The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin.

Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.

Protein Kinase A Downregulation Delays the Development and Progression of Polycystic Kidney Disease.

BACKGROUND: Upregulation of cAMP-dependent and cAMP-independent PKA signaling is thought to promote cystogenesis in polycystic kidney disease (PKD). PKA-I regulatory subunit RIα is increased in kidneys of orthologous mouse models. Kidney-specific knockout of RIα upregulates PKA activity, induces cystic disease in wild-type mice, and aggravates it in Pkd1RC/RC mice. METHODS: PKA-I activation or inhibition was compared with EPAC activation or PKA-II inhibition using Pkd1RC/RC metanephric organ cultures. The effect of constitutive PKA (preferentially PKA-I) downregulation in vivo was ascertained by kidney-specific expression of a dominant negative RIαB allele in Pkd1RC/RC mice obtained by crossing Prkar1αR1αB/WT, Pkd1RC/RC , and Pkhd1-Cre mice (C57BL/6 background). The effect of pharmacologic PKA inhibition using a novel, selective PRKACA inhibitor (BLU2864) was tested in mIMCD3 3D cultures, metanephric organ cultures, and Pkd1RC/RC mice on a C57BL/6 × 129S6/Sv F1 background. Mice were sacrificed at 16 weeks of age. RESULTS: PKA-I activation promoted and inhibition prevented ex vivo P-Ser133 CREB expression and cystogenesis. EPAC activation or PKA-II inhibition had no or only minor effects. BLU2864 inhibited in vitro mIMCD3 cystogenesis and ex vivo P-Ser133 CREB expression and cystogenesis. Genetic downregulation of PKA activity and BLU2864 directly and/or indirectly inhibited many pro-proliferative pathways and were both protective in vivo. BLU2864 had no detectable on- or off-target adverse effects. CONCLUSIONS: PKA-I is the main PKA isozyme promoting cystogenesis. Direct PKA inhibition may be an effective strategy to treat PKD and other conditions where PKA signaling is upregulated. By acting directly on PKA, the inhibition may be more effective than or substantially increase the efficacy of treatments that only affect PKA activity by lowering cAMP.

The genetic background significantly impacts the severity of kidney cystic disease in the Pkd1RC/RC mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), primarily due to PKD1 or PKD2 mutations, causes progressive kidney cyst development and kidney failure. There is significant intrafamilial variability likely due to the genetic background and environmental/lifestyle factors; variability that can be modeled in PKD mice. Here, we characterized mice homozygous for the PKD1 hypomorphic allele, p.Arg3277Cys (Pkd1RC/RC), inbred into the BALB/cJ (BC) or the 129S6/SvEvTac (129) strains, plus F1 progeny bred with the previously characterized C57BL/6J (B6) model; F1(BC/B6) or F1(129/B6). By one-month cystic disease in both the BC and 129 Pkd1RC/RC mice was more severe than in B6 and continued with more rapid progression to six to nine months. Thereafter, the expansive disease stage plateaued/declined, coinciding with increased fibrosis and a clear decline in kidney function. Greater severity correlated with more inter-animal and inter-kidney disease variability, especially in the 129-line. Both F1 combinations had intermediate disease severity, more similar to B6 but progressive from one-month of age. Mild biliary dysgenesis, and an early switch from proximal tubule to collecting duct cysts, was seen in all backgrounds. Preclinical testing with a positive control, tolvaptan, employed the F1(129/B6)-Pkd1RC/RC line, which has moderately progressive disease and limited isogenic variability. Magnetic resonance imaging was utilized to randomize animals and provide total kidney volume endpoints; complementing more traditional data. Thus, we show how genetic background can tailor the Pkd1RC/RC model to address different aspects of pathogenesis and disease modification, and describe a possible standardized protocol for preclinical testing.

Robotic Ultrasound and Novel Collagen Analyses for Polycystic Kidney Disease Research Using Mice.

BACKGROUND: 3D imaging and histology are critical tools for assessing polycystic kidney disease ( PKD ) in patients and animal models. Magnetic resonance ( MR ) imaging provides micron resolution, but is time consuming, expensive, and access to equipment and expertise is limiting. Robotic ultrasound ( US ) imaging has lower spatial resolution but is faster, more cost effective, and accessible. Similarly, Picrosirius red ( PSR ) staining and brightfield microscopy is commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. METHODS: In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume ( TKV ) measurements using US and MR. We additionally compared PSR imaging and quantification using standard brightfield with that by circularly polarized light with hue analysis, and fluorescence imaging analyzed using CT-FIRE software for automatic detection of individual collagen fibers. RESULTS: Increased TKV was detected by US in Pkd1RC/RC vs wild type ( WT ) at timepoints spanning early to established disease. US inter-observer variability was greater but allowed scanning in 2-5 minutes/mouse while MR required 20-30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield, polarized light showed fibers skewed thinner in Pkd1RC/RC vs WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1RC/RC vs WT, and fibers were thinner and curvier with no change in length. Additionally, fiber density was higher in both glomeruli and tubules in Pkd1RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1RC/RC , and trended higher in WT. CONCLUSIONS: These studies show robotic ultrasound is a rigorous imaging tool for pre-clinical PKD research. Additionally, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.