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. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared with other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants. In addition, we nominate 17 variants (including 11 previously unreported) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies in primary and immortalized B cells. Thus, we uncover important insights into the mechanistic relationships among genotype, epigenetics, and gene expression in SLE and AI disease phenotypes.

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. Methods: We designed a massively parallel reporter assay (MPRA) containing ~32K variants and transfected it into an Epstein-Barr virus transformed B cell line generated from an SLE case. Results: Our study expands our understanding of hQTLs, illustrating that epigenetic QTLs are more likely to contribute to functional mechanisms than eQTLs and other variant types, and a large proportion of hQTLs overlap transcription start sites (TSS) of noncoding RNAs. In addition, we nominate 17 variants (including 11 novel) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies primary and immortalized B cells. Conclusion: We uncover important insights into the mechanistic relationships between genotype, epigenetics, gene expression, and SLE and AI disease phenotypes.

Systemic lupus erythematosus variants modulate the function of an enhancer upstream of TNFAIP3.

TNFAIP3/A20 is a prominent autoimmune disease risk locus that is correlated with hypomorphic TNFAIP3 expression and exhibits complex chromatin architecture with over 30 predicted enhancers. This study aimed to functionally characterize an enhancer ∼55 kb upstream of the TNFAIP3 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. Co-immunoprecipitation, ChIP-qPCR, and 3C-qPCR were performed on patient-derived EBV B cells homozygous for the non-risk or SLE risk TNFAIP3 haplotype to assess haplotype-specific effects on transcription factor binding and chromatin regulation at the TNFAIP3 locus. This study found that the TNFAIP3 locus has a complex chromatin regulatory network that spans ∼1M bp from the promoter region of IL20RA to the 3' untranslated region of TNFAIP3. Functional dissection of the enhancer demonstrated co-dependency of the RelA/p65 and CEBPB binding motifs that, together, increase IL20RA and IFNGR1 expression and decreased TNFAIP3 expression in the context of the TNFAIP3 SLE risk haplotype through dynamic long-range interactions up- and downstream. Examination of SNPs in linkage disequilibrium (D' = 1.0) with rs10499197 identified rs9494868 as a functional SNP with risk allele-specific increase in nuclear factor binding and enhancer activation in vitro. In summary, this study demonstrates that SNPs carried on the ∼109 kb SLE risk haplotype facilitate hypermorphic IL20RA and IFNGR1 expression, while suppressing TNFAIP3 expression, adding to the mechanistic potency of this critically important locus in autoimmune disease pathology.

Mutated KLF4(K409Q) in meningioma binds STRs and activates FGF3 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 (KLF4K409Q) in human meningiomas. However, the molecular mechanism of this tumor-specific KLF4 mutation is unknown. Using genome-wide high-throughput and focused quantitative transcriptional approaches in human cell lines, primary meningeal cells, and meningioma tumor tissue, we found that a change in the evolutionarily conserved DNA-binding domain of KLF4 alters its DNA recognition preference, resulting in a shift in downstream transcriptional activity. In the KLF4K409Q-specific targets, the normally silent fibroblast growth factor 3 (FGF3) is activated. We demonstrated a neomorphic function of KLF4K409Q in stimulating FGF3 transcription through binding to its promoter and in using short tandem repeats (STRs) located within the locus as enhancers.

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. Chromatin conformation capture with quantitative polymerase chain reaction (3C-qPCR), chromatin immunoprecipitation (ChIP)-qPCR, and small interfering RNA (siRNA) knockdown assays were performed on patient-derived Epstein-Barr virus-transformed B cells homozygous for the UBE2L3/YDJC nonrisk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. RESULTS: Of the 7 prioritized variants, 5 demonstrated allele-specific increases in nuclear protein binding affinity and regulatory activity. High-throughput sequencing of chromosome conformation capture coupled with ChIP (HiChIP) and 3C-qPCR uncovered a long-range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and the downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3/YDJC risk haplotype, and correlated with the loss of CCCTC-binding factor (CTCF) and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long-range interaction between the 2 promoters and reduced UBE2L3 expression. CONCLUSION: The UBE2L3/YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1-mediated interaction between the UBE2L3 and YDJC promoters.

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. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFß and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/Treg differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets.

Role of Systemic Lupus Erythematosus Risk Variants With Opposing Functional Effects as a Driver of Hypomorphic Expression of TNIP1 and Other Genes Within a Three-Dimensional Chromatin Network.

OBJECTIVE: Genetic variants in the region of tumor necrosis factor-induced protein 3-interacting protein 1 (TNIP1) are associated with autoimmune disease and reduced TNIP1 gene expression. The aim of this study was to define the functional genetic mechanisms driving TNIP1 hypomorphic expression imparted by the systemic lupus erythematosus-associated TNIP1 H1 risk haplotype. METHODS: Dual luciferase expression and electrophoretic mobility shift assays were used to evaluate the allelic effects of 11 risk variants on enhancer function and nuclear protein binding in immune cell line models (Epstein-Barr virus [EBV]-transformed human B cells, Jurkat cells, and THP-1 cells), left in a resting state or stimulated with phorbol 12-myristate 13-acetate/ionomycin. HiChIP was used to define the regulatory 3-dimensional (3-D) chromatin network of the TNIP1 haplotype by detecting in situ long-range DNA contacts associated with H3K27ac-marked chromatin in EBV B cells. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression of genes within the 3-D chromatin network. RESULTS: Bioinformatics analyses of 50 single-nucleotide polymorphisms on the TNIP1 H1 risk haplotype identified 11 non-protein-coding variants with a high likelihood of influencing TNIP1 gene expression. Eight variants in EBV B cells, 5 in THP-1 cells, and 2 in Jurkat cells exhibited various allelic effects on enhancer activation, resulting in a cumulative suppressive effect on TNIP1 expression (net effect of risk variants -7.14 fold, -6.80 fold, and -2.44 fold, respectively; n > 3). Specifically, in EBV B cells, only 2 variants (rs10057690 and rs13180950) exhibited allele-specific loss of both enhancer activity and nuclear protein binding (each P < 0.01 relative to nonrisk alleles). In contrast, the rs10036748 risk allele reduced binding affinities of the transcriptional repressors basic helix-loop-helix family member 40/differentially expressed in chondrocytes 1 (bHLHe40/DEC1) (P < 0.05 relative to nonrisk alleles) and CREB-1 (P not significant) in EBV B cells, resulting in a gain of enhancer activity (P < 0.05). HiChIP and qRT-PCR analyses revealed that overall transcriptional repression of the TNIP1 haplotype extended to the neighboring genes DCTN4 and GMA2, both of which also showed decreased expression in the presence of the TNIP1 risk haplotype (P < 0.001 and P < 0.01, respectively, relative to the nonrisk haplotype); notably, it was found that these genes share a 3-D chromatin network. CONCLUSION: Hypomorphic TNIP1 expression results from the combined concordant and opposing effects of multiple risk variants carried on the TNIP1 risk haplotype, with the strongest regulatory effect in B lymphoid lineage cells. Furthermore, the TNIP1 risk haplotype effect extends to neighboring genes within a shared chromatin network.

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-1MET 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. SHetA2 and PRIMA-1MET were tested in ovarian cancer cell lines and fallopian tube secretory epithelial cells using isobolograms, fluorescent cytometry, Western blots and ELISAs. Drugs were administered to mice after peritoneal injection of MESOV mutant p53 ovarian cancer cells and prior to tumor establishment, which was evaluated by logistic regression. Fifty-eight percent of TP53 mutations were missense and there were no mortalin mutations in TCGA high-grade serous ovarian cancers. Mortalin levels were sequentially increased in serous benign, borderline and carcinoma tumors. SHetA2 caused p53 nuclear and mitochondrial accumulation in cancer, but not in healthy, cells. Endogenous or exogenous mutant p53 increased SHetA2 resistance. PRIMA-1MET decreased this resistance and interacted synergistically with SHetA2 in mutant and wild type p53-expressing cell lines in association with elevated reactive oxygen species/ATP ratios. Tumor-free rates in animals were 0% (controls), 25% (PRIMA1MET ), 42% (SHetA2) and 67% (combination). SHetA2 (p = 0.004) and PRIMA1MET (p = 0.048) functioned additively in preventing tumor development with no observed toxicity. These results justify the development of SHetA2 and PRIMA-1MET alone and in combination for ovarian cancer maintenance therapy.

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. We show that the epigenome regulates HLA class II genes differently in individuals who carry HLA-DR3 or HLA-DR15 haplotypes, resulting in differential 3D chromatin conformation and gene expression. Finally, we identify significant expression QTL (eQTL) x hQTL interactions that reveal substructure within eQTL gene expression, suggesting potential implications for functional genomic studies that leverage eQTL data for subject selection and stratification.

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 ß transcripts of single CD4+CD45RA- T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and ß sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and ß sequences enables further work toward identification of target antigens and development of novel therapies.

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. Cleft palate in these mice was associated with delay/failure of palatal shelf elevation caused by tongue malposition and micrognathia. Osr2-Cre;Erk2(fl/fl) mice, in which the Erk2 deletion is restricted to the palatal mesenchyme, did not display cleft palate, suggesting that palatal clefting in Wnt1-Cre;Erk2(fl/fl) mice is a secondary defect. Tongues in Wnt1-Cre;Erk2(fl/fl) mice exhibited microglossia, malposition, disruption of the muscle patterning and compromised tendon development. The tongue phenotype was extensively rescued after culture in isolation, indicating that it might also be a secondary defect. The primary malformations in Wnt1-Cre;Erk2(fl/fl) mice, namely micrognathia and mandibular asymmetry, are linked to an early osteogenic differentiation defect. Collectively, our study demonstrates that mutation of Erk2 in neural crest derivatives phenocopies the human Pierre Robin sequence and highlights the interconnection of palate, tongue and mandible development. Because the ERK pathway serves as a crucial point of convergence for multiple signaling pathways, our study will facilitate a better understanding of the molecular regulatory mechanisms of craniofacial development.

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

Molecules involved in WNT/ß-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/ß-catenin signaling regulates each step of myogenesis. Here, we show that WNT/ß-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/ß-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. We found that a blockade of WNT/ß-catenin signaling in the proliferating cells decreases proliferation activity, but does not induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C (Cdc25c). During muscle differentiation, the inhibition of WNT/ß-catenin signaling blocks myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. Blocking WNT/ß-catenin signaling in the well-differentiated myofibers results in the failure of maintenance of their structure by disruption of cadherin/ß-catenin/actin complex formation, which plays a crucial role in connecting a myofiber's cytoskeleton to the surrounding extracellular matrix. Thus, our results indicate that WNT/ß-catenin signaling can regulate multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by targeting step-specific molecules.

ALK5-mediated transforming growth factor ß 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. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

TGFß 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 TGFß signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-ß-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-ß-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFß signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

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

Mutations in transforming growth factor beta (TGFß) 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 TGFß signaling activation. However, little is known about metabolic processes downstream of TGFß signaling during palatogenesis. Here, we show that Tgfbr2 mutant palatal mesenchymal cells spontaneously accumulate lipid droplets, resulting from reduced lipolysis activity. Tgfbr2 mutant palatal mesenchymal cells failed to respond to the cell proliferation stimulator sonic hedgehog, derived from the palatal epithelium. Treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor or telmisartan, a modulator of p38 MAPK activation and lipid metabolism, blocked abnormal TGFß-mediated p38 MAPK activation, restoring lipid metabolism and cell proliferation activity both in vitro and in vivo. Our results highlight the influence of alternative TGFß signaling on lipid metabolic activities, as well as how lipid metabolic defects can affect cell proliferation and adversely impact palatogenesis. This discovery has broader implications for the understanding of metabolic defects and potential prevention of congenital birth defects.

Noncanonical transforming growth factor ß (TGFß) 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 ß (TGFß) 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. However, it is still unclear how TGFß signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2(fl/fl);Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFß-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2(fl/fl);Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFß-mediated regulation of FGF and BMP signaling during tongue development.

Smad4-Irf6 genetic interaction and TGFß-mediated IRF6 signaling cascade are crucial for palatal fusion in mice.

Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFß) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFß signaling and IRF6 activity during palate formation. Here, we show that TGFß signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFß signaling mediator Smad4 (Smad4(fl/fl);K14-Cre;Irf6(+/R84C)) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2(fl/fl);K14-Cre mice, although the secondary palate of Irf6(+/R84C) and Smad4(fl/fl);K14-Cre mice form normally. Furthermore, Smad4(fl/fl);K14-Cre;Irf6(+/R84C) mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFß/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2(fl/fl);K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFß-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.

Identification of candidate downstream targets of TGFß 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 (TGFß) signaling can cause craniofacial abnormalities, including cleft palate, in mammals. To elucidate the downstream targets of TGFß 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. Relative to controls, palatal tissues obtained from Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos at embryonic day 14.5 (E14.5) of gestation have a robust gene expression signature reflective of known defects in CNC-derived mesenchymal cell proliferation. Groups of differentially expressed genes (DEGs) were involved in diverse cellular processes and components associated with orofacial clefting, including the extracellular matrix, cholesterol metabolism, ciliogenesis, and multiple signaling pathways. A subset of the DEGs are known or suspected to be associated with an increased risk of orofacial clefting in humans and/or genetically engineered mice. Based on bioinformatics analyses, we highlight the functional relationships among differentially expressed transcriptional regulators of palatogenesis as well as transcriptional factors not previously associated with this process. We suggest that gene expression profiling studies of mice with TGFß signaling defects provide a valuable approach for identifying candidate mechanisms by which this pathway controls cell fate during palatogenesis and its role in the etiology of human craniofacial abnormalities.

Fibroblast growth factor 9 (FGF9)-pituitary homeobox 2 (PITX2) pathway mediates transforming growth factor ß (TGFß) 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 ß (TGFß) signaling plays crucial functions in regulating craniofacial development, and loss of TGFß 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 TGFß signaling during palatal formation. These target genes were selected based on combining results from gene expression profiles of embryonic day 14.5 palates from Tgfbr2(fl/fl);Wnt1-Cre mice and previously identified cleft palate phenotypes in genetically engineered mouse models. We found that fibroblast growth factor 9 (Fgf9) and transcription factor pituitary homeobox 2 (Pitx2) expressions are significantly down-regulated in the palate of Tgfbr2(fl/fl);Wnt1-Cre mice, and Fgf9 and Pitx2 loss of function mutations result in cleft palate in mice. Pitx2 expression is down-regulated by siRNA knockdown of Fgf9, suggesting that Fgf9 is upstream of Pitx2. We detected decreased expression of both cyclins D1 and D3 in the palates of Tgfbr2(fl/fl);Wnt1-Cre mice, consistent with the defect in cell proliferation. Significantly, exogenous FGF9 restores expression of cyclins D1 and D3 in a Pitx2-dependent manner and rescues the cell proliferation defect in the palatal mesenchyme of Tgfbr2(fl/fl);Wnt1-Cre mice. Our study indicates that a TGFß-FGF9-PITX2 signaling cascade regulates cranial neural crest cell proliferation during palate 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). The population received little dental care and was deemed at high risk for childhood caries. Findings of the study indicate that models incorporating both microbial and proteomic data are superior to models of only microbial or salivary data alone. Comparison of results for the combined and independent data suggests that the combination of proteomic and microbial sources is beneficial for the classification accuracy and that combined data lead to improved predictive models for caries-active and caries-free patients. The best predictive model had a 6% test error, >92% sensitivity, and >95% specificity. These findings suggest that further characterization of the oral microflora and the salivary proteome associated with health and caries may provide clinically useful biomarkers to better predict future caries experience.