Histone locus bodies: a paradigm for how nuclear biomolecular condensates control cell cycle regulated gene expression.

Histone locus bodies (HLBs) are biomolecular condensates that assemble at replication-dependent (RD) histone genes in animal cells. These genes produce unique mRNAs that are not polyadenylated and instead end in a conserved 3' stem loop critical for coordinated production of histone proteins during S phase of the cell cycle. Several evolutionarily conserved factors necessary for synthesis of RD histone mRNAs concentrate only in the HLB. Moreover, because HLBs are present throughout the cell cycle even though RD histone genes are only expressed during S phase, changes in HLB composition during cell cycle progression drive much of the cell cycle regulation of RD histone gene expression. Thus, HLBs provide a powerful opportunity to determine the cause-and-effect relationships between nuclear body formation and cell cycle regulated gene expression. In this review, we focus on progress during the last five years that has advanced our understanding of HLB biology.

Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos.

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.

Onset and duration of attenuation of exercise-induced bronchoconstriction in children by single-dose of montelukast.

Single-dose montelukast attenuates exercise-induced bronchoconstriction (EIB) in adults within 2 hours postdose and lasting through 24 hours. This study evaluated the onset and duration of EIB attenuation in children after a single dose of montelukast. A randomized, double-blind, placebo-controlled, two-period crossover study was performed. Patients (n = 66) aged 4-14 years, with preexercise forced expiratory volume in 1 second of (FEV(1)) ≥70% predicted and maximum percentage fall in FEV(1) of ≥20% at two screening exercise challenges were eligible. Patients were to receive single-dose montelukast (4 or 5 mg) or placebo before performing standardized exercise challenges at 2 and 24 hours postdose. A 3- to-7-day washout separated the two crossover periods. The primary end point was maximum percentage fall in FEV(1) after exercise challenge 2 hours postdose. Secondary end points included maximum percentage fall in FEV(1) after the 24-hour postdose challenge; each of the following at 2 and 24 hours postdose-maximum percentage fall in FEV(1) categorized as <10%, 10-20%, or >20%; area under the curve (AUC) during 60 minutes postchallenge; time to recovery of FEV(1) to within 5% of preexercise baseline; and need for rescue medication. The mean maximum percentage fall in FEV(1) after the 2-hour postdose exercise challenge was significantly attenuated after single-dose montelukast compared with placebo (15.35% versus 20.00%; p = 0.020). Montelukast was also significantly more effective than placebo for maximum percentage fall after the 24-hour challenge (12.92% versus 17.25%; p = 0.005), the categorized maximum percent fall in FEV(1) at 2 hours (p = 0.034), and AUC at 2 hours (p = 0.022) and 24 hours (p = 0.013). Single-dose montelukast provided rapid and sustained EIB attenuation in children. Clinicaltrials.gov identifier: NCT00534976.

Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes.

The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

CDK-Regulated Phase Separation Seeded by Histone Genes Ensures Precise Growth and Function of Histone Locus Bodies.

Many membraneless organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, our experiments identify a mechanism linking nuclear body growth and size with gene expression.

CRMP-1 enhances EVL-mediated actin elongation to build lamellipodia and the actin cortex.

Cells can control actin polymerization by nucleating new filaments or elongating existing ones. We recently identified CRMP-1 as a factor that stimulates the formation of Listeria monocytogenes actin comet tails, thereby implicating it in actin assembly. We now show that CRMP-1 is a major contributor to actin assembly in epithelial cells, where it works with the Ena/VASP family member EVL to assemble the actin cytoskeleton in the apical cortex and in protruding lamellipodia. CRMP-1 and EVL bind to one another and together accelerate actin filament barbed-end elongation. CRMP-1 also stimulates actin assembly in the presence of VASP and Mena in vitro, but CRMP-1-dependent actin assembly in MDCK cells is EVL specific. Our results identify CRMP-1 as a novel regulator of actin filament elongation and reveal a surprisingly important role for CRMP-1, EVL, and actin polymerization in maintaining the structural integrity of epithelial sheets.

Potential effects of fluticasone propionate on bone mineral density in patients with asthma: a 2-year randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To evaluate the effects of treatment with fluticasone propionate vs placebo on bone, hypothalamic-pituitary-adrenal (HPA) axis function, and the eyes in patients with asthma. PATIENTS AND METHODS: This randomized, double-blind, placebo-controlled study of 160 patients with asthma who had minimal previous exposure to corticosteroids was conducted from July 1994 through June 1997. Patients received fluticasone at 88 microg twice daily, fluticasone at 440 microg twice daily, or placebo twice daily for 2 years. Bone mineral density (BMD) was evaluated every 6 months by lumbar spine, proximal femur, and total body scans. Measurements of HPA axis function and ophthalmic evaluations were conducted at similar intervals. RESULTS: Among the 3 groups, no significant differences were observed in BMD at week 104 (at any anatomical site). Mean percent change from baseline in the lumbar spine was less than 1% for all 3 groups. At all time points, HPA axis function was similar in the 88-microg fluticasone group compared with the placebo group. For mean change from baseline in corticotropin-stimulated peak cortisol (P = .003 and P = .02 at weeks 24 and 52, respectively) and area under the stimulated plasma cortisol vs time curve (P = .002 and P = .02 at weeks 24 and 52, respectively), statistically significant reductions from baseline were observed in the 440-microg fluticasone group compared with the placebo group. These reductions of 10% to 13% from baseline were not accompanied by other signs of systemic effect and did not persist with continued treatment (at weeks 76 and 104). No important ocular changes were observed. CONCLUSION: Long-term treatment with 88 microg of fluticasone twice daily was comparable to placebo in all skeletal, ophthalmic, and HPA axis function assessments. Treatment with fluticasone at 440 microg twice daily resulted in no significant effects on BMD and a statistically significant but not clinically important temporary reduction in cortisol production.

Exercise-induced bronchoconstriction: The effects of montelukast, a leukotriene receptor antagonist.

Exercise-induced bronchoconstriction (EIB) is very common in both patients with asthma and those who are otherwise thought to be normal. The intensity of exercise as well as the type of exercise is important in producing symptoms. This may make some types of exercise such as swimming more suitable and extended running more difficult for patients with this condition. A better understanding of EIB will allow the physician to direct the patient towards a type of exercise and medications that can result in a more active lifestyle without the same concern for resulting symptoms. This is especially important for schoolchildren who are usually enrolled in physical education classes and elite athletes who may desire to participate in competitive sports. Fortunately several medications (short- and long-acting beta(2)-agonists, cromolyn, nedocromil, inhaled corticosteroids, and more recently leukotriene modifiers) have been shown to be effective in preventing or attenuating the effects of exercise in many patients. In addition, inhaled beta(2)-agonists have been shown to quickly reverse the airway obstruction that develops in patients and continue to be the reliever medications of choice. Inhaled corticosteroids are increasingly being recommended as regular therapy now that the role of inflammation and airway injury has been identified in EIB. With the discovery that there is a release of mediators such as histamine and leukotrienes from cells in the airway following exercise with resulting airway obstruction in susceptible individuals, interest has turned to attenuating their effects with mediator antagonists especially those that block the effects of leukotrienes. Studies with an oral leukotriene antagonist, montelukast, have shown beneficial effects in adults and children aged as young as 6 years with EIB. These effects can be demonstrated as soon as two hours and as long as 24 hours after administration without a demonstrated loss of a protective effect after months of treatment. The studies leading up to and resulting in an approval of montelukast for EIB for patients aged 15 years and older are reviewed in this paper.

Advances in the management of pediatric asthma: a review of recent FDA drug approvals and label updates.

Children have the highest prevalence of asthma of any age group. In the United States during 2001, there were 12.6 million physician and hospital outpatient visits for asthma treatment, of which almost 5 million involved children 18 years and younger. Therapeutic advances in pediatric asthma could improve patient outcomes and potentially reduce the burden on health care systems. Efforts to obtain efficacy and safety data in pediatric populations and develop pediatric formulations of asthma treatments have been encouraged by the FDA and clinicians. This article reviews the newest additions to asthma therapies approved for use in children, including an inhaled corticosteroid, some long-acting beta2-agonists, some leukotriene-receptor blockers, and a single-isomer, short-acting beta2-agonist.

Recent advances in the management of asthma using leukotriene modifiers.

Asthma is a chronic inflammatory disease of the airways that affects approximately 100 million people worldwide. In order to reduce symptoms, improve pulmonary function, and decrease morbidity, current treatment guidelines emphasize the importance of controlling the underlying inflammation in patients with asthma. Leukotrienes are leukocyte-generated lipid mediators that promote airway inflammation. Recognition of the importance of leukotrienes in the pathogenesis of asthma has led to the development of leukotriene modifiers, the first new class of drugs for the treatment of asthma to become available in 25 years. Controlled clinical trials with the four currently used leukotriene modifiers (montelukast, zafirlukast, and zileuton in the US and pranlukast in Japan) have established their efficacy in improving pulmonary function, reducing symptoms, decreasing night-time awakenings, and decreasing the need for rescue medications. They exert anti-inflammatory effects that attenuate cellular infiltration and bronchial hyperresponsiveness and complement the anti-inflammatory properties of inhaled corticosteroids. In patients with moderate and severe asthma, they permit tapering of the corticosteroid dose. In patients with exercise-induced asthma, leukotriene modifiers limit the decline in and quicken the recovery of pulmonary functions without the tolerance issues seen with chronic long-acting beta(2)-adrenoceptor agonist use. In patients with aspirin (acetylsalicylic acid)-induced asthma, they improve pulmonary function and shift the dose response curve to the right, reducing the patient's response to aspirin. In patients with seasonal allergic rhinitis, with or without concomitant asthma, they improve nasal, eye, and throat symptoms as well as quality of life. Leukotriene modifiers are generally safe and well tolerated with adverse effect profiles similar to that of placebo. The one safety issue raised with leukotriene modifiers, Churg-Strauss Syndrome, appears to be the unmasking of an already present syndrome that is manifested when the leukotriene modifiers permit corticosteroid doses to be reduced. Although current treatment guidelines recommend their use in patients with mild persistent asthma, these guidelines were developed just as leukotriene modifiers were coming to the market, before much of the clinical efficacy data were published. Because asthma is a heterogeneous disease, the different asthma phenotypes respond differently to therapies; consequently asthma therapy needs to be individualized. Leukotriene modifiers increase the therapeutic options for patients with asthma and, based on recent data, it is expected that future guidelines will describe expanded uses for these agents in clinical circumstances where these drugs are effective.