2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Abstract

During the progression of type 2 diabetes, β-cells fail and a subset lose features associated with their identity including reduced expression of key transcription factors (TFs). Among these is Pdx1, which is necessary for maintaining β-cell function and identity by controlling the expression of numerous β-cell genes including insulin, MafA, Glut2, and Ucn3. Pdx1, like most TFs, depends on the recruitment of coregulators to modulate its transcriptional activity. Numerous Pdx1-interacting proteins have been identified including Chd4, an ATP-dependent helicase of the chromatin remodeling NuRD complex. NuRD controls cellular processes including maintenance of cellular identity and mitochondrial function in a variety of tissues. The role Chd4 plays in modulating Pdx1 activity in the β-cell has yet to be explored. In this study, we performed co-immunoprecipitation and proximity ligation assay (PLA) in the Min6 murine insulinoma β-cell line and PLA in 8-week old C57BL/6 mouse pancreatic tissue, confirming Chd4:Pdx1 interactions in the nuclei of β-cells in both cell lines and tissue. Transient, RNAi-mediated knockdown of Chd4 in mouse β-cell lines altered the expression of Pdx1-regulated genes involved in β-cell function and identity including the MafA TF, which was reduced at both the RNA (41% ± 3 (p < 0.05)) and protein level. Moreover, the nuclear-encoded mitochondrial Tfam TF (reduced 30% ± 10 (p = 0.09)), the mitochondrial Mipep enzyme (reduced 35% ± 18 (p = 0.10)), and pre-Ins2, the unprocessed Ins2 transcript (reduced 38% ± 9 (p = 0.13)) were also altered, although they did not reach significance. These data suggest that Chd4 plays an important role in modulating the expression of genes controlled by Pdx1 in the β-cell. Future efforts are focused on further characterizing the role of Chd4 in the β-cell with CRISPR/Cas9 Chd4 knockout β-cell lines in vitro and β-cell specific Chd4 knockout mice. Disclosure R.K. Davidson: None. N. Casey: None. J. Spaeth: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K01DK115633)