Chromatin remodeling through histone posttranslational modifications (PTMs) and DNA methylation has recently been implicated in cognitive functions, but the mechanisms involved in such epigenetic regulation remain poorly understood. Here, we show that protein phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Our data show that PP1 is present at the chromatin in brain cells and interacts with enzymes of the epigenetic machinery including HDAC1 (histone deacetylase 1) and histone demethylase JMJD2A (jumonji domaincontaining protein 2A). The selective inhibition of the nuclear pool of PP1 in forebrain neurons in transgenic mice is shown to induce several histone PTMs that include not only phosphorylation but also acetylation and methylation. These PTMs are residue-specific and occur at the promoter of genes important for memory formation like CREB (cAMP response element-binding protein) and NF-B (nuclear factor-B). These histone PTMs further co-occur with selective binding of RNA polymerase II and altered gene transcription, and are associated with improved long-term memory for objects and space. Together, these findings reveal a novel mechanism for the epigenetic control of gene transcription and long-term memory in the adult brain that depends on PP1.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Isolated subunits of factor VIIIa were examined for their ability to accelerate the factor IXa-catalyzed activation of factor X. The A2 subunit enhanced the k cat for this conversion by 100-fold whereas the K m for factor X was unaffected. The apparent K d for the interaction of A2 subunit with factor IXa was ϳ300 nM. Similar results were obtained using purified A2 expressed as the isolated domain in Chinese hamster ovary cells, although this material was less stable than the factor VIIIa-derived material. Isolated A1 and A3-C1-C2 subunits showed no effect on the rate of factor X conversion. A2 subunit increased the fluorescence anisotropy of fluoresceinPhe-Phe-Arg-factor IXa (⌬r ‫؍‬ 0.015) and markedly increased anisotropy in the presence of factor X (⌬r ‫؍‬ 0.057), suggesting that it contributes to the orientation of the factor IXa active site and its relation to substrate. A synthetic peptide to A2 residues 558 -565 inhibited the A2-dependent enhancement of factor X activation with an IC 50 ‫؍‬ 40 M, a value similar to its K i for inhibition of the intrinsic factor Xase (105 M). These results indicate that the isolated A2 subunit modulates the active site of factor IXa and identifies a functional role for this subunit in factor VIIIa.Factors VIII and IX are essential plasma proteins that when deficient or defective result in hemophilia A and B, respectively. The activated forms of these proteins, factor IXa (a serine protease) and factor VIIIa 1 (a protein cofactor), form a Ca 2ϩ -and anionic phospholipid-dependent complex referred to as the intrinsic factor Xase complex that efficiently converts zymogen factor X to the serine protease, factor Xa (see Ref. 1 for review). The role of the phospholipid surface is to primarily reduce the K m for substrate, whereas factor VIIIa increases the k cat for this reaction by several orders of magnitude (2).Factor VIIIa is a non-covalent heterotrimer composed of the A1, A2, and A3-C1-C2 subunits (3, 4). The A1 and A3-C1-C2 subunits retain the divalent metal ion-dependent linkage responsible for the association of the heavy and light chains in heterodimeric factor VIII and can be isolated as a stable dimer (5, 6). The A2 subunit is weakly associated with the dimer (K d ϳ260 nM (7, 8)) primarily through electrostatic interaction (4), and at physiologic pH it readily dissociates, resulting in the loss of factor VIIIa activity (4, 7-9). Under the appropriate reaction conditions of slightly acidic pH and low ionic strength, factor VIIIa activity can be efficiently reconstituted from the isolated A2 subunit and the A1/A3-C1-C2 dimer (4, 8 -11).Factor Xase activity is labile and has been referred to as self-damping (12), whereas factor VIIIa-independent conversion of factor X by factor IXa is relatively stable. Association of factor VIIIa with factor IXa in the presence of phospholipid and Ca 2ϩ stabilizes cofactor activity (13). Furthermore, reconstitution of heterotrimeric factor ...

Emotional memory is a rapidly acquired and persistent form of memory, and its robustness is in part determined by the initial strength of the memory. Here, we provide new evidence that the protein phosphatase calcineurin (CaN), a potent negative regulator of neuronal signaling that is known to constrain learning and memory, critically regulates the establishment of emotional memory through mechanisms involving the immediate early gene Zif268 (also known as Egr1). We found that CaN is inhibited in the amygdala during the establishment of aversive memory, but Zif268 is activated. Using inducible transgenesis in mice, we further saw that CaN inhibition and Zif268 overexpression during memory establishment strengthen the memory trace and enhance its resistance to extinction. We found that CaN inhibition correlates with increased Zif268 expression and that a common pool of proteins is regulated in the amygdala after CaN inhibition and Zif268 overexpression. Together, these findings reveal a previously unknown mechanism for the control of emotional memory that depends on CaN and Zif268.

To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.

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Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and phosphatases (PPs) are important players in these mechanisms. Protein serine/threonine phosphatase 1 (PP1), in particular, was recently shown to be important for transcription-dependent memory by regulating chromatin remodeling. However, the impact of PP1 on gene transcription in adult neurons remains not fully delineated. Here, we demonstrate that the nuclear pool of PP1 is associated with transcriptional events involving molecular components of signaling cascades acting as positive and negative regulators of memory and brain plasticity. The data show that inhibiting this pool selectively in forebrain neurons improves memory performance, enhances long-term potentiation (LTP), and modulates gene transcription. These findings highlight an important role for PP1 in the regulation of gene transcription in LTM and synaptic plasticity in the adult brain.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the k cat for factor IXa-catalyzed activation of factor X by ϳ100-fold (ϳ1 min ؊1 ), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049 -19054). However, A1 subunit increased the A2-dependent stimulation by ϳ10-fold. The K m for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the k cat observed in the presence of saturating A1 and A2 subunits (ϳ15 min ؊1 ) represented 5-10% of the value observed for native factor VIIIa (ϳ200 min ؊1 ). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in k cat of over 1000-fold compared with factor IXa alone.The proteolytically activated form of factor VIII, factor VIIIa, serves as a cofactor for the serine protease factor IXa in the conversion of factor X to factor Xa. This complex of enzyme and cofactor, assembled on an anionic phospholipid surface, is referred to as the intrinsic factor Xase. The role of factor VIIIa is to increase the catalytic rate constant (k cat ) by several orders of magnitude. The phospholipid surface is primarily involved in reducing molecular interactions to a two-dimensional space, thereby markedly decreasing the K m for factor X. The association of factor VIIIa and factor IXa and the mechanism(s) by which factor VIIIa stimulates reaction rate are not fully understood. The importance of this interaction is indicated by defects or deficiency in factor VIII that result in hemophilia A.Factor VIII circulates as a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains) associated in a divalent metal ion-dependent heterodimer. Proteolysis by thrombin yields factor VIIIa, a trimer of A1, A2, and A3-C1-C2 subunits 1 (1, 2). The A1 and A3-C1-C2 subunits retain the divalent metal ion-dependent linkage and can be isolated as a stable dimer. Conversely, the A2 subunit is associated with the A1/A3-C1-C2 dimer in a primarily electrostatic interaction and readily dissociates from the dimer at physiological pH and ionic strength. However under appropriate reaction conditions, factor VIIIa can be reconstituted from isolated A1/A3-C1-C2 dimer and A2 subunit (2-4). The affinity of A2 subunit for the A1/A3-C1-C2 has been measured following functional assay (4, 5) as well as physical assay employing surface plasmon resonance (6). In human factor VIIIa, the affinity of A2 subunit for the A1/A3-C1-C2 dimer (K d ϳ260 ...