Epigenetic Regulation of Protein Kinase C Expression in the Hematopoietic Niche

The bone marrow sinusoidal vascular niche is important for HSC engraftment and regeneration of hematopoiesis after transplantation, but the epigenetic factors that control these biological functions are undefined. Using bulk RNA-seq on sorted zebrafish endothelial cell populations, we identified prkcda as a gene that is transcriptionally downregulated in the sinusoidal endothelial cells of the caudal hematopoietic territory (CHT) relative to arteriovenous endothelial cells. Dysregulated expression of prkcda in CHT endothelial cells increased the number of phenotypic HSPCs colonizing the CHT at 72 hpf and increased the number of long-term HSC clones as defined by genetic barcoding. In order to understand the epigenetic mechanisms controlling these expression patterns, we performed single cell RNA- and ATAC-sequencing on zebrafish kidney marrow. Single cell RNA-sequencing showed highly specific expression of prkcda in neutrophils. Cell identities were transferred to scATAC-seq data using gene activity scores; the scATAC neutrophil population showed a strong peak of chromatin accessibility in an intronic region approximately 6 kb from the transcriptional start site. Bulk ATAC-seq in E4-HUVEC sinusoidal endothelial cells showed a peak of chromatin accessibility in the same relative position of the PRKCD gene. Analysis of the zebrafish data using Cicero showed linkage between peaks of accessibility at the prkcda promoter and within the intron, identifying the latter a putative cis-acting enhancer sequence. We generated an mClover reporter construct driven by this sequence and microinjected it into zebrafish embryos. Fluorescence was observed in the endocardium and bulbus arteriosus of 72 hpf embryos, consistent with the prkcda in situ, confirming this as an enhancer sequence. We also observed fluorescence in the CHT of the prkcda enhancer-reporter transgenic animals, a pattern that is inconsistent with the prkcda in situ and implies active transcriptional repression at the native gene locus. FIMO analysis of the enhancer sequence identified putative transcription factor binding sites for WT1, KLF4, and KLF9. Ongoing work aims to clearly identify the transcriptional enhancers and repressors controlling expression of prkcda in the vascular niche. By understanding the epigenetic code controlling expression of important niche factors such as prkcda, we hope to gain a better understanding of how the niche is programmed to support hematopoiesis.