT-PCR confirmed rhythmic expression that had initially been indicated by
microarray analysis (),
providing evidence that the two-point night/day sampling strategy is a
productive and reliable approach, as a first step toward the identification of
night/day rhythmically expressed genes. In most cases, the amplitude of the
rhythm was greater using qRT-PCR. The use of multiple time points in the
qRT-PCR study provided a more complete profiling of the pattern of transcript
abundance. Comparison of these patterns reveals differences in the timing of
the peak in transcript abundance, consistent with previous reports
( 65,
66). In some cases there was a comparatively large variation at transition times
between low and high expression (e.g. Fosl2 and Cited4),
which may reflect individual variation
(). In the case of
Ttr and Mfrp, such variation does not appear to be
associated with a distinct 24-h pattern of expression; the similar pattern of
variation might reflect a common biological link (i.e. regulation)
not shared by the other genes examined. qRT-PCR was also used to confirm the microarray results from organ culture
studies. The results confirm the microarray results, providing additional
support for the conclusion that night/day differentially expressed genes are
controlled by a NE/cAMP mechanism ( Table
6). The effects of forskolin were also examined; forskolin
increases adenylate cyclase activity
( 67,
68), resulting in an increase
in cAMP. It was found to mimic the effects of Bt 2cAMP and NE,
providing further support for the conclusion that cAMP mediates the effects of
NE. Detection of cis-Regulatory Elements Computational detection of enriched cis-regulatory elements (PWMs)
obtained via submission of gene groups to ModuleMiner
( 63) showed that multiple,
diverse PWMs were enriched in the submitted groups (see supplemental Tables S7
and S8). Individual gene groups were found to be associated with distinct
collections of high scoring PWMs. Notable enrichments of PWM families in
particular gene groups included CREB (rhythmic > 8, <1/8), SP1 (rhythmic
> 8), Oct1 (retina), and Hox (Crx) (pineal and retina, pineal). None of
these were enriched in the control group, which included genes with low rEx
values (<1.5) and night/day expression differences between ½ and 2;
this group was, however, enriched in the Pax4 PWM. Also notable was the
relative absence of PWMs of regulatory transcription factor (TF) sites
compared with basal transcriptional sites in the pineal group. The results of these studies provide the most comprehensive profile of the
pineal transcriptome available, contributing to a more meaningful
understanding of tissue function and providing new evidence of metabolic
pathways and functional capacities of the pineal gland that have been
overlooked or unrecognized. These advances include a greater than 10-fold
increase in the number of genes known to be night/day differentially expressed
in the pineal gland; in addition, this study has revealed that night/day
differential expression is regulated by NE/cAMP signaling. Moreover, this
effort has identified for the first time a large set of highly expressed genes
that may or may not be differentially expressed on a night/day basis. The
lists of night/day differentially expressed genes and highly expressed genes
provide a valuable new data base for future studies of the pineal gland. Gene Expression Considered in Light of the Cellular Composition of
the Pineal Gland Discussion of the results of microarray studies is appropriately preceded
by consideration of the organization and composition of the tissue.
Pinealocytes are the dominant cell in the rodent pineal gland (~95%) and
are recognized to function as a melatonin factory
( 5). Interstitial cells are
located between pinealocytes and generally resemble brain fibrillary
astrocytes. In addition, the mammalian pineal gland contains a dense
vasculature composed of endothelial cells and pericytes of the capillaries in
fairly large perivascular spaces (), with a ground substance consisting of acid
mucopolysaccharides. Some arterioles are also present, which adds the smooth
muscle cell to the list of minor cellular components of the pineal gland. In
addition, minor populations of phagocytes are located in the pineal
perivascular spaces and in the parenchyma of the gland
( 69). Finally, T-cells and
other members of the immune family of cells can be transiently present in the
gland or located in a cluster just outside the gland
( 5). This cellular complexity makes it clear that some of the highly or
night/day differentially expressed genes may be located in cells other than
the pinealocyte. This possibility is made clear by the example of
Esm1, an endothelial cell marker that is known to be highly expressed
in the pineal gland ( 70,
71). Comparison of the
patterns of expression of Esm1 and of pinealocyte markers as revealed
by in situ hybridization histology makes it clear that Esm1
is not expressed in pinealocytes and is likely to mark endothelial cells.
Previous studies with another gene, Id1, have provided a reason of a
different nature that also argues for the importance of consideration of the
cellular localization of transcripts. These studies found that expression of
Id1 follows a daily rhythm
( 26) and is expressed at
levels that are ~8-fold higher than in other tissues. Id1 is
expressed in the pineal gland at very high levels in a small population of
glial-like cells and at lower levels in pinealocytes
( 72). Similarly, in evaluating
the conditional changes in gene expression (night/day, NE,
Bt 2cAMP), it is clear that genes may be differentially expressed in
non-pinealocyte sites and that evidence from histological studies or gene
profiling of purified cell preparations or both is required to establish the
site of expression of a gene of interest to provide the cellular context in
which a gene is expressed. Large Number of Genes That Exhibit Daily Changes in Expression in the
Pineal Gland An unexpected finding from this effort was the large number of genes that
exhibit night/day differences in expression; more than 600 genes are
differentially expressed on a night/day basis more than 2-fold, with ~70%
increasing at night. The far greater number of night/day differentially
expressed genes seen in this study as compared with the less than 40 genes
seen in previous studies ( 26,
55) may reflect several
factors, including the larger number of probe sets interrogated by the RAE230A
microarray (15,923 probe sets, 10,174 genes) and the Rat230_2 microarray
(31,099 probe sets, 13,663 genes), as compared with the two platforms used in
the previous studies, including the Affymetrix RG_U34A microarray (8,799 probe
sets, 4,996 genes) and the Atlas Rat 1.2 cDNA expression array (1,176 genes).
Other factors that may have contributed to the differences are the larger
number of replicates in this study, which is based on a total of 10 pools of
night and of day glands; previous studies used less. In addition, technical
differences and differences in statistical analyses may have contributed to
the number of genes detected. A striking feature of the global change in gene expression is that many
genes are differentially expressed on a night/day basis with a greater than
10-fold amplitude. This characteristic is consistent with the dedicated role
that the pineal gland has in time-keeping. It is likely that the number of night/day differentially expressed genes
will grow in the future for several reasons. One is that the two-point
sampling (mid-day versus midnight) used here may not have revealed
daily rhythms in expression that peak closer to dawn or dusk, which might be
revealed by more frequent sampling. For example, recent studies have revealed
such a rhythm in expression of Pax4
( 73). In addition, the
night/day differentially expressed genes identified in
Table 1 only include those that
have been assigned Entrez Gene identifiers and exceed a 2-fold night/day
threshold. Approximately 500 of these have not been assigned Entrez Gene
identifiers, and although these may represent noncoding RNAs of unrecognized
genes, some may represent unidentified genes. Accordingly, the complete
annotation of the rat genome will further expand the number of genes that are
expressed differentially on a 24-h basis in the pineal gland, as will more
frequent time sampling. Furthermore, the precise nature of the transcripts
encoded by genes cannot be reliably predicted from the results of microarray
studies, because the microarray probes are based on 3′ sequence; this
may not detect differential splicing and/or the actions of alternative
promoters, which can markedly alter the expressed transcript, as seen with
Crem, Slc15a1, Atp7b, and Pde4b
( 21,
23,
24,
74). It is important to note that changes in mRNA may be large and unequivocal,
but one cannot reliably predict that such changes will be translated into
changes in protein. Such a relationship is seen in the pineal gland in the
case of Mat2a, Slc15a1, Pde4b, and Fcer1a
( 22,
23,
75,
76); in these cases, the
transcripts and encoded proteins appear to have similar stability. However,
the daily changes in gene expression in other cases may be only slowly
translated into changes in proteins over a period of days or weeks, not hours,
thereby providing an integrated measure of prior levels of activity,
i.e. changes in the duration of the periods of expression of some
genes may lead to very gradual changes in the level of the encoded protein as
has been shown to be the case in the pineal gland with the Crem
splice variant Icer
( 24,
77) and the adrenergic
receptor Adra1b ( 19).
Likewise, post-translational regulation can cause very rapid changes in
protein, without the encoding mRNA changing as seen with Aanat
( 78,
79). Adrenergic/cAMP Signaling Plays a Dominant Role in Controlling the
Global Changes in Pineal Gene Expression As indicated in the Introduction, previous studies of night/day
differentially expressed genes have provided convincing evidence that NE
controls cAMP accumulation, which in turn controls gene expression. These
studies have established clearly that NE is released at night in the dark and
that when release is blocked, night/day differences in gene expression are
blocked. Moreover, it has been shown repeatedly that the night/day differences
in gene expression can be mimicked in organ or cell culture by treatment with
NE, which elevates cyclic AMP in this tissue. Furthermore, it has been
established that in all cases, the effects of NE on gene expression are
mimicked by elevation of cAMP or by cAMP protagonists. The results of this
study markedly expand the list of adrenergically regulated genes and in doing
so demonstrate that a single regulatory signal can have a profound effect on
the transcriptome of one tissue. A previous study did not find a correlation between night/day
differentially expressed genes and those induced by NE treatment
( 26,
55,
80). It is likely that this
difference is because of the short NE treatment period (1 h) used in the
previous study ( 80). The
current set of experiments used a 6-h treatment period, which more closely
reflects the period animals were in the dark in the in vivo
studies. As discussed above, the number of genes controlled by this signaling
cascade is likely to increase because of annotation issues and also because
only a single 6-h treatment period was studied, which might not detect changes
that occur rapidly and transiently or those that are slow to develop. In
addition, another reason that gene expression might not increase in some cases
is the artificial conditions of organ culture, including use of a defined
minimal medium. The absence of hormones and other factors critical for
expression of some genes might preclude a response to NE or
Bt2cAMP. Accordingly, it is possible that future investigations
will find that NE induction of some genes may require one or more coregulating
factors that are absent from the current organ culture medium. As a result,
the number of genes regulated by NE would increase. The effects of cAMP, as addressed in the Introduction, are likely to be
mediated by cAMP-dependent protein kinase and reflect either a specific
pCREB/CRE interaction or more general epigenetic mechanisms, including
regulation by histone H3 phosphorylation and acetylation. Epigenetic
modulation of chromatin organization could influence access of transcription
factors to regulatory elements in genes. In this case, cAMP can be seen as a
transcriptional regulator acting through epigenetic mechanisms that do not
involve pCREB/CRE interactions. The finding that cAMP suppresses expression of some genes may be explained
by the induction of inhibitory transcription factors, as recently discussed
( 81), and also along
epigenetic lines, because cAMP may act to block access to a regulatory element
by altering chromatin structure. The finding that most of the genes that are differentially expressed on a
night/day basis are also regulated by NE provides evidence for concluding that
these genes are expressed on a circadian basis, i.e. a daily rhythm
will be seen in constant darkness and does not require light/dark transitions.
This conclusion is supported by the fact that the release of NE into the
pineal extracellular space is controlled by the SCN. As discussed in the
Introduction, the SCN/SCG/NE/cAMP regulatory system has been found to regulate
a small number of genes in the pineal gland based on classical biochemical and
physiological evidence. The results of this study provide reason to conclude
that all genes found in this study to be controlled by NE and
Bt2cAMP are physiologically controlled by the endogenous circadian
oscillator in the SCN and that their rhythms can be correctly described as
circadian in nature. Genes That Are Spontaneously Up- or Down-regulated in Organ
Culture The relative level of expression of most genes compared with total gene
expression does not change remarkably after pineal glands are placed in
culture. However, we discovered a subset of genes that exhibited greater than
10-fold positive and negative changes during culture. Decreased gene
expression may reflect the absence from organ culture of a regulatory factor
or hormone normally present in the circulation or of a transmitter
(e.g. dopamine or neuroactive peptides) that is normally released
from the sympathetic nerve endings. Gene expression may also change in
response to the culture environment (95% O2 and defined medium) or
because physiologically relevant local control mechanisms do not function
in vitro. For example, these changes could reflect the absence of
interactions between pinealocytes and the vasculature. It is of special interest to note that there was a marked decrease in the
expression of one of the more important genes in the melatonin synthesis
pathway, Asmt, which encodes the last enzyme in melatonin synthesis.
This has not been reported previously. Studies with rodents have revealed that
expression of this enzyme can be regulated by adrenergic mechanisms
( 82,
83). Other studies of this
gene in Y79 cells, a human retinoblastoma-derived line, indicate that
expression of the gene is controlled by 9- cis-retinoic acid
( 84). However, we have not
been able to prevent the decrease in Asmt expression in the cultured
rat pineal gland by treatment with 9- cis-retinoic acid, NE, or
Bt 2cAMP. 16
Accordingly, the factors controlling expression of this gene remain
unknown. Studies with retinoblastoma cells have also found that
9- cis-retinoic acid regulates expression of Crx and a set of
genes expressed in cones ( 85),
pointing to the need for further studies on 9- cis-retinoic acid and
the role it plays in pineal biology. Similarly, expression of the opsin gene
Opn1sw falls more than 50-fold in culture; its expression is
controlled by T3 signaling
( 86). Accordingly, it is
possible that treatment of pineal glands with T3 and retinoic acid, which are
known to act in concert through heterodimeric receptor complexes, may prevent
some of the spontaneous large changes in gene expression that occur during
culture and that media used for pineal organ cultures should be supplemented
accordingly. As mentioned under “Results,” it is also likely that the
disappearance of some genes reflects the loss of blood cells normally present
in the vasculature, as is probably the case for Hba-a1 and
Hbb, which are expressed in the red blood cells. Functional Implications Functional clustering (see under “Experimental Procedures”) of
the genes selected for inclusion in Table
7 (rhythmically expressed, highly pineal enriched, or both) places
these genes into two broad functional categories. These are as follows: first,
groups of genes that participate in specialized functions; and second, genes
that have a nonspecialized, more common role in cell biology. Specialized Functional Gene Groups Two of these gene groups are predictable from previous knowledge (melatonin
production and phototransduction), whereas two other groups (immune response
and T3/retinoic acid signaling) are less predictable. Melatonin Production—Most predictable are those genes that
code for proteins involved in melatonin production, including both enzymes
that function directly in the melatonin synthesis pathway and cofactors
required for these enzymes. It is also very likely that many of the genes
encoding proteins dedicated to protein phosphorylation and to cAMP and calcium
signaling are involved in the control of melatonin synthesis, in part through
regulation of Aanat expression and processing of AANAT protein. Phototransduction—Gene expression similarities between the
pinealocyte and the retina have been documented before, but they were limited
to less than 20 genes. This study greatly extends the number of genes known to
be highly expressed primarily in pineal/retina to over 55 genes (63 probe
sets). A common expression pattern reflects well documented evolutionary
relationships between the pineal gland and retina
( 54,
87,
88); but by revealing the real
extent of pineal gland/retina coexpression, we have freshly questioned the
functional relevance of phototransduction-related genes in the mammalian
pineal gland (see below). Highly expressed pineal gland/retina genes also
include transcription factors ( Otx2, Crx, Neurod1, and Pax6)
that likely drive the common transcriptional outputs in these tissues. The
work done here also led to the finding that both tissues express high levels
of Pax4, an ortholog of Pax6
( 89). Importantly, adult
expression of these transcription factor genes implies roles in maintenance of
cellular phenotype, in addition to roles during development. It is of interest to note that the expression of one opsin gene in the
pineal gland is higher than that in the retina; Opn1sw is expressed
4-fold higher in the pineal gland as compared with the retina. In contrast,
Opn1mw and Rho are expressed at 18- or >400-fold higher
in the retina, respectively. This and the finding that the pineal gland
expresses many genes involved in phototransduction provide genetic evidence
that this tissue might detect light; however, there is no evidence that the
adult pineal gland has this capacity. Photodetection by neonatal pineal gland
has been reported
( 90– 92);
however, the underlying mechanism involved has not been elucidated, and it is
not clear whether this involves the phototransduction system that operates in
the retina or another mechanism. Pineal Opn1sw may be functionally
vestigial in the adult pinealocyte as regards detection of light. However, it
is also possible that it plays a passive role in signal transduction that does
not involve detection of light; for example, it might influence signal
transduction by binding to receptors and other proteins involved in adrenergic
signaling. Immune/Inflammation Response—Our identification of a large
cluster of immune/inflammation-associated genes expressed in the rat pineal
gland reflects similar findings in an avian species
( 88) and is therefore of
interest. A potential, immune-related, functional specialization for the
pineal gland is indicated by the presence of perivascular phagocytes that act
as antigen-presenting cells
( 69), and also by the strong
expression of Fcera1 (a receptor dedicated to IgE signaling), and a
related gene in the pineal gland
( 76). Further study of these
genes may lead to a better understanding of the role of the pineal gland in
the immune response. T3/Retinoic Acid Signaling—Our finding of high levels of
transcripts associated with T3/retinoic acid signaling suggests a potential
functional specialization related to this signaling pathway. Previously there
has been a report of effects of T3 on melatonin synthesis and a substantial
body of evidence indicating that Dio2 is night/day differentially
expressed in this tissue
( 93– 95).
Together with reports of effects of T3 on retinal function
( 86,
96– 99),
the accumulated evidence argues for future studies that involve specific
functional interventions of this signaling pathway in both the pineal gland
and retina. Nonspecialized Functional Gene Groups Cellular Signaling—A functional cluster of “cellular
signaling” genes derived from the genes of
Table 7 is consistent, at least
in part, with the evidence of adrenergic control of pineal function, which
involves a broad range of signal transduction-related proteins. As indicated
above, many of these are likely involved in regulating melatonin production.
The high rEx of Drd4 and known rhythmic pattern of expression
( 26) point to a related role
for dopamine in pineal function because dopamine is colocalized in pineal
nerve processes as a precursor of NE; as such, it is likely to be released
with NE. The role of dopamine in the pineal gland remains to be fully
established. It is of interest that Drd4 expression was not elevated
by NE or Bt 2cAMP in organ culture. This raises the question of how
expression of this gene is regulated in vivo. This study has also highlighted groups of genes that have not received
significant attention. These include genes related to prostaglandin synthesis
and the lipoxygenase 15 pathway, which leads to production of hepoxilins and
related compounds
( 100– 106).
The regulation and function of these pathways and their cellular sources and
targets requires further investigation. Other less well studied genes in a
pineal context included the receptors for prolactin, acetylcholine, GABA,
glutamate, and interleukin, suggesting roles in pineal gland signaling.
Acetylcholine receptor expression is consistent with anatomical evidence
( 5) that reveals the presence
of nonsympathetic, probably parasympathetic, nerve fibers in the pineal gland
of the rat and with biochemical studies that have shown the presence of both
muscarinic ( 107) and
nicotinic ( 108) cholinergic
receptors. Furthermore, it has been shown that a cholinergic input to the rat
pineal gland causes the release of glutamate, which has been reported to act
via glutamate receptors on the pinealocyte membrane to inhibit melatonin
synthesis ( 109,
110). Genes Dedicated to Small Molecule Biology—Included among
this cluster of genes is the taurine transporter Slc6a6, perhaps
explaining the high concentration of taurine in the pineal gland
( 111); this gene is also
highly expressed in the retina, another tissue with a high concentration of
taurine ( 112). Genes that
regulate metal homeostasis are also clustered here. Zn 2+ is
essential for the synthesis of melatonin, because it is required by Gch to
generate biopterin ( 113,
114), the cofactor for Tph1,
the first enzyme in melatonin synthesis. Another essential role of metals of
special relevance to pineal physiology is Sn +, which is essential
for T3 signaling because it is required by Dio2
( 115). Cell:Cell and Cell:Extracellular Matrix Contacts—Genes
encoding proteins involved in cell:cell and cell:extracellular matrix contacts
represent another group of genes that have not been well studied in the
context of the pineal gland. This includes several members of the cadherin
family, which form homophilic Ca2+-dependent associations.
Knowledge of the site of expression of these genes may be of practical utility
in identifying, purifying and immobilizing populations of cells recovered from
the pineal gland. Circadian Clock Genes—Generally absent from the list of
strongly rhythmic or highly expressed
( Table 7) genes are circadian
clock genes, exceptions being Per2 and Rorb. Expression
levels of Arntl, Clock, Per1, Per2, Per3, Cry1, Cry2, and
Arnt are less than 4-fold the median tissue level of expression.
Reports in the literature have established that these genes are expressed in
the pineal gland, some following a 24-h pattern
( 116– 119).
Their absence from the lists of highly expressed or highly rhythmic genes may
reflect a relatively unimportant role that the circadian clock system plays in
the mammalian pinealocyte in generating daily rhythms. The strong rhythm of
Per2 may reflect a related role in biological time-keeping, in that
it might influence the dynamics (intensity, duration) of the responses of
genes with E-boxes, which includes Aanat
( 120,
121). cis-Regulatory Elements A question raised by our finding of large groups of pineal gland-specific
and night/day differentially expressed genes is whether these groups of genes
share common transcriptional regulatory DNA sequences. This question was
addressed using ModuleMiner
( 63) using multiple groups of
genes as inputs. In general, the results of this effort did not provide an
indication that pineal specificity or night/day differences can be explained
by the presence of a few dominant regulatory elements. This may relate to
inherent limitations of this analysis, which include the analysis of only 10
kb 5′ to the transcription start site and filtering that eliminates
genes that do not exhibit human-mouse conservation of regulatory elements.
Conversely, it is possible that rhythmic or relatively high expression of
genes in the pineal gland reflects multiple parallel/hierarchical regulatory
cascades of such complexity that they would be impenetrable to this mode of
analysis. However, some of the results of this analysis (see supplemental
Table S7) are worthy of further consideration. Enrichment of a CREB family PWM in the group of highly rhythmic genes
(rhythmic >8, <1/8) is consistent with known, cAMP-driven, mechanisms of
gene regulation in the pineal gland, as discussed in the Introduction. The
NE/cAMP cascade is evident from the results of the experiments presented here,
in which gene expression was broadly enhanced by treatment with
Bt 2cAMP, a stable cAMP analog; with forskolin, an activator of
adenylate cyclase; or with NE, which elevates cAMP in the pineal gland.
Conversely, the absence of a similar enrichment in less highly rhythmic genes
suggests the presence of alternative mechanisms. The enrichment of SP1 PWMs in
moderately rhythmic and highly rhythmic groups is interesting in this context
and may warrant further investigation because a previous study has indicated a
role for SP1 in pineal gene rhythmicity
( 122). Surprisingly, we did not observe widespread enrichment of Hox family PWMs
across the tissue-specific (pineal and retina) groups of genes; this is
surprising because the Crx and Otx transcription factors are known to regulate
pineal gland- and retina-specific gene expression, as discussed in the
Introduction ( 31,
32,
123). Our results show the
Crx PWM to be enriched only in the combined submission of pineal only and
pineal/retina groups. This finding may indicate a more limited (pineal gland-
and retina-related) role for Hox family-related mechanisms than is currently
believed. With respect to the retina, our analysis has revealed strong
enrichment of another PWM family (Oct1) that is recognized as one PWM
family/transcription factor that is associated with the visual perception GO
term (GO:0007601; Matbase, Genomatix). With respect to the pineal gland, an
intriguing finding of the ModuleMiner analysis was the relative absence of
conserved regulatory transcription factor sites in the “pineal
only” group. This is an interesting result in which ModuleMiner detects
only a few enriched sequences, and these are primarily either core promoter
elements such as initiator and cap or CEBP, all of which are among the most
common sites found in eukaryotic promoters
( 124). This finding taken
together with the Pineal only and Pineal/retina result is suggestive of common
cis-regulatory rules for pineal/retinal tissues but the absence of a
pineal only mechanism, indicating either a novel cis or
trans ( e.g. microRNA) level of control not detected by this
analysis or that the pineal gland-specific cascade is controlled by multiple
unrelated mechanisms. Another surprising finding of the ModuleMiner analysis was the enrichment
of Pax4 sites within the control group of pineal genes, genes expressed in
this tissue that are neither rhythmic nor highly expressed relative to other
tissues. Given the high relative expression of Pax4 in the rat pineal gland
( 73), this may point to a
negative mode of regulation in which the “control” gene group is
suppressed via a Pax4-related mechanism. In this respect, previous studies
have provided evidence of repressive actions of Pax4
( 125). Final Statement The results of these studies provide investigators with a rich and
comprehensive genetic profiling of the rodent pineal gland and should provide
a sound foundation for future investigations of this tissue focused on the
factors controlling developmental and rhythmic gene expression. The evidence
provided here also provides a basis for future research on the role of
T3/retinoic acid signaling in the pineal gland and on the role of this tissue
in the immune/inflammation response. The finding that most genes that exhibit
daily changes in expression are regulated by NE/cAMP signaling raises
intriguing questions regarding the mechanisms involved, especially the role of
epigenetic events. Analysis of the factors regulating these large changes in
gene expression and expression of genes at relatively high levels in the
pineal gland may be of interest to investigators studying these genes in other
tissues, especially the retina. The indication from the results of analysis of
cis-regulatory elements that transcript abundance in the pineal gland
involves PWMs, which have not previously appeared in the pineal literature,
opens new doors to the analysis of the molecular control of gene expression in
this tissue. The support of John Walker and Andrew Su (Novartis Research Foundation) in
facilitating the use of data in the Novartis Research Foundation Data Base is
greatly appreciated, as is the expert histological assistance of Ursula
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