Unique, Intersecting, and Overlapping Roles of C/EBP β and CREB in Cells of the Innate Immune System
Unique, Intersecting, and Overlapping Roles of C/EBP ? and CREB in Cells of the Innate Immune System
Jason L. Larabee
Jimmy D. Ballard
OPEN CREB and C/EBP ? signaling pathways are modulated during inflammation and also targeted by Bacillus anthracis edema toxin (ET), but how these factors individually and jointly contribute to changes in immune cell function is poorly understood. Using CRISPR/Cas9 gene editing, macrophage cell lines lacking CREB and isoforms of C/EBP ? were generated and analyzed for changes in responses to LPS, ET, and IL-4. Macrophages lacking C/EBP ? suppressed induction of IL-10 and Arg1, while IL-6 was increased in these cells following exposure to LPS. Examination of C/EBP ? isoforms indicated the 38 kDa isoform was necessary for the expression of IL-10 and Arg1. ChIP-Seq analysis of CREB and C/EBP ? binding to targets on the chromosome of human PBMC identified several regions where both factors overlapped in their binding, suggesting similar gene targeting or cooperative effects. Based on the ChIPSeq data, a panel of previously unknown targets of CREB and C/EBP ? was identified and includes genes such as VNN2, GINS4, CTNNBL1, and SULF2. Isoforms of a transcriptional corepressor, transducin-like enhancer of Split (TLE), were also found to have CREB and C/EBP ? binding their promoter and were up regulated by ET. Finally, we explore a possible layer of C/EBP ? regulation by a protein complex consisting of adenomatous polyposis coli (APC) and PKA. Collectively, these data provide new insights into the role of CREB and C/EBP ? as immunosignaling regulators and targets of an important bacterial virulence factor.
Edema toxin (ET), the binary combination of Bacillus anthracis protective antigen (PA) and edema factor (EF),
intoxicates cells and generates high levels of intracellular cAMP through the calmodulin-dependent adenylate
cyclase activity of EF1,2. Studies have shown that a variety of intracellular signaling molecules (APC, Notch,
GSK3, PKA) and transcriptional regulators (TLE, CREB, C/EBP?, RBP-J, ?-catenin) are modulated in cells intoxicated
by ET, yet little is known about how the collective network of ET-related targets leads to changes in immune cell
function3?6. Based on the current understanding of these signaling molecules, their modulation could lead to a
combination of both pro-inflammatory and anti-inflammatory events in ET-intoxicated cells7?10. Moreover, the
extent to which these molecules function independently, in parallel, or intersect to drive transcriptional changes
in response to cAMP remains to be defined.
ET activates signaling cascades primarily through the actions of PKA wherein cAMP binds the regulatory
subunits of the PKA holoenzyme leading to the dissociation of the catalytic subunits from regulatory subunits11. The
released catalytic subunits then phosphorylates CREB, the prototypical cAMP responsive transcription factor12,
as well as a multitude of other cellular factors13. PKA activation results in transcriptional changes through both
the canonical PKA/CREB axis as well as through non-canonical signaling involving PKA interfacing with factors
such as those commonly associated with Wnt or Notch signaling3?6. As an example of non-canonical signaling,
ET activates GSK-3? in the nucleus of macrophages leading to the phosphorylation of transcriptional
regulators such as ?-catenin and C/EBP ?3,6. Interestingly, ET-activated GSK-3? phosphorylates CREB at Ser 1294,14 in
addition to PKA phosphorylation at Ser 133, thus demonstrating a point of convergence between canonical and
non-canonical signaling. Further study of ET-activated GSK-3? found GSK-3? phosphorylates C/EBP ? within a
scaffolding complex supported by adenomatous polyposis coli (APC)6, a large multi-domain protein important
for tumor suppression and Wnt signaling. Intriguingly, increased CREB activity promotes the expression of
C/EBP ?15, which is yet another point where canonical and non-canonical signaling converges.
Despite the recent progress in dissecting the immunomodulatory activity of ET, several unanswered questions
remain. For example, whether ET exploits physiologically relevant signaling events to promote
immunosuppression or if ET causes aberrant signaling events leading to cellular dysfunction is not known. This stems in part
from an incomplete understanding of the roles CREB and C/EBP ? play in modulating immune responses. To
this end, in the current study we took a multi-pronged approach to assess the general characteristics of activation
of these pathways in response to both inflammatory stimuli and ET. CRISPR/Cas9 gene editing generated stable
macrophage cell lines lacking CREB and isoforms of C/EBP ?, and these cells were tested for changes in responses
to a variety of factors. In the second part of this study, ChIP-seq analyses were performed on peripheral blood
mononuclear cells (PBMC) to determine the profiles of CREB and C/EBP ? localization throughout the genome.
Finally, using a combination of co-immunoprecipitation approaches, we show that PKA binds and interacts with
the APC complex.
Contributions of CREB and C/EBP ? to the expression of immune modulating factors. In the
first part of this study, macrophage cell lines lacking CREB or C/EBP ? expression were generated using CRISPR/
Cas9 gene editing in RAW 264.7 cells. As shown in Fig.?1A, CREB was undetectable in cells having undergone
CRISPR/Cas9 gene editing of the CREB encoding gene. Likewise, all 3 isoforms of C/EBP ? were undetectable in
macrophages subjected to CRISPR/Cas9-mediated editing of the C/EBP ?-encoding gene (Fig.?1C). This CRISPR/
Cas9 genetic manipulation yielded two stable cell lines termed RAW?CREB and RAW?total C/EBP?. RAW?CREB cells
were examined for changes in levels of C/EBP ?, and RAW?total C/EBP? cells were examined for changes in levels
of CREB. Neither transcription factor appeared to impact the expression of the other in these cells (Fig.?1B,C).
To examine the contributions of CREB and C/EBP ? responses to inflammatory stimuli in macrophages,
we treated RAW?CREB, RAW?total C/EBP?, and the parental cell line with 1 ?g/ml of LPS for 6 h and then measured
transcript levels from Il6, Tnfa, Inos, and Il10. As shown in Fig.?1D, relative to the parental cells Il6 transcripts
were modestly (2-fold) increased in RAW?CREB, whereas RAW?total C/EBP? showed about a 7-fold increase in Il6
transcripts. As shown in Supplementary Fig.?S1, ELISA data demonstrated RAW?total C/EBP? produced about 10
fold more IL-6 than parental cells after 2 to 6 h of LPS treatment. Moreover, Tnfa transcripts were slightly (2-fold)
increased in RAW?total C/EBP? and were not significantly changed in RAW?CREB (Fig.?1E). Inos transcript levels
were reduced in RAW?CREB, but slightly increased in RAW?total C/EBP? (Fig.?1F). Finally, as shown in Fig.?1G, LPS
stimulated an increase in Il10 transcripts in RAW?CREB macrophages whereas Il10 transcripts were almost
completely suppressed in RAW? total C/EBP? when compared to the parental cell line. These results suggest that C/EBP
? may be more prominently involved than CREB in producing anti-inflammatory cytokines and attenuating the
production of pro-inflammatory cytokines.
C/EBP ? isoforms differentially influence expression of IL-10 and IL-6. C/EBP ? can be expressed
as 3 protein isoforms generated from a single mRNA containing 3 translation start sites (Fig.?2A). Whether each
isoform has unique contributions to transcriptional responses following exposure of macrophages to LPS is not
known. Each of the three isoforms share a common carboxy-terminal DNA binding and dimerization domain,
but depending on where translation is initiated differ in the extent of transactivating and regulatory domains that
determine binding partners and regulation (Fig.?2A). To examine these different isoforms, two additional gRNA
directed towards the C/EBP ? gene were chosen and used to perform CRISPR/Cas9 editing. One gRNA targeted
a sequence near the first start site on the C/EBP ? mRNA in order to edit out the 38 kDA form of C/EBP ?. The
other gRNA disrupted the second start site and produced a clone lacking both the 38 kDa and 36 kDa isoforms
of C/EBP ?. Isoform-specific disruption of C/EBP ? was confirmed by immunoblot (Fig.?2B) and yielded two cell
lines termed RAW?38 C/EBP ? and RAW?38/36 C/EBP ?. As shown in Fig.?2D, RAW?38 C/EBP ? and RAW?38/36 C/EBP ? did
not produce detectable Il10 transcripts in response to LPS. RAW?38 C/EBP ? and RAW?38/36 C/EBP ? expressed elevated
levels of Il6 transcripts following LPS treatment; however, these increases in Il6 transcripts were not as high as
RAW?total C/EBP? and were not statistically significant (Fig.?2C). These results indicate the 38 kDA form of C/EBP ?
is necessary for the induction of IL-10 while all 3 isoforms contribute to increases in IL-6.
C/EBP ? and CREB contribute to cAMP-induced expression of IL-10 and Arg1. Next, experiments
examined how C/EBP ? isoforms contributes to anti-inflammatory responses induced by stimuli such as IL-4
and ET. IL-10 production was analyzed in the CRISPR/Cas9 edited RAW 264.7 cells exposed to ET, IL-4, or a
combination of ET and IL-4. As shown in Fig.?3A, Il10 transcript levels in RAW?CREB were reduced by about 25%
compared to the parental RAW 264.7 cells. Evaluation of RAW?total C/EBP? and the isoform specific knockout cell
lines (RAW?38/36 C/EBP ? and RAW?38 C/EBP ?) revealed that Il10 transcript levels were suppressed in each treatment
condition by approximately 75% (Fig.?3A).
Arg1 is a marker of anti-inflammatory M2 macrophages and we were curious to know if CREB and/or C/EBP
? might influence the expression of Arg1. As shown in Fig.?3B, treatment with ET increased levels of Arg1
transcripts in RAW 264.7 and this was further enhanced in RAW?CREB cells. Conversely, Arg1 transcript levels were
nearly undetectable in RAW?38 C/EBP ?, RAW?38/36 C/EBP ?, and RAW? total C/EBP ? cells treated with ET (Fig.?3B). To
determine if this effect was unique to treatment with ET, Arg1 transcript levels were also measured in cells treated
with IL-4. Arg1 levels were not repressed in RAW?CREB, RAW?38 C/EBP ? and RAW?38/36 C/EBP ?, but were reduced by
almost 80% in RAW? total C/EBP ? cells (Fig.?3B). In agreement with work from other groups16, the combination of
IL-4 and ET resulted in a synergistic increase in Arg1 (Figs.?3B,C). Following the trend of the ET only treatment,
Arg1 induced by ET/IL-4 was abolished in all C/EBP ? knockout cell lines (RAW?38 C/EBP ?, RAW?38/36 C/EBP ?, and
RAW? total C/EBP ?), while a slight increase in Arg1 was observed in RAW?CREB (Fig.?3C).
C/EBP ? binds IL-6 and IL-10 genes in human PBMC. Because C/EBP ? responds to ET and is
critical for mediating macrophage responses, in the next experiments a ChIP-seq approach was used to investigate
C/EBP ? localization on chromosomes in human PBMC. Using ChIP- validated antibodies specific for C/EBP ?,
we identified ?35,400 peaks (i.e. putative sites of interaction with the chromosome). Motif analysis demonstrated
the most intense peaks center on C/EBP ? targeting DNA binding motifs. C/EBP ? binding was detected in both
the promoter and enhancer of the IL6 gene and in the promoter of the IL10 gene (Figs.?4A,B). Interestingly,
C/EBP ? was not detected in association with ARG1 (Fig.?4C), suggesting a possible alternative mechanism of
ARG1 regulation by C/EBP ?. ChIP-seq was also performed on PBMC with ChIP-validated antibodies against
CREB and ?21,464 peaks were detected. Analysis of the most intense peaks found these centered on the CREB
DNA binding motif (TGACGTCA). Examining the IL10 and ARG1 genes did not reveal CREB binding in
proximity to these genes (Figs.?4B?C). For the IL6 gene, CREB binding was observed within the gene at?a position also
occupied by C/EBP ? (Fig.?4A).
cAMP enhances recruitment of C/EBP ? and CREB to a subset of PBMC genes. We also
performed the ChIP-seq experiment on PBMC treated with cAMP elevating agents to determine if we could detect
the recruitment of C/EBP ? or CREB to DNA binding sites. As shown in Supplementary Table?S1, few sites were
found that recruited C/EBP ? during elevated cAMP in PMBC, with only 141 regions enriched for C/EBP ? under
this treatment condition. In the case of CREB, cAMP elevations in PBMC also induced the recruitment of CREB
to a limited number of genes with 249 regions enriched for CREB (Supplementary Table?S2). Figure?5 shows some
selected examples of genes that have C/EBP ? and/or CREB recruited to putative enhancer regions after cAMP
elevations in PBMC. As shown in Fig.?5A, cAMP induces C/EBP ?, but not CREB, to bind the VNN2 gene
downstream (+17,355) of the transcription start site. Experiments next determined if the cAMP-dependent enrichment
of C/EBP ? at the VNN2 gene in PBMC correlated with increased gene expression in purified human monocytes
with increased cAMP. Thus, in human monocytes treated with ET or 6MB-cAMP, RT-qPCR results revealed
robust expression of VNN2 by cAMP (Fig.?6A). A common profile in these PBMC was the cAMP-dependent
recruitment of CREB to regions that are constitutively occupied by C/EBP ?. In Fig.?5B?D, examples of this
phenomenon in PBMC are provide by 3 genes (GINS4, CTNNBL1, and SULF2). In these examples, cAMP increases
result in CREB enrichment at sites downstream of the transcription start site (GINS4 +15,935; CTNNBL1 +7994;
and SULF2 +45,412) (Fig.?5B?D). As a demonstration of the possible consequence of recruiting CREB, transcript
levels were evaluated in human monocytes and found that cAMP elevations (ET or 6MB-cAMP) induce the
expression of GINS4, CTNNBL1, and SULF2 (Fig.?6B?D).
CREB and C/EBP ? bind TLE promoters in human PBMC. The ChIP-seq analysis also identified genes
with CREB and C/EBP ? constitutively occupying promoter regions. TLE1 had such a profile as shown by C/
EBP ? binding at position ?295 and CREB binding at position ?300 (Fig.?7A) in the absence of elevated cAMP.
Interestingly, we have previously demonstrated that TLE1 is induced by cAMP in human monocytes5 suggesting
some cAMP responsive promoters are primed with cAMP transcription factors under basal conditions. This
observation is also noteworthy because recent work by other groups has revealed that TLE1 is a negative regulator
In addition to TLE1, the TLE family of transcriptional corepressors is composed of other members such as
TLE3 and TLE4. As such, experiments next evaluated TLE3 and TLE4 to determine if C/EBP ? or CREB bind
the promoter of these genes in a similar fashion as TLE1. As shown in Fig.?7B?C, TLE3 and TLE4 had C/EBP ?
and CREB in their promoters. Transcript levels of TLE3 and TLE4 were also examined in human monocytes in
response to elevated cAMP. As shown by RT-qPCR, both ET and 6MB-cAMP induced TLE3 and TLE4 transcripts
similarly to TLE1 (Fig.?7D?F). Considering the role of TLE1 in immunosuppression, we also asked whether
activating inflammatory processes in monocytes could alter how cAMP impacts TLE expression. Thus, human
monocytes were exposed to LPS in the presence and absence of ET or 6MB-cAMP and expression levels of TLE1,
TLE3, and TLE4 were measured. As shown in Fig.?7D,F, LPS treatment increased TLE1 transcript levels and
caused a modest increase in TLE4 expression. Conversely, LPS completely reversed the cAMP-mediated
induction of TLE3 in human monocytes (Fig.?7E). Thus, while each of the three TLE genes contain C/EBP ? and CREB
in their promoter regions and can be modulate by cAMP, TLE3 appears to differ in its susceptibility to modulation
PKA intersects with the non-canonical cAMP pathway through interactions with APC. In pre
vious work, we found that C/EBP ? was part of a cAMP responsive protein complex including APC and GSK-36.
Within this complex, GSK-3 phosphorylates and activates C/EBP ? in an APC dependent manner6. Considering
this, we were curious to know if components of the cAMP pathway such as PKA also associated with APC.
Therefore, the catalytic subunit of PKA (PKA cat ?) was immunoprecipitated from RAW 264.7 cells and the
sample was probed using antibody to APC. In the reciprocal analysis, APC was immunoprecipitated and the
sample was probed using antibody to the catalytic subunit of PKA. As shown in Fig.?8A, the results of this analysis
suggest PKA and APC interact within the cell. Next an analysis was performed to determine if increased levels
of cAMP could alter binding interactions between APC and PKA cat ?. Thus, RAW 264.7 cells were treated
with ET or 6MB-cAMP and binding interactions between APC and PKA cat ? was examined. Interestingly
when macrophages were treated with ET or 6-MB-cAMP, PKA cat ? was released from APC as shown in the
co-immunoprecipitation experiment in Fig.?8B.
To further define the binding mechanism between PKA and APC, interactions between APC and the PKA
regulatory subunit (PKA R) were analyzed. In this experiment, an immunoprecipitation was performed that
determined whether PKA RI or RII was able to bind APC. Results from this experiment demonstrated that the
RII subunit of PKA bound APC (Fig.?8C) while the RI subunit did not bind APC. This interaction was observed
in both RAW 264.7 cells (mouse macrophage cell line) and primary human macrophages (Fig.?8C). Because
RII commonly binds to A-Kinase Anchor Proteins (AKAP), we tested the possibility that APC bound the RII
through an AKAP like mechanism. As shown in Fig.?8D, PKA RII has a N-terminal docking and dimerization
(D/D) domain responsible for binding AKAP. Thus, we determined if APC was able to bind a mutant form of the
PKA RII that lacks the D/D domain (PKA RII ?D/D). In these experiments, PKA RII and PKA RII ?D/D were
expressed in RAW264.7 cells by transducing these cells with a retrovirus containing a HA-tagged form of PKA RII
or PKA RII ?D/D. As shown in Fig.?8E, each form of HA-tagged RII expressed at comparable levels in transduced
RAW 264.7 cells. Next, we immunoprecipitated APC from the cells transduced with HA-tagged RII or HA-tagged
RII ?D/D and then performed an immunoblot probing with antibodies against the HA-tag. As shown in Fig.?8E,
HA-tagged RII was detected but HA-tagged RII ?D/D was not in these APC immunoprecipitations. These data
indicate that the D/D domain is necessary for PKA R II to bind APC, suggesting APC/PKA interactions are
similar to AKAP/PKA interactions. Overall, these results indicate PKA, like other ET-modulated factors, interacts
with APC and this may be a crucial site for localized targets of cAMP signaling.
While much is known about how B. anthracis as well as other pathogens elevate cAMP within host cells2, less is
known about how cAMP reprograms cell such as macrophages. Many of the signaling activities of cAMP are
carried out by the activation of PKA and the basic region leucine zipper transcription factors CREB and C/EBP ?4,6,10.
These cAMP effectors are used ubiquitously by cells, but limited information is available describing how these
proteins affect macrophage phenotypes after cAMP is elevated during infection. Here, a multi-pronged approach
was undertaken to examine 3 critical nodes of the vast cAMP signaling network in monocytic cells. First,
CRISPR/Cas9 gene editing in a macrophage cell lines was used to examine the contributions of CREB and
isoforms of C/EBP ? to inflammatory responses and cAMP elevations (Figs?1?3). In the second group of studies,
Results from the ChIP-seq also indicate several unexpected layers of complexity in the CREB, C/EBP ?
regulatory network. In contrast to past models describing CREB function and in agreement with recent ChIP-seq
data23,24, few DNA regions exhibit cAMP dependent recruitment of CREB or C/EBP ?. Most of the DNA binding
regions is constitutively occupied by CREB, C/EBP ?, or both. CREB appears to be more responsive than C/
EBP ? in regards to cAMP-inducible binding, as CREB was recruited to 249 regions and C/EBP ? recruited to
149 regions (Supplementary Information). The cAMP-inducible binding of CREB and C/EBP ? is located
outside regions that are considered promoters and are most likely enhancers, which is observed with genes such as
CTNNBL1, SULF2, GINS4, and VNN2 (Fig.?5). Interestingly, two of these genes (SULF2 and VNN2) lack CREB
or C/EBP ? binding prototypical promoters (Fig.?5). Because the ChIP-seq data is from PBMC, we determined if
these 4 genes were also induced in purified human monocytes; therefore, we measured transcript levels and found
that these 4 genes were induced in a cAMP dependent manner (Fig.?6). Of these 4 genes, VNN2 is recognized as
a factor involved in inflammation and leukocyte migration25 as well as can be upregulated by cAMP increases26.
The ChIP-Seq data in PBMC also corroborated our previous biochemical findings that increased cAMP
leads to the up regulation of TLE1 in human monocytes. Both CREB and C/EBP ? were found to bind at
promoter regions of TLE1 as well as the promoters of other TLE isoforms, TLE3 and TLE4 (Fig.?7). The binding
of CREB and C/EBP ? to these promoters was not induced by increased cAMP; however, we confirmed that
TLE3 and TLE4 respond to cAMP in monocytes by demonstrating that transcript levels of these TLE isoforms
were upregulated in purified human monocytes (Fig.?7). Transcript levels were also measured under
inflammatory conditions created by LPS stimulation, and results revealed that cAMP-induced TLE1 and TLE4 were
not reduced by LPS (Fig.?7). However, cAMP-induced TLE3 was reduced to basal levels after LPS stimulation,
suggesting an unexplored layer of regulation in which LPS-induced signaling represses transcription of TLE3
(Fig.?7). TLE are a family transcription corepressor proteins (TLE1-4) recruited to genes through interaction
with DNA binding proteins such as Hes and TCF/LEF27. Recently other groups have discovered that TLE1 and
TLE4 help control inflammation and are involved in anti-inflammatory phenotypes17,18,28,29. Our observations
that TLE is upregulated by increased cAMP suggest that TLE contributes to the mechanism of cAMP mediated
In the final group of experiments, we examined the mechanism of how cAMP may regulate the activity of C/
EBP ?. Our previous work had shown that C/EBP ? is part of a protein complex that included APC and GSK-36;
however, we were unsure how this complex connected to cAMP signaling. We were surprised to discover that
APC was able to bind both the catalytic and regulatory subunits of PKA (Fig.?8). Further work demonstrated that
APC demonstrated characteristics of a AKAP, such as releasing the catalytic subunit of PKA after cAMP
stimulation and binding the regulatory subunit through the D/D domain (Fig.?8). APC is known to scaffold interactions
between a range of signaling proteins and was recently linked to inflammatory activities30. Thus, APC functioning
as a center for cAMP signaling and regulating inflammation is probable and a subject of future studies.
Institutional compliance. Experimental protocols, environmental and biological safety, and research
involving materials from human subjects were reviewed and approved by the institutional biosafety committee
and institutional review board. All experiments were carried out in accordance with relevant guidelines and
Reagents. The membrane-permeable cAMP analog, N6-monobutyryladenosine 3?,5?-cyclic monophosphate
(6MB-cAMP), was obtained from Biolog (Bremen, Germany). LPS (catalog number tlrl-eblps) was acquired
from Invivogen (San Diego, CA). IL-4 (catalog number 214-14) was purchased from Peprotech (Rocky Hill,
NJ). PA (catalog number 171E) and EF (catalog number 178a) were purchased from List Biological Laboratories
Cell culture and isolation of primary human cells. RAW 264.7 were obtained from the ATCC and
cultured in DMEM with 10% FBS. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats
derived from deidentified human whole blood obtained from the Oklahoma Blood Institute under a protocol
categorized as exempt by the Institutional Review Board at the University of Oklahoma Health Sciences Center. To
isolate PBMC, buffy coats were diluted in complete RPMI 1640 medium (10% FBS plus penicillin/streptomycin),
and then applied to density gradient centrifugation with Histopaque 1077 (Sigma-Aldrich). In some experiments,
monocytes were purified from PBMC using a monocyte isolation kit acquired from Miltenyi Biotec (Auburn,
CA). This kit utilizes a negative selection method in which all non-monocytes are labeled with magnetic beads
and separated from monocytes. The isolated monocytes were diluted into complete RPMI 1640 medium at a
concentration of 1.0 ? 106 cells/ml and then exposed to experimental conditions. In other experiments, macrophages
were cultured from the human PBMC. To culture human macrophages, monocytes were first isolated by adhesion
to tissue culture plastic and the resulting monocytes were added to 6-well plates in RPMI 1640 media containing
7.5% heat inactivated autologous human fibrin-depleted plasma, penicillin/streptomycin, and 500 ng/ml M-CSF
(Peprotech). The culture was continued for a week as the monocytes differentiated into macrophages and then
used in immunoprecipitation experiments.
Generation of cell lines with CRISPR/Cas9 gene editing. Lentivirus particles containing Cas9,
gRNA, and a puromycin resistant gene (pac gene) were prepared by the following method using these 3
plasmids: pCMV-VSV-G (Addgene plasmid 8454 gift from Bob Weinberg)31 psPAX2 (Addgene plasmid 12260
gift from Didier Trono), and lentiCRISPRv1 (Addgene plasmid 52961 gift from Feng Zhang)32. With the
lentiCRISPRv1 plasmid, gRNA directed towards CREB or C/EBP ? were cloned into the plasmid. The sequences
of the gRNA were based on the sequences used in the mouse GeCKO (Genome-Scale CRISPR Knock-Out)
lentiviral pooled library32 and are as follows: CREB, TGTACTGCCCACTGCTAGTT; total C/EBP ?,
GCGCAGGGCGAACGGGAAAC; ?38 C/EBP ?, CGCGTTCATGCACCGCCTGC; and ?38/36 C/EBP ?,
CGGCTTGGCGCCGTAGTCGT. These plasmids were cotransfected into 293FT cells (ThermoFisher Scientific;
catalog number: R70007) using the calcium phosphate transfection method. 293FT cells were cultured in DMEM
media containing 10% FBS and supplemented with 0.1 mM MEM Non-Essential Amino Acids, 1 mM sodium
pyruvate and 2 mM L-glutamine. Forty-eight hours after transfection, lentivirus particles released into the media
were harvested and used to transduce RAW 264.7 cells. Lentivirus transduction was accomplished by adding
lentivirus particles plus 8 ?g/ml Polybrene to cells plated at 2.5 ? 104 cells/well in a 12-well plate and then
centrifuging the lentivirus mixture with cells for 2 h at 1200 ? g at 25 ?C. After incubating the cells with lentivirus overnight
at 37 ?C, the lentivirus particles were removed and the cells were placed under antibiotic selection with 3.5 ?g/
ml of puromycin for 3?4 days to kill non-transduced cells. Single cell colonies were then isolated and grown in
the presence of puromycin. The colonies were then screened for CREB or C/EBP ? gene disruption by PCR of
genomic DNA and immunoblotting.
Reverse Transcriptase qPCR Analysis. RNA was isolated from RAW 264.7 cells or human monocytes and
then converted to cDNA in reverse transcription reactions using SuperScript III (Invitrogen). Equal amounts of
cDNA for each sample were combined with a SYBR Green PCR master mix (Qiagen) and gene-specific primers.
Amplification reactions were then performed with an Applied Biosystems 7500 real-time PCR system. Relative
changes in levels of the mRNA of the gene of interest were compared with the levels of mouse Actb or human
ACTB mRNA using the 2???Ct method.
Immunoblot. Total proteins were extracted from RAW 264.7 cells by removing culture medium and then
adding 4 ?C lysis buffer containing 1% SDS, 50 mM Tris (pH 7.4), 5 mM EDTA, and a protease inhibitor
mixture (Sigma, catalog no. P8340). The cells were incubated in this lysis buffer on ice for 15min, passed through
a 22-gauge needle 10 times, and centrifuged for 5 min at 20,000 ? g. For immunoblot analysis, these proteins
extracts (10 ?g/well) were combined with sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5%
?-mercaptoethanol, and 0.001% bromphenol blue) and heated at 95 ?C for 7 min. Proteins were then separated
using 10?12% SDS-PAGE and transferred to a PVDF membrane by electroblotting. The membrane was blocked
with 5% nonfat milk in a wash buffer consisting of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20.
The membranes were then probed with rabbit monoclonal antibody against CREB (Cell Signaling Tech; catalog
number 9197), rabbit antibodies against C/EBP ? (Santa Cruz Biotechnology; catalog number sc-150), rabbit
monoclonal antibody against PKA cat (Cell Signaling Tech, catalog number 5842), rabbit antibodies against PKA
RII (Santa Cruz Biotechnology; catalog number sc-908 and sc-909), mouse monoclonal antibody against APC
(clone FE9; Millipore Sigma; catalog no. OP44), rabbit monoclonal antibody against HA-tag (Cell Signaling Tech,
catalog number 3724) or a mouse monoclonal antibody against GAPDH (Abcam; catalog no. ab8245). The
membrane was then washed and incubated with a secondary antibody conjugated to horseradish peroxidase. The
immunoblots were developed with an enhanced chemiluminescent protein development system (GE Healthcare)
and exposed to film or imaged with a ChemiDoc MP Imaging System (Bio-Rad).
ChIP-Seq and data processing. PBMC were plated at 2 million cells per ml in RPMI containing 10%
FBS, 100 units/ml penicillin, and 100 ?g/ml streptomycin. These cells were then treated with 10nM ET (10 nM
EF and 10 nM PA) or 500 ?M 6MB-cAMP for 4.5 h and then isolated by centrifugation followed by snap
freezing in liquid N2 as recommended by Active Motif. For each condition, 60 million PBMC were prepared from 3
donors (20 million PBMC per donor). ChIP was performed by Active Motif with antibodies validated for ChIP
against CREB (Cell Signaling Tech; catalog number 9197) or against C/EBP ? (Santa Cruz Biotechnology, catalog
number sc-150). After ChIP DNA libraries were constructed, 75-nt sequence reads were generated by Illumina
Coimmunoprecipitation. For the described coimmunoprecipitation analyses, cells were lysed by
incubating for 5 min in lysis buffer (20 mM HEPES (pH 7.9), 350 mM NaCl, 30 mM MgCl2, 10% glycerol, 0.5% Nonidet
P-40, 200 ?M DTT, and protease inhibitor mixture), passing the lysates through a 22-gauge needle 10 times, and
then centrifuging the lysates at 18,000 ? g for 5 min. Next, 500 ?g of total protein from the lysate was diluted into
a binding buffer (20 mM HEPES (pH 7.9), 30 mM MgCl2, 10% glycerol, 0.2% Nonidet P-40, 200 ?M DTT, and
protease inhibitor mixture) to produce a final volume of 300 ?l. The following antibodies were then added to the
immunoprecipitations: rabbit antibodies against APC (Santa Cruz Biotechnology, catalog number sc-896), rabbit
monoclonal antibody against PKA cat (Cell Signaling Tech, catalog number 5842), or control rabbit IgG (Santa
Cruz Biotechnology, catalog number sc-2027). The immunoprecipitations were subsequently incubated for 2h at
4 ?C. Then, protein G-conjugated magnetic beads were added, and the incubation was continued for an additional
30 min. After removing the input protein, the magnetic beads were washed three times in 200 ?l of binding buffer,
and the immunoprecipitated proteins were eluted and subjected to immunoblot analysis.
Expression of PKA RII in RAW 264.7 cells. PKA RII is the product of the Prkar2a gene. Thus, PCR was
used to amplify the Prkar2a gene from mouse cDNA and to fuse DNA sequence to the gene in order to
produce a HA tag on its C-terminal (RII HA-tagged). PCR primers were also designed to amply the portion of the
Prkar2a gene that does not produce the N-terminal D/D domain (?D/D RII HA-tagged). Each of these PCR
products was cloned into the pENTR/D-TOPO vector (Invitrogen) and subsequently transferred into expression
vectors through a recombination reaction using LR Clonase (Invitrogen). The retrovirus expression vector used
to express RII HA-tagged or ?D/D RII HA-tagged was pQCXIB (Addgene plasmid 17487, originating from E.
Campeau)33. For retrovirus-mediated expression of these proteins, pQCXIB along with murine leukemia virus
gag/pol and vesicular stomatitis virus G expression plasmids were each cotransfected into 293 T cells (cultured in
DMEM with 10% FBS) using the calcium phosphate transfection method. Forty-eight hours after transfection,
retroviral particles were harvested and used to transduce RAW 264.7 cells. For RAW 264.7 cell transduction, the
retrovirus particles plus 8 ?g/ml Polybrene were added to cells plated at 5.0 ? 104 cells/well in a 12-well plate. The
plate containing the cell and the retrovirus mixture were then centrifuged for 2 h at 1200 ? g at 25 ?C and placed in
the tissue culture incubator overnight. Retrovirus was then removed and replaced with fresh medium containing
2 ?g/ml of blasticidin in order to kill non-transduced cells.
J.D.B. and J.L.L. designed experiments, analyzed data, and wrote the manuscript. J.L.L. and G.H. performed the
experiments. All authors read and approved the final manuscript.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-35184-y.
Competing Interests: The authors declare no competing interests. Publisher?s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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