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Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1(Enh/Enh)). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia—even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1(Enh/−) mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

Summary Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells. For complete details on the use and execution of this protocol, please refer to Brigidi et al. (2019), Carlin et al. (2018), Heinz et al. (2018), Nott et al. (2019), Sakai et al. (2019), and Seidman et al. (2020)., Graphical abstract, Highlights • Cost-effective rapid, high-quality ChIP-seq prep (∼$40/library) • 8-well tube strip format for medium- to high-throughput ChIP-seq • Wide input range (0.1–5 million cells per IP) • Recommendations for antibody choice, assay optimization, and troubleshooting, Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells.

The sensing and clearing of microbial infections by multicellular organisms is accomplished through specialized germ line-encoded receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are a group of PRRs that recognize conserved molecules or products derived from a plethora of pathogens such as bacteria, fungi, protozoa, and viruses. Following ligand recognition, TLRs function to induce acute inflammatory responses that contribute to the killing of invading pathogens. In addition, TLR activation also induces dendritic cell maturation and proinflammatory cytokine secretion, which act synergistically to activate and enhance pathogen-specific adaptive immune responses. The initiation of the functional TLR response depends upon the recruitment of TLR-signaling adaptor molecules containing Toll Interleukin-1 receptor domains. These TLR-signaling adaptors then initiate signaling cascades leading to the activation of AP-1, which are the transcription factor(s) activated by MAP kinases, nuclear translocation of NF-kB-light chain enhancer of activated B cells, and interferon-regulatory factor transcription factor families.