Massively parallel reporter assay confirms regulatory potential of hQTLs and reveals important variants in lupus and other autoimmune diseases.

We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele-dependent manner.

Massively Parallel Reporter Assay Confirms Regulatory Potential of hQTLs and Reveals Important Variants in Lupus and Other Autoimmune Diseases.

Objective: To systematically characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate gene expression in an allele dependent manner.

Systemic lupus erythematosus variants modulate the function of an enhancer upstream of .

/A20 is a prominent autoimmune disease risk locus that is correlated with hypomorphic expression and exhibits complex chromatin architecture with over 30 predicted enhancers. This study aimed to functionally characterize an enhancer ∼55 kb upstream of the promoter marked by the systemic lupus erythematosus (SLE) risk haplotype index SNP, rs10499197. Allele effects of rs10499197, rs58905141, and rs9494868 were tested by EMSA and/or luciferase reporter assays in immune cell types.

Mutated KLF4(K409Q) in meningioma binds STRs and activates gene expression.

Krüppel-like factor 4 (KLF4) is a transcription factor that has been proven necessary for both induction and maintenance of pluripotency and self-renewal. Whole-genome sequencing defined a unique mutation in KLF4 (KLF4) in human meningiomas. However, the molecular mechanism of this tumor-specific KLF4 mutation is unknown.

Variants on the UBE2L3/YDJC Autoimmune Disease Risk Haplotype Increase UBE2L3 Expression by Modulating CCCTC-Binding Factor and YY1 Binding.

Objective: Genetic variants spanning UBE2L3 are associated with increased expression of the UBE2L3-encoded E2 ubiquitin-conjugating enzyme H7 (UbcH7), which facilitates activation of proinflammatory NF-κB signaling and susceptibility to autoimmune diseases. We undertook this study to delineate how genetic variants carried on the UBE2L3/YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression.

Methods: We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele-specific effects of risk variants positioned in chromatin accessible regions of immune cells.

Single Cell Transcriptomics Implicate Novel Monocyte and T Cell Immune Dysregulation in Sarcoidosis.

Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls.

Novel ovarian cancer maintenance therapy targeted at mortalin and mutant p53.

Current ovarian cancer maintenance therapy is limited by toxicity and no proven impact on overall survival. To study a maintenance strategy targeted at missense mutant p53, we hypothesized that the release of mutant p53 from mortalin inhibition by the SHetA2 drug combined with reactivation of mutant p53 with the PRIMA-1 drug inhibits growth and tumor establishment synergistically in a mutant-p53 dependent manner. The Cancer Genome Atlas (TCGA) data and serous ovarian tumors were evaluated for TP53 and HSPA9/mortalin status.

Enhancer histone-QTLs are enriched on autoimmune risk haplotypes and influence gene expression within chromatin networks.

Genetic variants can confer risk to complex genetic diseases by modulating gene expression through changes to the epigenome. To assess the degree to which genetic variants influence epigenome activity, we integrate epigenetic and genotypic data from lupus patient lymphoblastoid cell lines to identify variants that induce allelic imbalance in the magnitude of histone post-translational modifications, referred to herein as histone quantitative trait loci (hQTLs). We demonstrate that enhancer hQTLs are enriched on autoimmune disease risk haplotypes and disproportionately influence gene expression variability compared with non-hQTL variants in strong linkage disequilibrium.

Single-cell analysis of glandular T cell receptors in Sjögren's syndrome.

CD4 T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4CD45RA T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype.

Disruption of the ERK/MAPK pathway in neural crest cells as a potential cause of Pierre Robin sequence.

Disrupted ERK1/2 signaling is associated with several developmental syndromes in humans. To understand the function of ERK2 (MAPK1) in the postmigratory neural crest populating the craniofacial region, we studied two mouse models: Wnt1-Cre;Erk2(fl/fl) and Osr2-Cre;Erk2(fl/fl). Wnt1-Cre;Erk2(fl/fl) mice exhibited cleft palate, malformed tongue, micrognathia and mandibular asymmetry.

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets.

Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle development and homeostasis, we used in vitro culture systems for both primary mouse myoblasts and C2C12 cells, which can differentiate into myofibers.

ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation.

TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate.

Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFβ signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice.

Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice.

Mutations in transforming growth factor beta (TGFβ) receptor type II (TGFBR2) cause Loeys-Dietz syndrome, characterized by craniofacial and cardiovascular abnormalities. Mice with a deletion of Tgfbr2 in cranial neural crest cells (Tgfbr2(fl/fl);Wnt1-Cre mice) develop cleft palate as the result of abnormal TGFβ signaling activation. However, little is known about metabolic processes downstream of TGFβ signaling during palatogenesis.

Noncanonical transforming growth factor β (TGFβ) signaling in cranial neural crest cells causes tongue muscle developmental defects.

Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor β (TGFβ) type II receptor in CNC cells in mice (Tgfbr2(fl/fl);Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development.

Identification of candidate downstream targets of TGFβ signaling during palate development by genome-wide transcript profiling.

Nonsyndromic orofacial clefts are common birth defects whose etiology is influenced by complex genetic and environmental factors and gene-environment interactions. Although these risk factors are not yet fully elucidated, it is known that alterations in transforming growth factor-beta (TGFβ) signaling can cause craniofacial abnormalities, including cleft palate, in mammals. To elucidate the downstream targets of TGFβ signaling in palatogenesis, we analyzed the gene expression profiles of Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos with cleft palate and other craniofacial deformities resulting from the targeted inactivation of the Tgfbr2 gene in their cranial neural crest (CNC) cells.

Fibroblast growth factor 9 (FGF9)-pituitary homeobox 2 (PITX2) pathway mediates transforming growth factor β (TGFβ) signaling to regulate cell proliferation in palatal mesenchyme during mouse palatogenesis.

Cleft palate represents one of the most common congenital birth defects. Transforming growth factor β (TGFβ) signaling plays crucial functions in regulating craniofacial development, and loss of TGFβ receptor type II in cranial neural crest cells leads to craniofacial malformations, including cleft palate in mice (Tgfbr2(fl/fl);Wnt1-Cre mice). Here we have identified candidate target genes of TGFβ signaling during palatal formation.

Identification of microbial and proteomic biomarkers in early childhood caries.

The purpose of this study was to provide a univariate and multivariate analysis of genomic microbial data and salivary mass-spectrometry proteomic profiles for dental caries outcomes. In order to determine potential useful biomarkers for dental caries, a multivariate classification analysis was employed to build predictive models capable of classifying microbial and salivary sample profiles with generalization performance. We used high-throughput methodologies including multiplexed microbial arrays and SELDI-TOF-MS profiling to characterize the oral flora and salivary proteome in 204 children aged 1-8 years (n = 118 caries-free, n = 86 caries-active).

Automatic selection of preprocessing methods for improving predictions on mass spectrometry protein profiles.

Mass spectrometry proteomic profiling has potential to be a useful clinical screening tool. One obstacle is providing a standardized method for preprocessing the noisy raw data. We have developed a system for automatically determining a set of preprocessing methods among several candidates.

Efficient peak-labeling algorithms for whole-sample mass spectrometry proteomics.

Whole-sample mass spectrometry (MS) proteomics allows for a parallel measurement of hundreds of proteins present in a variety of biospecimens. Unfortunately, the association between MS signals and these proteins is not straightforward. The need to interpret mass spectra demands the development of methods for accurate labeling of ion species in such profiles.

Peptide identification in whole-sample mass spectrometry proteomics.

Peptide identification for whole-sample mass spectrometry (MS) proteomics is in its infancy. While sophisticated tandem MS/MS instrumentation exists for accurate peptide identification after sample separation, there are few options for those who produce data from intact protein samples. We present a novel algorithm which uses available information from the literature and online protein databases to provide reliable labeling of features in whole-sample MS proteomic data.

Inter-session reproducibility measures for high-throughput data sources.

High-throughput biological assays such as micro-arrays and mass spectrometry (MS) have risen as potential clinical tools for disease detection. Multiple potential biomarkers can be rapidly and cheaply evaluated for a large number of patients. Typical research and evaluation studies in these fields have focused primarily on data that were generated from samples in a single data-generation session.

Intersession reproducibility of mass spectrometry profiles and its effect on accuracy of multivariate classification models.

Motivation: The 'reproducibility' of mass spectrometry proteomic profiling has become an intensely controversial topic. The mere mention of concern over the 'reproducibility' of data generated from any particular platform can lead to the anxiety over the generalizability of its results and its role in the future of discovery proteomics. In this study, we examine the reproducibility of proteomic profiles generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) across multiple data-generation sessions.