Since CREB has pleiotropic effects on cell function and potentially activates several genes in hematopoietic and leukemia cells, we performed microarray analysis with total RNA isolated from K562 chronic myeloid leukemia cells transduced with CREB or control shRNA. The comparison of transcriptional profiles in wild type and CREB shRNA transduced K562 cells revealed a large number of differentially expressed genes (see Additional file 2
). Among these genes, some are direct targets of CREB, while others are indirect targets. To infer which of these genes was potentially directly regulated by CREB, we combined the expression data with the ChIP-chip data of CREB bound promoters as demonstrated by Marc Montminy[14
]. As was previously observed CREB binding sites are highly conserved across different tissues. However, these sites are activated by cAMP in a tissues specific manner. Therefore by combining these two datasets we attempted to uncover the functional CREB sites in hematopoietic tissues.
Our hypothesis for discovering functional CREB sites in hematopoietic cells is that if a gene is found to be differentially expressed in the CREB shRNA K562 transduced cells, and bound by CREB it is likely to be a direct target. To identify these genes we developed a metric that accounts for both the significance of the expression change and binding data for each gene (described in detail in Methods).
Since CREB has been described as both a transcriptional activator (when phosphorylated) and a repressor, we were interested in genes that were both up and downregulated in CREB shRNA transduced cells. The resulting rank ordered list allows us to sort genes by their likelihood of being functional CREB targets in K562 cells. It is difficult to determine, however, where to draw a threshold between the true and false targets. We have decided to restrict our analysis to the top several hundred targets that had both significant changes in expression and binding, as we deemed these to be highly enriched for true versus false targets. However, we do not claim that these are the only functional CREB targets in K562 cells, as the exact number of true targets is difficult to determine. The top down and upregulated genes revealed by this analysis are listed in Tables and , and the full list is found in the supplementary materials.
Potential CREB target genes.
Potential CREB target genes.
Genes within the downregulated list were BECLIN 1, UBE2B. Both these genes have a cAMP responsive element binding site(s) in their promoters. These genes were selected for further validation because they are known to be involved in autophagy/apoptosis (BECLIN 1), cell cycle/DNA repair (UBE2B) [25
]. Quantitative real time-polymerase chain reaction (qRT-PCR) with mRNA from AML cell lines (K562 and TF-1) and primary leukemic blasts from a patient with M4-AML was performed. UBE2B expression was significantly reduced in CREB shRNA transduced TF-1 and K562 myeloid leukemia cells compared to controls (Figure , p < 0.05). BECLIN and UBE2B were downregulated in primary AML cells transduced with CREB shRNA (Figure , p < 0.05).
Figure 1 Expression of potential target genes downstream of CREB in myeloid leukemia cells. Primers specific for the UBE2B, BECLIN1, and CREB genes were generated and utilized for quantitative real-time PCR by SyberGreen method (Bio-Rad Inc.) Relative gene expression (more ...)
Having confirmed the validity of our microarray results in these two test cases we set out to characterize the function of the complete list of CREB target genes using two annotation schemes. The first utilizes the annotation contained in the Ingenuity Pathway Analysis software (IPA). This analysis showed that there is a significant enrichment for cell cycle (P < 1e-3) and cancer (P < 1e-3) genes. The full list of genes associated with cancer is shown in Table . Many of these genes regulate cell cycle, signaling, DNA repair, or metabolism, which are consistent with previously published results [5
]. Furthermore, the role of CREB in the pathogenesis of leukemias has also been described in the literature [2
The subset of CREB target genes associated with cancer according to Ingenuity Pathways Analysis.
IPA also allows us to study CREB target genes in the context of protein-protein interactions networks. A network for downregulated genes interacting with CREB is shown in Figure , with a subset of the downregulated targets shown in grey, while other genes not in the target list that interact with these, shown in white. Here we see that there is prior literature supporting our analysis that CREB1 regulates PTGS2 (COX2), NR4A3 and TOM1, as depicted by the blue lines. Interestingly, COX2 is an important drug target, and suggests that commonly used COX2 inhibitors may provide a target for acute leukemia.
Figure 2 A network depicting interactions between direct CREB targets (shown in grey) and proteins that these interact with (shown in white). PTGS2, NR4A3 and TOM1 are direct CREB targets whose regulation by CREB was previously described in the literature (clue (more ...)
The second analysis that we performed used the terms from Gene Ontology to identify common characteristics among the top K562 CREB targets. Here we find the striking and unexpected result that ten percent of the downregulated targets code for histone genes (P < 1e-10, Table ). We also performed an analysis of the top upregulated genes but did not find any significant GO terms. Although there is some prior literature indicating that CREB or CREB-related pathways may play a role in regulating histone modifications primarily through the histone acetylase CREB Binding Protein (CBP)[5
], the fact that CREB directly regulates the transcription of histone genes in these cells is unexpected.
Gene Ontology terms that are enriched among the top CREB targets.
To further validate the hypothesis that CREB is an activator of these 20 histone genes, we utilized previously published analyses of the gene promoters to identify consensus CREB binding sequences. The results shown in Table demonstrate that nearly all the histone genes contain CREB half sites along with a TATA box in the vicinity of these. Thus three lines of evidence support the assignment of these 20 histone genes as CREB targets in K562 cells: expression, binding and sequence based.
We examined the distribution of expression of these 20 histone genes across human tissues. The expression data were obtained from the GNF body atlas. We were able to extract expression profiles for 81 histone genes contained in the human genome. Fifteen of these overlapped with the 20 histone CREB targets. We show the expression of all 81 histone genes in Figure , where the identity of the 15 CREB target genes is shown in the last row. We see that the 15 genes are clustered into two groups containing more than one gene, with a third group consisting of a single histone HIST1H1C. One of the groups contains histones that are broadly expressed across human tissues, and particularly in all hematopoietic tissues. The second group is instead expressed in a very narrow range of tissues including K562 cells, bone marrow, prostate and thymus.
Figure 3 The tissue specific expression of histone genes. Each row of the figure represents a tissue from the GNF Body Atlas (see methods). We show only the top 30 tissues with highest variance of expression of histone genes. Each column represents a histone gene. (more ...)
We examined the expression of three histones that are putative targets of CREB by real time PCR with mRNA from K562, TF-1, and primary cells from patients with AML. The three histones selected were based on our microarray analyses. Our results demonstrated a statistically significant decrease in histonesHIST1H2Bj, HIST1H3B, and HIST2H2AA in K562 and TF-1 cells (Figure ). Interestingly, in primary cells from a patient with AML, only HIST1H3B and HIST2H2AA, but not HIST1H2BJ expression was decreased with CREB knockdown. These results suggest that histones are differentially expressed in AML and that specific histones are potential targets of CREB. This analysis supports the hypothesis that CREB regulates a subset of histone genes that are normally expressed in a small set of rapidly dividing tissues. These genes are presumably aberrantly activated in K562 and other leukemia cells, and could potentially contribute to the malignant phenotype.
Figure 4 Expression of target histone genes is decreased in CREB knockdown myeloid leukemia cells. Primers specific for HIST1H2BJ, HIST1H3B, and HIST2H2AA were generated and utilized for quantitative real-time PCR by the SYBR Green method (Applied Biosystems). (more ...)