Gene-set analysis identifies master transcription factors in developmental courses

This article reports on the author s study of developmental mathematics courses at two-year colleges in the Virginia Community College System in the United States. The study was in part to determine the effectiveness of developmental courses in preparing students for college-level work. The author also investigated the influence on success of certain pedagogical factors. In this study, success was measured by number of students who took and passed a developmental mathematics course and went on to pass the college-level course for which it was a prerequisite. A recent report by the Institute for Higher Education Policy debunks (1998, p. v) the misconceptions that remediation is too expensive and is an inappropriate function for colleges. Instead the report argues that remediation is a core function of higher education and a good investment for society, as well as for colleges and universities. The report proposes strategies for the future - two mutually reinforcing goals (Institute, 1998, p. ix): (1) Reducing the need for remediation in higher education, and (2) improving the effectiveness of remedial education in higher education. This study concentrates on the latter of these two charges - the effectiveness of the developmental mathematics programs in higher education. Descriptive data include credit hours, enrollment, attendance, class size, classroom participation, student success, and retention rates. Classroom observations and discussions with mathematics faculty are also included.

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

Purpose The aim of this longitudinal study was to describe developmental courses of childhood dysarthria against the background of typical speech motor development by collecting auditory-perceptual data. Method Fourteen children (four girls, 10 boys; 5;1-8;4 [years;months] at Time 1) with neurological conditions (CNC) and 14 typically developing children (CTD) matched for age and gender were assessed at three points in time over an 18-month period. Speech samples were collected using the Bogenhausener Dysarthrie Skalen-Kindliche Dysarthrien (BoDyS-KiD; in English: Bogenhausen Dysarthria Scales-Childhood Dysarthria), a German tool for the assessment of childhood dysarthria, and analyzed by means of nine perceptual scales covering all clinically relevant speech components. Age normalization was performed according to a method published recently. Data from the matched controls were used to estimate whether the gradients of the CNC group's developmental trajectories exceeded those of typical development. Results The children with neurological conditions presented heterogeneous speech profiles with a wide range of severity. At the group level, relatively stable trajectories of the age-normalized dysarthria total score were found over the observation period. The nine perceptual scales showed more or less parallel developments. All patients except two followed the growth curve describing the developmental course of the typically developing children. Conclusions Most children took advantage of the developmental dynamics as they developed parallel to the age norm. With its comprehensive description of the developmental courses of 14 children with neurological conditions, this study may contribute to a more valid, statistically verified clinical assessment of the course of childhood dysarthria.

A key problem in understanding transcriptional regulatory networks is deciphering what cis regulatory logic is encoded in gene promoter sequences and how this sequence information maps to expression. A typical computational approach to this problem involves clustering genes by their expression profiles and then searching for overrepresented motifs in the promoter sequences of genes in a cluster. However, genes with similar expression profiles may be controlled by distinct regulatory programs. Moreover, if many gene expression profiles in a data set are highly correlated, as in the case of whole organism developmental time series, it may be difficult to resolve fine-grained clusters in the first place. We present a predictive framework for modeling the natural flow of information, from promoter sequence to expression, to learn cis regulatory motifs and characterize gene expression patterns in developmental time courses. We introduce a cluster-free algorithm based on a graph-regularized version of partial least squares (PLS) regression to learn sequence patterns—represented by graphs of k-mers, or “graph-mers”—that predict gene expression trajectories. Applying the approach to wildtype germline development in Caenorhabditis elegans, we found that the first and second latent PLS factors mapped to expression profiles for oocyte and sperm genes, respectively. We extracted both known and novel motifs from the graph-mers associated to these germline-specific patterns, including novel CG-rich motifs specific to oocyte genes. We found evidence supporting the functional relevance of these putative regulatory elements through analysis of positional bias, motif conservation and in situ gene expression. This study demonstrates that our regression model can learn biologically meaningful latent structure and identify potentially functional motifs from subtle developmental time course expression data.

While behavioral and educational data characterize a fourth grade shift in reading development, neuroscience evidence is relatively lacking. We used the N400 component of the event-related potential waveform to investigate the development of single word processing across the upper elementary years, in comparison to adult readers. We presented third graders, fourth graders, fifth graders, and college students with a well-controlled list of real words, pseudowords, letter strings, false font strings, and animal name targets. Words and pseudowords elicited similar N400s across groups. False font strings elicited N400s similar to words and letter strings in the three groups of children, but not in college students. The pattern of findings suggests relatively adult-like semantic and phonological processing by third grade, but a long developmental time course, beyond fifth grade, for orthographic processing in this context. Thus, the amplitude of the N400 elicited by various word-like stimuli does not reflect some sort of shift or discontinuity in word processing around the fourth grade. However, the results do suggest different developmental time courses for the processes that contribute to automatic single word reading and the integrative N400.

In eukaryotes, chromatin provides a way to compact the genetic material into the confined space of a nucleus. It is also a means to store the same genetic information in different chromatin states. Alteration of these states is enabled by chromatin modifying and remodeling machineries – enzymes that utilize a diverse range of structural changes to chromatin. Despite their apparent importance in gene regulation, it is unclear how they facilitate the transition between chromatin states. Within two distinct projects, we aimed to (1) decipher how chromatin modifying complexes, namely the Polycomb group proteins, are targeted to chromatin and (2) how chromatin remodelers, specifically the ISWI remodeling complexes, change chromatin structure. Polycomb group proteins assemble as chromatin-modifying complexes that maintain the memory of the silent transcriptional state, in part through methylation of lysine 27 on histone H3. Despite their established importance during development, it is largely unclear how these complexes are recruited to specific target genes and how they impair transcription. In flies, Polycomb is recruited by Polycomb response elements that are abundant in various DNA-binding factor motifs. However, the contribution of individual motifs is not yet resolved. In mammals, equivalents of Polycomb response elements are not yet characterized. Here, we aimed to dissect Polycomb-mediated silencing in the mouse genome by identifying DNA determinants of Polycomb recruitment and investigating the role of Polycomb recruitment in transcriptional silencing. More specifically, we developed an assay to test many DNA sequences with various sequence properties for their ability to drive PRC2 recruitment in mouse embryonic stem cells. The assay enabled integration of hundreds of sequences into a defined genomic location in parallel. We found that high density of unmethylated CG motifs within a synthetic backbone sequence is sufficient to recruit PRC2. Furthermore, to link PRC2 recruitment with transcriptional repression, we used CRISPR/Cas9 technology to delete the core PRC2 (Eed) component and monitored the transcriptional response by RNA-seq. Upon depletion of global H3K27me3 levels, we observed no significant changes in gene expression in mouse embryonic stem cells but global deregulation of PRC2 targets during differentiation into neuronal progenitors. These results indicate that recruitment of PRC2 and subsequent H3K27 methylation is important for cell-fate transition, but not required for gene repression in mouse embryonic stem cells. For the second project, we were interested in chromatin remodelers (ISWI) and their role in regulating chromatin structure. Chromatin remodelers are known to use the energy of ATP hydrolysis to evict, slide and reposition nucleosomes, yet we do not fully understand how nucleosome positioning and occupancy affects transcription factor binding. To this aim, we deleted Snf2h, the ATPase subunit of the ISWI chromatin remodeling family, in mouse embryonic stem cells. The Snf2h knockout mouse embryonic stem cells are viable with unchanged expression of pluripotency markers, which is exciting as this is the first viable knockout of an ATPase remodeler subunit. To determine global changes upon deletion of Snf2h, we monitored nucleosome positioning, chromatin accessibility and transcriptional response in Snf2h knockout cells using MNase, ATAC and RNA sequencing, respectively. Extensive data analysis revealed global changes in nucleosome positioning proximal to transcription start sites and transcription factor motifs. Analyzing nucleosome positioning and chromatin accessibility data, we identified transcription factors that require Snf2h to bind their target sites, such as CTCF. It seems that in the absence of Snf2h, nucleosomes cannot be evicted from CTCF motifs, which in turn results in loss of CTCF binding. Taken together, these results indicate that ISWI complexes enable transcription factor binding, at both promoters and distal regulatory regions, by sliding of motif-bound nucleosomes.

Introduction: Inflammatory oxidative microenvironments can alter heme oxygenase-1 (HO-1) homeostasis. Dysregulation of HO-1 favors proinflammatory signals, while transferrin receptor 1 (TfR1) regulates anti-inflammatory signal transduction. Previously, we have shown that chorioamnionitis (CHORIO) induces sustained elevations in HO-1/TfR1 at postnatal day (P)2 and mononuclear cell-driven inflammation at term age equivalent P7. Here, we hypothesized that an altered HO-1/TfR1 developmental time course would coincide with inflammatory/immune signal changes in the brain. Methods: To induce CHORIO in rats, we performed a laparotomy followed by bilateral transient uterine artery occlusion and intra-amniotic injection of lipopolysaccharide (LPS) at E18. The control group received laparotomy only with equivalent duration of anesthesia. We used real-time polymerase chain reaction (RT-PCR), multiplex electrochemiluminescent immunoassay (MECI), and flow cytometry (FC) to study changes in pro- and anti-inflammatory gene expression, immune cell secretome, and immune cells at critical and clinically relevant timepoints following CHORIO. Results: We found an acute reduction in anti-inflammatory signals in the cortex on embryonic day (E)19. This was followed by an increased proinflammatory signature on postnatal day (P)2. There were also significant alterations in the phenotypic distribution of splenic T cells on P7, a key organ in immune function. Furthermore, we showed that the microenvironment of the cortex at P21 was skewed toward a proinflammatory state by significant increases in IL-6. The prominence of T-helper cells (Th) in brain concomitant with a proinflammatory state at P21 suggests emerging inflammation and potential for neural injury. Conclusions: Defining how these and other inflammatory/immune signatures contribute to perinatal brain injury and investigating distinct immune signatures at more developmental time courses will be beneficial for targeting emerging therapies.

Dopamine D1 Receptor Changes Due to Caesarean Section Birth: Effects of Anesthesia, Developmental Time Course, and Functional Consequences

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

BackgroundThe domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues.ResultsRandomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development.ConclusionExpression profiles obtained from public RNA-seq datasets – despite being generated by different laboratories using different methodologies – can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species.

This study investigated the developmental time course of spoken word recognition in older children using eye tracking to assess how the real-time processing dynamics of word recognition change over development. We found that 9-year-olds were slower to activate the target words and showed more early competition from competitor words than 16-year-olds; however, both age groups ultimately fixated targets to the same degree. This contrasts with a prior study of adolescents with language impairment (McMurray, Samelson, Lee, & Tomblin, 2010) that showed a different pattern of real-time processes. These findings suggest that the dynamics of word recognition are still developing even at these late ages, and developmental changes may derive from different sources than individual differences in relative language ability.

The auditory middle latency response (MLR) seems to have a relatively long developmental time course, extending through the first decade of life. Characteristics of each MLR component change developmentally not only with respect to waveform morphology but also with respect to response reliability, dependence on awareness state, and stimulus rate. Both human and animal studies indicate that these complex changes may be a result of multiple generating systems that show multiple time courses of development. This framework has practical ramifications in that clinical and research studies of MLR in young children must take into account the development sequence. Furthermore, it cannot be assumed a priori that research results obtained from adults will apply to young children. The complexity of the process raises intriguing questions regarding the functional development of auditory perception.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Brown adipose tissues (BAT) regulate homeostatic energy balances in response to physiological changes such as nutrition intake, calorie restriction, exercise, and environmental temperature by consuming energy to generate heat, and thus serve as an important organ for obesity and metabolic diseases. We performed an integrated transcriptomic and metabolomic characterization of developing mouse BAT from embryo to adult to obtain a time-resolved picture of BAT development. We demonstrated that there are two distinct developmental changes that are BAT-specific. We also examined transcription factor binding sites and discovered key transcription factors in the developmental time course. A comparison of our data with other organ development transcriptome and metabolome data revealed BAT-specific transcriptome and metabolome patterns. Our findings provide an overview of mouse BAT development as well as implications for developmental and functional BAT controls.

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.