BK Polyomavirus microRNA in Kidney Transplant Recipient.

BK polyomavirus-associated nephropathy (PyVAN) remains a serious threat for renal dysfunction and graft loss in kidney transplant recipients on immunosuppressive medication. In this study, a pilot cohort of 16 kidney transplant recipients were recruited of which eight were with significant BKPV viremia (sBKPV) and the rest were controls matched to age, gender, and time since transplant. We used next-generation sequencing to characterize the miRNA expression profile in urine samples. In total, the expression of 8 miRNAs (miR-16-5p,miR-200c-3p,bkv-miR-B1-3p,let-7b-3p,miR-1269b,bkv-miR-B1-5p,miR-193a-3p,miR-944) were upregulated whereas 21miRNAs (miR-134-5p,miR-4724-5p,miR-127-3p,miR-6500-3p,miR-507,miR-378b,miR-3911,miR-211-5p,miR-486-5p,miR-143-3p,miR-3195,miR-1307-5p,miR-29a-5p,miR-378f,miR-12136,miR-378g,miR-144-3p,miR-378a-3p,let-7i-5p,miR-204-5p,miR-146a-5p) were downregulated with fold change > 2. We found that bkv-miR-B1-5p and bkv-miR-B1-3p have 19-fold and 5-fold higher expression values in BKPV viremia patient samples, respectively. A few earlier studies have reported BKV miRNA in urine and serum samples using the RT- PCR from PyVAN patients. Our results corroborated findings from earlier studies and highlighted the need for additional evaluation of the role of sequencing approaches for monitoring BKPV specific and host miRNAs to better understand the viral reactivation and disease pathogenesis.

Single-nucleus multi-omic profiling of polyploid heart nuclei identifies fusion-derived cardiomyocytes in the human heart.

Understanding the mechanisms of polyploidization in cardiomyocytes is crucial for advancing strategies to stimulate myocardial regeneration. Although endoreplication has long been considered the primary source of polyploid human cardiomyocytes, recent animal work suggests the potential for cardiomyocyte fusion. Moreover, the effects of polyploidization on the genomic-transcriptomic repertoire of human cardiomyocytes have not been studied previously. We applied single-nuclei whole genome sequencing, single nuclei RNA sequencing, and multiome ATAC + gene expression (from the same nuclei) techniques to nuclei isolated from 11 healthy hearts. Utilizing post-zygotic non-inherited somatic mutations occurring during development as "endogenous barcodes," to reconstruct lineage relationships of polyploid cardiomyocytes. Of 482 cardiomyocytes from multiple healthy donor hearts 75.7% can be sorted into several developmental clades marked by one or more somatic single-nucleotide variants (SNVs). At least ~10% of tetraploid cardiomyocytes contain cells from distinct clades, indicating fusion of lineally distinct cells, whereas 60% of higher-ploidy cardiomyocytes contain fused cells from distinct clades. Combined snRNA-seq and snATAC-seq revealed transcriptome and chromatin landscapes of polyploid cardiomyocytes distinct from diploid cardiomyocytes, and show some higher-ploidy cardiomyocytes with transcriptional signatures suggesting fusion between cardiomyocytes and endothelial and fibroblast cells. These observations provide the first evidence for cell and nuclear fusion of human cardiomyocytes, raising the possibility that cell fusion may contribute to developing or maintaining polyploid cardiomyocytes in the human heart.

High-quality nuclei isolation from postmortem human heart muscle tissues for single-cell studies.

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

High-Quality Nuclei Isolation from Postmortem Human Heart Muscle Tissues for Single-Cell Studies.

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

Effects of intron conversion in the human CYP11B2 gene on its transcription and blood pressure regulation in transgenic mice.

The human cytochrome P450 family 11 subfamily B member 2 (hCYP11B2) gene encodes aldosterone synthase, the rate-limiting enzyme in the biosynthesis of aldosterone. In some humans, hCYP11B2 undergoes a unique intron conversion whose function is largely unclear. The intron conversion is formed by a replacement of the segment of DNA within intron 2 of hCYP11B2 with the corresponding region of the hCYP11B1 gene. We show here that the intron conversion is located in an open chromatin form and binds more strongly to the transcriptional regulators histone acetyltransferase P300 (p300), NFκB, and CCAAT enhancer-binding protein α (CEBPα). Reporter constructs containing the intron conversion had increased promoter activity on transient transfection in H295R cells compared with WT intron 2. We generated humanized transgenic (TG) mice containing all the introns, exons, and 5'- and 3'-flanking regions of the hCYP11B2 gene containing either the intron conversion or WT intron 2. We found that TG mice containing the intron conversion have (a) increased plasma aldosterone levels, (b) increased hCYP11B2 mRNA and protein levels, and (c) increased blood pressure compared with TG mice containing WT intron 2. Results of a ChIP assay showed that chromatin obtained from the adrenals of TG mice containing the intron conversion binds more strongly to p300, NFκB, and CEBPα than to WT intron 2. These results uncover a functional role of intron conversion in hCYP11B2 and suggest a new paradigm in blood pressure regulation.

A polymorphism in intron I of the human angiotensinogen gene (hAGT) affects binding by HNF3 and hAGT expression and increases blood pressure in mice.

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin II. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with the hepatocyte nuclear factor 3 (HNF3)-binding site than +1164G. Here we found that an oligonucleotide containing +1164A binds HNF3ß more strongly than +1164G and that Hap-I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the hypoxanthine-guanine phosphoribosyltransferase locus, we generated a transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor, CCAAT enhancer-binding protein ß, and HNF3ß bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that, unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure.