RNF12 is an X-Encoded dose-dependent activator of X chromosome inactivation.

In somatic cells of female placental mammals, one X chromosome is inactivated to minimize sex-related dosage differences of X-encoded genes. Random X chromosome inactivation (XCI) in the embryo is a stochastic process, in which each X has an independent probability to initiate XCI, triggered by the nuclear concentration of one or more X-encoded XCI-activators. Here, we identify the E3 ubiquitin ligase RNF12 as an important XCI-activator. Additional copies of mouse Rnf12 or human RNF12 result in initiation of XCI in male mouse ES cells and on both X chromosomes in a substantial percentage of female mouse ES cells. This activity is dependent on an intact open reading frame of Rnf12 and correlates with the transgenic expression level of RNF12. Initiation of XCI is markedly reduced in differentiating female heterozygous Rnf12(+/-) ES cells. These findings provide evidence for a dose-dependent role of RNF12 in the XCI counting and initiation process.

Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings.

The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC.

Identifying cystogenic paracrine signaling molecules in cyst fluid of patients with polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD) paracrine signaling molecules in cyst fluid can induce proliferation and cystogenesis of neighboring renal epithelial cells. However, the identity of this cyst-inducing factor is still unknown. The aim of this study was to identify paracrine signaling proteins in cyst fluid using a 3D in vitro cystogenesis assay. We collected cyst fluid from 15 ADPKD patients who underwent kidney or liver resection (55 cysts from 13 nephrectomies, 5 cysts from 2 liver resections). For each sample, the ability to induce proliferation and cyst formation was tested using the cystogenesis assay (RPTEC/TERT1 cells in Matrigel with cyst fluid added for 14 days). Kidney cyst fluid induced proliferation and cyst growth of renal epithelial cells in a dose-dependent fashion. Liver cyst fluid also induced cystogenesis. Using size exclusion chromatography, 56 cyst fluid fractions were obtained of which only the fractions between 30 and 100 kDa showed cystogenic potential. Mass spectrometry analysis of samples that tested positive or negative in the assay identified 43 candidate cystogenic proteins. Gene ontology analysis showed an enrichment for proteins classified as enzymes, immunity proteins, receptors, and signaling proteins. A number of these proteins have previously been implicated in ADPKD, including secreted frizzled-related protein 4, S100A8, osteopontin, and cysteine rich with EGF-like domains 1. In conclusion, both kidney and liver cyst fluids contain paracrine signaling molecules that drive cyst formation. Using size exclusion chromatography and mass spectrometry, we procured a candidate list for future studies. Ultimately, cystogenic paracrine signaling molecules may be targeted to abrogate cystogenesis in ADPKD.

RNF12 activates Xist and is essential for X chromosome inactivation.

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12⁻/⁻ knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.