Correlation of total serum immunoglobulin E level, sputum, and peripheral eosinophil count in assessing the clinical severity in bronchial asthma.

CONTEXT: Asthma is a chronic inflammatory disorder of the airway with involvement of various cellular populations and release of many inflammatory mediators. Eosinophils and serum immunoglobulin E (IgE) are considered a good marker of airway inflammation in asthma. The correlation of clinical assessment with various markers of airway inflammation in asthma is not well established in the Indian population. AIMS: This study aims to study the correlation of serum IgE, sputum eosinophil count, and peripheral eosinophil count with clinical severity of Asthma. METHODS: This is a cross-sectional study involving 76 stable asthmatic patients of 18-60 years of age attending the pulmonary medicine OPD. Spirometry measured at baseline. Participants were categorized according to the GINA criteria based on clinical symptoms and pulmonary function test. Blood samples were collected for peripheral eosinophil count, serum IgE levels, and sputum samples for eosinophil count. All three parameters were compared with severity of asthma. The correlation of sputum eosinophil count, peripheral eosinophil count, and serum IgE with severity of asthma was analyzed by Pearson's Chi-square test, Fisher's exact test, and the correlation coefficient was reported together with standard error of the estimate. RESULTS: The mean age of patients in our study was 37.42 years and 56.6% were male. There was a significant inverse correlation between serum IgE levels and predicted forced expiratory volume 1 s (FEV1). Sputum eosinophilia was significantly seen in severe persistent asthma patients (19.7%). There was a significant inverse correlation between sputum eosinophil count and predicted FEV1and forced vital capacity. We also found there was a significant association between peripheral eosinophil count, sputum eosinophil count, and elevated serum IgE (g100 IU/mL) with severe persistent asthma. CONCLUSIONS: The assessment of sputum eosinophil count is simple, inexpensive, noninvasive, and direct measurement of airway inflammation. It could be the preferred method in monitoring airway inflammation and guided management in day-to-day practice.

Deconstructing transcriptional heterogeneity in pluripotent stem cells.

Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.

Gene networks of fully connected triads with complete auto-activation enable multistability and stepwise stochastic transitions.

Fully-connected triads (FCTs), such as the Oct4-Sox2-Nanog triad, have been implicated as recurring transcriptional motifs embedded within the regulatory networks that specify and maintain cellular states. To explore the possible connections between FCT topologies and cell fate determinations, we employed computational network screening to search all possible FCT topologies for multistability, a dynamic property that allows the rise of alternate regulatory states from the same transcriptional network. The search yielded a hierarchy of FCTs with various potentials for multistability, including several topologies capable of reaching eight distinct stable states. Our analyses suggested that complete auto-activation is an effective indicator for multistability, and, when gene expression noise was incorporated into the model, the networks were able to transit multiple states spontaneously. Different levels of stochasticity were found to either induce or disrupt random state transitioning with some transitions requiring layovers at one or more intermediate states. Using this framework we simulated a simplified model of induced pluripotency by including constitutive overexpression terms. The corresponding FCT showed random state transitioning from a terminal state to the pluripotent state, with the temporal distribution of this transition matching published experimental data. This work establishes a potential theoretical framework for understanding cell fate determinations by connecting conserved regulatory modules with network dynamics. Our results could also be employed experimentally, using established developmental transcription factors as seeds, to locate cell lineage specification networks by using auto-activation as a cipher.

Making a noisy gene: HDACs turn up the static.

In this issue of Molecular Cell, Weinberger et al. (2012) find that particular histone deacetylases (HDACs) regulate distinct stages of transcription, implicating chromatin dynamics in the generation of gene-specific noise within populations of genetically identical cells.

Cellular signal processing: out of one, many.

Cells can make fate decisions in response to information from the environment. In this issue of Molecular Cell, Chen et al. (2012) describe how the design of a signal-processing pathway allows a homogenous population of cells to display diverse responses to uniform growth factor cues.

Master transcription factors determine cell-type-specific responses to TGF-ß signaling.

Transforming growth factor beta (TGF-ß) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-ß signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-ß signaling.

Evidence that gene activation and silencing during stem cell differentiation requires a transcriptionally paused intermediate state.

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3' transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98-99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.

The Msx1 Homeoprotein Recruits Polycomb to the Nuclear Periphery during Development.

Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropriate regulation of gene expression programs during development.

Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells.

Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3'UTR, the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unscheduled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation.

Myogenic factors that regulate expression of muscle-specific microRNAs.

Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.

Control of developmental regulators by Polycomb in human embryonic stem cells.

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters.

We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

Core transcriptional regulatory circuitry in human embryonic stem cells.

The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.

A modular, bifunctional RNA that integrates itself into a target RNA.

Nature often combines independent functional domains to achieve complex function, but this approach has not been extensively explored with artificial enzymes. Here, a group I ribozyme, which can act as an endoribonuclease, was partnered with the R3C ribozyme, which catalyzes the ligation of RNA molecules. The conjoined ribozymes have the potential to perform successive RNA cleavage and joining reactions, resulting in their mutual integration into a target RNA substrate. When simply joined together, however, the ribozymes were unable to achieve this outcome because of inefficient transfer of the substrate between the two catalytic subunits. In vitro evolution was used to optimize the behavior of the conjoined ribozymes, resulting in bifunctional molecules with substantially improved integration activity. The ligase subunit of these molecules was unchanged, whereas the group I subunit acquired several mutations, mostly in peripheral regions. The generation and study of this bifunctional assembly helps shed light on the evolution of modular enzymes and the obstacles that must be overcome in bringing together independent functional domains. These molecules also may be useful as tools for the insertional mutagenesis of target mRNAs.