Ubiquitin-proteasomal regulation of chromatin remodeler INO80 in the nucleus accumbens mediates persistent cocaine craving.

Neuroadaptations in the nucleus accumbens (NAc) underlie cue-induced cocaine craving that intensifies ("incubates") during abstinence and is believed to contribute to persistent relapse vulnerability. Changes in gene expression often govern perpetual behavioral abnormalities, but epigenetic plasticity during prolonged abstinence from drug exposure is poorly understood. We examined how E3 ubiquitin ligase TRIM3 dysregulates chromatin remodeler INO80 to mediate cocaine craving during prolonged abstinence. We found that INO80 expression increased in the NAc on abstinence day 30 (AD30) but not on AD1 following cocaine self-administration. Furthermore, TRIM3, which mediates degradation of INO80, was reduced on AD30, along with TRIM3-INO80 interaction. Viral-mediated gene transfer of INO80 or TRIM3 governed cocaine craving during prolonged abstinence. Lastly, chromatin immunoprecipitation followed by massively parallel DNA sequencing identified INO80-mediated transcriptional regulation of predicted pathways associated with cocaine plasticity. Together, these results demonstrate a novel ubiquitin-proteasomal-epigenetic mechanism by which TRIM3-INO80 mediates cocaine craving during prolonged abstinence.

Systemic and acute administration of parathyroid hormone-related peptide(1-36) stimulates endogenous beta cell proliferation while preserving function in adult mice.

AIMS/HYPOTHESIS: A major focus in the treatment of diabetes is to identify factors that stimulate endogenous beta cell growth while preserving function. The first 36 amino acids of parathyroid hormone-related protein (PTHrP) are sufficient to enhance proliferation and function in rodent and human beta cells in vitro. This study examined whether acute and systemic administration of the amino-terminal PTHrP(1-36) peptide can achieve similar effects in rodent beta cells in vivo. METHODS: Adult male mice were injected with 40, 80 or 160 µg of PTHrP(1-36) per kg body weight or with vehicle for 25 days. Glucose and beta cell homeostasis, as well as expression of differentiation markers and cell cycle genes were analysed. RESULTS: All three doses of PTHrP(1-36) significantly enhanced beta cell proliferation in vivo at day 25, with 160 µg/kg PTHrP(1-36) increasing proliferation as early as day 5. Importantly, the two higher doses of PTHrP(1-36) caused a significant 30% expansion of beta cell mass, with a short-term improvement in glucose tolerance. PTHrP(1-36) did not cause hypercalcaemia, or change islet number, beta cell size, beta cell death or expression of differentiation markers. Analysis of islet G1/S cell cycle proteins revealed that chronic overabundance of PTHrP(1-139) in the beta cell significantly increased the cell cycle activator cyclin D2 and decreased levels of cyclin-dependent kinase 4 inhibitor (p16( Ink4a ) [Ink4a also known as Cdkn2a]), but acute treatment with PTHrP(1-36) did not. CONCLUSIONS/INTERPRETATION: Acute and systemic administration of PTHrP(1-36) increases rodent beta cell proliferation and mass without negatively affecting function or survival. These findings highlight the future potential therapeutic effectiveness of this peptide under diabetes-related pathophysiological conditions.

Glucose stimulates human beta cell replication in vivo in islets transplanted into NOD-severe combined immunodeficiency (SCID) mice.

AIMS/HYPOTHESIS: We determined whether hyperglycaemia stimulates human beta cell replication in vivo in an islet transplant model METHODS: Human islets were transplanted into streptozotocin-induced diabetic NOD-severe combined immunodeficiency mice. Blood glucose was measured serially during a 2 week graft revascularisation period. Engrafted mice were then catheterised in the femoral artery and vein, and infused intravenously with BrdU for 4 days to label replicating beta cells. Mice with restored normoglycaemia were co-infused with either 0.9% (wt/vol.) saline or 50% (wt/vol.) glucose to generate glycaemic differences among grafts from the same donors. During infusions, blood glucose was measured daily. After infusion, human beta cell replication and apoptosis were measured in graft sections using immunofluorescence for insulin, and BrdU or TUNEL. RESULTS: Human islet grafts corrected diabetes in the majority of cases. Among grafts from the same donor, human beta cell proliferation doubled in those exposed to higher glucose relative to lower glucose. Across the entire cohort of grafts, higher blood glucose was strongly correlated with increased beta cell replication. Beta cell replication rates were unrelated to circulating human insulin levels or donor age, but tended to correlate with donor BMI. Beta cell TUNEL reactivity was not measurably increased in grafts exposed to elevated blood glucose. CONCLUSIONS/INTERPRETATION: Glucose is a mitogenic stimulus for transplanted human beta cells in vivo. Investigating the underlying pathways may point to mechanisms capable of expanding human beta cell mass in vivo.

Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.

Mammalian dosage compensation involves silencing of one of the two X chromosomes in females and is controlled by the X-inactivation center (Xic). The Xic, which includes Xist and its antisense transcription unit Tsix/Xite, somehow senses the number of X chromosomes and triggers Xist up-regulation from one of the two X chromosomes in females. We found that a segment of the mouse Xic lying several hundred kilobases upstream of Xist brings the two Xics together before the onset of X inactivation. This region can autonomously drive Xic trans-interactions even as an ectopic single-copy transgene. Its introduction into male embryonic stem cells is strongly selected against, consistent with a possible role in trans-activating Xist. We propose that homologous associations driven by this novel X-pairing region (Xpr) of the Xic enable a cell to sense that more than one X chromosome is present and coordinate reciprocal Xist/Tsix expression.

Identification of human homologues of the mouse mammary tumor virus receptor.

Human breast tumors contain DNA sequences with high homology to the Mouse Mammary Tumor Virus (MMTV) env gene. env encodes the membrane glycoprotein that binds to the MMTV receptor (Mtvr) in mouse tissues and is required for infection. If humans become infected with MMTV, identification of a human Mtvr might shed light on the mechanism of infection. Here, we identified two human genes, Mtvr1 and Mtvr2, encoding proteins highly related to the mouse receptor. Mtvr-related sequences were also detected in other mammalian species. Thus, zoonotic transmission of MMTV is a real possibility given the existence of highly homologous MMTV receptors.

Codon-improved Cre recombinase (iCre) expression in the mouse.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.

Alteration of Cre recombinase site specificity by substrate-linked protein evolution.

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.

The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Parathyroid hormone-related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets.

The cloning of the so-called 'parathyroid hormone-related protein' (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N-terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly-hormone which comprises a family of distinct peptide hormones arising from post-translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N-terminal, mid-region and C-terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1-34) and PTHrP(1-36), is the only cloned receptor so far. PTHrP is a PTH-like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo-vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium-sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Efficient FLP recombination in mouse ES cells and oocytes.

We report an improved vector, pCAGGS-FLPe, for transient expression of the enhanced FLP recombinase in mouse ES cells and oocytes. In standard transfection experiments, about 6% of total ES colonies showed FLP recombination, albeit with mosaicism within each colony. After microinjection of pCAGGS-FLPe into oocytes, about one-third of heterozygotic mice born showed complete FLP recombination. Thus pCAGGS-FLPe presents two practical options for removal of FRT cassettes in mice.

A CamKIIalpha iCre BAC allows brain-specific gene inactivation.

We describe the generation of transgenic mouse lines expressing the Cre recombinase enzyme in brain under control of the CamKIIalpha gene present in a BAC expression vector. The CamKIIalpha BAC transgene gave a faithful expression pattern resembling the pattern of the endogenous CamKIIalpha gene. Specifically, high levels of CamKIIalpha Cre were detected in hippocampus, cortex, and amygdala, and lower levels were detected in striatum, thalamus, and hypothalamus. As expected, no expression was detected in the cerebellum or outside of the brain. The expression level of the BAC CamKIIalpha driven Cre was shown to be copy number dependent. To test the activity of the Cre recombinase, the transgenic mice were crossed with mice harbouring the CREB (cAMP response element binding protein) allele with the 10th exon flanked by two loxP sites, and recombination was monitored by the disappearance of the CREB protein. Finally, evaluation of the developmental postnatal expression of the CamKIIalpha Cre BAC revealed the expression of the Cre recombinase as early as P3.

Self-deleting suicide vectors (SDSV): selective killing of p53-deficient cancer cells.

A self-deleting retrovirus vector carrying a herpes simplex virus (HSV)-thymidine kinase suicide gene has been developed to selectively kill cancer cells expressing a dysfunctional p53 tumor suppressor protein. When cells containing functional p53 are infected with the virus, the integrated provirus and the HSV-thymidine kinase gene are deleted from the genome by site-specific recombination (Cre/loxP). In contrast, cells without p53 or cells expressing a DNA-binding mutant of p53 retain the provirus and become susceptible to killing by ganciclovir. This strategy provides a new concept for the selective killing of cancer cells that can be adapted to any other dysfunctional transcription factor expressed by different tumors.

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA.

Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including some which are virtually impossible by conventional methods. These approaches do not rely on the presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position on a target molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences from complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any size, including very large ones, and present powerful new avenues for DNA manipulation in general.

NSD3, a new SET domain-containing gene, maps to 8p12 and is amplified in human breast cancer cell lines.

We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor alpha. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf-Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.

Parathyroid hormone-related protein-(1--36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.

All would agree that hypercalcemia occurs among patients with humoral hypercalcemia of malignancy (HHM) as a result of osteoclastic bone resorption. Some studies suggest that enhanced renal calcium reabsorption, which plays an important pathophysiological role in the hypercalcemia occurring in primary hyperparathyroidism, is also important pathophysiologically in HHM. Other studies have not agreed. In large part, these differences result from the inability to accurately assess creatinine and calcium clearance in critically ill subjects with HHM. To circumvent these issues, we have developed steady state 48-h PTH-related protein (PTHrP) infusion and 8-h hypercalcemic calcium clamp protocols. These techniques allow assessment of the effects of steady state PTHrP and calcium infusions in normal healthy volunteers in a setting in which renal function is stable and measurable and in which the filtered load of calcium can be matched in PTHrP- and calcium-infused subjects. Normal subjects were infused with saline (placebo), PTHrP, or calcium. Subjects receiving PTHrP, as expected, displayed mild hypercalcemia (10.2 mg/dL), suppression of endogenous PTH-(1--84), and phosphaturia. Subjects receiving the hypercalcemic calcium clamp displayed indistinguishable degrees of hypercalcemia and PTH suppression. Despite their matched degrees of hypercalcemia and PTH suppression, the two groups differed importantly with regard to fractional calcium excretion (FECa). The hypercalcemic calcium clamp group was markedly hypercalciuric (FECa averaged 6.5%), whereas FECa in the PTHrP-infused subjects was approximately 50% lower (between 2.5--3.7%), and no different from that in the normal controls, which ranged from 1.5--3.0%. These studies demonstrate that PTHrP is able to stimulate renal calcium reabsorption in healthy volunteers. These studies suggest that PTHrP-induced renal calcium reabsorption, in concert with the well established acceleration of osteoclastic bone resorption, contributes in a significant way to the hypercalcemia observed in patients with HHM.