Clustered PHD domains in KMT2/MLL proteins are attracted by H3K4me3 and H3 acetylation-rich active promoters and enhancers.

Histone lysine-specific methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukemia (MLL1-4) proteins, mediate positive transcriptional memory. Acting as the catalytic subunits of human COMPASS-like complexes, KMT2A-D methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered (multiple) PHDs localize to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His). The mutations cause a domain loss-of-function. Taken together, our data suggest that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons.

Nuclear localization of ß-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that ß-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying ß-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear ß-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with ß-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3ß, components of the ß-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic ß-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of ß-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in ß-catenin labeling for ubiquitination and subsequent degradation.

Novel ß-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability.

BACKGROUND: LEF1/TCF transcription factors and their activator ß-catenin are effectors of the canonical Wnt pathway. Although Wnt/ß-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by ß-catenin in neurons. We recently showed that ß-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new ß-catenin targets in the adult thalamus. RESULTS: We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear ß-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between ß-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of ß-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca(2+)-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by ß-catenin, although the binding of ß-catenin to the regulatory sequences of these genes could not be confirmed. CONCLUSIONS: In the thalamus, ß-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of ß-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies.

Adar-mediated A-to-I editing is required for embryonic patterning and innate immune response regulation in zebrafish.

Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery reveals pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3'-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning are found to contain multiple editing sites. Adar knockdown or overexpression affect gene expression by 12 hpf. Analysis of adar-/- zygotic mutants further reveals that the previously described role of Adar in mammals in regulating the innate immune response is conserved in zebrafish. Our study therefore establishes distinct maternal and zygotic functions of RNA editing by Adar in embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes, and in the regulation of the innate immune response, respectively.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

LEF1/beta-catenin complex regulates transcription of the Cav3.1 calcium channel gene (Cacna1g) in thalamic neurons of the adult brain.

beta-Catenin, together with LEF1/TCF transcription factors, activates genes involved in the proliferation and differentiation of neuronal precursor cells. In mature neurons, beta-catenin participates in dendritogenesis and synaptic function as a component of the cadherin cell adhesion complex. However, the transcriptional activity of beta-catenin in these cells remains elusive. In the present study, we found that in the adult mouse brain, beta-catenin and LEF1 accumulate in the nuclei of neurons specifically in the thalamus. The particular electrophysiological properties of thalamic neurons depend on T-type calcium channels. Cav3.1 is the predominant T-type channel subunit in the thalamus, and we hypothesized that the Cacna1g gene encoding Cav3.1 is a target of the LEF1/beta-catenin complex. We demonstrated that the expression of Cacna1g is high in the thalamus and is further increased in thalamic neurons treated in vitro with LiCl or WNT3A, activators of beta-catenin. Luciferase reporter assays confirmed that the Cacna1G promoter is activated by LEF1 and beta-catenin, and footprinting analysis revealed four LEF1 binding sites in the proximal region of this promoter. Chromatin immunoprecipitation demonstrated that the Cacna1g proximal promoter is occupied by beta-catenin in vivo in the thalamus, but not in the hippocampus. Moreover, WNT3A stimulation enhanced T-type current in cultured thalamic neurons. Together, our data indicate that the LEF1/beta-catenin complex regulates transcription of Cacna1g and uncover a novel function for beta-catenin in mature neurons. We propose that beta-catenin contributes to neuronal excitability not only by a local action at the synapse but also by activating gene expression in thalamic neurons.

Expression of STIM1 in brain and puncta-like co-localization of STIM1 and ORAI1 upon depletion of Ca(2+) store in neurons.

Recent findings indicate that Store Operated Ca(2+) Entry (SOCE) in non-excitable cells is based on the interaction of ER calcium sensor STIM1 with the plasma membrane Ca(2+) channel protein ORAI1. However, despite physiological evidence for functional SOCE in neurons, its mechanism is not known. Using PCR, immunoblotting and immunohistochemical methods we show that STIM1 protein is present in the mouse brain. The protein and mRNA levels of STIM1 are similar in the thalamus, the hippocampus, the cortex and the amygdala and the higher level is observed in the cerebellum. Immunohistochemistry of the cortex and the hippocampus of brain sections shows that STIM1 is present in cell bodies and dendrites of pyramidal neurons. In the cerebellum STIM1 is present in Purkinje and granule cells. The same immunostaining pattern is observed in cultured hippocampal and cortical neurons. Localization of YFP-STIM1 and ORAI1 changes from a dispersed pattern in untreated cortical neurons to puncta-like pattern in cells with a Ca(2+) store depleted by thapsigargin treatment. The YFP-STIM1(D76A) dominant positive mutant, which is active regardless of the Ca(2+) level in ER, concentrates as puncta even without depletion of the neuronal Ca(2+) store. Also, this mutant forces ORAI1 redistribution to form puncta-like staining. We suggest that in neurons, just as in non-excitable cells, the STIM1 and ORAI1 proteins are involved in SOCE.

Cell motility affects the intensity of gap junctional coupling in prostate carcinoma and melanoma cell populations.

Connexins and gap junctions play a crucial role during carcinogenesis. While diverse regulatory systems have been shown to modulate their function, the influence of cell motility on the intensity of gap junctional intercellular coupling has yet to be systematically addressed. Since cancer cell motility and intercellular coupling determine cancer development, we aimed at elucidating how mutual cell translocation modulates the intensity of gap junctional coupling in cell populations. Time-lapse analyses of the motility of connexin43 (Cx43)-coupled rat prostate carcinoma (MAT-LyLu) and mouse melanoma (B16) cells cultured on hyper-adhesive substrata revealed a reduced intensity of intercellular translocations in the two cell populations compared to the control conditions. While no detectable effects on the architecture of the actin cytoskeleton and Cx43 expression and phosphorylation were observed, the inhibition of cell motility was paralleled by an increase in the abundance of Cx43-positive plaques in cell-to-cell contacts and an enhancement of gap junctional coupling in cell populations cultured on hyper-adhesive substrata. Thus, a direct correlation between two cellular parameters crucial for carcinogenesis, i.e. cell motility and gap junctional coupling intensity exists in cancer cell populations.

TCF7L2 mediates the cellular and behavioral response to chronic lithium treatment in animal models.

The mechanism of lithium's therapeutic action remains obscure, hindering the discovery of safer treatments for bipolar disorder. Lithium can act as an inhibitor of the kinase GSK3α/ß, which in turn negatively regulates ß-catenin, a co-activator of LEF1/TCF transcription factors. However, unclear is whether therapeutic levels of lithium activate ß-catenin in the brain, and whether this activation could have a therapeutic significance. To address this issue we chronically treated mice with lithium. Although the level of non-phospho-ß-catenin increased in all of the brain areas examined, ß-catenin translocated into cellular nuclei only in the thalamus. Similar results were obtained when thalamic and cortical neurons were treated with a therapeutically relevant concentration of lithium in vitro. We tested if TCF7L2, a member of LEF1/TCF family that is highly expressed in the thalamus, facilitated the activation of ß-catenin. Silencing of Tcf7l2 in thalamic neurons prevented ß-catenin from entering the nucleus, even when the cells were treated with lithium. Conversely, when Tcf7l2 was ectopically expressed in cortical neurons, ß-catenin shifted to the nucleus, and lithium augmented this process. Lastly, we silenced tcf7l2 in zebrafish and exposed them to lithium for 3 days, to evaluate whether TCF7L2 is involved in the behavioral response. Lithium decreased the dark-induced activity of control zebrafish, whereas the activity of zebrafish with tcf7l2 knockdown was unaltered. We conclude that therapeutic levels of lithium activate ß-catenin selectively in thalamic neurons. This effect is determined by the presence of TCF7L2, and potentially contributes to the therapeutic response.

Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.