 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 30, 2008 |
| Title |
Expression Profile of CREB knockdown in Myeloid Leukemia Cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
|
| Summary |
Background. The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. Therefore, we propose CREB to be a potential target for therapy. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line.
Results.
By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. The transcription factor Elk-1 was upregulated in response to CREB deletion.
Conclusions.
We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. In particular, we speculate that the regulation of histone genes may play an important role in this process, by possibly altering the regulation of DNA replication during the cell cycle.
|
| |
|
| Overall design |
Cell lines. The following human leukemia cell lines were transduced with shRNAs: K562 (Iscoves + 10% FCS) and TF-1 (RPMI + 10%FCS + rhGM-CSF. Cells were cultured at 37oC, 5% CO2 and split every 3 to 4 days. Primary AML bone marrow samples were processed as previously described [12]. All human samples were obtained with approval from the Institutional Review Board and consents were signed, according to the Helsinki protocol.
shRNA sequence design and constructs. The CREB specific shRNA sequences were selected and validated based on accepted parameters established by Tuschl et al. [25-27]; CREB shRNA-1, CREB shRNA-2, CREB shRNA-3. Controls included empty vector, luciferase shRNA, and scrambled shRNA. shRNA sequences are: CREB shRNA-1(5’GCAAATGACAGTTCAAGCCC3’), shRNA-2 (5’GTACAGCTGGCTAACAATGG3’), shRNA-3 (5’GAGAGAGGTCCGTCTAATG3’), Luciferase shRNA (5’GCCATTCTATCCTCTAGAGGA3’), Scramble shRNA (5’GGACGAACCTGCTGAGATAT3’). Short-hairpin sequences were synthesized as oligonucleotides and annealed according to standard protocol. Annealed shRNAs were then subcloned into pSICO-R shRNA vectors from the Jacks laboratory at MIT [28]. The second generation SIN vector HIV-CSCG was used to produce human shRNA vectors [29].
Microarray analysis. Total RNA (10 μg) was extracted from K562 cells transduced with vector alone or CREB shRNA was submitted to the UCLA DNA Microarray Facility. RNA samples were labeled and hybridized by standard protocol to Affymetrix GeneChip Human Genome U133+ Array Set HG-U133A array. Gene expression values were calculated using the MAS5 software. The expression values are quantile normalized across all arrays. We obtained the expression profiles for a control set and CREB downregulated K562 cells. These are first quantile normalized, and then a t-test is performed between the two groups to identify significantly differentially regulated genes. The analysis was performed using Matlab (Mathworks, Inc.). A normal quantile plot of T is shown in figure qqplot.tiff. Data points with t-scores < 1/(2N) display with red circles, were N is the total number of genes. We therefore see that there are a significant number of differentially expressed genes, which are either direct or indirect targets of CREB.
|
| |
|
| Contributor(s) |
Pellegrini M, Cheng JC, Voutila J, Judelson D, Nelson SF, Sakamoto KM |
| Citation(s) |
18801183 |
| |
| Submission date |
Jul 09, 2008 |
| Last update date |
Mar 25, 2019 |
| Contact name |
matteo pellegrini |
| E-mail(s) |
matteop@mcdb.ucla.edu
|
| Phone |
310 825-0012
|
| Organization name |
UCLA
|
| Street address |
610 charles young drive east
|
| City |
los angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
|
| Samples (20)
|
| GSM304480 |
K562 cells control replicate 5 |
| GSM304481 |
K562 cells control replicate 6 |
| GSM304482 |
K562 cells control replicate 7 |
| GSM304483 |
K562 cells control replicate 8 |
| GSM304484 |
K562 cells control replicate 9 |
| GSM304486 |
K562 cells control replicate 10 |
| GSM304487 |
K562 cells CREB Knock-out replicate 1 |
| GSM304488 |
K562 cells CREB Knock-out replicate 2 |
| GSM304489 |
K562 cells CREB Knock-out replicate 3 |
| GSM304490 |
K562 cells CREB Knock-out replicate 4 |
| GSM304491 |
K562 cells CREB Knock-out replicate 5 |
| GSM304492 |
K562 cells CREB Knock-out replicate 6 |
| GSM304493 |
K562 cells CREB Knock-out replicate 7 |
| GSM304494 |
K562 cells CREB Knock-out replicate 8 |
| GSM304495 |
K562 cells CREB Knock-out replicate 9 |
| GSM304496 |
K562 cells CREB Knock-out replicate 10 |
| GSM304498 |
K562 cells control replicate 4 |
|
| Relations |
| BioProject |
PRJNA113387 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE12056_RAW.tar |
100.3 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...