Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification.

The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). Here, we used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities. Furthermore, clustering the top-scoring classifier sequence features identified novel cardiac and cell type-specific regulatory motifs. For example, we found that the Myb motif learned by the classifier is crucial for CC activity, and the Myb TF acts in concert with two forkhead domain TFs and Polo kinase to regulate cardiac progenitor cell divisions. In addition, differential motif enrichment and cis-trans genetic studies revealed that the Notch signaling pathway TF Suppressor of Hairless [Su(H)] discriminates PC from CC enhancer activities. Collectively, these studies elucidate molecular pathways used in the regulatory decisions for proliferation and differentiation of cardiac progenitor cells, implicate Su(H) in regulating cell fate decisions of these progenitors, and document the utility of enhancer modeling in uncovering developmental regulatory subnetworks.

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos.

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, previously uncharacterized mesodermal transcription factors. Naive Bayes classifiers using transcription factor binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded in them.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.

LOESS correction for length variation in gene set-based genomic sequence analysis.

MOTIVATION: Sequence analysis algorithms are often applied to sets of DNA, RNA or protein sequences to identify common or distinguishing features. Controlling for sequence length variation is critical to properly score sequence features and identify true biological signals rather than length-dependent artifacts. RESULTS: Several cis-regulatory module discovery algorithms exhibit a substantial dependence between DNA sequence score and sequence length. Our newly developed LOESS method is flexible in capturing diverse score-length relationships and is more effective in correcting DNA sequence scores for length-dependent artifacts, compared with four other approaches. Application of this method to genes co-expressed during Drosophila melanogaster embryonic mesoderm development or neural development scored by the Lever motif analysis algorithm resulted in successful recovery of their biologically validated cis-regulatory codes. The LOESS length-correction method is broadly applicable, and may be useful not only for more accurate inference of cis-regulatory codes, but also for detection of other types of patterns in biological sequences. AVAILABILITY: Source code and compiled code are available from http://thebrain.bwh.harvard.edu/LM_LOESS/

Reducing false alarm rates for critical arrhythmias using the arterial blood pressure waveform.

BACKGROUND: Over the past two decades, high false alarm (FA) rates have remained an important yet unresolved concern in the Intensive Care Unit (ICU). High FA rates lead to desensitization of the attending staff to such warnings, with associated slowing in response times and detrimental decreases in the quality of care for the patient. False arrhythmia alarms are commonly due to single channel ECG artifacts and low voltage signals, and therefore it is likely that the FA rates may be reduced if information from other independent signals is used to form a more robust hypothesis of the alarm's etiology. METHODS: A large multi-parameter ICU database (PhysioNet's MIMIC II database) was used to investigate the frequency of five categories of false critical ("red" or "life-threatening") ECG arrhythmia alarms produced by a commercial ICU monitoring system, namely: asystole, extreme bradycardia, extreme tachycardia, ventricular tachycardia and ventricular fibrillation/tachycardia. Non-critical ("yellow") arrhythmia alarms were not considered in this study. Multiple expert reviews of 5386 critical ECG arrhythmia alarms from a total of 447 adult patient records in the MIMIC II database were made using the associated 41,301 h of simultaneous ECG and arterial blood pressure (ABP) waveforms. An algorithm to suppress false critical ECG arrhythmia alarms using morphological and timing information derived from the ABP signal was then tested. RESULTS: An average of 42.7% of the critical ECG arrhythmia alarms were found to be false, with each of the five alarm categories having FA rates between 23.1% and 90.7%. The FA suppression algorithm was able to suppress 59.7% of the false alarms, with FA reduction rates as high as 93.5% for asystole and 81.0% for extreme bradycardia. FA reduction rates were lowest for extreme tachycardia (63.7%) and ventricular-related alarms (58.2% for ventricular fibrillation/tachycardia and 33.0% for ventricular tachycardia). True alarm (TA) reduction rates were all 0%, except for ventricular tachycardia alarms (9.4%). CONCLUSIONS: The FA suppression algorithm reduced the incidence of false critical ECG arrhythmia alarms from 42.7% to 17.2%, where simultaneous ECG and ABP data were available. The present algorithm demonstrated the potential of data fusion to reduce false ECG arrhythmia alarms in a clinical setting, but the non-zero TA reduction rate for ventricular tachycardia indicates the need for further refinement of the suppression strategy. To avoid suppressing any true alarms, the algorithm could be implemented for all alarms except ventricular tachycardia. Under these conditions the FA rate would be reduced from 42.7% to 22.7%. This implementation of the algorithm should be considered for prospective clinical evaluation. The public availability of a real-world ICU database of multi-parameter physiologic waveforms, together with their associated annotated alarms is a new and valuable research resource for algorithm developers.

Context-specific adaptation of saccade gain is enhanced with rest intervals between changes in context state.

Dual-state adaptation of motor responses has been known for some time. A more recent development is a form of dual-state adaptation known as "context-specific adaptation," which was explored through the use of saccade gain adaptation. In this model, two different adapted saccade gains are associated with two different states of a context cue, and the gain switches between the two adapted states when the context cue changes state. Such adaptation is imposed by alternating context/adaptation states over the course of an adaptation session. Here, vertical eye position as a context cue for adaptation of horizontal saccade gain is used: gain increase is induced with the eyes up 10 degrees, and gain decrease with the eyes down 10 degrees. This context cue is not very effective: there is interference between context/adaptation conditions such that gain-decrease adaptation with eyes down transfers to the eyes-up (gain-increase) context. It was hypothesized that the juxtaposition in time of the alternating adaptation states exacerbated this interference. In order to test this, one-minute rest breaks were inserted between each change in context/adaptation state. The resulting context-specific adaptation improved dramatically: gain-increase and gain-decrease adaptations were more rapid and more complete. This resembles consolidation of motor learning, which, however, occurs over much longer time spans (hours rather than minutes). Thus, the results may reflect the operation of a novel "short-term" motor consolidation process.

Acquisition of context-specific adaptation is enhanced with rest intervals between changes in context state, suggesting a new form of motor consolidation.

We previously showed that the saccadic system could be adapted in a context-specific manner: two different adapted gains could be associated with two different context cues, with the gain state switched when the context state was switched. This was accomplished by alternating context/adaptation states several times over the course of an adaptation session, and assessing saccade gain in each context state before and after adaptation. One context cue we studied was vertical eye position; an adaptive gain increase was induced with the eyes up 10 degrees, and an adaptive gain decrease with the eyes down 10 degrees. This context cue was only partially effective: there was considerable undesired transfer of adaptation from the eyes-down condition (gain-decrease) to the eyes-up condition (gain-increase), with the result that there was little or no gain-increase adaptation. One explanation for this is that the two context/adaptation states, presented one after the other, interfered with each other. In the present study, we tested this hypothesis by interposing one-minute rest intervals between each alternation in context/adaptation state. The resulting context-specific adaptation is greatly improved (relative to the case when there are no rest intervals): both gain-increase and gain-decrease adaptations are stronger and occur more rapidly. This effect resembles that found in studies on the consolidation of motor learning, although such consolidation is believed to occur over much longer time spans (hours rather than minutes).

5'RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy.

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5' start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5' start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5'RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X-encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5'RNA-Seq has the potential to identify genome-wide changes in 5' start-site usage that are associated with pathogenic phenotypes.

iSyTE: integrated Systems Tool for Eye gene discovery.

PURPOSE: To facilitate the identification of genes associated with cataract and other ocular defects, the authors developed and validated a computational tool termed iSyTE (integrated Systems Tool for Eye gene discovery; http://bioinformatics.udel.edu/Research/iSyTE). iSyTE uses a mouse embryonic lens gene expression data set as a bioinformatics filter to select candidate genes from human or mouse genomic regions implicated in disease and to prioritize them for further mutational and functional analyses. METHODS: Microarray gene expression profiles were obtained for microdissected embryonic mouse lens at three key developmental time points in the transition from the embryonic day (E)10.5 stage of lens placode invagination to E12.5 lens primary fiber cell differentiation. Differentially regulated genes were identified by in silico comparison of lens gene expression profiles with those of whole embryo body (WB) lacking ocular tissue. RESULTS: Gene set analysis demonstrated that this strategy effectively removes highly expressed but nonspecific housekeeping genes from lens tissue expression profiles, allowing identification of less highly expressed lens disease-associated genes. Among 24 previously mapped human genomic intervals containing genes associated with isolated congenital cataract, the mutant gene is ranked within the top two iSyTE-selected candidates in approximately 88% of cases. Finally, in situ hybridization confirmed lens expression of several novel iSyTE-identified genes. CONCLUSIONS: iSyTE is a publicly available Web resource that can be used to prioritize candidate genes within mapped genomic intervals associated with congenital cataract for further investigation. Extension of this approach to other ocular tissue components will facilitate eye disease gene discovery.

An evolutionarily conserved enhancer regulates Bmp4 expression in developing incisor and limb bud.

To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.

Mutations in the RNA granule component TDRD7 cause cataract and glaucoma.

The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis.