Hematopoietic Transcription Factor PU.1 and Enhancer RNA Loup Establish a Feedforward Regulatory Loop Governing Monocyte/Macrophage Biology

The ETS transcription factor PU.1, known for its precise expression pattern, plays a crucial role in blood development. Deviations from its normal graded levels in hematopoietic lineages are associated with blood-related neoplasms and malignancies. As a transcription factor, PU.1 binds to purine-rich DNA sequences in enhancers and promoters as well as other transcription factors to regulate gene expression. While noncoding genes are increasingly reported to control fundamental biological processes in both normal and malignant hematopoiesis, most of our understanding of PU.1's role comes from its myeloid coding target genes. Furthermore, the role of PU.1 in enhancer regulation has not been systematically explored, highlighting a critical area for future research.

In this study, we investigated the role of PU.1 in enhancer regulation. To that end, we first compiled a comprehensive catalog of putative and verified enhancers from databases and published sources. We then interrogated this catalog with a genome-wide PU.1 occupancy profile in a myeloid cell line. In doing so, we identified a repertoire of PU.1-bound enhancers in myeloid cells. Given that enhancer-associated noncoding RNAs (eRNAs) are increasingly reported to promote enhancer activities, we focused our attention on this group of noncoding genes. By interrogating the list of PU.1-bound enhancers with the PU.1-mediated transcript profile, we revealed a group of eRNAs regulated by PU.1. Among these noncoding genes, to our surprise, was the eRNA LOUP, which we had previously discovered as an RNA inducer of PU.1. We further demonstrated that PU.1 occupies a genomic region within the LOUP locus that exhibits the epigenetic features of a myeloid-specific superenhancer. In addition to its previously described role in regulating myeloid differentiation and inflammatory signaling, we demonstrated that LOUP regulates the expression of surface markers and cytokines tailored to monocyte/macrophage biology. Furthermore, our data also indicated the role of LOUP in microbial clearance.

Taken together, our findings suggest that LOUP and PU.1 are partner regulatory biomolecules within an enhancer-mediated feedforward regulatory loop that induces the expression of each other, thereby regulating monocytic/macrophage differentiation and functions. We are currently investigating RNA and protein biomolecules that control this feedforward regulatory loop and its role in myeloid neoplasms and malignancies. Our study could provide a fundamental mechanistic understanding of hematopoietic enhancer specificity and lay the groundwork for further translational studies aimed at developing new therapeutic approaches for the treatment of inflammatory diseases and hematopoietic malignancies.

No relevant conflicts of interest to declare.