Differential regulation of MYC expression by PKHD1/Pkhd1 in human and mouse kidneys: phenotypic implications for recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is a severe, hereditary, hepato-renal fibrocystic disorder that leads to early childhood morbidity and mortality. Typical forms of ARPKD are caused by pathogenic variants in the PKHD1 gene, which encodes the fibrocystin/polyductin (FPC) protein. MYC overexpression has been proposed as a driver of renal cystogenesis, but little is known about MYC expression in recessive PKD. In the current study, we provide the first evidence that MYC is overexpressed in kidneys from ARPKD patients and confirm that MYC is upregulated in cystic kidneys from cpk mutant mice. In contrast, renal MYC expression levels were not altered in several Pkhd1 mutant mice that lack a significant cystic kidney phenotype. We leveraged previous observations that the carboxy-terminus of mouse FPC (FPC-CTD) is proteolytically cleaved through Notch-like processing, translocates to the nucleus, and binds to double stranded DNA, to examine whether the FPC-CTD plays a role in regulating MYC/Myc transcription. Using immunofluorescence, reporter gene assays, and ChIP, we demonstrate that both human and mouse FPC-CTD can localize to the nucleus, bind to the MYC/Myc P1 promoter, and activate MYC/Myc expression. Interestingly, we observed species-specific differences in FPC-CTD intracellular trafficking. Furthermore, our informatic analyses revealed limited sequence identity of FPC-CTD across vertebrate phyla and database queries identified temporal differences in PKHD1/Pkhd1 and CYS1/Cys1 expression patterns in mouse and human kidneys. Given that cystin, the Cys1 gene product, is a negative regulator of Myc transcription, these temporal differences in gene expression could contribute to the relative renoprotection from cystogenesis in Pkhd1-deficient mice. Taken together, our findings provide new mechanistic insights into differential mFPC-CTD and hFPC-CTD regulation of MYC expression in renal epithelial cells, which may illuminate the basis for the phenotypic disparities between human patients with PKHD1 pathogenic variants and Pkhd1-mutant mice.

Pkhd1cyli/cyli mice have altered renal Pkhd1 mRNA processing and hormonally sensitive liver disease.

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd1cyli/cyli (cyli) mice exhibit a severe liver pathology but lack renal disease. Further analysis revealed that several alternatively spliced Pkhd1 mRNA, all containing exon 48, were expressed in cyli kidneys, but in lower abundance than in wild-type kidneys, suggesting that these transcripts escaped from nonsense-mediated decay (NMD). We identified an AAAAAT motif in exon 48 upstream of the cyli mutation which could enable ribosomal frameshifting, thus potentially allowing production of sufficient amounts of FPC for renoprotection. This mechanism, expressed in a species-specific fashion, may help explain the disparities in the renal phenotype observed between Pkhd1 mutant mice and patients with PKHD1-related disease. KEY MESSAGES: The Pkhd1cyli/cyli mouse expresses cystic liver disease, but no kidney phenotype. Pkhd1 mRNA expression is decreased in cyli liver and kidneys compared to wild-type. Ribosomal frameshifting may be responsible for Pkhd1 mRNA escape from NMD. Pkhd1 mRNA escape from NMD could contribute to the absent kidney phenotype.

Cystin is required for maintaining fibrocystin (FPC) levels and safeguarding proteome integrity in mouse renal epithelial cells: A mechanistic connection between the kidney defects in cpk mice and human ARPKD.

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in congenital polycystic kidney (cpk) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r-cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

Transcription factor Ap2b regulates the mouse autosomal recessive polycystic kidney disease genes, Pkhd1 and Cys1.

Transcription factor Ap2b (TFAP2B), an AP-2 family transcription factor, binds to the palindromic consensus DNA sequence, 5'-GCCN3-5GGC-3'. Mice lacking functional Tfap2b gene die in the perinatal or neonatal period with cystic dilatation of the kidney distal tubules and collecting ducts, a phenotype resembling autosomal recessive polycystic kidney disease (ARPKD). Human ARPKD is caused by mutations in PKHD1, DZIP1L, and CYS1, which are conserved in mammals. In this study, we examined the potential role of TFAP2B as a common regulator of Pkhd1 and Cys1. We determined the transcription start site (TSS) of Cys1 using 5' Rapid Amplification of cDNA Ends (5'RACE); the TSS of Pkhd1 has been previously established. Bioinformatic approaches identified cis-regulatory elements, including two TFAP2B consensus binding sites, in the upstream regulatory regions of both Pkhd1 and Cys1. Based on reporter gene assays performed in mouse renal collecting duct cells (mIMCD-3), TFAP2B activated the Pkhd1 and Cys1 promoters and electromobility shift assay (EMSA) confirmed TFAP2B binding to the in silico identified sites. These results suggest that Tfap2b participates in a renal epithelial cell gene regulatory network that includes Pkhd1 and Cys1. Disruption of this network impairs renal tubular differentiation, causing ductal dilatation that is the hallmark of recessive PKD.

Cystin genetic variants cause autosomal recessive polycystic kidney disease associated with altered Myc expression.

Mutation of the Cys1 gene underlies the renal cystic disease in the Cys1cpk/cpk (cpk) mouse that phenocopies human autosomal recessive polycystic kidney disease (ARPKD). Cystin, the protein product of Cys1, is expressed in the primary apical cilia of renal ductal epithelial cells. In previous studies, we showed that cystin regulates Myc expression via interaction with the tumor suppressor, necdin. Here, we demonstrate rescue of the cpk renal phenotype by kidney-specific expression of a cystin-GFP fusion protein encoded by a transgene integrated into the Rosa26 locus. In addition, we show that expression of the cystin-GFP fusion protein in collecting duct cells down-regulates expression of Myc in cpk kidneys. Finally, we report the first human patient with an ARPKD phenotype due to homozygosity for a deleterious splicing variant in CYS1. These findings suggest that mutations in Cys1/CYS1 cause an ARPKD phenotype in mouse and human, respectively, and that the renal cystic phenotype in the mouse is driven by overexpression of the Myc proto-oncogene.

Molecular signatures of differential responses to exercise trainings during rehabilitation.

The loss and recovery of muscle mass and function following injury and during rehabilitation varies among individuals. While recent expression profiling studies have illustrated transcriptomic responses to muscle disuse and remodeling, how these changes contribute to the physiological responses are not clear. In this study, we quantified the effects of immobilization and subsequent rehabilitation training on muscle size and identified molecular pathways associated with muscle responsiveness in an orthopaedic patient cohort study. The injured leg of 16 individuals with ankle injury was immobilized for a minimum of 4 weeks, followed by a 6-week rehabilitation program. The maximal cross-sectional area (CSA) of the medial gastrocnemius muscle of the immobilized and control legs were determined by T1-weighted axial MRI images. Genome-wide mRNA profiling data were used to identify molecular signatures that distinguish the patients who responded to immobilization and rehabilitation and those who were considered minimal responders. RESULTS: Using 6% change as the threshold to define responsiveness, a greater degree of changes in muscle size was noted in high responders (-14.9 ± 3.6%) compared to low responders (0.1 ± 0.0%) during immobilization. In addition, a greater degree of changes in muscle size was observed in high responders (20.5 ± 3.2%) compared to low responders (2.5 ± 0.9%) at 6-week rehabilitation. Microarray analysis showed a higher number of genes differentially expressed in the responders compared to low responders in general; with more expression changes observed at the acute stage of rehabilitation in both groups. Pathways analysis revealed top molecular pathways differentially affected in the groups, including genes involved in mitochondrial function, protein turn over, integrin signaling and inflammation. This study confirmed the extent of muscle atrophy due to immobilization and recovery by exercise training is associated with distinct remodeling signature, which can potentially be used for evaluating and predicting clinical outcomes.

Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy.

Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

DUX4 differentially regulates transcriptomes of human rhabdomyosarcoma and mouse C2C12 cells.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p<0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.

miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function. METHODS: To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells to determine the effect of miR-411 on myogenic markers. RESULTS: Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary (2.1 fold, p<0.01) and immortalized (2.7 fold, p<0.01) myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting (-3.7 fold, p<0.01). C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411. CONCLUSIONS: The study demonstrated that miR-411 was differentially expressed in FSHD myoblasts and may play a role in regulating myogenesis.

Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives.

The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.

A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Genome-wide identification of tissue-specific enhancers in the Ciona tadpole.

Less than 100 cis-regulatory DNAs have been characterized in the context of transgenic metazoan embryos. Here we investigate the feasibility of conducting a genome-wide search for tissue-specific enhancers in the ascidian Ciona intestinalis. A total of 138 random genomic DNA fragments with an average size of 1.7 kb were separately placed 5' of a lacZ reporter gene. Eleven of the lacZ fusion genes displayed localized patterns of expression in tadpole-stage Ciona embryos. At least five of these transgenes appear to contain bona fide tissue-specific enhancers that direct expression in the cerebral vesicle, neural tube, primordial adhesive organ, notochord, and tail epidermis. One of the enhancers maps near Distalless (Ci-Dll-A) and recapitulates most aspects of the endogenous expression pattern, including localized expression in the anterior-most regions of the neurogenic ectoderm. We discuss the prospects of creating a regulatory atlas of the Ciona genome, whereby every enhancer is identified for every gene.