Functional Genomic and Proteomic Characterization of Normal and Oncogenic CEBPA Variants In Myeloid Cells
Authors: Grebien Florian , Giambruno Roberto , Bennett Keiryn L , Colinge Jacques , Superti-Furga Giulio 
Published: October 2019

Abstract 3873

The transcription factor CCAAT/enhancer binding protein alpha (CEBPA) plays diverse, yet crucial roles in the differentiation of various cell types. In the hematopoietic system, CEBPA activity is required for the terminal maturation of granulocytes from committed granulocyte-macrophage progenitors. The main functions of CEBPA in this process include the induction of lineage-specific genes but also the establishment of cell cycle arrest. It has been hypothesized that CEBPA executes such diverse functions through cooperation and interaction with other lineage-specific transcription factors, chromatin modifying enzymes but also the cell cycle machinery.

CEBPA has been found to be mutated in about 9% of patients with acute myeloid leukemia (AML). The most prominent alterations represent frame-shift mutations in the N-terminal part of the protein, leading to increased expression of a shorter isoform that is translated from an alternative AUG initiation codon. This truncated CEBPA variant (p30) has lost the ability to block cellular differentiation. Alternatively, mutations in the C-terminal portion of the protein have also been identified. These mutations are mainly in-frame insertions of one or more amino acids in the basic region/leucine zipper part of the protein and abolish DNA binding of CEBPA. C-terminally mutated CEBPA might exert dominant negative effects, as it retains the ability to dimerize with other CEBP proteins. Generally, it is assumed that mutations in the CEBPA gene lead to de-regulation of gene expression and altered interactions of the CEBPA protein with its binding partners. However, a systematic, multilayered comparison of cellular changes that are induced upon CEBPA mutation is lacking.

To systematically assess functional differences between wild-type (WT) and mutated CEBPA isoforms, we have generated affinity-tagged variants of CEBPA WT, the p30 isoform and CEBPA carrying a duplication of Lys313 in the basic region/leucine zipper domain (CEBPA KK). These constructs were expressed in different myeloid progenitor cell lines. Ectopic expression of CEBPA WT led to downregulation of immature markers and the attainment of more differentiated cellular morphology, while expression of CEBPA KK and p30 variants did not have this effect. To study how oncogenic mutations alter the genome-wide gene expression program of myeloid cells, we have performed chromatin-immunoprecipitation followed by deep sequencing (ChIP-seq) analysis of tagged forms of CEBPA WT, CEBPA p30 and CEBPA KK. Together with mRNA expression profiling of the same cells using microarrays, this analysis yields the identification of novel direct and indirect CEBPA target genes. Moreover, it will allow to discern genes that are de-regulated in CEBPA-mutated AML. In parallel, we are characterizing protein interaction partners of CEBPA WT and the p30 and KK mutant by affinity purification and mass spectrometry. Integration of these datasets will greatly improve our knowledge and understanding of molecular pathways that are critical for CEBPA-mutated AML.

Disclosures:

No relevant conflicts of interest to declare.