GRADED PU.1 LEVELS ACTIVATE GRANULOCYTE VS. MACROPHAGE GENES VIA MULTIPLE (SUPER)ENHANCER ELEMENTS

Precise levels of transcription factor PU.1 are critical for differentiation to distinct blood lineages, and even modest decreases leads to leukemogenesis. However, the mechanism of how target genes sense different PU.1 levels is unknown. To address this, we used myeloid progenitors encoding inducible PU.1 transgene. Intermediate PU.1 activity induced differentiation into granulocyte-like cells, while high PU.1 produced macrophages. Comparative analysis of regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Myeloperoxidase (Mpo) and Gelatinase B (Mmp9) identified 14 Mpo and 16 Mmp9 putative enhancers resembling superenhancers. Interestingly, the PU.1 binding motifs within these regions have an intermediate to high affinity (log ods score). To test their functionality we assembled successively these regulatory sequences into luciferase vectors. Specifically, we identified novel enhancer elements in -3.4 and -15 kb of Mpo which responded to intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of promoter while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at multiple Mmp9 enhancers. This model is supported by our Chromosome confirmation capture (3C) data. Native activity of tested enhancers was confirmed by expression of bidirectional enhancer RNAs. The mutation of affinity of PU.1 binding sites within enhancers lead to reprogramming of their activity. In conclusion, our data support the model that PU.1 at intermediate concentrations binds to high affinity binding sites in several enhancers of granulocyte genes, causing their successive interaction that leads to transcription activation. Collectively, this suggests that a cooperative assembly of several cell-type specific enhancers is required for optimal Mpo and Mmp9 activation.