Lineage Regulators Direct BMP and Wnt Pathways to Cell-Specific Programs During Differentiation and Regeneration,

Abstract 3387

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we have evaluated their function during hematopoietic regeneration after irradiation. Using heat-shock inducible transgenic zebrafish lines that overexpress BMP2 or Wnt8, we demonstrated accelerated marrow recovery following irradiation. Heat-shock induced overexpression of the respective inhibitors Chordin and DKK1 blunted the recovery. Surprisingly, gene expression profiling after induction of BMP or Wnt signaling in zebrafish marrow cells post-irradiation revealed increased expression of the key hematopoietic genes scl, runx1, and gata2. To determine if the effect of BMP and Wnt signaling on hematopoietic genes during regeneration was direct, we performed ChIP-PCR for Smad1 and the hematopoietic regulator Gata2 in murine lineage-negative progenitors seven days after a sublethal irradiation. We found that Smad1 and Gata2 co-occupy hematopoietic genes including Cd9, Il13, Mapk6, and Meis1. To examine the binding of SMAD1 and TCF7L2 throughout the genome of hematopoietic cells, we employed ChIP-seq in human erythroid and myeloid leukemia cell lines, K562 and U937, respectively. More than 70% of the genes bound by SMAD1 and TCF7L2 were co-occupied with the lineage transcription factors GATA1 and GATA2 in erythroid cells, and with C/EBPα in myeloid cells. This finding suggests that signaling transcription factors control hematopoietic gene programs by binding DNA adjacent to lineage-specific transcription factors. The transcriptional output of BMP and Wnt activity was tested on an LMO2 enhancer reporter construct. Expression of SMAD1 or TCF7L2 alone had little effect, but markedly increased reporter activity in conjunction with GATA2, indicating that BMP and Wnt signaling cooperate with lineage regulators to enhance transcription of cell-type specific target genes. To establish the order of transcription factor occupancy, we utilized estrogen-inducible C/EBPα-ER in K562 cells or GATA1 induction in murine G1ER cell lines, and assessed SMAD1 occupancy before and after induction of each respective lineage regulator. Induction of the myeloid lineage regulator C/EBPα in K562 cells shifted binding of SMAD1, such that SMAD1 co-occupancy with C/EBPα changed from 6% to 15% of C/EBPα targets. In contrast, expression of the erythroid regulator GATA1 promoted loss of SMAD1 on 82% of its targets, and restricted more than 98% of the remaining SMAD1 sites to erythroid targets adjacent to GATA1. Co-occupancy of signaling factors and lineage regulators was further tested in primary human CD34+ multipotent hematopoietic progenitors and CD34+ cells directed to the erythroid lineage. Both SMAD1 and TCF7L2 co-localized with GATA2 on greater than 75% of bound genes in multipotent CD34+ progenitor cells. Similar to our results following GATA1 induction in G1ER cells, SMAD1 occupancy shifted to 65% erythroid targets upon differentiation of progenitors to the erythroid lineage. These data provide strong evidence that the binding of signaling factors follows the genomic occupancy of the dominant lineage regulator during differentiation. Together, our findings demonstrate that hematopoietic regeneration is driven by collaboration of master regulators and signaling transcription factors to control the entire hematopoietic program.