ChIP-on-Chip Analysis Identified HLXB9 as a Transcription Factor In Hematopoietic Cells and Alters the Expression of Genes Involved In Cell-Adhesion
Authors: Wildenhain Sarah , Ruckert Christian , Daschkey Svenja , Dugas Martin , Hauer Julia , Borkhardt Arndt 
Published: October 2019

Abstract
Abstract 2486
Infants with t (7;12)/HLXB9-TEL positive Acute Myeloid Leukemia (AML) have an Event-Free Survival (EFS) of 0 % and are characterized by concomitant HLXB9 (MNX1) expression. However, the role of the homeobox protein HLXB9 on hematopoietic cell development remains unknown. Expression profiling of t (7;12) and t (11;X) positive leukemias revealed up-regulation of cell-cell interacting genes in t (7;12) positive leukemia (Wildenhain et al., 2010). Furthermore, no increased expression of HOX-Genes, like HOXA9 and MEIS1, could be observed in t (7;12) positive leukemia compared to t (11;X) positive leukemia. Based on the altered gene expression profile in t (7;12) positive leukemia we investigated the role of HLXB9 as a transcription factor in hematopoietic cells using ChIP-on-chip analysis and its impact on the cellular gene expression pattern using Affymetrix expression arrays. The myeloid cell line HL60 was stable transfected with a CMV-HLXB9 (HL60/HLXB9) expression vector or an empty vector control (HL60/control). Microarray analysis was performed using “Human Gene 1.0 ST Arrays” (Affymetrix) and data from the HL60/HLXB9 cells were normalized to HL60/control cells. ChIP-on-chip analysis was performed using the “SimpleChIP Enzymatic Chromatin IP Kit” (Cell Signaling Technologies). Hybridisation on “385K RefSeq Promoter arrays” and analysis of raw data were performed by NimbleGen using the NimbleScan software. Data were visualized with the SignalMap software. Altered expression analyses as well as enrichment of promoter regions were validated by quantitative RT-PCR. Expression analysis revealed 81 differentially expressed genes, whereof 63 were down-regulated indicating that HLXB9 acts as a transcriptional repressor, as characteristic for homeobox proteins. CLEC5A, normally expressed in mature myeloid cells, is the highest differentially repressed gene. Further, we identified several differentially expressed genes which interfere in cell-adhesion and/or angiogenesis (e.g. IL8, ZYX, SELL, SPP1, EMILIN2). Western blot analysis of nuclear extracts confirmed the translocation of HLXB9 into the nucleus. ChIP-on-chip analysis revealed binding of HLXB9 to several promoter regions, among them the promoters of ZYX and IL8. Binding of HLXB9 to those promoters results in a decreased gene expression.These data strengthens the hypothesis, that HLXB9 plays a major role in cell adhesion and/or cell interactions. Further we observed increased expression of the adhesion molecule CD11b, when culturing HL60/HLXB9 cells in All-Trans Retinoic Acid (ATRA) containing medium in contrast to HL60/control cells. In summary, this study shows that HLXB9 acts as a transcription factor in hematopoietic cells and has a repressive function on gene expression. HLXB9 target genes regulate cell-adhesion and angiogenesis. This study provides the first molecular results of HLXB9 function in hematopoietic cells and supports the previously published data showing the importance on altered gene expression of cell-cell interacting genes in the pathogenesis of t (7;12) positive leukemia.

Disclosures:
No relevant conflicts of interest to declare.