How does C/EBPa induce the activation and silencing of genes during the trans- differentiation of pre-B cells into macrophages?

Using our inducible pre-B cell line system carrying C/EBPaER we have shown that the cells become transgene independent about 24 hours after induction. During this time endogenous C/EBPb, a transcription factor that can also induce transdifferentiation, as well as PU.1, a partner required for C/EBP, become transcriptionally activated. Knockdown of C/EBPb and PU.1 almost completely inhibits the reprogramming, showing that they are required for the establishment of the myeloid fate.

In contrast, endogenous C/EBPa only becomes activated after commitment, and knockdown experiments showed that it is responsible for the maintenance of the macrophage state. These experiments suggest that C/ EBPa induces a stable myeloid transcription factor network consisting of the transcription factor triad C/ EBPa, C/EBPb and PU.1.

To study the underlying molecular mechanism we have embarked on a major effort to study transcription factor binding and chromatin modifications during the C/EBPa induced transdifferentiation. For this purpose we collected samples of uninduced (0h) as well as cells induced for 3h, 12h, 24h and 48h and performed ChIPseq experiments. These included the binding of the myeloid regulators C/EBPa, C/EBPb and PU.1 as well as "Pol II" to DNA and also the chromatin marks H3K4me1, HeK4me3, H3K27me3, H3K27Ac and H3K9me3.

Our data show that establishment of the macrophage program involves the activation of two enhancer pathways, depending on whether C/EBPα or its partner PU.1 binds to DNA first. These two enhancer-binding modes dictate distinct kinetics of histone modification and nucleosome positioning changes. Importantly, genes associated with the two enhancer classes differ in their upregulation kinetics, both during induced lineage reprogramming and normal differentia- tion, and may exert distinct biological functions.

We have also studied the silencing of the B cell program, and did ChIPseq analyses of DNA binding of the B cekll regulators E2A and Ebf1 during transdifferentiation, as well as Pax5 and Foxo1 in preB cells. We found that silencing of the B cell program correlates with the transient binding of C/EBPa to enhancers of key B cell regulators, manifesting itself with a rapid decrease in "Pol II" binding. We are now investigating how this works at the molecular level.