Interrogation of nonconserved human adipose lincRNAs identifies a regulatory role of linc-ADAL in adipocyte metabolism.

Long intergenic noncoding RNAs (lincRNAs) have emerged as important modulators of cellular functions. Most lincRNAs are not conserved among mammals, raising the fundamental question of whether nonconserved adipose-expressed lincRNAs are functional. To address this, we performed deep RNA sequencing of gluteal subcutaneous adipose tissue from 25 healthy humans. We identified 1001 putative lincRNAs expressed in all samples through de novo reconstruction of noncoding transcriptomes and integration with existing lincRNA annotations. One hundred twenty lincRNAs had adipose-enriched expression, and 54 of these exhibited peroxisome proliferator-activated receptor γ (PPARγ) or CCAAT/enhancer binding protein α (C/EBPα) binding at their loci. Most of these adipose-enriched lincRNAs (~85%) were not conserved in mice, yet on average, they showed degrees of expression and binding of PPARγ and C/EBPα similar to those displayed by conserved lincRNAs. Most adipose lincRNAs differentially expressed (n = 53) in patients after bariatric surgery were nonconserved. The most abundant adipose-enriched lincRNA in our subcutaneous adipose data set, linc-ADAL, was nonconserved, up-regulated in adipose depots of obese individuals, and markedly induced during in vitro human adipocyte differentiation. We demonstrated that linc-ADAL interacts with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) at distinct subcellular locations to regulate adipocyte differentiation and lipogenesis.

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

OBJECTIVE: To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. APPROACH AND RESULTS: We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA-/-) had barely detectable LAL enzymatic activity. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [3H]-cholesterol to apolipoprotein A-I was abolished in LIPA-/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [3H]-cholesterol-labeled AcLDL, [3H]-cholesterol efflux was, however, not different between control and LIPA-/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA-/- IPSDM. In nonlipid loaded state, LIPA-/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA-/- IPSDM. LIPA-/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA-/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human macrophages.