Omics Profiling of S2P Mutant Fibroblasts as a Mean to Unravel the Pathomechanism and Molecular Signatures of X-Linked MBTPS2 Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is an inherited skeletal dysplasia characterized by low bone density, bone fragility and recurrent fractures. The characterization of its heterogeneous genetic basis has allowed the identification of novel players in bone development. In 2016, we described the first X-linked recessive form of OI caused by hemizygous MBTPS2 missense variants resulting in moderate to severe phenotypes. MBTPS2 encodes site-2 protease (S2P), which activates transcription factors involved in bone (OASIS) and cartilage development (BBF2H7), ER stress response (ATF6) and lipid metabolism (SREBP) via regulated intramembrane proteolysis. In times of ER stress or sterol deficiency, the aforementioned transcription factors are sequentially cleaved by site-1 protease (S1P) and S2P. Their N-terminal fragments shuttle to the nucleus to activate gene transcription. Intriguingly, missense mutations at other positions of MBTPS2 cause the dermatological spectrum condition Ichthyosis Follicularis, Atrichia and Photophobia (IFAP) and Keratosis Follicularis Spinulosa Decalvans (KFSD) without clinical overlap with OI despite the proximity of some of the pathogenic variants. To understand how single amino acid substitutions in S2P can lead to non-overlapping phenotypes, we aimed to compare the molecular features of MBTPS2-OI and MBTPS2-IFAP/KFSD, with the ultimate goal to unravel the pathomechanisms underlying MBTPS2-OI. RNA-sequencing-based transcriptome profiling of primary skin fibroblasts from healthy controls (n = 4), MBTPS2-OI (n = 3), and MBTPS2-IFAP/KFSD (n = 2) patients was performed to identify genes that are differentially expressed in MBTPS2-OI and MBTPS2-IFAP/KFSD individuals compared to controls. We observed that SREBP-dependent genes are more downregulated in OI than in IFAP/KFSD. This is coupled to alterations in the relative abundance of fatty acids in MBTPS2-OI fibroblasts in vitro, while no consistent alterations in the sterol profile were observed. Few OASIS-dependent genes are suppressed in MBTPS2-OI, while BBF2H7- and ATF6-dependent genes are comparable between OI and IFAP/KFSD patients and control fibroblasts. Importantly, we identified genes involved in cartilage physiology that are differentially expressed in MBTPS2-OI but not in MBTPS2-IFAP/KFSD fibroblasts. In conclusion, our data provide clues to how pathogenic MBTPS2 mutations cause skeletal deformities via altered fatty acid metabolism or cartilage development that may affect bone development, mineralization and endochondral ossification.

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Skeletal cell YAP and TAZ redundantly promote bone development by regulation of collagen I expression and organization

10.1101/143982 ◽

2017 ◽

Cited By ~ 1

Author(s):

Christopher D. Kegelman ◽

Devon E. Mason ◽

James H. Dawahare ◽

Genevieve D. Vigil ◽

Scott S. Howard ◽

...

Keyword(s):

Osteogenesis Imperfecta ◽

Target Genes ◽

Bone Development ◽

Bone Matrix ◽

Cell Lineage ◽

Binding Domains ◽

Conditional Deletion ◽

Neonatal Lethality ◽

Allele Dosage

ABSTRACTThe functions of the transcriptional co-activators YAP and TAZ in bone are controversial. Each has been observed to either promote or inhibit osteogenesis in vitro, while their roles in bone development are unknown. Here we report that combinatorial YAP/TAZ deletion from skeletal cells in mice caused osteogenesis imperfecta with severity dependent on targeted cell lineage and allele dosage. Osteocyte-conditional deletion impaired bone accrual and matrix collagen, while allele dosage-dependent deletion from all osteogenic lineage cells caused spontaneous fractures, with neonatal lethality only in dual homozygous knockouts. We identified putative target genes whose mutation in humans causes osteogenesis imperfecta and which contain promoter-proximate binding domains for the YAP/TAZ co-effector, TEAD4. Two candidates, Col1a1 and SerpinH1, exhibited reduced expression upon either YAP/TAZ deletion or YAP/TAZ-TEAD inhibition by verteporfin. Together, these data demonstrate that YAP and TAZ redundantly promote bone matrix development and implicate YAP/TAZ-mediated transcriptional regulation of collagen in osteogenesis imperfecta.

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Altered microRNAs in C3H10T1/2 cells induced by p.E95K mutant IHH signaling

Hereditas ◽

10.1186/s41065-021-00207-8 ◽

2021 ◽

Vol 158 (1) ◽

Author(s):

Wei Zhou ◽

Luan Chen ◽

Hao Wu ◽

Ting Wang ◽

Gang Ma ◽

...

Keyword(s):

Bone Development ◽

Differentially Expressed ◽

Regulatory Role ◽

Luciferase Reporter ◽

Missense Mutations ◽

Mirna Regulation ◽

Luciferase Reporter Gene ◽

Differentially Expressed Mirnas ◽

And Control

Abstract Background Indian Hedgehog (IHH), an important cell signaling protein, plays a key regulatory role in development of cartilage and chondrogenesis. Earlier studies have shown that heterozygous missense mutations in IHH gene may cause brachydactyly type A1 (BDA1), an autosomal dominant inheritance disease characterized by apparent shortness or absence of the middle phalanges of all digits. MicroRNAs (miRNAs) have been found to be significant post-transcriptional regulators of gene expression and significantly influence the process of bone-development. Therefore, it is possible that miRNAs are involved in the mechanism underlying the development of BDA1. However, the relationship between miRNAs and the pathogenesis of BDA1 remains unclear. Methods In this study, we used microarray-based miRNA profiling to investigate the role of miRNAs in BDA1 by characterization of differentially expressed miRNAs in C3H10T1/2 cell line induced by wild type (WT) and p.E95K mutant (MT) IHH signaling. Results Our results identified 6 differentially expressed miRNAs between WT and control (CT) group and 5 differentially expressed miRNAs between MT and CT groups. In particular, miR-135a-1-3p was found to be a significantly differentially expressed miRNA between WT and CT group. Results of dual-luciferase reporter gene experiment successfully discovered Hoxd10 was one of the target gene of miR-135a-1-3p. Additionally, our pathway analysis revealed that the targets of these miRNAs of interest were highly involved with Runx1/2, Notch and collagen-related pathways. Conclusions Taken together, our findings provided important clue for future study of the process of miRNA-regulation in IHH signaling and novel insights into the regulatory role of miRNA in pathogenesis of BDA1.

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Plastin 3 in X-Linked Osteoporosis: Imbalance of Ca2+-Dependent Regulation Is Equivalent to Protein Loss

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.635783 ◽

2021 ◽

Vol 8 ◽

Author(s):

Christopher L. Schwebach ◽

Elena Kudryashova ◽

Dmitri S. Kudryashov

Keyword(s):

Osteogenesis Imperfecta ◽

Actin Cytoskeleton ◽

Bone Development ◽

Genetic Disorder ◽

Cellular Structures ◽

Protein Loss ◽

Pathogenic Variants ◽

Recent Advances ◽

Intracellular Ca

Osteogenesis imperfecta is a genetic disorder disrupting bone development and remodeling. The primary causes of osteogenesis imperfecta are pathogenic variants of collagen and collagen processing genes. However, recently variants of the actin bundling protein plastin 3 have been identified as another source of osteogenesis imperfecta. Plastin 3 is a highly conserved protein involved in several important cellular structures and processes and is controlled by intracellular Ca2+ which potently inhibits its actin-bundling activity. The precise mechanisms by which plastin 3 causes osteogenesis imperfecta remain unclear, but recent advances have contributed to our understanding of bone development and the actin cytoskeleton. Here, we review the link between plastin 3 and osteogenesis imperfecta highlighting in vitro studies and emphasizing the importance of Ca2+ regulation in the localization and functionality of plastin 3.

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120. OXYGEN REGULATED GENE EXPRESSION IN MOUSE CUMULUS CELLS

Reproduction Fertility and Development ◽

10.1071/srb09abs120 ◽

2009 ◽

Vol 21 (9) ◽

pp. 39

Author(s):

K. Tam ◽

K. Banwell ◽

D. Froiland ◽

D. Russell ◽

K. Kind ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Oxygen Concentration ◽

In Vitro Maturation ◽

Target Genes ◽

Cumulus Cells ◽

Differentially Expressed ◽

Oxygen Levels ◽

Regulated Gene Expression

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that mediate the expression of a range of genes in response to low oxygen. Previously we showed that subsequent developmental outcomes were influenced by oxygen levels during in vitro maturation. The aim of the current study was to examine the effects of varying oxygen concentration during in vitro maturation of mouse COCs on expression of HIF target genes in the cumulus cells. I mmature COCs were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 d) and cultured for 17-18 h under 2, 5 or 20% O2. Hyaluronidase-treated and recovered cumulus cells were collected and mRNA extracted for analysis. A microarray approach (Affymetrix 430_2) was used to identify genes in cumulus cells that were differentially expressed under varying oxygen concentrations (2, 5, 10 and 20%). This revealed 218 differentially expressed probes, of which 34 were up-regulated with decreasing oxygen levels. The great majority of these were classified as HIF-regulated genes. Specific analysis from real time RT-PCR of HIF regulated target genes Slc2a1, Ldha, Pgk1, Eno1, Ndrg1, Bnip3 were all significantly up-regulated (by at least 5–fold) when cells were cultured at 2% or 5% oxygen, when compared to 20% oxygen. Hif-1a mRNA decreased when cumulus cells were cultured in 2%, compared to 20% oxygen. This study demonstrates that cumulus cell gene expression is influenced by oxygen concentration, and suggests that these effects are mediated by the HIF transcription factors.

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Tumor-suppressive function and mechanism of HOXB13 in right-sided colon cancer

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-019-0086-1 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 3

Author(s):

Binbin Xie ◽

Bingjun Bai ◽

Yuzi Xu ◽

Yunlong Liu ◽

Yiming Lv ◽

...

Keyword(s):

Colon Cancer ◽

Tissue Sample ◽

Suppressive Effect ◽

Gene Expression Omnibus ◽

Differentially Expressed ◽

Antitumor Effects ◽

Molecular Features ◽

Induced Apoptosis

AbstractRight-sided colon cancer (RCC) and left-sided colon cancer (LCC) differ in their clinical and molecular features. An investigation of differentially expressed genes (DEGs) between RCC and LCC could contribute to targeted therapy for colon cancer, especially RCC, which has a poor prognosis. Here, we identified HOXB13, which was significantly less expressed in RCC than in LCC and associated with prognosis in RCC, by using 5 datasets from the Gene Expression Omnibus (GEO). Tissue sample analysis showed that HOXB13 was differentially expressed between normal and only RCC tumor tissues. HOXB13 inhibited colon cancer cell proliferation and induced apoptosis both in vitro and in vivo. Furthermore, we found that HOXB13 might be regulated by DNMT3B and suppress C-myc expression to exert antitumor effects via β-catenin/TCF4 signals in RCC. In conclusion, the current study is the first to demonstrate that HOXB13 has a tumor-suppressive effect in RCC. High expression levels of HOXB13 are associated with prolonged overall survival in patients with RCC. The DNMT3B-HOXB13-C-myc signaling axis might be a molecular target for the treatment of RCC.

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Zebrafish mafbb Mutants Display Osteoclast Over-Activation and Bone Deformity Resembling Osteolysis in MCTO Patients

Biomolecules ◽

10.3390/biom11030480 ◽

2021 ◽

Vol 11 (3) ◽

pp. 480

Author(s):

Yujie Han ◽

Weihao Shao ◽

Dan Zhong ◽

Cui Ma ◽

Xiaona Wei ◽

...

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

Bone Development ◽

Myeloid Differentiation ◽

Missense Mutations ◽

Wild Type ◽

Bone Deformity ◽

Tarsal Bones

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia with osteolysis at the carpal and tarsal bones. Heterozygous missense mutations in the transcription factor MAFB are found in patients with MCTO. MAFB is reported to negatively regulate osteoclastogenesis in vitro. However, the in vivo function of MAFB and its relation to MCTO remains unknown. In this study, we generated zebrafish MAFB homolog mafbb mutant utilizing CRISPR/Cas9 technology. Mafbb deficient zebrafish demonstrated enhanced osteoclast cell differentiation and abnormal cartilage and bone development resembling MCTO patients. It is known that osteoclasts are hematopoietic cells derived from macrophages. Loss of mafbb caused selective expansion of definitive macrophages and myeloid cells, supporting that mafbb restricts myeloid differentiation in vivo. We also demonstrate that MAFB MCTO mutations failed to rescue the defective osteoclastogenesis in mafbb−/− embryos, but did not affect osteoclast cells in wild type embryos. The mechanism of MCTO mutations is likely haploinsufficiency. Zebrafish mafbb mutant provides a useful model to study the function of MAFB in osteoclastogenesis and the related MCTO disease.

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BATF2 and FOXJ1 are differentially expressed in coronavirus infections.

10.31219/osf.io/psd97 ◽

2020 ◽

Author(s):

Shahan Mamoor

Keyword(s):

Transcription Factors ◽

Viral Infections ◽

Transcriptional Response ◽

Human Cells ◽

Differentially Expressed ◽

In Vivo Models ◽

Microarray Datasets ◽

Coronavirus Infections

Unraveling the host transcriptional response to viral infections is important for understanding host-pathogen interactions. We mined published microarray datasets (1-5) to identify conserved and specific differentially expressed genes in in vitro and in vivo models of coronavirus infections. We found significant transcriptional induction of the transcription factors BATF2 and FOXJ1 in Middle East Respiratory Syndrome (MERS) coronavirus infection in human cells in vitro; BATF2 was also differentially expressed in the lungs of mice infected with the Severe Acute Respiratory Syndrome (SARS) coronavirus 1 (SARS-CoV-1) but not in human cells infected with the human coronavirus HCoV-229E. These data highlight specific host induction of transcription factors by different members of the coronavirus family.

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Evaluation of CRISPR gene-editing tools in zebrafish

BMC Genomics ◽

10.1186/s12864-021-08238-1 ◽

2022 ◽

Vol 23 (1) ◽

Author(s):

José M. Uribe-Salazar ◽

Gulhan Kaya ◽

Aadithya Sekar ◽

KaeChandra Weyenberg ◽

Cole Ingamells ◽

...

Keyword(s):

Differentially Expressed Genes ◽

Gene Editing ◽

Differentially Expressed ◽

Transient Effects ◽

Cytoskeleton Organization ◽

Functional Studies ◽

Network Analyses ◽

Molecular Features

Abstract Background Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. Methods To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. Results We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used ‘mock’ CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the ‘mock’ injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. Conclusion Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

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A mutant form of ERα associated with estrogen insensitivity affects the coupling between ligand binding and coactivator recruitment

Science Signaling ◽

10.1126/scisignal.aaw4653 ◽

2020 ◽

Vol 13 (650) ◽

pp. eaaw4653

Author(s):

Yin Li ◽

Laurel A. Coons ◽

René Houtman ◽

Kathryn E. Carlson ◽

Teresa A. Martin ◽

...

Keyword(s):

Ligand Binding ◽

Binding Affinity ◽

Transcriptional Response ◽

Estrogen Receptor Α ◽

Mutant Form ◽

Missense Mutations ◽

Gene Encoding ◽

Molecular Features ◽

Transcriptional Regulatory

A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα–coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.

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Antifibrotic factor KLF4 is repressed by the miR-10/TFAP2A/TBX5 axis in dermal fibroblasts: insights from twins discordant for systemic sclerosis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-221050 ◽

2021 ◽

pp. annrheumdis-2021-221050

Author(s):

Maya Malaab ◽

Ludivine Renaud ◽

Naoko Takamura ◽

Kip D Zimmerman ◽

Willian A da Silveira ◽

...

Keyword(s):

Transcription Factors ◽

Systemic Sclerosis ◽

Complex Disease ◽

Ex Vivo ◽

Stem Cell Differentiation ◽

Dermal Fibroblasts ◽

Conditional Knockout ◽

In Vivo Models ◽

Molecular Features

ObjectivesSystemic sclerosis (SSc) is a complex disease of unknown aetiology in which inflammation and fibrosis lead to multiple organ damage. There is currently no effective therapy that can halt the progression of fibrosis or reverse it, thus studies that provide novel insights into disease pathogenesis and identify novel potential therapeutic targets are critically needed.MethodsWe used global gene expression and genome-wide DNA methylation analyses of dermal fibroblasts (dFBs) from a unique cohort of twins discordant for SSc to identify molecular features of this pathology. We validated the findings using in vitro, ex vivo and in vivo models.ResultsOur results revealed distinct differentially expressed and methylated genes, including several transcription factors involved in stem cell differentiation and developmental programmes (KLF4, TBX5, TFAP2A and homeobox genes) and the microRNAs miR-10a and miR-10b which target several of these deregulated genes. We show that KLF4 expression is reduced in SSc dFBs and its expression is repressed by TBX5 and TFAP2A. We also show that KLF4 is antifibrotic, and its conditional knockout in fibroblasts promotes a fibrotic phenotype.ConclusionsOur data support a role for epigenetic dysregulation in mediating SSc susceptibility in dFBs, illustrating the intricate interplay between CpG methylation, miRNAs and transcription factors in SSc pathogenesis, and highlighting the potential for future use of epigenetic modifiers as therapies.

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