The TCF4/ß-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer.

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/ß-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/ß-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/ß-catenin mRNA levels and activated TCF4/ß-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/ß-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and ß-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/ß-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/ß-catenin complex activity.

Histone H3 Acetylation is Asymmetrically Induced Upon Learning in Identified Neurons of the Food Aversion Network in the Mollusk Helix Lucorum.

Regulation of gene expression is an essential step during long-term memory formation. Recently, the involvement of DNA-binding transcription factors and chromatin remodeling in synaptic plasticity have been intensively studied. The process of learning was shown to be associated with chromatin remodeling through histone modifications such as acetylation and phosphorylation. We have previously shown that the MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) regulatory cascade plays a key role in the food aversion conditioning in the mollusk Helix. Specifically, command neurons of withdrawal behavior exhibit a learning-dependent asymmetry (left-right) in MAPK/ERK activation. Here, we expanded our molecular studies by focusing on a potential MAPK/ERK target - histone H3. We studied whether there is a learning-induced MAPK/ERK-dependent acetylation of histone H3 in command neurons RPa(2/3) and LPa(2/3) of the right and left parietal ganglia and whether it is asymmetrical. We found a significant learning-dependent increase in histone H3 acetylation in RPa(2/3) neurons but not in LPa(2/3) neurons. Such an increase in right command neurons depended on MAPK/ERK activation and correlated with a lateralized avoidance movement to the right visible 48 h after training. The molecular changes found in a selective set of neurons could thus represent a lateralized memory process, which may lead to consistent turning in one direction when avoiding a food that has been paired with an aversive stimulus.

Learning-induced lateralized activation of the MAPK/ERK cascade in identified neurons of the food-aversion network in the mollusk Helix lucorum.

The MAPK/ERK pathway plays an important role in the regulation of gene expression during memory formation both in vertebrates and invertebrates. In the mollusk Helix lucorum, serotonin induces activation of MAPK/ERK in the central nervous system (CNS) upon food aversion learning. Such learning depends on a neuronal network in which specialized neurons play distinct roles so that they may exhibit different activation levels of the MAPK/ERK pathway. Here we performed a comparative analysis of MAPK/ERK activation in single neurons of the food-aversion network, focusing both on command neurons, which mediate withdrawal behavior and process information pertaining to the unconditioned stimulus, and on neurons of the procerebrum, the mollusk's olfactory center, which process information from the conditioned stimulus. By means of Western blots designed to detect micro amounts of proteins, we determined MAPK/ERK activation in these neurons and found that after food aversion learning phospho-ERK levels increased significantly in RPa(2/3) command neurons of the right parietal ganglia and in the procerebrum. Such an increase was prevented by injection of PD98095, an inhibitor of the ERK upstream kinase (MEK-1). In contrast, no activation of MAPK/ERK was detected in similar conditions in the corresponding neurons of the left parietal ganglia LPa(2/3). This asymmetry was verified after serotonin application to the CNS in order to mimic learning. Our results thus show that learning involves synchronous and asymmetric serotonin-dependent MAPK/ERK activation. Such an asymmetry may reflect lateralization of memory processes in the mollusk brain.

Expression of MAP/ERK kinase cascade corresponds to the ability to develop food aversion in terrestrial snail at different stages of ontogenesis.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) cascade plays an important role in gene expression regulation during memory formation in both vertebrates and invertebrates. MAPK/ERK regulates gene expression through phosphorylation of transcription factors binding to the regulatory elements SRE and CRE of target genes. Previously we reported that juvenile snails Helix lucorum differ from adult animals in a spectrum of transcription factors binding to DNA regulatory elements SRE and AP-1. In this study we analyzed the expression and activation of MAPK/ERK in CNS of H. lucorum during formation of the conditioned avoidance reflex at different stages of postnatal ontogenesis. Under conditions of learning, juvenile snails (aged 2-3 months) possessing immature mechanisms of avoidance reflex plasticity showed dramatically low level of phosphorylation and, correspondingly, low activation of MAPK/ERK in comparison to adult animals. Beside this, the MAPK/ERK cascade was not activated after 10 and 60 min after learning in juvenile snails in contrast to adults, while basal expression level of this kinase was similar in juveniles and adults. Low activation of MAPK/ERK cascade can cause a deficiency in phosphorylation of downstream transcription factors binding to SRE and thereby influence the expression of early response genes (particularly, of the family AP-1) and late response genes necessary for cellular and synaptic plasticity. These observations suggest that the MAPK/ERK regulatory cascade plays an essential role in the formation of conditioned avoidance reflexes in Helix. Low activation of this cascade might be one of the reasons for deficiency of long-term memory formation during avoidance learning in juvenile animals.