Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.

Interplay among GATA transcription factors is an important determinant of cell fate during hematopoiesis. Although GATA-2 regulates hematopoietic stem cell function, mechanisms controlling GATA-2 expression are undefined. Of particular interest is the repression of GATA-2, because sustained GATA-2 expression in hematopoietic stem and progenitor cells alters hematopoiesis. GATA-2 transcription is derepressed in erythroid precursors lacking GATA-1, but the underlying mechanisms are unknown. Using chromatin immunoprecipitation analysis, we show that GATA-1 binds a highly restricted upstream region of the Ϸ70-kb GATA-2 domain, despite >80 GATA sites throughout the domain. GATA-2 also binds this region in the absence of GATA-1. Genetic complementation studies in GATA-1-null cells showed that GATA-1 rapidly displaces GATA-2, which is coupled to transcriptional repression. GATA-1 also displaces CREB-binding protein (CBP), despite the fact that GATA-1 binds CBP in other contexts. Repression correlates with reduced histone acetylation domain-wide, but not altered methylation of histone H3 at lysine 4. The GATA factor-binding region exhibited cell-type-specific enhancer activity in transient transfection assays. We propose that GATA-1 instigates GATA-2 repression by means of disruption of positive autoregulation, followed by establishment of a domain-wide repressive chromatin structure. Such a mechanism is predicted to be critical for the control of hematopoiesis.

One of the most common methods for providing postoperative analgesia is via patient-controlled analgesia (PCA). Although the typical approach is to administer opioids via a programmable infusion pump, other drugs and other modes of administration are available. This article reviews the history and practice of many aspects of PCA and provides extensive guidelines for the practice of PCA-administered opioids. In addition, potential adverse effects and recommendations for their monitoring and treatment are reviewed.

The aim of this randomized controlled trial was to determine the effect of jaw relaxation, music and the combination of relaxation and music on postoperative pain after major abdominal surgery during ambulation and rest on postoperative days 1 and 2. Opioid medication provided for pain, following abdominal surgery, does not always give sufficient relief and can cause undesired side effects. Thus, additional interventions such as music and relaxation may provide more complete relief. Previous studies have found mixed results due to small sample sizes and other methodological problems. In a rigorous experimental design, 500 subjects aged 18-70 in five Midwestern hospitals were randomly assigned by minimization to a relaxation, music, relaxation plus music, or control group. Interventions were taught preoperatively and tested postoperatively. The same amount of time was spent with subjects in the control group. Pain was measured with the visual analogue sensation and distress of pain scales. Demographic and surgical variables, and milligrams of parenteral or oral opioids in effect at the time of testing were not significantly different between the groups, nor did they correlate with pain scores. Controlling for pretest sensation and distress, orthogonal a priori contrasts and multivariate analysis of covariance indicated that the three treatment groups had significantly less pain than the controls, (P = 0.028-0.000) which was confirmed by the univariate analysis of covariance (P = 0.018-0.000). Post hoc multivariate analysis revealed that the combination group had significantly less sensation and distress of pain than the control group on all post-tests (P = 0.035-0.000), and the relaxation and music groups had significantly less on all tests (P = 0.022-0.000) except after ambulation. At post ambulation those using relaxation did not have significantly less pain than the controls on both days and those using music did not on day 1, although there were some univariate effects. A corresponding significant decrease in mastery of the interventions from pre to post ambulation suggests the need for reminders to focus on the intervention during this increased activity. Physicians and nurses preparing patients for surgery and caring for them afterward, should encourage patients to use relaxation and music as adjuvants to medication for postoperative pain.

Increased ␣-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant Parkinson's disease (PD). Variation in SNCA expression may be critical in common, genetically complex PD but the underlying regulatory mechanism is unknown. We show that SNCA and the heme metabolism genes ALAS2, FECH, and BLVRB form a block of tightly correlated gene expression in 113 samples of human blood, where SNCA naturally abounds (validated P ‫؍‬ 1.6 ؋ 10 ؊11 , 1.8 ؋ 10 ؊10 , and 6.6 ؋ 10 ؊5 ). Genetic complementation analysis revealed that these four genes are co-induced by the transcription factor GATA-1. GATA-1 specifically occupies a conserved region within SNCA intron-1 and directly induces a 6.9-fold increase in ␣-synuclein. Endogenous GATA-2 is highly expressed in substantia nigra vulnerable to PD, occupies intron-1, and modulates SNCA expression in dopaminergic cells. This critical link between GATA factors and SNCA may enable therapies designed to lower ␣-synuclein production.␣-synuclein dosage ͉ GATA-1 ͉ GATA-2 ͉ gene expression ͉ microarray

The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at ؉9.5 kb. Despite indistinguishable GATA-1 occupancy at ؊77 kb and ؉9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the ؊77 kb region. The ؊77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1-and GATA-2-expressing cells. By contrast, the ؉9.5 kb region exhibited considerably stronger enhancer activity in GATA-2-than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1-or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the ؊77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine ␤-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the ␤major promoter. ER-GATA-1 increased erythroid Krü ppel-like factor and SWI͞SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of ␤-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial͞temporal regulation and underlies the multistep activation mechanism.erythropoiesis ͉ globin ͉ histone ͉ epigenetic ͉ erythoid Krü ppel-like factor

The hematopoietic transcription factor GATA-1 regulates erythropoiesis and ␤-globin expression. Although consensus GATA-1 binding sites exist throughout the murine ␤-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the ␤-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner. E ukaryotic DNA wraps Ϸ1.7 times around a core histone octamer to form nucleosomes, which in turn, fold into highly condensed chromatin. The role of chromatin structure in regulating gene expression is established through extensive analysis in diverse systems (1-8). An important consequence of chromatin folding is the regulation of cis-element accessibility, thereby preventing the constitutive loading of trans-acting factors and RNA polymerase II (pol II). Coactivators and corepressors are recruited to chromosomal sites via interactions with transcription factors and catalyze chromatin remodeling (9, 10). Although certain factors can recognize binding sites on nucleosomal DNA, others are occluded (11,12). Factors capable of binding nucleosomal sites would not likely be able to access sites in condensed chromatin. Thus, site occupancy in vivo cannot be predicted by sequence analysis, but rather requires analysis of binding in living cells.We use the murine ␤-globin locus to investigate how transcriptional control occurs within chromatin domains (13,14). The ␤-globin locus consists of several genes arrayed in the order of their developmental expression (15). High-level transcription of these genes requires an upstream locus control region (LCR) (16-19), comprised of four DNaseI hypersensitive sites, 21). Besides an erythroid-specific enhancer function, the LCR counteracts transgene silencing (16,22).A common feature of active chromatin is core histone acetylation (23). We hypothesized that LCRs function by recruiting histone acetyltransferases that establish broad acetylation patterns (24). Analysis of the human GH domain in transgenic mice has provided strong evidence for the hypothesis that LCRs can establish broad acetylation patterns (25). Analysis of acetylation at the murine ␤-globin locus in adult erythroid cells revealed enrichment of acetylated histones H3 and H4 at the LCR and the adult globin genes, ␤major and ␤minor (24, 26). Much less acetylation was evident over a Ϸ30-kb region spanning the silent embryonic ␤-globin genes Ey and ␤H1, and between the adult ␤-globin genes. In embryonic erythroid cells, acetylation was high at the LCR, the embryonic ␤H1 promoter and the inactive ␤minor promoter (24). Although the LCR confers high-level ␤-globin expression, deletion of HS1-HS4 from the murine locus did not abrogate hyperacetylatio...