Kidney Week

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

• PKD Cellular Pathogenesis, ARPKD, ADTKD, Ciliopathies
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

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

Santiago Cuevas,

• Yang, Chaozhe, Children's National Medical Center, Bowie, Maryland, United States

Chaozhe Yang,

• Harafuji, Naoe, Children's National Health System, Washington, District of Columbia, United States

Naoe Harafuji,

• Odinakachukwu, Maryanne, Children's National Medical Center, Bowie, Maryland, United States

Maryanne Odinakachukwu,

• Germino, Gregory G., Deputy Director, NIDDK/NIH, Bethesda, Maryland, United States

Gregory G. Germino,

• Outeda, Patricia, University of Maryland, Baltimore, Maryland, United States

Patricia Outeda,

• Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States

Terry J. Watnick,

• Guay-Woodford, Lisa M., Children's National Health System, Washington, District of Columbia, United States

Lisa M. Guay-Woodford,

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 Pkhd1^cyli, Pkhd1^del67, Pkhd1^del3-67, Cys1^cpk 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 Cys1^cpk cystic kidneys, but not in Pkhd1^cyli, Pkhd1^del67, or Pkhd1^del3-67 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.