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SOX4, POU2F2 and BACH2, transcription factors in normal and malignant lymphopoiesis Malene Krag Kjeldsen, Karen Dybkær and Hans E. Johnsen Department of Hematology, Aalborg Hospital www.blodet.dk Mail: [email protected] Introduction The stem cell derived transcription factors SOX4, POU2F2 and BACH2 are known to be important in B-cell differentiation and B-cell malignancies. In silico analysis (1) of these transcription factors indicate that they are differentially expressed in patients with diffuse large B-cell lymphoma (DLBCL)(2-4). Furthermore, murine studies on retroviral insertional mutagenesis demonstrate that these transcription factors are common integration sites in B-cell lymphomas in general, personal communication with Professor Finn Skou Petersen. Both of these observations lead to the hypothesis that transcription factors, central in B-cell lymphoiesis and embryogenesis, regulate target genes with important impact on oncogenesis of DLBCL. Only few target genes for SOX4, POU2F2 and BACH2 are known. Therefore, the objectives of this study are I) to identify new target genes and II) determine dysregulation of specific target genes in DLBCL patients compared to normal B-cell subpopulations. Chromatin immunoprecipitation (ChIP) is a technique used to determine DNA and protein interactions and it is therefore a strong tool to determine genes regulated by specific transcription factors(5). ChIP combined with global techniques such as micro array and sequencing and the gene specific technique quantitative real time RT-PCR (qPCR) are used to identify and determine dysregulation of target genes in DLBCL and normal B-cell subsets. ChIP on chip Experimental setup DNA fragments analysed utilising Affymetrix human promoter Array. A single array contains 25,500 human promoters. ChIP – sequencing ChIP - qPCR DNA fragments analysed using sequencing gives all binding sites on the whole genome, both promoters and enhancer/ repressor elements. Furthermore consensus binding sequence can be determined. DNA fragments analysed using qPCR only allows predetermined genes to be studied. Preliminary results In silico analysis Transcription factors Figure 1: The experimental setup of the project Samples In silico analysis of selected transcription factors in DLBCL and normal B-cells. Chromatin immunprecipitation (ChIP) The stem cell derived transcription factors, SOX4, POU2F2 and BACH2 are differentially expressed in DLBCL. SOX4 BACH2 POU2F2 Figure 3: Gene expression profile of specific transcription factors in DLBCL and normal B-cells. Preliminary results – verifying ChIP 1) Proper sonication resulting in DNA fragment 100bp-1kb 0,02 CD20p1 ChIP2 - relative beads 8,0 0,02 CD20p2 7,0 relatif to Input P2 5,0 4,0 3,0 2,0 1,0 0,0 Oct2a CD20: Binding of SOX4 and BACH2 only promoter 2 and response element. Oct2b SOX4 BACH2a BACH2b IgG ChIP2 - relative beads Beads Figure 4: ChIP-qPCR with CD20 as target gene. CD20 has two alternative promoters, the long form is P1 and the short form is P2 in the figure. Re is consensus binding site, located in 3’ UTR. 0,02 CD44pp 7,0 6,0 CD44: Binding of SOX4, BACH2 and POU2F2 (Oct2) to the promoter. P1 0,02 CD20re 6,0 relatif to Input Critical steps in ChIP Verifying whether antibodies are suited for ChIP analysis includes ChIP-qPCR. Negative controls; beads only and unspecific IgG. 0,02 CD44e2 5,0 4,0 3,0 2,0 1,0 0,0 Oct2a Oct2b SOX4 BACH2a BACH2b IgG Beads Figure 5: ChIP-qPCR with CD44 as target gene. pp is the promoter and e2 is exon 2. 2) An antibody capable of precipitating the protein-DNA complex • Determine genes regulated by the stem cell derived transcription factors, SOX4, POU2F2 and BACH2 using ChIP on chip in human DLBCL cell lines 3) Analysing the DNA fragment • Determine consensus binding sites for the stem cell derived transcription factors SOX4, POU2F2 and BACH2 using ChIP-sequencing Future perspectives • Utilising ChIP-qPCR in normal and malignant B-cells determining a potential dysregulation of target genes of interest ? Is CD44 a target gene of SOX4 and BACH2, and what does it mean? Figure 2: The principles of the ChIP technique. First protein and DNA is cross-linked, then chromatin is sheared and the protein-DNA complexes are isolated. After reversing the cross-link the DNA fragments are identified and quantitated. ? Is CD20 a target gene of SOX4, BACH2 and POU2F2 and what does it mean? ? Does the antibodies work in ChIP? 1) Bioinformatician A.D. Højfeldt, Department of Haematology, Aalborg Hospital, Århus University Hospital 2) Saez,A.I. et al. Analysis of octamer-binding transcription factors Oct2 and Oct1 and their coactivator BOB.1/OBF.1 in lymphomas. Mod. Pathol. 15, 211-220 (2002). 3) Sasaki,S. et al. Cloning and expression of human B cell-specific transcription factor BACH2 mapped to chromosome 6q15. Oncogene 19, 37393749 (2000). 4) Liao,Y.L. et al. Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma. Oncogene(2008). 5) Collas,P. & Dahl,J.A. Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front Biosci. 13, 929-943 (2008).