Modifiable comorbidities and disability in multiple sclerosis.

Multiple sclerosis is a common neurological disease that results in disability in early adulthood that may progress through a relatively normal lifespan. Other comorbid health conditions can increase the likelihood of progression of MS and independently contribute to limitations in activities and social participation. We examine common modifiable health conditions and behaviors, including hypertension, obesity, diabetes mellitus, alcohol, and smoking to determine their impacts on MS and disability. Appropriate identification and treatment can improve health status, diminish disability, and improve quality of life.

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration.

Lens epithelium-derived growth factor (LEDGF) fusion proteins can direct HIV-1 DNA integration to novel sites in the host genome. The C terminus of LEDGF contains an integrase binding domain (IBD), and the N terminus binds chromatin. LEDGF normally directs integrations to the bodies of expressed genes. Replacing the N terminus of LEDGF with chromatin binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. We chose two well-characterized CBDs: the plant homeodomain (PHD) finger from ING2 and the chromodomain from heterochromatin binding protein 1alpha (HP1alpha). The ING2 PHD finger binds H3K4me3, a histone mark that is associated with the transcriptional start sites of expressed genes. The HP1alpha chromodomain binds H3K9me2,3, histone marks that are widely distributed throughout the genome. A fusion protein in which the ING2 PHD finger was linked to the LEDGF IBD directed integrations near the start sites of expressed genes. A similar fusion protein in which the HP1alpha chromodomain was linked to the LEDGF IBD directed integrations to sites that differed from both the PHD finger fusion-directed and LEDGF-directed integration sites. The ability to redirect HIV-1 DNA integration may help solve the problems associated with the activation of oncogenes when retroviruses are used in gene therapy.

Decreased replication origin activity in temporal transition regions.

In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains.

The tumor suppressor Cdc73 functionally associates with CPSF and CstF 3' mRNA processing factors.

The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.

Phosphorylation of the menin tumor suppressor protein on serine 543 and serine 583.

Multiple endocrine neoplasia type 1 (MEN-1) is a heritable syndrome typified by tumors in multiple endocrine organs, including the pituitary, parathyroids, and pancreatic islets. MEN-1 is attributable to mutations in the MEN1 tumor-suppressor gene that encodes the menin protein. Recent studies have implicated menin in transcriptional regulation and in covalent histone modification; however, little is known about modifications of the menin protein. Here, we report that menin is subject to phosphorylation on serine residues, including Ser543 and Ser583. Phosphorylation-defective mutants of either or both of these residues retain the associated histone methyltransferase activity of menin, as well as binding to the trithorax complex members Ash2L, Rbbp5, and MLL2 and to RNA polymerase II. Chromatin immunoprecipitation experiments reveal that binding of menin to the Hoxc8 locus is not affected by phosphorylation on Ser543 or Ser583.

Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c.

Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclin-dependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.

Nursing time allocation and other considerations for staffing.

The purpose of this project was to evaluate how nursing staff on the telemetry unit at Merle West Medical Center, a rural Pacific northwest community medical center in the USA, spend their time on selected nursing tasks, and how they believed staffing and patient acuity levels could best be determined. A self-report survey tool was developed, piloted and administered. Of the tasks that were listed in the survey, registered nurses (RNs) reported spending 39% of their time performing tasks that RNs only can perform. RNs reported spending 12% of their time performing activities that certified nursing assistants (CNAs) could perform alone and 49% of their time was spent on tasks that both RNs and CNAs must perform. Results from the quantitative portion of the survey indicated that RNs may not be used efficiently due to task overlap. Administering a modified survey which includes a balance of direct and indirect patient care tasks, as well as only those tasks that are truly performed frequently, will increase the relevance of the results.

The parafibromin tumor suppressor protein is part of a human Paf1 complex.

Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit. Immunofluorescence experiments show that parafibromin is a nuclear protein. In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4. Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process.

Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors.

Mutations in the MEN1 gene are associated with the multiple endocrine neoplasia syndrome type 1 (MEN1), which is characterized by parathyroid hyperplasia and tumors of the pituitary and pancreatic islets. The mechanism by which MEN1 acts as a tumor suppressor is unclear. We have recently shown that menin, the MEN1 protein product, interacts with mixed lineage leukemia (MLL) family proteins in a histone methyltransferase complex including Ash2, Rbbp5, and WDR5. Here, we show that menin directly regulates expression of the cyclin-dependent kinase inhibitors p27Kip1 and p18Ink4c. Menin activates transcription by means of a mechanism involving recruitment of MLL to the p27Kip1 and p18Ink4c promoters and coding regions. Loss of function of either MLL or menin results in down-regulation of p27Kip1 and p18Ink4c expression and deregulated cell growth. These findings suggest that regulation of cyclin-dependent kinase inhibitor transcription by cooperative interaction between menin and MLL plays a central role in menin's activity as a tumor suppressor.

Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus.

The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser 5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells.

RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.