CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding.

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil's major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core "VKEEP" sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein-protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis.

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Aurora kinase A is required for hematopoiesis but is dispensable for murine megakaryocyte endomitosis and differentiation.

Aurora kinase A (AURKA) is a therapeutic target in acute megakaryocytic leukemia. However, its requirement in normal hematopoiesis and megakaryocyte development has not been extensively characterized. Based on its role as a cell cycle regulator, we predicted that an Aurka deficiency would lead to severe abnormalities in all hematopoietic lineages. Here we reveal that loss of Aurka in hematopoietic cells causes profound cell autonomous defects in the peripheral blood and bone marrow. Surprisingly, in contrast to the survival defects of nearly all hematopoietic lineages, deletion of Aurka was associated with increased differentiation and polyploidization of megakaryocytes both in vivo and in vitro. Furthermore, in contrast to other cell types examined, megakaryocytes continued DNA synthesis after loss of Aurka. Thus, like other cell cycle regulators such as Aurkb and survivin, Aurka is required for hematopoiesis, but is dispensable for megakaryocyte endomitosis. Our work supports a growing body of evidence that the megakaryocyte endomitotic cell cycle differs significantly from the proliferative cell cycle.

ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize hematopoietic progenitor cells.

ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis.

Novel combinatorial interactions of GATA-1, PU.1, and C/EBPepsilon isoforms regulate transcription of the gene encoding eosinophil granule major basic protein.

GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPepsilon isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPepsilon(27) isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPepsilon(27) are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPepsilon(32/30) and epsilon(27) physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPepsilon isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPepsilon(27).