Elsevier

Blood Cells, Molecules, and Diseases

Volume 39, Issue 3, November–December 2007, Pages 278-286
Blood Cells, Molecules, and Diseases

Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitation and c-Myb knockdown

https://doi.org/10.1016/j.bcmd.2007.05.007Get rights and content

Abstract

The transcription factor c-Myb is an important regulator of hematopoiesis required for proper development of most blood cell lineages in vertebrates. An increasing number of target genes for c-Myb are being published, although with little or no overlap between the lists of genes reported. This raises the question of which criteria a bona fide c-Myb-target gene should satisfy. In the present paper, we have analyzed a set of previously reported target genes using chromatin immunoprecipitation (ChIP) and siRNA-mediated knockdown. Among the seven well-studied c-Myb target genes that we analyzed by ChIP, only ADA, c-MYC and MAT2A seemed to be occupied by c-Myb under our experimental settings in the Myb-positive cell lines Jurkat and HL60. After siRNA-mediated knockdown of c-Myb expression, the expression levels of two out of three ChIP positive Myb target genes, ADA and c-MYC, were strongly affected. These results clearly demonstrate the importance of combining different methods for target gene validation and suggest that a combination of ChIP and c-Myb knockdown may represent a powerful approach to identify a core collection of c-Myb target genes.

Introduction

The transcription factor c-Myb controls complex biological functions during hematopoiesis, presumably through activation (or repression) of critical target genes (reviewed in [1]). A longstanding puzzle, well-known also for other transcriptional activators, is how a modest sequence specificity of c-Myb [2], [3], [4] is compatible with its highly specific biological functions. Studies in mice have shown that c-Myb is essential for normal hematopoiesis to take place [5]. The transcription factor controls crucial steps at several stages during T- and B-cell development [6], [7], [8], and progression through key stages of hematopoiesis is dependent on distinct threshold levels of c-Myb [9]. Given such intricate Myb-dependent functions, an important and challenging task is the identification of the relevant and direct target genes of c-Myb that mediate these functions in vivo. Despite several reports [10], [11], [12], [13] on this issue, we are still far from being able to explain the biology of c-Myb on the basis of the target genes reported.

A classical approach to asses the validity of a proposed Myb-target gene has been (i) to identify MRE sequences in the promoter, (ii) analyze DNA-binding of c-Myb to these MREs in vitro and (iii) test the promoter segment with and without the MRE in a reporter assay (iv) to study whether c-Myb is able to activate the potential target gene when embedded in chromatin. A final criterion, less often fulfilled, is (v) to confirm that the target gene is directly activated by c-Myb, i.e. by demonstration of a rapid induction response or by comparing induction in the presence and absence of protein synthesis inhibitors.

Recently, the methodological repertoire available to assess target gene status has expanded. Chromatin immunoprecipitation (ChIP) can be used to measure binding of an endogenous transcription factor to the regulatory regions of its target genes, reflecting occupancy in living cells and thus provide evidence for a potential direct regulation of the gene. ChIP has been successfully used for analyses of activators like c-Myc, E2F and USF (reviewed in [14]). The use of RNA interference (RNAi) [15] has also been shown to be a powerful tool for exploring gene function and identifying target genes. Since the expression of a target gene is expected to diminish as a consequence of activator knockdown, this represents another potentially useful method for target gene validation. Recently, siRNAs directed against specific transcription factors with subsequent ChIP analysis have been used to identify direct target genes regulated by the transcription factors glucocorticoid receptor [16], HNF-1β [17] and ELF1 [18]. An important advantage of both methods is that the common ectopic overexpression of the transcription factor under study is avoided.

Over the last decade, several putative target genes for c-Myb have been identified [1], [19], [20] and, with the use of global expression arrays, the number of genes that appears to be Myb regulated has increased [10]. It is therefore important to have at hand easy, reliable methods for target gene verification, based on some well-defined, generally accepted criteria. In this work, we have addressed the question of whether ChIP and siRNA knockdown assays when combined could be used as a more reliable approach for target gene validation than the classical assays, in order to define a core collection of c-Myb target genes. To our knowledge, only one very recent Myb-target report has combined these two criteria in the validation analysis of the proposed target gene [21]. Our approach was to select a test set of seven genes previously reported to be c-Myb targets, and analyze whether these could be confirmed by ChIP analyses and siRNA-mediated knockdown of c-Myb. Of the target genes tested, only ADA, c-MYC and MAT2A seemed to be occupied by c-Myb under our experimental settings in the Myb-positive cell lines Jurkat and HL60. After siRNA-mediated knockdown of c-Myb expression, the expression levels of two out of the three ChIP-positive genes, ADA and c-MYC, were strongly affected. Implications of these observations for target gene validation are discussed.

Section snippets

Cells

HL60, Jurkat and K562 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, glutamine, penicillin, streptomycin and incubated at 37 °C in 5% CO2. For ChIP analysis, HL60 and Jurkat cells were cultured in spinner flasks to a density of approximately 1 × 106 cells/ml.

Formaldehyde cross-linking and chromatin immunoprecipitation

Formaldehyde cross-linking and ChIP were performed as described [22], [23] using the following antibodies: anti-c-Myb H-141 (sc-7874, Santa Cruz), anti-acetylated histone H3 (06-599, Upstate

c-Myb binds to the c-MYC and MAT2A promoters and to the locus control region of ADA in vivo

To critically assess the validity of the rising number of proposed c-Myb target genes, we decided first to perform ChIP analyses with a c-Myb specific antibody on a selection of reported Myb-target genes. The Myb positive human cell lines Jurkat (human T cell line) and HL60 (human promyelocytic leukemia cell line) were selected for these experiments. After treatment of the cells with formaldehyde and shearing of chromatin to an average length of 400 nucleotides, purified cross-linked chromatin

Discussion

Despite many reports on putative target genes for c-Myb, it is still questionable how many of these genes are valid, since the criteria for being a bona fide target gene are not well established. It is a matter of concern that despite an increasing number of target genes for c-Myb coming out of global search approaches, little or no overlap is seen between the lists of genes reported. Although some of this difference may be explained by requirements for diverse combinatorial sets of

Acknowledgments

This paper is based on a presentation at a Focused Workshop, the 4th Myb Workshop, sponsored by The Leukemia & Lymphoma Society, Civitella Alfedena, Italy, 20–24 May 2007. This work was supported by the Norwegian Research Council (T.B. & O.S.G.), including its FUGE-program (E.M.B & O.S.G), The Norwegian Cancer Society (V.M., T.S. & O.S.G) and by grants from the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie to B.L. We thank Olliver Dittrich for helpful assistance in

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    1

    Present address: Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, N-0317 Oslo, Norway.

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