Differential Effects of Sumoylation on the Activities of CCAAT Enhancer Binding Protein alpha (C/EBPα) p42 versus p30 May Contribute in Part, to Aberrant C/EBPα Activity in Acute Leukemias

William Hankey,Nancy Berliner,Arati Khanna-Gupta,Hong Sun,Terry Zibello,Matthew Silver

doi:10.4081/hr.2011.e5

Abstract

In this study, we have examined the role of post-translational modification of the myeloid master regulator C/EBPα by small ubiquitin-related modifier (SUMO). We have used transient transfection analysis, oligonucleotide pulldown assays and chromatin immuno-precititation in all-trans retinoic acid (ATRA)-inducible promyelocytic cell lines MPRO and NB4. We demonstrate that sumoylated wild-type p42-C/EBPα is associated with negative regulation of the myeloid specific lactoferrin (LF) gene in early myeloid cells and that a reduction in p42-C/EBPα sumoylation coincides with expression of the LF gene in maturing myeloid cells. In the acute promyelocytic leukemia cell line NB4 however, sumoylated p42 remains persistently bound to the LF promoter following ATRA-induction. This correlates with lack of lactoferrin expression in these cells. Changes in sumoylation status of C/EBPα thus appear to contribute to a switch that regulates transcriptional activity of this master regulator during normal neutrophil development. We also demonstrate that sumoylation of the AML associated dominant negative p30-C/EBPα isoform does not alter transactivation activity of the LF promoter. This may be because the p30 C/EBPα isoform binds to the LF promoter much less efficiently than its full length counterpart. Our data suggest that the activity of p42-C/EBPα in the developing neutrophil is more sensitive to changes in sumoylation than the p30 isoform. This difference may contribute to the leukemogenic potential of p30-C/EBPα.

Highlights

C/EBPa is the founding member of a family of basic region/leucine zipper transcription factors that is a master regulator of granulopoiesis.[1]. It is expressed at high levels throughout myeloid differentiation and binds to the promoters of multiple myeloid- specific genes at different stages of myeloid matura
Only Abstract e In this study, we have examined the role of s post-translational modification of the myeloid u master regulator C/EBPa by small ubiquitinl related modifier (SUMO)
We demonstrate that sumoylated wildm type p42-C/EBPa is associated with negative regulation of the myeloid specific lactoferrin m (LF) gene in early myeloid cells and that a o reduction in p42-C/EBPa sumoylation coincides with expression of the LF gene in matur

Summary

Introduction

C/EBPa is the founding member of a family of basic region/leucine zipper (bzip) transcription factors that is a master regulator of granulopoiesis.[1]. Medical Genetics, Columbus, OH, USA 2Division of Hematology, Brigham and tein This domain harbors the conserved basicregion leucine-zipper responsible for DNA. In the acute promyelocytic n leukemia cell line NB4 sumoylated p42 remains persistently bound to the LF proo moter following ATRA-induction. This correN lates with lack of lactoferrin expression in these cells. Chromatin immunoprecipitation studies from our laboratory have previously demonstrated that C/EBPa associates with the promoters of late myeloid genes such as lactoferrin (LF) in immature neutrophils where LF is not expressed. We chose to examine the impact of sumoylation on the activity of both isoforms of C/EBPa during myeloid differentiation and to ascertain their potential impact on leukemogenesis

Materials and Methods

Chromatin immunoprecipitation analysis

Findings

Sumoylation or Sumo modification is a post