zDHHC9 Regulates Cardiomyocyte Rab3a Activity and Atrial Natriuretic Peptide Secretion Through Palmitoylation of Rab3gap1.

Production and release of natriuretic peptides by the stressed heart reduce cardiac workload by promoting vasodilation, natriuresis, and diuresis, which has been leveraged in the recent development of novel heart-failure pharmacotherapies, yet the mechanisms regulating cardiomyocyte exocytosis and natriuretic peptide release remain ill defined. We found that the Golgi S-acyltransferase zDHHC9 palmitoylates Rab3gap1 resulting in its spatial segregation from Rab3a, elevation of Rab3a-GTP levels, formation of Rab3a-positive peripheral vesicles, and impairment of exocytosis that limits atrial natriuretic peptide release. This novel pathway potentially can be exploited for targeting natriuretic peptide signaling in the treatment of heart failure.

An Intact Krüppel-like factor 9 Gene Is Required for Acute Liver Period 1 mRNA Response to Restraint Stress.

The clock protein period 1 (PER1) is a central component of the core transcription-translation feedback loop governing cell-autonomous circadian rhythms in animals. Transcription of Per1 is directly regulated by the glucocorticoid (GC) receptor (GR), and Per1 mRNA is induced by stressors or injection of GC. Circulating GCs may synchronize peripheral clocks with the central pacemaker located in the suprachiasmatic nucleus of the brain. Krüppel-like factor 9 (KLF9) is a zinc finger transcription factor that, like Per1, is directly regulated by liganded GR, and it associates in chromatin at clock and clock-output genes, including at Per1. We hypothesized that KLF9 modulates stressor-dependent Per1 transcription. We exposed wild-type (WT) and Klf9 null mice (Klf9-/-) of both sexes to 1 hour restraint stress, which caused similar 2- to 2.5-fold increases in plasma corticosterone (B) in each genotype and sex. Although WT mice of both sexes showed a 2-fold increase in liver Per1 mRNA level after restraint stress, this response was absent in Klf9-/- mice. However, injection of B in WT and Klf9-/- mice induced similar increases in Per1 mRNA. Our findings support that an intact Klf9 gene is required for liver Per1 mRNA responses to an acute stressor, but a possible role for GCs in this response requires further investigation.

Krüppel-Like Factors 9 and 13 Block Axon Growth by Transcriptional Repression of Key Components of the cAMP Signaling Pathway.

Krüppel-like factors (KLFs) are zinc finger transcription factors implicated in diverse biological processes, including differentiation of neural cells. The ability of mammalian neurons to elongate axons decreases during postnatal development in parallel with a decrease in cAMP, and increase in expression of several Klf genes. The paralogous KLFs 9 and 13 inhibit neurite outgrowth, and we hypothesized that their actions are mediated through repression of cAMP signaling. To test this we used the adult mouse hippocampus-derived cell line HT22 engineered to control expression of Klf9 or Klf13 with doxycycline, or made deficient for these Klfs by CRISPR/Cas9 genome editing. We also used primary hippocampal cells isolated from wild type, Klf9 -/- and Klf13 -/- mice. Forced expression of Klf9 or Klf13 in HT22 changed the mRNA levels of several genes involved with cAMP signaling; the predominant action was gene repression, and KLF13 influenced ∼4 times more genes than KLF9. KLF9 and KLF13 repressed promoter activity of the protein kinase a catalytic subunit alpha gene in transfection-reporter assays; KLF13, but not KLF9 repressed the calmodulin 3 promoter. Forskolin activation of a cAMP-dependent promoter was reduced after forced expression of Klf9 or Klf13, but was enhanced in Klf gene knockout cells. Forced expression of Klf9 or Klf13 blocked cAMP-dependent neurite outgrowth in HT22 cells, and axon growth in primary hippocampal neurons, while Klf gene knockout enhanced the effect of elevated cAMP. Taken together, our findings show that KLF9 and KLF13 inhibit neurite/axon growth in hippocampal neurons, in part, by inhibiting the cAMP signaling pathway.

Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain.

Thyroid hormone (T3) plays pivotal roles in vertebrate development, acting via nuclear T3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T3-dependent developmental process, caused us to hypothesize that T3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3). Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T3 response elements within the DNA methyltransferase 3a and Krüppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T3 response element. Our findings show that T3 can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes.

Molecular Mechanisms for Krüppel-Like Factor 13 Actions in Hippocampal Neurons.

Krüppel-like factors (KLFs) play key roles in nervous system development and function. Several KLFs are known to promote, and then maintain neural cell differentiation. Our previous work focused on the actions of KLF9 in mouse hippocampal neurons. Here we investigated genomic targets and functions of KLF9's paralog KLF13, with the goal of understanding how these two closely related transcription factors influence hippocampal cell function, proliferation, survival, and regeneration. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf13 expression with doxycycline. We also generated HT22 Klf13 knock out cells, and we analyzed primary hippocampal cells from wild type and Klf13-/- mice. RNA sequencing showed that KLF13, like KLF9, acts predominantly as a transcriptional repressor in hippocampal neurons and can regulate other Klf genes. Pathway analysis revealed that genes regulated by KLF13 are involved in cell cycle, cell survival, cytoarchitecture regulation, among others. Chromatin-streptavidin sequencing conducted on chromatin isolated from HT22 cells expressing biotinylated KLF13 identified 9506 genomic targets; 79% were located within 1-kb upstream of transcription start sites. Transfection-reporter assays confirmed that KLF13 can directly regulate transcriptional activity of its target genes. Comparison of the target genes of KLF9 and KLF13 found that they share some functions that were likely present in their common ancestor, but they have also acquired distinct functions during evolution. Flow cytometry showed that KLF13 promotes cell cycle progression, and it protects cells from glutamate-induced excitotoxic damage. Taken together, our findings establish novel roles and molecular mechanisms for KLF13 actions in mammalian hippocampal neurons.

The Paralogous Krüppel-like Factors 9 and 13 Regulate the Mammalian Cellular Circadian Clock Output Gene Dbp.

An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 on Dbp transcription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL.

Coordinated transcriptional regulation by thyroid hormone and glucocorticoid interaction in adult mouse hippocampus-derived neuronal cells.

The hippocampus is a well-known target of thyroid hormone (TH; e.g., 3,5,3'-triiodothyronine-T3) and glucocorticoid (GC; e.g., corticosterone-CORT) action. Despite evidence that TH and GC play critical roles in neural development and function, few studies have identified genes and patterns of gene regulation influenced by the interaction of these hormones at a genome-wide scale. In this study we investigated gene regulation by T3, CORT, and T3 + CORT in the mouse hippocampus-derived cell line HT-22. We treated cells with T3, CORT, or T3 + CORT for 4 hr before cell harvest and RNA isolation for microarray analysis. We identified 9 genes regulated by T3, 432 genes by CORT, and 412 genes by T3 + CORT. Among the 432 CORT-regulated genes, there were 203 genes that exhibited an altered CORT response in the presence of T3, suggesting that T3 plays a significant role in modulating CORT-regulated genes. We also found 80 genes synergistically induced, and 73 genes synergistically repressed by T3 + CORT treatment. We performed in silico analysis using publicly available mouse neuronal chromatin immunoprecipitation-sequencing datasets and identified a considerable number of synergistically regulated genes with TH receptor and GC receptor peaks mapping within 1 kb of chromatin marks indicative of hormone-responsive enhancer regions. Functional annotation clustering of synergistically regulated genes reveal the relevance of proteasomal-dependent degradation, neuroprotective effect of growth hormones, and neuroinflammatory responses as key pathways to how TH and GC may coordinately influence learning and memory. Taken together, our transcriptome data represents a promising exploratory dataset for further study of common molecular mechanisms behind synergistic TH and GC gene regulation, and identify specific genes and their role in processes mediated by cross-talk between the thyroid and stress axes in a mammalian hippocampal model system.

The Krüppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn.

Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools. SIGNIFICANCE STATEMENT: Although information transfer at central synapses via sustained high-frequency neural activity requires coordinated synaptic vesicle (SV) recycling, the mechanism(s) by which synapses sense and dynamically modify SV pools to match network demands remains poorly defined. To advance understanding, we quantified SV pool sizes and their sensitivity to neural activity while altering Tomo1 expression, a putative regulator of the presynaptic Readily Releasable Pool. Remarkably, we find Tomo1 actions to extend beyond the Readily Releasable Pool to mediate the Total Recycling Pool and SV Resting Pool distribution, and this action is sensitive to neural activity through Cdk5 phosphorylation of Tomo1. Moreover, Tomo1 appears to exert these actions through interaction with Rab3A-GTP and synapsin proteins. Together, our results argue that Tomo1 is a central mediator of SV availability for neurotransmission.

Liganded Thyroid Hormone Receptors Transactivate the DNA Methyltransferase 3a Gene in Mouse Neuronal Cells.

Thyroid hormone (T3) is essential for proper neurological development. The hormone, bound to its receptors, regulates gene transcription in part by modulating posttranslational modifications of histones. Methylation of DNA, which is established by the de novo DNA methyltransferase (DNMT)3a and DNMT3b, and maintained by DNMT1 is another epigenetic modification influencing gene transcription. The expression of Dnmt3a, but not other Dnmt genes, increases in mouse brain in parallel with the postnatal rise in plasma [T3]. We found that treatment of the mouse neuroblastoma cell line Neuro2a[TRß1] with T3 caused rapid induction of Dnmt3a mRNA, which was resistant to protein synthesis inhibition, supporting that it is a direct T3-response gene. Injection of T3 into postnatal day 6 mice increased Dnmt3a mRNA in the brain by 1 hour. Analysis of two chromatin immunoprecipitation-sequencing datasets, and targeted analyses using chromatin immunoprecipitation, transfection-reporter assays, and in vitro DNA binding identified 2 functional T3-response elements (TREs) at the mouse Dnmt3a locus located +30.3 and +49.3 kb from the transcription start site. Thyroid hormone receptors associated with both of these regions in mouse brain chromatin, but with only 1 (+30.3 kb) in Neuro2a[TRß1] cells. Deletion of the +30.3-kb TRE using CRISPR/Cas9 genome editing eliminated or strongly reduced the Dnmt3a mRNA response to T3. Bioinformatics analysis showed that both TREs are highly conserved among eutherian mammals. Thyroid regulation of Dnmt3a may be an evolutionarily conserved mechanism for modulating global changes in DNA methylation during postnatal neurological development.

Efficient transfection of dissociated mouse chromaffin cells using small-volume electroporation.

We have developed an improved procedure for isolating and transfecting a chromaffin cell-enriched population of primary cells from adult mouse adrenal glands. Significantly, the parameters of a novel electroporation transfection technique were optimized to achieve an average transfection efficiency of 45 % on the small number of cells derived from the mouse glands. Such transfection efficiency was previously unachievable with the electroporation protocols conventionally used with bovine chromaffin cells, even with use of large cell numbers. Our small scale technique now makes feasible the use of genetically homogenous inbred mouse models for investigations on the exocytotic pathway without the time, expense, and cellular changes associated with viral approaches. High fidelity co-expression of multiple plasmids in individual cells is a further advantage of the procedure. To assess whether the biophysical characteristics of mouse adrenal chromaffin cells were altered by this process, we examined structural integrity using immunocytochemistry and functional response to stimuli using calcium imaging, amperometry, and whole-cell capacitance and current clamp recordings. We conclude these parameters are minimally affected. Finally, we demonstrate that high transfection efficiency makes possible the use of primary mouse adrenal chromaffin cells, rather than a cell line, in human growth hormone secretion assays for high throughput evaluation of secretion.

Distinct actions of Rab3 and Rab27 GTPases on late stages of exocytosis of insulin.

Rab GTPases associated with insulin-containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic ß-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase-activating protein overexpression in ß-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready SGs in ß-cells, they also direct unique kinetic and functional properties of the exocytotic pathway.