Functional Elucidation Of RNA Binding Protein QKI In The Process Of Myeloid Haematopoiesis

Myeloid lineage derived haematopoiesis, which produces monocyte/macrophage and granulocyte, is essential for the innate immunity and the wound healing. Although great advances have been made in the understandings of the myeloid differentiation process, the underlying mechanism is not fully understood. It has been widely accepted that cytokine related signal pathway influenced by both intrinsic and external stimuli is fundamental for normal hemopoietic differentiation. Aberrent expression of cytokine related signals leads to leukemia initiation. To this end, further exploring the underlying regulatory mechanism involved in the hemopoietic differentiation process will be valuable for leukemia prevention and treatment.RNA binding protein QKI encoded by the quaking gene locus was designated from the quaking viable mice. Quaking viable (qkv) is an autosomal recessive mutation due to a deletion of the 5'flanking region of the qki gene, leading to diminished expression of the selective RNA-binding protein QKI in myelin producing cells and subsequently dysmyelination in the CNS. QKI harbors amino acid domains characteristic of RNA-binding and interaction with Src homology 3 (SH3)-containing signaling molecules, therefore belongs to a fast-growing family denoted as signal transduction activators of RNA (STAR). Currently, all the findings related with QKI are mainly focused on CNS, esp. related with MBP and MAG mRNA stability, splicing, translocation and translational repression during oligodentrocytes differentiation.. As RNA-binding protein, its interaction with target mRNA is mainly through binding with cis-element in target mRNA 3'UTR (Quaking Response Element, QRE).However, the wide expression pattern of QKI in numerous tissues besides CNS implicates a fundamental role of QKI in other systems. In fact, the lethal phenotype in ENU-induced homozygous mutant qki mice highly proposed that QKI has a critical role in embryonic development prior to the start of myelination. It is now accepted that QKI plays an essential role in vascular development Interestingly, QKI is also expressed in the yolk sac endoderm, adjacent to the mesodermal site of developing blood islands, where early hemopoietic and endothelial cells originat from. All these data suggest a potential role of QKI in haematopoiesis. According to the predicted 1400 candidate target mRNAs published from Nat Stuct Mol Bio, many putative downstream target mRNAs are related with myeloid differentiation. Thus we focused on the role of QKI on the myeloid differentiation.For the first time, in the leukemic cells HL-60 and THP-1, QKI expression level was down-regulated both at mRNA and protein levels during the myeloid differentiation towards monocyte/macrophage and granulocyte. To test whether transcriptional regulation is causal for such reduction,. QKI promoter with size of 2kb upstream to ATG start codon was analyzed by Bioinformatic methods, the results revealed that multiple haematopoietic differentiation related transcriptional factors are putative regulators of QKI. Among them, C/EBPαdisplayed a similar expression pattern with QKI. In addition, C/EBPαexpression level declined prior to the drop of QKI level, highly suggesting that C/EBPαis responsible for QKI transcriptional regulation. Then QKI promoter driven luciferase reporter readings and positive ChIP assay confirmed the direct transcriptional regulation of C/EBPαon QKI promoter region. Reduced expression of C/EBPαin the differentiated cells led to less C/EBPαoccupation on the QKI promoter and end up with down-regulation of QKI.During myeloid differentiation process, there's a reversed relationship between QKI and CSF1 R (Macrophage-colony stimulating factor receptor,M-CSFR) which is critical factor for the lineage decision. RNAi knocking down of QKI or over-expressing QKI, CSF1R showed reverse changes too. Analysis of the QREs located in the 3'UTR of CSF1R among multiple species disclosed that the QREs are highly conserved, suggesting a conservative regulation between QKI and CSF1R. CSF1R mRNA 3'UTR reporter assay and RNA-IP test further confirmed the direct interaction between QKI and CSF1R. The lengthened half-life of CSF1R supported that QKI mediated negative regulation on CSF1R is via mRNA stability alterations.In the process of granulocyte differentiation, under 10% serum, ATRA induced HL-60 differentiation is not so dramatic but shows cell cycle arrest changes, at which time both C/EBPαand QKI showed a relative stable expression. While at 2% serum, the induced granulocyte showed more mature differentiation according to the marker gene level changes and nuclear staining pattern. Transfection of QKI RNAi acccelerated the differentiation process by the Giemmsa nuclear stainings. Further in-depth mechanism is undergoing.In order to successfully over-express QKI in blood cells, lentivirus system encoding exogeneous QKI and EGFP was constructed and packaged. Purification of this lentivirus is now undergoing.In summary, we first defined an important role of QKI in myeloid differentiation process.esp. it's one of downstream target gene of C/EBPα., which belong to be a fate-decision factor. Further more, our findings provided the first direct evidence that QKI is responsible for destabilize CSF1R by direct interaction with its 3'UTR. Since cEBPαis also important for cell cycle regulation and adipocyte differentiation. Such regulation may also occur in other system. While CSF1R expression level changes is closely related with other types of cancer, such as breast cancer. QKI mediated regulation may be more practical for other systems.