Research Report 2010 - MDC

type but not in C/EBP deficient or L/L osteoclasts thatboth can not switch to “LAP” expression. Profiling analysisof the transcriptome showed that rapamycin treatmentor ectopic expression of “LAP” activated expressionof MafB, a negative regulator of osteoclastogenesis.This suggested that restriction of osteoclastogenesisby “LAP” or rapamycin is dependent on MafB. Inaccordance, knock-down of MafB induced osteoclastogenesis,regardless of rapamycin treatment or C/EBPisoform expression. Altogether, the data showed thatdifferential regulation of MafB gene expression byC/EBPβ isoforms determines the balance of boneturnover and that the control of C/EBPβ isoform translationrepresents a target for osteoporosis treatment.Post-translational C/EBP modifications andepigenetic functionsElisabeth Kowenz-Leutz and Ole PlessSeveral years ago, we found that C/EBPα,β may instructeven non-hematopoietic cells, such as skin fibroblasts,to express myeloid genes. Others have shown thatC/EBPs may reprogram lymphocytes into myeloid cells.Accordingly, C/EBPs entail epigenetic competence andgene regulatory functions to determine cell fate. Wehad also shown that cellular signaling cascades regulatethe activity of C/EBPβ to convert extracellular informationinto gene regulation. C/EBPβ is a ras/ MAP kinasesignal sensitive transcription factor that regulatesgenes involved in metabolism, proliferation, differentiation,immunity, senes cence, and tumorigenesis. Thefunctional capacity of C/EBPβ is governed by proteininteractions that depend on post-translational C/EBPβmodifications. In a proteome-wide interaction screen,the histone-lysine N-methyl trans ferase, H3 lysine-9specific 3 (G9a) was found to directly inter act with theC/EBPβ transactivation domain (TAD). G9a, but not adefective G9a mutant abrogated the transactivationpotential of wild type C/EBPβ. Metabolic labelingshowed that C/EBPβ is post-translationally modified bymethylation of its TAD. A conserved lysine residue (K39)in the C/EBPβ-TAD served as a substrate for G9a mediatedmethylation. A C/EBPβ K39 alanine exchangemutant was resistant to G9a mediated inhibition andconferred super-activation of myeloid genes. These dataidentified C/EBPβ as a direct substrate of G9a thatalters the functional properties of C/EBPβ by posttranslational lysine methylation.Mass spectrometry of cell derived C/EBPβ (in collaborationwith Gunnar Dittmar, MDC) revealed extensivemethylation of N-terminal arginine residues in C/EBPβ.The protein arginine methyl-transferase 4 (PRMT4) wasfound to interact with C/EBPβ and to di-methylate theconserved arginine residue (R3) in the C/EBPβ TAD.Phosphorylation of the regulatory domain of C/EBPβ byras/ MAP kinase signaling abrogated the inter actionbetween C/EBPβ and PRMT4. Differential proteomicscreening with R3-methylated and un-methylatedC/EBPβ peptides, protein interaction studies, and mutationalanalysis revealed that methylation of R3 constrainedthe interaction between C/EBPβ with SWI/SNFand Mediator complexes. Both complexes play essentialroles in chromatin remodeling and transcription initiationby polymerase II and were previously implicated inC/EBPβ functions. Mutation of the R3-C/EBPβ methylationsite alters the ability of C/EBPβ to induce myeloidand adipogenic differentiation. Thus, phospho ry lationof the transcription factor C/EBPβ couples ras signalingto arginine methylation and regulates the interactionof C/EBPβ with epigenetic gene regulatory protein complexesduring cell differentiation.A number of implications and conceptual advances arecontained in these results. An “indexing code” of posttranslationaltranscription factor modifications hasrecently been suggested (Sims & Reinberg, 2008),although experimental evidence is currently still scarce.Covalent modifications by “writers” (here, R-methylationby PRMT4, and K-methylation by G9a) determinemodification dependent docking of “readers” (SWI/SNF;Mediator; G9a). Our results imply that writing/readingsuch an indexing code is downstream of receptor tyrosinekinase signaling, relaying signals to epigeneticevents that finally determine cell fate. This concept is animportant extension of the Histone Code hypothesis tonon-histone transcription factor proteins, which comefirst in gene regulation and before chromatin modificationsoccur. Many mechanistic (gene regulation indevelopment and disease) and medical issues (pharmacology)are implied.Functional interactions between C/EBPalpha andE2F-DP complexesKatrin Zaragoza and Qingbin LiuC/EBPα coordinates proliferation arrest and differentiationin many cell types. C/EBPα transactivates lineagespecific differentiation genes and inhibits proliferationby repressing E2F-regulated genes. The myeloproliferativeC/EBPαBRM2 mutant serves as a paradigm forrecurrent human C-terminal bZip C/EBPα mutationsthat are involved in acute myeloid leukemogenesis.BRM2 fails to repress E2F and fails to induce adipogen-92 Cancer Research