Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries

Most biological processes—such as embryonic development, differentiation, or cellular responses to external stimuli—are in essence dynamic, which requires the underlying transcriptional networks to be tightly temporally coordinated. Several large-scale temporal studies have focused on the comprehensive mapping of dynamic changes of gene expression but have not revealed how coordination is achieved on the transcriptional level (e.g., van Wageningen et al. 2010). To address this question globally, the binding of regulatory proteins has been examined using methods such as chromatin immunoprecipitation with microarrays (ChIP-chip) or sequencing (ChIP-seq) (e.g., Sandmann et al. 2006a; Johnson et al. 2007; Vogel et al. 2007). Still, many such studies have operated with just one or two conditions. The exceptions include temporal examinations in the model systems Drosophila and yeast (Sandmann et al. 2006b; Jakobsen et al. 2007; Ni et al. 2009; Zinzen et al. 2009). So far, very few in vivo, multi-time point investigations of transcription factor (TF) binding have been undertaken in higher vertebrates. Mammalian liver regeneration is a well-studied process, in which the large majority of mature hepatocytes rapidly and in a highly synchronized manner re-enter the cell cycle upon injury (Fausto et al. 2006; Michalopoulos 2007; Malato et al. 2011). Serial transplantation of liver tissue has demonstrated a very high “repopulating” capacity of hepatocytes (Overturf et al. 1997), which lends hope to using these cells in regenerative medicine. The ability of mature liver cells to proliferate is reminiscent of specific, differentiated cells of the immune system (naive T cells, B cells) that are kept in quiescence until exposed to specific stimuli (Glynne et al. 2000; Yusuf and Fruman 2003; Feng et al. 2008). However, it remains open whether similar programs control the proliferation of hepatocytes and immune cells. In the liver, a number of studies have mapped temporal changes in mRNA levels during regeneration (e.g., White et al. 2005). This, coupled with functional studies, has led to the identification of several TFs involved in the regenerative response (for review, see Kurinna and Barton 2011). Still, knowledge about how these factors are coordinated temporally throughout the regenerative process is limited. CEBPA (C/EBPalpha) and CEBPB (C/EBPbeta) are two key hepatocyte TFs known to have divergent roles in liver function and regeneration. The two factors belong to the same basic region leucine zipper-family (bZIP), and several studies have shown that they bind the same core DNA sequence, acting as either homo- or heterodimers (Diehl and Yang 1994; Rana et al. 1995; Osada et al. 1996). While CEBPA is highly expressed in the quiescent condition (before injury) and regulates many metabolic liver genes, CEBPB is up-regulated during liver regrowth and is required for a full regenerative response (Greenbaum et al. 1995; Wang et al. 1995). In many tissues, CEBPA is observed to be an anti-proliferative factor facilitating differentiation, while CEBPB has been found to be either pro- or anti-proliferative in different settings (Porse et al. 2001; Nerlov 2007). In the skin, the two factors appear to act redundantly to limit epidermal stem cell activity (Lopez et al. 2009). In the current study, we have examined dynamic TF binding during liver regeneration in the mouse by performing a time course of ChIP-seq experiments to map and quantify binding of CEBPA and CEBPB on a genome-wide scale at a high level of temporal resolution. We find that CEBPA and CEBPB generally occupy the same positions in the genome of hepatocytes in vivo, but they do so with quantitatively divergent temporal patterns during regeneration. To further dissect the dynamic transcriptional network behind liver regeneration, we interrogated the temporally defined groups of CEBP-bound elements for regulatory properties, both with respect to sequence composition and differential expression of associated genes.