CEBPA-regulated lncRNAs, new players in the study of acute myeloid leukemia
© Hughes et al.; licensee BioMed Central Ltd. 2014
Received: 1 September 2014
Accepted: 16 September 2014
Published: 25 September 2014
CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation. Disruption of CEBPA function contributes to the development of acute myeloid leukemia (AML). CEBPA regulates a large number of protein coding genes of which several were shown to contribute to CEBPA function. In this study, we expand the analysis of CEBPA transcriptional targets to the newly identified class of long non-coding RNAs. We show that lncRNAs are a main component of the transcriptional program driven by C/EBPα and that many of these are also induced during granulocytic differentiation of AML cell lines supporting their relevance in proliferation arrest and differentiation.
To the Editor
LncRNAs participate in multiple networks controlling cell differentiation and development , with their expression already associated with cancer and several disorders . To what degree C/EBPα regulates the expression of lncRNAs is still largely unknown.
In order to annotate the presence of putative C/EBPα binding sites in the promoter of differentially expressed genes, we used previously generated ChIP data sets for CEBPB (C/EBPα) and CEBPD (C/EBPα) in K562 cells , which exhibit identical DNA-binding specificities with C/EBPα . We found several coding and non-coding differentially expressed genes bound by either CEBPB or CEBPD in their putative promoter region within a distance of -5 kb from the TSS (Figure 1B and Additional file 1, Additional file 7: Figure S3, Additional file 8: Table S4, Additional file 9: Table S5, Additional file 10: Table S6 and Additional file 11: Table S7).
Different AML cell lines are widely used to study the block of differentiation in AML because they can be differentiated in mature and functional myeloid cells by treatment with specific agents. Thus, we analysed the expression of selected lncRNAs in NB4 cells, which are able to undergo granulocytic differentiation by treatment with all-trans retinoic acid (ATRA) . Notably, the majority of validated C/EBPα-induced lncRNAs in K562 are also significantly upregulated by ATRA in NB4 (21 out of 26), suggesting that they may play a role in the differentiation process (Figure 2B). Nevertheless, upon validated lncRNAs repressed by C/EBPα treatment in K562, 6 out of 8 showed opposite trend while 2 were not significantly expressed in NB4 (data not shown). This behaviour still remains to be explained and extended to the study of more lncRNAs in NB4 cell line: we speculate it could be due to silencing of diverse cellular settings between K562 and NB4 cell lines.
In summary, this study shows that lncRNAs are a main component of the transcriptional program driven by C/EBPα. We identified more than 900 lncRNAs regulated by C/EBPα in K562. We confirmed that the majority of these are also induced during granulocytic differentiation of AML cell lines supporting their relevance in proliferation arrest and differentiation. How many of the lncRNAs identified in this study are directly involved in regulating differentiation programmes of AML is an interesting question that warrants further investigations.
Moreover, regardless of function, this work indicates that changes in lncRNAs expression might also have diagnostic applications in AML with CEBPA mutations.
JMH and BS conducted experiments, participated in research design and interpretation of data. FMG performed data analysis. IB participated in research design and provided financial support. AF designed research, wrote the manuscript, and provided financial support. All authors read and approved the final manuscript.
The authors would like to thank Prof K. Nerlov for CEBPA plasmid, Dr A. Rosa and Dr A. Brivanlou for the ePiggyBac inducible transposon system, M. Arceci and M. Marchioni for technical assistance. This work was supported by FP7-PEOPLE-2011-ITN Project HemID (289611), Italian Epigenomics Flagship Project (EPIGEN) and “Research Projects of National Interest” (PRIN).
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