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Abstract: FR-PO990

Nuclear Complexes Containing FPC-CTD/Mcm3/Mcm5/STAT1 Define a Myc-Regulatory Pathway in Mouse with Implications for Renal Cystogenesis

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic

Authors

  • Cuevas, Santiago, Children’s National Health System, Washington, District of Columbia, United States
  • Yang, Chaozhe, Children's National Medical Center, Bowie, Maryland, United States
  • Harafuji, Naoe, Children's National Health System, Washington, District of Columbia, United States
  • Odinakachukwu, Maryanne, Children's National Medical Center, Bowie, Maryland, United States
  • Germino, Gregory G., Deputy Director, NIDDK/NIH, Bethesda, Maryland, United States
  • Outeda, Patricia, University of Maryland, Baltimore, Maryland, United States
  • Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Guay-Woodford, Lisa M., Children's National Health System, Washington, District of Columbia, United States
Background

ARPKD (MIM 263200) typically results from mutations in PKHD1. The intracellular C-terminal domain (FPC-CTD), encoded by exon 67, undergoes regulated membrane-release (Kaimori, 2007). Using single particle EM, we have shown that affinity-captured, natively-formed FPC-CTD nuclear assemblies integrate into DNA networks in ring-shaped structures reminiscent of known DNA-binding proteins (Harafuji, 2016). The current study was designed to examine FPC-CTD binding partners and their nuclear function.

Methods

Stable cell lines expressing V5-tagged FPC-CTD were generated; cell lysates fractionated on glycerol gradients; and immunoprecipitation (IP) were performed. Proteins were identified by mass spectrometry (IP-MS) and interactions confirmed by co-IP. Kidneys from ARPKD patients as well as the Pkhd1cyli, Pkhd1del67, Pkhd1del3-67, Cys1cpk mouse models were examined by RT-PCR, immunohistochemistry, and IB. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were conducted using the Myc P1 promoter.

Results

IP-MS identified 34 proteins, including four members of the minichromosome maintenance protein complex (Mcm3, Mcm5, Mcm6, Mcm7). IP confirmed FPC-CTD-Mcm3, FPC-CTD-Mcm5, and Mcm5-STAT1 interactions. In FPC-CTD stable cell lines, Myc is upregulated. FPC-CTD binds to the P1 promoter (ChIP) and enhances Myc P1 promoter activity (luciferase assay). When compared to controls, we observed Myc overexpression in human ARPKD and Cys1cpk cystic kidneys, but not in Pkhd1cyli, Pkhd1del67, or Pkhd1del3-67 kidneys, none of which express cystic disease.

Conclusion

Trudel (2015) has proposed that Myc overexpression defines a causative pathway in renal cystic disease. In this study, we show that: 1) in vitro, FPC-CTD upregulates Myc expression; and 2) FPC-CTD nuclear complexes contain Mcm3/Mcm5. Mcm5 interacts with STAT1, which transcriptionally represses Myc expression (Ramana, 2000). Our data suggest a reciprocal mechanism in which FPC-CTD activates, while STAT1 represses Myc expression. This model may explain the lack of Myc upregulation in mouse kidneys with reduced or absent FPC-CTD. Given that the mouse and human FPC-CTD share only 41% identity, we speculate that there are species-specific differences in Myc translational regulation.