Identification of Human STAT5-dependent Gene Regulatory Elements Based on Interspecies Homology

Erik A Nelson,Sarah R Walker,Wei Li,X Shirley Liu,David A Frank

doi:10.1074/jbc.m605001200

Abstract

STAT5 is a transcription factor essential for hematopoietic physiology. STAT5 functions to transduce signals from cytokines to the nucleus where it regulates gene expression. Although several important transcriptional targets of STAT5 are known, most remain unidentified. To identify novel STAT5 targets, we searched chromosomes 21 and 22 for clusters of STAT5 binding sites contained within regions of interspecies homology. We identified four such regions, including one with tandem STAT5 binding sites in the first intron of the NCAM2 gene. Unlike known STAT5 binding sites, this site is found within a very large intron and resides approximately 200 kb from the first coding exon of NCAM2. We demonstrate that this region confers STAT5-dependent transcriptional activity. We show that STAT5 binds in vivo to the NCAM2 intron in the NKL natural killer cell line and that this binding is induced by cytokines that activate STAT5. Neither STAT1 nor STAT3 bind to this region, despite sharing a consensus binding sequence with STAT5. Activation of STAT4 and STAT5 causes the accumulation of both of these STATs to the NCAM2 regulatory region. Therefore, using an informatics based approach to identify STAT5 targets, we have identified NCAM2 as both a STAT4- and STAT5-regulated gene, and we show that its expression is regulated by cytokines essential for natural killer cell survival and differentiation. This strategy may be an effective way to identify functional binding regions for transcription factors with known cognate binding sites anywhere in the genome.

Highlights

Well studied example is chronic myelogenous leukemia, in which the Bcr/Abl oncogene causes the constitutive activation of STAT5, which directly leads to continuous up-regulation of antiapoptotic genes, such as bcl-x [4]
Since functional STAT5 regulatory regions are likely to be conserved among diverse species, we further considered only those that had 100% homology at both STAT5 consensus sites between humans, chimpanzees, mice, and rats
To determine if genes known to be important in the biology of NK cells showed a similar response to IL-2 and IFN-␣, we examined expression of IFN-␥, which is expressed by natural killer cells and is essential for the immunomodulatory function of these cells [22]

Summary

Introduction

Well studied example is chronic myelogenous leukemia, in which the Bcr/Abl oncogene causes the constitutive activation of STAT5, which directly leads to continuous up-regulation of antiapoptotic genes, such as bcl-x [4]. Given that STAT5 can bind to cognate DNA sequences in tandem [13,14,15], we searched for regions of high homology that contained two STAT5 consensus binding sites Using this strategy, we identified such a sequence in the first intron of NCAM2 (neural cell adhesion molecule 2). Combining bioinformatic techniques with knowledge of STAT5 binding sites can be used to identify novel STAT5 targets and to better understand STAT-mediated transcriptional regulation. This technique should be applicable to any transcription factor for which binding sequences have been defined

Methods

Results

Conclusion