Krüppel-like factor 9 (Klf9) is a zinc finger transcription factor that functions in neural cell differentiation, but little is known about its genomic targets or mechanism of action in neurons. We used the mouse hippocampus-derived neuronal cell line HT22 to identify genes regulated by Klf9, and we validated our findings in mouse hippocampus. We engineered HT22 cells to express a Klf9 transgene under control of the tetracycline repressor, and used RNA sequencing to identify genes modulated by Klf9. We found 217 genes repressed and 21 induced by Klf9. We also engineered HT22 cells to co-express biotin ligase and a Klf9 fusion protein containing an N-terminal biotin ligase recognition peptide. Using chromatin-streptavidin precipitation (ChSP) sequencing we identified 3,514 genomic regions where Klf9 associated. Seventy-five percent of these were within 1 kb of transcription start sites, and Klf9 associated in chromatin with 60% of the repressed genes. We analyzed the promoters of several repressed genes containing Klf9 ChSP peaks using transient transfection reporter assays and found that Klf9 repressed promoter activity, which was abolished after mutation of Sp/Klf-like motifs. Knockdown or knockout of Klf9 in HT22 cells caused dysregulation of Klf9 target genes. Chromatin immunoprecipitation assays showed that Klf9 associated in chromatin from mouse hippocampus with genes identified by ChSP sequencing on HT22 cells, and expression of Klf9 target genes was dysregulated in the hippocampus of neonatal Klf9-null mice. Gene ontology analysis revealed that Klf9 genomic targets include genes involved in cystokeletal remodeling, Wnt signaling and inflammation. We have identified genomic targets of Klf9 in hippocampal neurons and created a foundation for future studies on how it functions in chromatin, and regulates neuronal morphology and survival across the lifespan.

Cardiac hypertrophy is a common adaptation to cardiovascular stress and often a prelude to heart failure. We examined how S -palmitoylation of the small GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1), impacts cardiomyocyte stress signaling. Mutation of the Cys-178 palmitoylation site impaired activation of Rac1 when overexpressed in cardiomyocytes. Cardiomyocyte-specific Rac1 conditional knockin (Rac1 ^cKI ) mice expressing a Rac1 ^C178S mutant protein exhibited normal cardiac structure and function but developed more severe cardiac hypertrophy in response to angiotensin II (AngII) infusion, cardiomyocyte-specific overexpression of AngII type 1 receptor (AT1R), and cardiac pressure overload. Moreover, pressure overload and AT1R overexpression evoked cardiac failure phenotypes in Rac1 ^cKI mice not observed in controls. Mechanistically, Rac1 ^cKI hearts and cardiomyocytes genetically resistant to Rac1 S -palmitoylation had a profound increase in protein kinase A (PKA) substrate phosphorylation in response to acute β-adrenergic stimulation, as did Rac1 ^cKI hearts subjected to chronic AngII treatment, AT1R overexpression, or pressure overload that correlates with more advanced heart failure phenotypes. This was not associated with increased PKA enzymatic activity, suggesting potential deficits in phosphatase activity at PKA-regulated phospho-sites. Taken together, this study suggests Rac1 S -palmitoylation dampens adrenergic drive and PKA-dependent modulation of the phospho-proteome in response to cardiovascular stress, revealing essential functions for S -acylated Rac1 in cardiac adaptation.