Supplementary Materials01. from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBP in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBP, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity. Introduction Cells sense and respond to their cellular environment through SKI-606 kinase activity assay signal transduction pathways, which can deliver information to the genome in the form of activated transcription factors. These factors tend to occupy specific genomic regions and associate with different co-activators and chromatin remodeling complexes to direct their response. This occurs by either activating or repressing transcription or by changing the chromatin architecture, thus reforming the accessibility of certain genomic loci (Mosimann et al., 2009; Moustakas and Heldin, 2009). This combination of actions allow for the same signaling pathways to be used in multiple cellular environments eliciting different responses. The BMP and Wnt signaling pathways are two highly conserved signaling pathways that interact Prp2 during many developmental processes, ultimately through regulation of transcription via SMAD and TCF/LEF transcription factors (Clevers, 2006; Larsson and Karlsson, 2005; Staal and Luis, 2010). Both pathways participate in the formation of the hematopoietic system during development, but appear to be expendable during adult steady state hematopoiesis (Cheng et al., 2008; Goessling et al., 2009; Jeannet et al., 2008; Koch et al., 2008; Lengerke et al., 2008; McReynolds et al., 2007; Nostro et al., 2008; Singbrant et al., 2010; Tran et al., 2010). In both development and regeneration, hematopoietic stem cells divide and differentiate in response to cell-intrinsic and extrinsic signals to produce all of the SKI-606 kinase activity assay hematopoietic lineages. Here we show that the BMP and Wnt signaling pathways are critical for efficient regeneration of the adult hematopoietic system, as they are in development. Additionally, BMP and Wnt have been implicated in differentiation into erythroid and myeloid lineages. Specifically, in culture, BMP treatment can augment erythroid, megakaryocytic, and granulocytic-monocytic output of CD34+ progenitors (Detmer and Walker, 2002; Fuchs et al., 2002; Jeanpierre et al., 2008). Similarly, Wnt3a ligand can regulate the production of erythroid and myeloid cells from ESC and myeloid progenitors from adult HSC (Cheng et al., 2008; Nostro et al., 2008; Staal and Luis, 2010). The underlying mechanism for BMP and SKI-606 kinase activity assay Wnt regulation of regeneration and differentiation resides in the cell-type specific targets of the SMAD and TCF transcription factors, respectively. Based on previous findings, SMAD and TCF proteins can couple with other transcription factors to regulate a small number of cell-specific genes (Clevers, 2006; Massague et al., 2005; Mosimann et al., 2009). Signaling-mediated transcription factors have recently begun to be studied in a genome-wide manner, and these studies have revealed that Smad1 and Tcf7l1/Tcf3 can co-occupy target sites with the Oct4/Nanog/Sox2 transcriptional complex on pluripotency target genes in embryonic stem cells (ESCs) (Chen et al., 2008; Cole et al., 2008) and TCF7L2 can co-localize with CDX2 in colonic cells (Verzi et al., 2010). This led us to consider the possibility that BMP and Wnt signaling factors couple with distinct transcription factors important for lineage identity during hematopoietic regeneration and differentiation. To determine how SMAD and TCF transcription factors select their targets in distinct lineages during regeneration and differentiation, we explored their genome-wide DNA binding in various hematopoietic environments across multiple species. Initially, co-binding of Smad1 with Gata2 at individual genes in regenerating progenitors was observed. Genome-wide analysis revealed that SMAD1 and TCF7L2 selectively bind in concert with cell-specific master regulators at lineage distinctive genes in erythroid and myeloid cell populations. In addition, the expression of a myeloid master regulator in erythroid cells is sufficient to redirect a fraction of Smad1 binding. During differentiation, the binding of signaling factors shifts from genes of multiple hematopoietic lineages in progenitor cells to genes specific for differentiated cells guided by the dominant lineage factor. Together, these data support a mechanism by which lineage regulators direct SMAD and TCF proteins to tissue-specific enhancers. The selective use of these pathways during regeneration suggests that coordinated binding of SMAD1 and TCF7L2 with lineage-restricted regulators is the underlying mechanism for BMP and Wnt effects during hematopoietic differentiation and regeneration. Results Wnt and BMP play essential.