Proteomic identification of the MYST domain histone acetyltransferase TIP60 (HTATIP) as a co-activator of the myeloid transcription factor C/EBPα

Abstract

The transcription factor C/EBPa (CEBPA) is a key player in granulopoiesis and leukemogenesis. We have previously reported the interaction of C/EBPa with other proteins (utilizing mass spectrometry) in transcriptional regulation. In the present study, we characterized the association of the MYST domain histone acetyltransferase Tat-interactive protein (TIP) 60 (HTA-TIP) with C/EBPa. We show in pull-down and co-precipitation experiments that C/EBPa and HTATIP interact. A chromatin immunoprecipitation (ChIP) and a confirmatory Re-ChIP assay revealed in vivo occupancy of the C/EBPa and GCSF-R promoter by HTATIP. Reporter gene assays showed that HTATIP is a coactivator of C/EBPa. The co-activator function of HTATIP is dependent on its intact histone acetyltransferase (HAT) domain and on the C/EBPa DNA-binding domain. The resulting balance between histone acetylation and deacetylation at the C/EBPa promoter might represent an important mechanism of C/EBPa action. We observed a lower expression of HTATIP mRNA in undifferentiated U937 cells compared to retinoic acid-induced differentiated U937 cells, and correlated expression of CEBPA and HTATIP mRNA levels were observed in leukemia samples. These findings point to a functional synergism between C/EBPa and HTATIP in myeloid differentiation and suggest that HTATIP might be an important player in leukemogenesis.

Proteomics of AML1/ETO Target Proteins: AML1–ETO Targets a C/EBP–NM23 Pathway

“…We have previously shown that AML1-ETO blocks myeloid transcription factor C/EBPα by downregulating its mRNA, protein, and DNA binding activity in t(8;21) myeloid leukemia [20], and inactivates the myeloid master regulator PU.1 by direct protein-protein interaction in myeloid differentiation [21]. Our group has routinely applied proteomic approach to analyze proteomic profile of various AML subtypes, target, and interacting protein partner identification in AML and APL cells [22][23][24][25][26]. Because AML1-ETO disrupts and blocks the normal function of myeloid transcription factors and requires other cooperating factors to induce leukemia, we hypothesized that the systematic identification of AML1-ETO target proteins on a global proteome-wide level might lead to novel insights into the pathogenesis and systems biology of AML1-ETOinduced leukemia on a post-genomic functional level.…”

C/EBPα, do not forget your TIP60

“…The paper by Bararia et al, 1 published in this issue of Leukemia has brought TIP60, another cellular acetyltransferase under the spotlight of myeloid leukemogenesis. Transcriptional deregulation is a recurring theme in human acute myeloid leukemia (AML).…”

“…7 To gain further insights into the functional regulation of C/EBPa, Bararia et al have biochemically purified and validated another acetyltransferase, TIP60 as a C/EBPa-interacting partner. 1 They show that TIP60 directly interacts with C/EBPa, and acts as a transcriptional co-activator in an acetyltransferase-dependent manner. TIP60 complexes with C/EBPa to enhance acetylation of histones 3 and 4, and regulates expression of downstream targets including C/EBPa itself ( Figure 1).…”

“…It is interesting to note that TIP60 can be recruited by either the N-terminal or the DNA-binding domain of C/EBPa, while a maximal binding will only be achieved with the wild-type full-length protein. 1 Thus, C/EBPa generated by mutated alleles such as the N-terminal truncated form will potentially have reduced ability to interact with TIP60, which will partially compromise its activities and functions. Given the tumor suppressor function of TIP60, it will be interesting to determine its interaction with different C/EBPa mutants, and the functional consequence of its reduction in normal and malignant hematopoiesis in particular in the context of C/EBPa-mediated leukemogenesis.…”

“…Consistently, the expression of TIP60 correlates with C/EBPa expression in human leukemia samples and U937 cells upon retinoic acid-induced differentiation, suggesting TIP60 as a direct co-activator for C/EBPa in hematopoietic cells. 1 TIP60 is a founding member of the MYST family of lysine acetyltransferases, which also include MOZ and MORF. Notably, both MOZ and MORF are directly involved in chromosomal translocations in AML to form chimeric fusion proteins with CBP and/or p300.…”