Complex Regulation of Protocadherin Epigenetics on Aging-Related Brain Health.

Life expectancy continues to increase in the high-income world due to advances in medical care; however, quality of life declines with increasing age due to normal aging processes. Current research suggests that various aspects of aging are genetically modulated and thus may be slowed via genetic modification. Here, we show evidence for epigenetic modulation of the aging process in the brain from over 1800 individuals as part of the Framingham Heart Study. We investigated the methylation of genes in the protocadherin (PCDH) clusters, including the alpha (PCHDA), beta (PCDHB), and gamma (PCDHG) clusters. Reduced PCDHG, elevated PCDHA, and elevated PCDHB methylation levels were associated with substantial reductions in the rate of decline of regional white matter volume as well as certain cognitive skills, independent of overall accelerated or retarded aging as estimated by a DNA clock. These results are likely due to the different effects of the expression of genes in the alpha, beta, and gamma PCHD clusters and suggest that experience-based aging processes related to a decline in regional brain volume and select cognitive skills may be slowed via targeted epigenetic modifications.

Mitotic Block and Epigenetic Repression Underlie Neurodevelopmental Defects and Neurobehavioral Deficits in Congenital Heart Disease.

Poor neurodevelopment is often observed with congenital heart disease (CHD), especially with mutations in chromatin modifiers. Here analysis of mice with hypoplastic left heart syndrome (HLHS) arising from mutations in Sin3A associated chromatin modifier Sap130 , and adhesion protein Pcdha9, revealed neurodevelopmental and neurobehavioral deficits reminiscent of those in HLHS patients. Microcephaly was associated with impaired cortical neurogenesis, mitotic block, and increased apoptosis. Transcriptional profiling indicated dysregulated neurogenesis by REST, altered CREB signaling regulating memory and synaptic plasticity, and impaired neurovascular coupling modulating cerebral blood flow. Many neurodevelopmental/neurobehavioral disease pathways were recovered, including autism and cognitive impairment. These same pathways emerged from genome-wide DNA methylation and Sap130 chromatin immunoprecipitation sequencing analyses, suggesting epigenetic perturbation. Mice with Pcdha9 mutation or forebrain-specific Sap130 deletion without CHD showed learning/memory deficits and autism-like behavior. These novel findings provide mechanistic insights indicating the adverse neurodevelopment in HLHS may involve cell autonomous/nonautonomous defects and epigenetic dysregulation and suggest new avenues for therapy.

Spatial transcriptome profiling uncovers metabolic regulation of left-right patterning.

Left-right patterning disturbance can cause severe birth defects, but it remains least understood of the three body axes. We uncovered an unexpected role for metabolic regulation in left-right patterning. Analysis of the first spatial transcriptome profile of left-right patterning revealed global activation of glycolysis, accompanied by right-sided expression of Bmp7 and genes regulating insulin growth factor signaling. Cardiomyocyte differentiation was left-biased, which may underlie the specification of heart looping orientation. This is consistent with known Bmp7 stimulation of glycolysis and glycolysis suppression of cardiomyocyte differentiation. Liver/lung laterality may be specified via similar metabolic regulation of endoderm differentiation. Myo1d , found to be left-sided, was shown to regulate gut looping in mice, zebrafish, and human. Together these findings indicate metabolic regulation of left-right patterning. This could underlie high incidence of heterotaxy-related birth defects in maternal diabetes, and the association of PFKP, allosteric enzyme regulating glycolysis, with heterotaxy. This transcriptome dataset will be invaluable for interrogating birth defects involving laterality disturbance.

Single-cell transcriptomic analysis identifies murine heart molecular features at embryonic and neonatal stages.

Heart development is a continuous process involving significant remodeling during embryogenesis and neonatal stages. To date, several groups have used single-cell sequencing to characterize the heart transcriptomes but failed to capture the progression of heart development at most stages. This has left gaps in understanding the contribution of each cell type across cardiac development. Here, we report the transcriptional profile of the murine heart from early embryogenesis to late neonatal stages. Through further analysis of this dataset, we identify several transcriptional features. We identify gene expression modules enriched at early embryonic and neonatal stages; multiple cell types in the left and right atriums are transcriptionally distinct at neonatal stages; many congenital heart defect-associated genes have cell type-specific expression; stage-unique ligand-receptor interactions are mostly between epicardial cells and other cell types at neonatal stages; and mutants of epicardium-expressed genes Wt1 and Tbx18 have different heart defects. Assessment of this dataset serves as an invaluable source of information for studies of heart development.

Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with NKX2-5 deficiency.

Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been fully studied, which hinders their wide spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart diseases with chamber defects. We show that our systems generate chamber-specific organoids comprising of the major cardiac cell types, and we use single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we developed a machine learning label transfer approach leveraging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebstein's anomaly associated genetic variant in NKX2-5, and we successfully recapitulated the disease's atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.

Uncompensated mitochondrial oxidative stress underlies heart failure in an iPSC-derived model of congenital heart disease.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease with 30% mortality from heart failure (HF) in the first year of life, but the cause of early HF remains unknown. Induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CM) from patients with HLHS showed that early HF is associated with increased apoptosis, mitochondrial respiration defects, and redox stress from abnormal mitochondrial permeability transition pore (mPTP) opening and failed antioxidant response. In contrast, iPSC-CM from patients without early HF showed normal respiration with elevated antioxidant response. Single-cell transcriptomics confirmed that early HF is associated with mitochondrial dysfunction accompanied with endoplasmic reticulum (ER) stress. These findings indicate that uncompensated oxidative stress underlies early HF in HLHS. Importantly, mitochondrial respiration defects, oxidative stress, and apoptosis were rescued by treatment with sildenafil to inhibit mPTP opening or TUDCA to suppress ER stress. Together these findings point to the potential use of patient iPSC-CM for modeling clinical heart failure and the development of therapeutics.

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability.

Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Common deletion variants causing protocadherin-α deficiency contribute to the complex genetics of BAV and left-sided congenital heart disease.

Bicuspid aortic valve (BAV) with ~1%-2% prevalence is the most common congenital heart defect (CHD). It frequently results in valve disease and aorta dilation and is a major cause of adult cardiac surgery. BAV is genetically linked to rare left-heart obstructions (left ventricular outflow tract obstructions [LVOTOs]), including hypoplastic left heart syndrome (HLHS) and coarctation of the aorta (CoA). Mouse and human studies indicate LVOTO is genetically heterogeneous with a complex genetic etiology. Homozygous mutation in the Pcdha protocadherin gene cluster in mice can cause BAV, and also HLHS and other LVOTO phenotypes when accompanied by a second mutation. Here we show two common deletion copy number variants (delCNVs) within the PCDHA gene cluster are associated with LVOTO. Analysis of 1,218 white individuals with LVOTO versus 463 disease-free local control individuals yielded odds ratios (ORs) at 1.47 (95% confidence interval [CI], 1.13-1.92; p = 4.2 × 10-3) for LVOTO, 1.47 (95% CI, 1.10-1.97; p = 0.01) for BAV, 6.13 (95% CI, 2.75-13.7; p = 9.7 × 10-6) for CoA, and 1.49 (95% CI, 1.07-2.08; p = 0.019) for HLHS. Increased OR was observed for all LVOTO phenotypes in homozygous or compound heterozygous PCDHA delCNV genotype comparison versus wild type. Analysis of an independent white cohort (381 affected individuals, 1,352 control individuals) replicated the PCDHA delCNV association with LVOTO. Generalizability of these findings is suggested by similar observations in Black and Chinese individuals with LVOTO. Analysis of Pcdha mutant mice showed reduced PCDHA expression at regions of cell-cell contact in aortic smooth muscle and cushion mesenchyme, suggesting potential mechanisms for BAV pathogenesis and aortopathy. Together, these findings indicate common variants causing PCDHA deficiency play a significant role in the genetic etiology of common and rare LVOTO-CHD.

Single-cell RNA sequencing reveals differential cell cycle activity in key cell populations during nephrogenesis.

The kidney is a complex organ composed of more than 30 terminally differentiated cell types that all are required to perform its numerous homeostatic functions. Defects in kidney development are a significant cause of chronic kidney disease in children, which can lead to kidney failure that can only be treated by transplant or dialysis. A better understanding of molecular mechanisms that drive kidney development is important for designing strategies to enhance renal repair and regeneration. In this study, we profiled gene expression in the developing mouse kidney at embryonic day 14.5 at single-cell resolution. Consistent with previous studies, clusters with distinct transcriptional signatures clearly identify major compartments and cell types of the developing kidney. Cell cycle activity distinguishes between the "primed" and "self-renewing" sub-populations of nephron progenitors, with increased expression of the cell cycle-related genes Birc5, Cdca3, Smc2 and Smc4 in "primed" nephron progenitors. In addition, augmented expression of cell cycle related genes Birc5, Cks2, Ccnb1, Ccnd1 and Tuba1a/b was detected in immature distal tubules, suggesting cell cycle regulation may be required for early events of nephron patterning and tubular fusion between the distal nephron and collecting duct epithelia.

Mitochondrial Respiration Defects in Single-Ventricle Congenital Heart Disease.

Background: Congenital heart disease (CHD) with single-ventricle (SV) physiology is now survivable with a three-stage surgical course ending with Fontan palliation. However, 10-year transplant-free survival remains at 39-50%, with ventricular dysfunction progressing to heart failure (HF) being a common sequela. For SV-CHD patients who develop HF, undergoing the surgical course would not be helpful and could even be detrimental. As HF risk cannot be predicted and metabolic defects have been observed in Ohia SV-CHD mice, we hypothesized that respiratory defects in peripheral blood mononuclear cells (PBMCs) may allow HF risk stratification in SV-CHD. Methods: SV-CHD (n = 20), biventricular CHD (BV-CHD; n = 16), or healthy control subjects (n = 22) were recruited, and PBMC oxygen consumption rate (OCR) was measured using the Seahorse Analyzer. Respiration was similarly measured in Ohia mouse heart tissue. Results: Post-Fontan SV-CHD patients with HF showed higher maximal respiratory capacity (p = 0.004) and respiratory reserve (p < 0.0001), parameters important for cell stress adaptation, while the opposite was found for those without HF (reserve p = 0.037; maximal p = 0.05). This was observed in comparison to BV-CHD or healthy controls. However, respiration did not differ between SV patients pre- and post-Fontan or between pre- or post-Fontan SV-CHD patients and BV-CHD. Reminiscent of these findings, heart tissue from Ohia mice with SV-CHD also showed higher OCR, while those without CHD showed lower OCR. Conclusion: Elevated mitochondrial respiration in PBMCs is correlated with HF in post-Fontan SV-CHD, suggesting that PBMC respiration may have utility for prognosticating HF risk in SV-CHD. Whether elevated respiration may reflect maladaptation to altered hemodynamics in SV-CHD warrants further investigation.

scds: computational annotation of doublets in single-cell RNA sequencing data.

MOTIVATION: Single-cell RNA sequencing (scRNA-seq) technologies enable the study of transcriptional heterogeneity at the resolution of individual cells and have an increasing impact on biomedical research. However, it is known that these methods sometimes wrongly consider two or more cells as single cells, and that a number of so-called doublets is present in the output of such experiments. Treating doublets as single cells in downstream analyses can severely bias a study's conclusions, and therefore computational strategies for the identification of doublets are needed. RESULTS: With scds, we propose two new approaches for in silico doublet identification: Co-expression based doublet scoring (cxds) and binary classification based doublet scoring (bcds). The co-expression based approach, cxds, utilizes binarized (absence/presence) gene expression data and, employing a binomial model for the co-expression of pairs of genes, yields interpretable doublet annotations. bcds, on the other hand, uses a binary classification approach to discriminate artificial doublets from original data. We apply our methods and existing computational doublet identification approaches to four datasets with experimental doublet annotations and find that our methods perform at least as well as the state of the art, at comparably little computational cost. We observe appreciable differences between methods and across datasets and that no approach dominates all others. In summary, scds presents a scalable, competitive approach that allows for doublet annotation of datasets with thousands of cells in a matter of seconds. AVAILABILITY AND IMPLEMENTATION: scds is implemented as a Bioconductor R package (doi: 10.18129/B9.bioc.scds). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Control of cytokinesis by ß-adrenergic receptors indicates an approach for regulating cardiomyocyte endowment.

One million patients with congenital heart disease (CHD) live in the United States. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. Here, analysis of heart tissue from an infant with tetralogy of Fallot with pulmonary stenosis (ToF/PS) labeled with isotope-tagged thymidine demonstrated that cardiomyocyte cytokinesis failure is increased in this common form of CHD. We used single-cell transcriptional profiling to discover that the underlying mechanism of cytokinesis failure is repression of the cytokinesis gene ECT2, downstream of ß-adrenergic receptors (ß-ARs). Inactivation of the ß-AR genes and administration of the ß-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the number of cardiomyocytes (endowment) and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes in vitro. These results suggest that ß-blockers could be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.

Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified 4-(phenylthio)butanoic acid (PTBA), a histone deacetylase inhibitor (HDI), as an enhancer of renal recovery, and showed that PTBA treatment increased RTEC proliferation and reduced renal fibrosis. Here, we investigated the regenerative mechanisms of PTBA in zebrafish models of larval renal injury and adult cardiac injury. With respect to renal injury, we showed that delivery of PTBA using an esterified prodrug (UPHD25) increases the reactivation of the renal progenitor gene Pax2a, enhances dedifferentiation of RTECs, reduces Kidney injury molecule-1 (Kim-1) expression, and lowers the number of infiltrating macrophages. Further, we found that the effects of PTBA on RTEC proliferation depend upon retinoic acid signaling and demonstrate that the therapeutic properties of PTBA are not restricted to the kidney but also increase cardiomyocyte proliferation and decrease fibrosis following cardiac injury in adult zebrafish. These studies provide key mechanistic insights into how PTBA enhances tissue repair in models of acute injury and lay the groundwork for translating this novel HDI into the clinic.This article has an associated First Person interview with the joint first authors of the paper.

The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric kidney development.

The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.

Airway ciliary dysfunction and respiratory symptoms in patients with transposition of the great arteries.

BACKGROUND: Our prior work on congenital heart disease (CHD) with heterotaxy, a birth defect involving randomized left-right patterning, has shown an association of a high prevalence of airway ciliary dysfunction (CD; 18/43 or 42%) with increased respiratory symptoms. Furthermore, heterotaxy patients with ciliary dysfunction were shown to have more postsurgical pulmonary morbidities. These findings are likely a reflection of the common role of motile cilia in both airway clearance and left-right patterning. As CHD comprising transposition of the great arteries (TGA) is commonly thought to involve disturbance of left-right patterning, especially L-TGA with left-right ventricular inversion, we hypothesize CHD patients with transposition of great arteries (TGA) may have high prevalence of airway CD with increased respiratory symptoms. METHODS AND RESULTS: We recruited 75 CHD patients with isolated TGA, 28% L and 72% D-TGA. Patients were assessed using two tests typically used for evaluating airway ciliary dysfunction in patients with primary ciliary dyskinesia (PCD), a recessive sinopulmonary disease caused by respiratory ciliary dysfunction. This entailed the measurement of nasal nitric oxide (nNO), which is typically low with PCD. We also obtained nasal scrapes and conducted videomicroscopy to assess respiratory ciliary motion (CM). We observed low nNO in 29% of the patients, and abnormal CM in 57%, with 22% showing both low nNO and abnormal CM. No difference was observed for the prevalence of either low nNO or abnormal ciliary motion between patients with D vs. L-TGA. Respiratory symptoms were increased with abnormal CM, but not low nNO. Sequencing analysis showed no compound heterozygous or homozygous mutations in 39 genes known to cause PCD, nor in CFTR, gene causing cystic fibrosis. As both are recessive disorders, these results indicate TGA patients with ciliary dysfunction do not have PCD or cystic fibrosis (which can cause low nNO or abnormal ciliary motion). CONCLUSIONS: TGA patients have high prevalence of abnormal CM and low nNO, but ciliary dysfunction was not correlated with TGA type. Differing from PCD, respiratory symptoms were increased with abnormal CM, but not low nNO. Together with the negative findings from exome sequencing analysis, this would suggest TGA patients with ciliary dysfunction do not have PCD but nevertheless may suffer from milder airway clearance deficiency. Further studies are needed to investigate whether such ciliary dysfunction is associated with increased postsurgical complications as previously observed in CHD patients with heterotaxy.

The complex genetics of hypoplastic left heart syndrome.

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

Novel modeling of combinatorial miRNA targeting identifies SNP with potential role in bone density.

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies.

High throughput determination of TGFß1/SMAD3 targets in A549 lung epithelial cells.

BACKGROUND: Transforming growth factor beta 1 (TGFß1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFß1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFß1/SMAD3 signaling in lung epithelial cells. METHODOLOGY: We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFß1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFß1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. RESULTS AND CONCLUSIONS: Known TGFß1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFß1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFß1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFß1 on FOXA2.

Finding subtypes of transcription factor motif pairs with distinct regulatory roles.

DNA sequences bound by a transcription factor (TF) are presumed to contain sequence elements that reflect its DNA binding preferences and its downstream-regulatory effects. Experimentally identified TF binding sites (TFBSs) are usually similar enough to be summarized by a 'consensus' motif, representative of the TF DNA binding specificity. Studies have shown that groups of nucleotide TFBS variants (subtypes) can contribute to distinct modes of downstream regulation by the TF via differential recruitment of cofactors. A TF(A) may bind to TFBS subtypes a(1) or a(2) depending on whether it associates with cofactors TF(B) or TF(C), respectively. While some approaches can discover motif pairs (dyads), none address the problem of identifying 'variants' of dyads. TFs are key components of multiple regulatory pathways targeting different sets of genes perhaps with different binding preferences. Identifying the discriminating TF-DNA associations that lead to the differential downstream regulation is thus essential. We present DiSCo (Discovery of Subtypes and Cofactors), a novel approach for identifying variants of dyad motifs (and their respective target sequence sets) that are instrumental for differential downstream regulation. Using both simulated and experimental datasets, we demonstrate how current motif discovery can be successfully leveraged to address this question.

In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach.

BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency. RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation. CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.