DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain.

Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.

To eat or not to eat: ontogeny of hypothalamic feeding controls and a role for leptin in modulating life-history transition in amphibian tadpoles.

Many animal life histories entail changing feeding ecology, but the molecular bases for these transitions are poorly understood. The amphibian tadpole is typically a growth and dispersal life-history stage. Tadpoles are primarily herbivorous, and they capitalize on growth opportunities to reach a minimum body size to initiate metamorphosis. During metamorphic climax, feeding declines, at which time the gastrointestinal (GI) tract remodels to accommodate the carnivorous diet of the adult frog. Here we show that anorexigenic hypothalamic feeding controls are absent in the tadpole, but develop during metamorphosis concurrent with the production of the satiety signal leptin. Before metamorphosis there is a large increase in leptin mRNA in fat tissue. Leptin receptor mRNA increased during metamorphosis in the preoptic area/hypothalamus, the key brain region involved with the control of food intake and metabolism. This corresponded with an increase in functional leptin receptor, as evidenced by induction of socs3 mRNA and phosphorylated STAT3 immunoreactivity, and suppression of feeding behaviour after injection of recombinant frog leptin. Furthermore, we found that immunoneutralization of leptin in tadpoles at metamorphic climax caused them to resume feeding. The absence of negative regulation of food intake in the tadpole allows the animal to maximize growth prior to metamorphosis. Maturation of leptin-responsive neural circuits suppresses feeding during metamorphosis to facilitate remodelling of the GI tract.

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.

Krüppel-like factors are effectors of nuclear receptor signaling.

Binding of steroid and thyroid hormones to their cognate nuclear receptors (NRs) impacts virtually every aspect of postembryonic development, physiology and behavior, and inappropriate signaling by NRs may contribute to disease. While NRs regulate genes by direct binding to hormone response elements in the genome, their actions may depend on the activity of other transcription factors (TFs) that may or may not bind DNA. The Krüppel-like family of transcription factors (KLF) is an evolutionarily conserved class of DNA-binding proteins that influence many aspects of development and physiology. Several members of this family have been shown to play diverse roles in NR signaling. For example, KLFs (1) act as accessory transcription factors for NR actions, (2) regulate expression of NR genes, and (3) as gene products of primary NR response genes function as key players in NR-dependent transcriptional networks. In mouse models, deletion of different KLFs leads to aberrant transcriptional and physiological responses to hormones, underscoring the importance of these proteins in the regulation of hormonal signaling. Understanding the functional relationships between NRs and KLFs will yield important insights into mechanisms of NR signaling. In this review we present a conceptual framework for understanding how KLFs participate in NR signaling, and we provide examples of how these proteins function to effect hormone action.

Stress hormones mediate predator-induced phenotypic plasticity in amphibian tadpoles.

Amphibian tadpoles display extensive anti-predator phenotypic plasticity, reducing locomotory activity and, with chronic predator exposure, developing relatively smaller trunks and larger tails. In many vertebrates, predator exposure alters activity of the neuroendocrine stress axis. We investigated predator-induced effects on stress hormone production and the mechanistic link to anti-predator defences in Rana sylvatica tadpoles. Whole-body corticosterone (CORT) content was positively correlated with predator biomass in natural ponds. Exposure to caged predators in mesocosms caused a reduction in CORT by 4 hours, but increased CORT after 4 days. Tadpoles chronically exposed to exogenous CORT developed larger tails relative to their trunks, matching morphological changes induced by predator chemical cue; this predator effect was blocked by the corticosteroid biosynthesis inhibitor metyrapone. Tadpole tail explants treated in vitro with CORT increased tissue weight, suggesting that CORT acts directly on the tail. Short-term treatment of tadpoles with CORT increased predation mortality, likely due to increased locomotory activity. However, long-term CORT treatment enhanced survivorship, likely due to induced morphology. Our findings support the hypothesis that tadpole physiological and behavioural/morphological responses to predation are causally interrelated. Tadpoles initially suppress CORT and behaviour to avoid capture, but increase CORT with longer exposure, inducing adaptive phenotypic changes.

Metamorphic gene regulation programs in Xenopus tropicalis tadpole brain.

Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs that underlie morphogenesis. Gene expression screens using tissues from premetamorphic tadpoles treated with TH identified some TH target genes, but few studies have analyzed genome-wide changes in gene regulation during spontaneous metamorphosis. We analyzed RNA sequencing data at four developmental stages from the beginning to the end of spontaneous metamorphosis, conducted on the neuroendocrine centers of Xenopus tropicalis tadpole brain. We also conducted chromatin immunoprecipitation sequencing (ChIP-seq) for TRs, and we compared gene expression changes during metamorphosis with those induced by exogenous TH. The mRNA levels of 26% of protein coding genes changed during metamorphosis; about half were upregulated and half downregulated. Twenty four percent of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks. Genes involved with neural cell differentiation, cell physiology, synaptogenesis and cell-cell signaling were upregulated, while genes involved with cell cycle, protein synthesis, and neural stem/progenitor cell homeostasis were downregulated. There is a shift from building neural structures early in the metamorphic process, to the differentiation and maturation of neural cells and neural signaling pathways characteristic of the adult frog brain. Only half of the genes modulated by treatment of premetamorphic tadpoles with TH for 16 h changed expression during metamorphosis; these represented 33% of the genes whose mRNA levels changed during metamorphosis. Taken together, our results provide a foundation for understanding the molecular basis for metamorphosis of tadpole brain, and they highlight potential caveats for interpreting gene regulation changes in premetamorphic tadpoles induced by exogenous TH.

Mechanisms of molecular mimicry of plant CLE peptide ligands by the parasitic nematode Globodera rostochiensis.

Nematodes that parasitize plant roots cause huge economic losses and have few mechanisms for control. Many parasitic nematodes infect plants by reprogramming root development to drive the formation of feeding structures. How nematodes take control of plant development is largely unknown. Here, we identify two host factors involved in the function of a receptor ligand mimic, GrCLE1, secreted by the potato cyst nematode Globodera rostochiensis. GrCLE1 is correctly processed to an active form by host plant proteases. Processed GrCLE1 peptides bind directly to the plant CLE receptors CLV2, BAM1, and BAM2. Involvement of these receptors in the ligand-mimicking process is also supported by the fact that the ability of GrCLE1 peptides to alter plant root development in Arabidopsis (Arabidopsis thaliana) is dependent on these receptors. Critically, we also demonstrate that GrCLE1 maturation can be entirely carried out by plant factors and that the availability of CLE processing activity may be essential for successful ligand mimicry.

Developmental reversal in neuropeptide Y action on feeding in an amphibian.

Neuropeptide Y (NPY) is expressed in the hypothalamus where it exerts orexigenic actions within the feeding control circuit. While NPY stimulates feeding in juvenile and adult animals, it is not known whether NPY influences food intake at earlier life stages. We investigated a role for NPY in regulating feeding at two stages of the life cycle of an amphibian, the Western spadefoot toad Spea hammondii. We administered NPY by intracerebroventricular (i.c.v.) injection to juvenile toads or prometamorphic tadpoles, and monitored locomotion, feeding behavior and/or food intake. Injection of NPY (20 or 200 ng/g BW) into juvenile toads decreased the latency to, and increased the number of strikes at prey, and the number of crickets eaten compared to uninjected or vehicle-injected controls. By contrast, injection of NPY (0.02-20 ng/g BW) into prometamorphic tadpoles caused a dose-dependent decrease in time spent foraging compared to controls. Blocking NPY signaling in the prometamorphic tadpole brain by i.c.v. injection of a general NPY receptor antagonist increased foraging, and partly blocked the action of exogenous NPY on foraging. Taken together, our findings show a developmental reversal in NPY actions on feeding in an amphibian, with the peptide having a characteristic orexigenic action in the juvenile toad, but an inhibitory action on foraging in the prometamorphic tadpole. The anorexigenic action of NPY in the tadpole correlates with a decrease in feeding that occurs at metamorphic climax when the tadpole's gut and cranium remodels for the transition to a carnivorous diet.

CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification.

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3-derived CLE ligand. The biochemical role of the receptor-like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2-CRN heteromultimeric and CLV1-BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane-localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3-derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand-binding receptor complexes controlling stem cell specification in Arabidopsis.

Chordate metamorphosis: ancient control by iodothyronines.

A new study shows that iodothyronines induce metamorphosis in the cephalochordate amphioxus by binding to a receptor homologous to vertebrate thyroid hormone receptors. Iodothyronine-induced metamorphosis may be an ancestral feature of the chordates.

Evolution of leptin structure and function.

Leptin, the protein product of the obese(ob or Lep) gene, is a hormone synthesized by adipocytes that signals available energy reserves to the brain, and thereby influences development, growth, metabolism and reproduction. In mammals, leptin functions as an adiposity signal: circulating leptin fluctuates in proportion to fat mass, and it acts on the hypothalamus to suppress food intake. Orthologs of mammalian Lep genes were recently isolated from several fish and two amphibian species, and here we report the identification of two Lep genes in a reptile, the lizard Anolis carolinensis. While vertebrate leptins show large divergence in their primary amino acid sequence, they form similar tertiary structures, and may have similar potencies when tested in vitro on heterologous leptin receptors (LepRs). Leptin binds to LepRs on the plasma membrane, activating several intracellular signaling pathways. Vertebrate LepRs signal via the Janus kinase (Jak) and signal transducer and activator of transcription (STAT) pathway. Three tyrosine residues located within the LepR cytoplasmic domain are phosphorylated by Jak2 and are required for activation of SH2-containing tyrosine phosphatase-2, STAT5 and STAT3 signaling. These tyrosines are conserved from fishes to mammals, demonstrating their critical role in signaling by the LepR. Leptin is anorexigenic in representatives of all vertebrate classes, suggesting that its role in energy balance is ancient and has been evolutionarily conserved. In addition to its integral role as a regulator of appetite and energy balance, leptin exerts pleiotropic actions in development, physiology and behavior.

Mechanisms and significance of nuclear receptor auto- and cross-regulation.

The number of functional hormone receptors expressed by a cell in large part determines its responsiveness to the hormonal signal. The regulation of hormone receptor gene expression is therefore a central component of hormone action. Vertebrate steroid and thyroid hormones act by binding to nuclear receptors (NR) that function as ligand-activated transcription factors. Nuclear receptor genes are regulated by diverse and interacting intracellular signaling pathways. Nuclear receptor ligands can regulate the expression of the gene for the NR that mediates the hormone's action (autoregulation), thus influencing how a cell responds to the hormone. Autoregulation can be either positive or negative, the hormone increasing or decreasing, respectively, the expression of its own NR. Positive autoregulation (autoinduction) is often observed during postembryonic development, and during the ovarian cycle, where it enhances cellular sensitivity to the hormonal signal to drive the developmental process. By contrast, negative autoregulation (autorepression) may become important in the juvenile and adult for homeostatic negative feedback responses. In addition to autoregulation, a NR can influence the expression other types of NRs (cross-regulation), thus modifying how a cell responds to a different hormone. Cross-regulation by NRs is an important means to temporally coordinate cell responses to a subsequent (different) hormonal signal, or to allow for crosstalk between hormone signaling pathways.

Stress hormones mediate developmental plasticity in vertebrates with complex life cycles.

The environment experienced by developing organisms can shape the timing and character of developmental processes, generating different phenotypes from the same genotype, each with different probabilities of survival and performance as adults. Chordates have two basic modes of development, indirect and direct. Species with indirect development, which includes most fishes and amphibians, have a complex life cycle with a free-swimming larva that is typically a growth stage, followed by a metamorphosis into the adult form. Species with direct development, which is an evolutionarily derived developmental mode, develop directly from embryo to the juvenile without an intervening larval stage. Among the best studied species with complex life cycles are the amphibians, especially the anurans (frogs and toads). Amphibian tadpoles are exposed to diverse biotic and abiotic factors in their developmental habitat. They have extensive capacity for developmental plasticity, which can lead to the expression of different, adaptive morphologies as tadpoles (polyphenism), variation in the timing of and size at metamorphosis, and carry-over effects on the phenotype of the juvenile/adult. The neuroendocrine stress axis plays a pivotal role in mediating environmental effects on amphibian development. Before initiating metamorphosis, if tadpoles are exposed to predators they upregulate production of the stress hormone corticosterone (CORT), which acts directly on the tail to cause it to grow, thereby increasing escape performance. When tadpoles reach a minimum body size to initiate metamorphosis they can vary the timing of transformation in relation to growth opportunity or mortality risk in the larval habitat. They do this by modulating the production of thyroid hormone (TH), the primary inducer of metamorphosis, and CORT, which synergizes with TH to promote tissue transformation. Hypophysiotropic neurons that release the stress neurohormone corticotropin-releasing factor (CRF) are activated in response to environmental stress (e.g., pond drying, food restriction, etc.), and CRF accelerates metamorphosis by directly inducing secretion of pituitary thyrotropin and corticotropin, thereby increasing secretion of TH and CORT. Although activation of the neuroendocrine stress axis promotes immediate survival in a deteriorating larval habitat, costs may be incurred such as reduced tadpole growth and size at metamorphosis. Small size at transformation can impair performance of the adult, reducing probability of survival in the terrestrial habitat, or fecundity. Furthermore, elevations in CORT in the tadpole caused by environmental stressors cause long term, stable changes in neuroendocrine function, behavior and physiology of the adult, which can affect fitness. Comparative studies show that the roles of stress hormones in developmental plasticity are conserved across vertebrate taxa including humans.

Stress hormone-mediated antipredator morphology improves escape performance in amphibian tadpoles.

Complete functional descriptions of the induction sequences of phenotypically plastic traits (perception to physiological regulation to response to outcome) should help us to clarify how plastic responses develop and operate. Ranid tadpoles express several plastic antipredator traits mediated by the stress hormone corticosterone, but how they influence outcomes remains uncertain. We investigated how predator-induced changes in the tail morphology of wood frog (Rana sylvatica) tadpoles influenced their escape performance over a sequence of time points when attacked by larval dragonflies (Anax junius). Tadpoles were raised with no predator exposure, chemical cues of dragonflies added once per day, or constant exposure to caged dragonflies crossed with no exogenous hormone added (vehicle control only), exogenous corticosterone, or metyrapone (a corticosteroid synthesis inhibitor). During predation trials, we detected no differences after four days, but after eight days, tadpoles exposed to larval dragonflies and exogenous corticosterone had developed deeper tail muscles and exhibited improved escape performance compared to controls. Treatment with metyrapone blocked the development of a deeper tail muscle and resulted in no difference in escape success. Our findings further link the predator-induced physiological stress response of ranid tadpoles to the development of an antipredator tail morphology that confers performance benefits.

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.

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis.

Thyroid hormone (T(3)) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T(3) receptors (TR) of which there are two subtypes, TRalpha and TRbeta. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRalpha and TRbeta in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T(3) until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T(3). Using double labeling fluorescent histochemistry with confocal microscopy we found TRalpha expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRbeta was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRbeta promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRalpha selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRbeta-selective agonists GC1 or GC24 did not. Our findings support the view that T(3) acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRalpha.

Molecular mechanisms of corticosteroid synergy with thyroid hormone during tadpole metamorphosis.

Corticosteroids (CS) act synergistically with thyroid hormone (TH) to accelerate amphibian metamorphosis. Earlier studies showed that CS increase nuclear 3,5,3'-triiodothyronine (T(3)) binding capacity in tadpole tail, and 5' deiodinase activity in tadpole tissues, increasing the generation of T(3) from thyroxine (T(4)). In the present study we investigated CS synergy with TH by analyzing expression of key genes involved in TH and CS signaling using tadpole tail explant cultures, prometamorphic tadpoles, and frog tissue culture cells (XTC-2 and XLT-15). Treatment of tail explants with T(3) at 100 nM, but not at 10 nM caused tail regression. Corticosterone (CORT) at three doses (100, 500 and 3400 nM) had no effect or increased tail size. T(3) at 10 nM plus CORT caused tails to regress similar to 100 nM T(3). Thyroid hormone receptor beta (TRbeta) mRNA was synergistically upregulated by T(3) plus CORT in tail explants, tail and brain in vivo, and tissue culture cells. The activating 5' deiodinase type 2 (D2) mRNA was induced by T(3) and CORT in tail explants and tail in vivo. Thyroid hormone increased expression of glucocorticoid (GR) and mineralocorticoid receptor (MR) mRNAs. Our findings support that the synergistic actions of TH and CS in metamorphosis occur at the level of expression of genes for TRbeta and D2, enhancing tissue sensitivity to TH. Concurrently, TH enhances tissue sensitivity to CS by upregulating GR and MR. Environmental stressors can modulate the timing of tadpole metamorphosis in part by CS enhancing the response of tadpole tissues to the actions of TH.

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.

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.