CONCLUSIONES, APORTES Y RECOMENDACIONES

The C/EBPα protein is of great interest because of its involvement in myeloid differentiation and leukemia. C/EBPα is abundant in early myeloid cells where it binds to the promoters of key myeloid target genes 124, including its own promoter 29. Ectopic

C/EBPα expression induces granulocytic differentiation and blocks erythroid

differentiation of human CD34+ cells 21. The identification of novel interacting partners of C/EBPα might lead to novel therapeutic approaches that specifically target leukemogenic mechanisms which depend on C/EBPα loss of function mutations 125. C/EBPα interacting proteins have previously been identified by 1-D SDS PAGE coupled with mass spectrometry in prostate cancer cell lines 126 , using GST-C/EBP-DBD in myeloid cell line 70 and by yeast two hybrid screens 127. Here, we describe two approaches, one based on 2-D gel electrophoresis and the other on 1-SDS PAGE/nano LC, to identify novel C/EBPα interacting proteins which will help in elucidating the complicated protein interaction network underlying normal C/EBPα function.

Several novel C/EBPα interacting proteins were identified by this approach (Table 6 and 7). These are proteins involved in DNA repair and cell cycle: SMC1, MCM5; Proteins involved in chromatin modification and remodeling VPS72 (which is a part of NuA4 complex containing TIP60), TIP60 and ASXL1; the Ras superfamily member RAB34; the MAX binding protein MNT; the nuclear pore complex protein RANBP2; the splice factors SFPQ and TRA2B; metabolic enzymes like KAD5 and PGAM1 and many other proteins including proteins like hnRNP, RB, E2F4, and C/EBPα, which had already been shown to interact with C/EBPα directly 51,80,86. To confirm the protein interaction results found by mass spectrometry, we have confirmed the interaction between C/EBPα and MCM5 and TIP60 and further characterized the functional significance of the interaction between C/EBPα and TIP60.

Our two approaches, the 2-D and the 1-D/nano LC, revealed different C/EBPα

interacting protein. This result is most likely due to the inherent differences in these two approaches which affect the coverage and the confidence of the protein identification procedure. In the 2-D gel approach, complex protein samples were separated over a larger defined pH range, increasing the resolution for each protein when compared to a 1-D SDS-PAGE gel. Furthermore, in contrast to the 2-D analyses, where differentially expressed spots were visually identified and manually excised, the identification of proteins from the 1-D gel was independent of their visualization and allowed for the identificationof proteins with high isoelectric points.

Using these two approaches we identified a large number of known and novel C/EBPα

interacting proteins. We could confirm the interaction of C/EBPα and two other proteins, namely MCM5 and TIP60, by alternative methods. Furthermore, we concentrated our efforts on analyzing the functional significance the interaction between C/EBPα and TIP60.

The identification of TIP60 as a novel interacting partner of C/EBPα was intriguing because TIP60 has been shown to be an interacting partner of a numbr of other transcription factors in mammalian cells and has been shown to function as a coactivator or a corepressor. TIP60-mediated repression might be effected through the recruitment of histone deacetylase HDAC7 128 or independent of HAT activity 106,129. TIP60-mediated activation is generally thought to require the HAT activity of TIP60, in particular through the acetylation of histones on target promoters. Transcription factors themselves might be substrates for TIP60-mediated acetylation 130.

TIP60 is also a very interesting protein because it might affect higher-order chromatin structure. The eukaryotic chromatin structure is very complex, highly dynamic and regulates virtually all DNA-associated processes, like transcription, replication and DNA repair 131. Chromatin is also the basis of epigenetic inheritance, by which the differential expression state of genes can be transmitted through successive cellular generations 132. There are two known broad classes of enzymes that regulate chromatin 133,134. First the chromatin remodeling enzymes that uses ATP hydrolysis to modify the histone

composition or positioning of nucleosomes, without introducing covalent modifications of histones. Second, histone-modifying enzymes modify chromatin by covalently adding a variety of chemical moieties to specific histone residues. These enzymes are highly specifc and belong to a variety of protein families like histone acetyltransferases (HATs), deacetylases (HDACs), methyltransferases, demethylases, kinases, ubiquitin ligases and others. Chromatin–modifying enzymes exist as a part of large macromolecular complexes that exercise various functions like enzymatic activity, substrate specificity, chromatin association, and site specific recruitment by DNA-bound transcription factors. TIP60 is a HAT and is recruited by many transcription factors to a number of target promoters, where it participates in histone acetylation and transcriptional activation. It is interesting to note that members of the MYST domain histone acetyltransferase family are also involved in long lasting modifications of chromatin functions such as dosage compensation of the Drosophila X chromosome and therefore might play a special role in more long lasting epigenetic modifications which might be associated with permanent lineage decisions in the hematopoietic differentiation program. This is the first report of the interaction of the transcription factor C/EBPα interacting with a MYST family member protein.