Analysis Of Transcription Factor Binding Sites Research Articles

Benzo[a]pyrene exposure causes exonal switch resulting in reduced surface CD5 expression in an AHR-dependent manner

The function of CD5 protein in T cells is well documented, but regulation of its surface-level expression has yet to be fully understood. However, variation in its surface expression is associated with various immunopathological conditions and haematological malignancies. Briefly, expression of an alternate exon E1B of a human endogenous retroviruses (HERV) origin directly downregulates the conventional transcript variant (E1A), as its expression leads to the retention of the resultant protein at the intracellular level (cCD5). A separate promoter governs the expression of E1B and may be influenced by different transcription factors. Hence, we performed in silico transcription factor binding site (TFBS) analysis of the 3 kb upstream region from TSS of exon E1B and found five putative DREs (Dioxin Response elements) with good similarity scores. Further, we observed the upregulation in E1B expression after the exposure of BaP (a dioxin) and the reduction of E1A expression and their respective protein, i.e. sCD5 and cCD5. The binding of AHR at the predicted DRE sites was confirmed by ChIP qPCR and AHR specific inhibitor and gene silencing studies suggested the involvement of AHR in exonal switch. This study indicates that the polycyclic aromatic hydrocarbon decreases the sCD5 expression by upregulating alternative exon expression, which may adversely affect the overall T cell functions.

Angular limb deformity associated with TSPAN18, NRG3 and NOVA2 in Rambouillet rams

Angular limb deformity (ALD) affects many species of livestock and companion animals. The mechanisms of ALD development are not well understood, but previous research suggests the involvement of genetic risk factors. A case-control genome-wide association study (GWAS) was conducted with 40 ALD-affected and 302 unaffected Rambouillet rams and 40,945 single nucleotide polymorphisms (SNPs). Forelimbs of 6 ALD-affected rams were examined and diagnosed with osteochondrosis. Genome-wide or chromosome-wide significant SNPs were positioned exonic, intronic or within the 3′UTR of genes TSPAN18, NRG3 and NOVA2, respectively. These genes have previously described roles related to angiogenesis and osteoblast, osteoclast and chondrocyte proliferation and differentiation, which suggests the possibility for their involvement in the pathogenesis of osteochondrosis. Functional consequences of SNPs were evaluated through transcription factor binding site analysis, which predicted binding sites for transcription factors of known importance to bone growth, including SOX6, SOX9 and RUNX2. The identification of genetic risk factors for ALD may help to improve animal welfare and production in Rambouillet, a breed known to be at risk for ALD development. This study proposes genes TSPAN18, NRG3 and NOVA2 as targets for further research towards understanding the etiology of ALD in Rambouillet sheep.

The role of FOXO4/NFAT2 signaling pathway in dysfunction of human coronary endothelial cells and inflammatory infiltration of vasculitis in Kawasaki disease.

The Ca+/NFAT (Nuclear factor of activated T cells) signaling pathway activation is implicated in the pathogenesis of Kawasaki disease (KD); however, we lack detailed information regarding the regulatory network involved in the human coronary endothelial cell dysfunction and cardiovascular lesion development. Herein, we aimed to use mouse and endothelial cell models of KD vasculitis in vivo and in vitro to characterize the regulatory network of NFAT pathway in KD. Among the NFAT gene family, NFAT2 showed the strongest transcriptional activity in peripheral blood mononuclear cells (PBMCs) from patients with KD. Then, NFAT2 overexpression and knockdown experiments in Human coronary artery endothelial cells (HCAECs) indicated that NFAT2 overexpression disrupted endothelial cell homeostasis by regulation of adherens junctions, whereas its knockdown protected HCAECs from such dysfunction. Combined analysis using RNA-sequencing and transcription factor (TF) binding site analysis in the NFAT2 promoter region predicted regulation by Forkhead box O4 (FOXO4). Western blotting, chromatin immunoprecipitation, and luciferase assays validated that FOXO4 binds to the promoter and transcriptionally represses NFAT2. Moreover, Foxo4 knockout increased the extent of inflamed vascular tissues in a mouse model of KD vasculitis. Functional experiments showed that inhibition NFAT2 relieved Foxo4 knockout exaggerated vasculitis in vivo. Our findings revealed the FOXO4/NFAT2 axis as a vital pathway in the progression of KD that is associated with endothelial cell homeostasis and cardiovascular inflammation development.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in >90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

PERM1 regulates genes involved in fatty acid metabolism in the heart by interacting with PPARα and PGC-1α

PERM1 (PGC-1/ERR-induced regulator in muscle 1) is a muscle-specific protein induced by PGC-1 and ERRs. Previous studies have shown that PERM1 promotes mitochondrial biogenesis and metabolism in cardiomyocytes in vitro. However, the role of endogenous PERM1 in the heart remains to be investigated with loss-of-function studies in vivo. We report the generation and characterization of systemic Perm1 knockout (KO) mice. The baseline cardiac phenotype of the homozygous Perm1 KO mice appeared normal. However, RNA-sequencing and unbiased pathway analyses showed that homozygous downregulation of PERM1 leads to downregulation of genes involved in fatty acid and carbohydrate metabolism in the heart. Transcription factor binding site analyses suggested that PPARα and PGC-1α are involved in changes in the gene expression profile. Chromatin immunoprecipitation assays showed that PERM1 interacts with the proximal regions of PPAR response elements (PPREs) in endogenous promoters of genes involved in fatty acid oxidation. Co-immunoprecipitation and reporter gene assays showed that PERM1 promoted transcription via the PPRE, partly in a PPARα and PGC-1α dependent manner. These results suggest that endogenous PERM1 is involved in the transcription of genes involved in fatty acid oxidation through physical interaction with PPARα and PGC-1α in the heart in vivo.

Genomic Aberrations Generate Fusion Gene FOXK2::TP63 and Activate NFKB1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24–26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.

Placental DNA methylation changes in gestational diabetes mellitus

ABSTRACT In this study, we investigated the association between altered methylation in the maternal placenta and hyperglycaemia and explored the epigenetic mechanisms underlying gestational diabetes mellitus (GDM). Reduced representation bisulphite sequencing (RRBS) and RNA sequencing (RNA-seq) were performed on placental tissues obtained from women with GDM and healthy controls. Further, pyrosequencing, correlation analyses, and linear regression analyses were performed to valuate relationships between aberrantly methylated-differentially expressed genes and clinical parameters. The EMBOSS and JASPAR databases were used for a computational analysis of CpG islands and transcription factor-binding sites in the TRIM67 promoter region. A CpG island with a length of 264 bp in the placental TRIM67 promoter region in the GDM group exhibited significant hypermethylation at four CpG sites. The hypermethylation of the TRIM67 promoter region in the maternal placenta showed a significant, positive correlation with the 1 h and 2 h oral glucose tolerance test (OGTT) values and a negative correlation with lipoprotein(a). Placental DNA methylation levels in the TRIM67 promoter region were markedly elevated in GDM and were associated with blood glucose and lipid levels during healthy pregnancy.

Targets for Renal Carcinoma Growth Control Identified by Screening FOXD1 Cell Proliferation Pathways.

Simple SummaryFOXD1 regulates the proliferation of clear cell renal cell carcinoma (ccRCC) cells, and ccRCC cells in which FOXD1 has been inactivated do not form tumors efficiently in an animal model. Reproducing growth inhibition in tumor cells by inhibiting FOXD1 pathways presents a possible therapeutic approach for ccRCC and other cancers. We have established an analysis strategy to identify FOXD1-regulated target pathways that may be therapeutically tractable, and compounds that modulate these pathways were selected for testing. Targets in three pathways were identified: FOXM1, PME1, and TMEM167A, which were inhibited by compounds FDI-6, AMZ-30, and silibinin, respectively. The effects of these compounds on the growth of tumor cells from patients cultured in a novel 3D tumor-replica culture environment revealed that FDI-6 and silibinin had strong growth inhibitory effects. This investigation informs new therapeutic targets to control ccRCC tumor growth, and provides a strategy to compare the responsiveness of individual patient tumor replicas to growth-inhibitory compounds.Clinical association studies suggest that FOXD1 is a determinant of patient outcome in clear cell renal cell carcinoma (ccRCC), and laboratory investigations have defined a role for this transcription factor in controlling the growth of tumors through regulation of the G2/M cell cycle transition. We hypothesized that the identification of pathways downstream of FOXD1 may define candidates for pharmacological modulation to suppress the G2/M transition in ccRCC. We developed an analysis pipeline that utilizes RNA sequencing, transcription factor binding site analysis, and phenotype validation to identify candidate effectors downstream from FOXD1. Compounds that modulate candidate pathways were tested for their ability to cause growth delay at G2/M. Three targets were identified: FOXM1, PME1, and TMEM167A, which were targeted by compounds FDI-6, AMZ-30, and silibinin, respectively. A 3D ccRCC tumor replica model was used to investigate the effects of these compounds on the growth of primary cells from five patients. While silibinin reduced 3D growth in a subset of tumor replicas, FDI-6 reduced growth in all. This study identifies tractable pathways to target G2/M transition and inhibit ccRCC growth, demonstrates the applicability of these strategies across patient tumor replicas, and provides a platform for individualized patient testing of compounds that inhibit tumor growth.

Emergence of sex-specific transcriptomes in a sexually dimorphic brain nucleus.

SUMMARYWe present the transcriptomic changes underlying the development of an extreme neuroanatomical sex difference. The robust nucleus of the arcopallium (RA) is a key component of the songbird vocal motor system. In zebra finch, the RA is initially monomorphic and then atrophies in females but grows up to 7-fold larger in males. Mirroring this divergence, we show here that sex-differential gene expression in the RA expands from hundreds of predominantly sex chromosome Z genes in early development to thousands of predominantly autosomal genes by the time sexual dimorphism asymptotes. Male-specific developmental processes include cell and axonal growth, synapse assembly and activity, and energy metabolism; female-specific processes include cell polarity and differentiation, transcriptional repression, and steroid hormone and immune signaling. Transcription factor binding site analyses support female-biased activation of pro-apoptotic regulatory networks. The extensive and sex-specific transcriptomic reorganization of RA provides insights into potential drivers of sexually dimorphic neurodevelopment.

AURKB, in concert with REST, acts as an oxygen-sensitive epigenetic regulator of the hypoxic induction of MDM2

The acute response to hypoxia is mainly driven by hypoxia-inducible factors, but their effects gradually subside with time. Hypoxia-specific histone modifications may be important for the stable maintenance of long-term adaptation to hypoxia. However, little is known about the molecular mechanisms underlying the dynamic alterations of histones under hypoxic conditions. We found that the phosphorylation of histone H3 at Ser-10 (H3S10) was noticeably attenuated after hypoxic challenge, which was mediated by the inhibition of aurora kinase B (AURKB). To understand the role of AURKB in epigenetic regulation, DNA microarray and transcription factor binding site analyses combined with proteomics analysis were performed. Under normoxia, phosphorylated AURKB, in concert with the repressor element-1 silencing transcription factor (REST), phosphorylates H3S10, which allows the AURKB–REST complex to access the MDM2 proto-oncogene. REST then acts as a transcriptional repressor of MDM2 and downregulates its expression. Under hypoxia, AURKB is dephosphorylated and the AURKB–REST complex fails to access MDM2, leading to the upregulation of its expression. In this study, we present a case of hypoxia-specific epigenetic regulation of the oxygen-sensitive AURKB signaling pathway. To better understand the cellular adaptation to hypoxia, it is worthwhile to further investigate the epigenetic regulation of genes under hypoxic conditions.

Distinct promoter regions of the oxytocin receptor gene are hypomethylated in Prader-Willi syndrome and in Prader-Willi syndrome associated psychosis

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder caused by a loss of usually paternally expressed, maternally imprinted genes located on chromosome 15q11-q13. Individuals with PWS display a specific behavioral phenotype and have a higher susceptibility than the general population for certain psychiatric conditions, especially psychosis. An impairment of the oxytocin system has been described in Prader-Willi syndrome, but has not yet been investigated in detail on the epigenetic level. Recent studies have pointed out altered methylation patterns of the oxytocin receptor gene (OXTR) in various psychiatric disorders, including psychosis. In this study, we investigated methylation rates of CpG dinucleotides in the promoter region of the oxytocin receptor gene via bisulfite-sequencing using DNA extracted from peripheral blood samples of 31 individuals with PWS and 14 controls matched for age, sex, and BMI. Individuals with PWS show significantly lower methylation in the intron 1 region of the OXTR than neurotypical controls (p = 0.012). Furthermore, male PWS subjects with psychosis show significantly lower methylation of the OXTR exon 1 region than those without psychosis (p = 0.002). Transcription factor binding site analysis revealed E2F1 as a transcription factor potentially binding to the exon 1 region. E2F1 is physiologically regulated by Necdin, an anti-apoptotic protein whose corresponding gene is located within the PWS locus. This study provides evidence of a disruption of the Oxytocin system on an epigenetic level in PWS in general and in individuals with PWS and psychosis.

Comparative genomic analyses reveal cis-regulatory divergence after polyploidization in cotton

Polyploidization has long been recognized as a driver for the evolutionary formation of superior plant traits coupled with gene expression novelty. However, knowledge of the effect of regulatory variation on expression changes following polyploidization remains limited. In this study, we characterized transcriptional regulatory divergence by comparing tetraploid cotton with its putative diploid ancestors. We identified 144,827, 99,609, and 219,379 Tn5 transposase-hypersensitive sites (THSs) in Gossypium arboreum, G. raimondii, and G. hirsutum, respectively, and found that the conservation of promoter THSs was associated with coordination of orthologous genes expression. This observation was consistent with analysis of transcription-factor binding sites (TFBS) for 262 known motifs: genes with higher TFBS conservation scores (CS) showed less change than those genes with lower TFBS CS in expression levels. TFBS influenced by genomic variation were involved in the novel regulation networks between transcriptional factors and target genes in tetraploid cotton. We describe an example showing that the turnover of TFBS was linked to expression pattern divergence of genes involved in fiber development (fiber-related genes). Our findings reveal the regulatory divergence of the transcriptional network in cotton after polyploidization and characterizes the regulatory relationships of genes contributing to desirable traits.

HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis.

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

Evaluation of candidate point mutation of Kisspeptin 1 gene associated with litter size in Indian Goat breeds and its effect on transcription factor binding sites

Kisspeptin gene (Kiss1) has a significant role in reproductive processes in mammals. However, only little information is available about the association of Kiss1 gene with litter size in Indian goat breeds. Thus, blood samples from 285 randomly selected animals were collected for DNA isolation and SNP profiling. The PCR product of 242 bp size harboring g.2540C>T mutation of Kiss1 gene was digested with the restriction enzyme Sac1. Least squares analysis revealed that Barbari goats showed significantly higher average litter size (2.86±0.08) compared to Beetal, Sirohi and Sojat breeds (P < 0.01). SNP locus g.2540C>T of Kiss1 gene also showed significant effect on litter size (P < 0.01). Goats with Genotype CT (2.66 ± 0.07) and TT (2.67 ± 0.26) had significantly higher (P < 0.01) litter size than CC (1.50 ± 0.05). From the transcription factor binding site analysis, it was predicted that due to g.2540C>T SNP, both native and mutant variant forms coded for putative binding sites for different transcription Factor. Allele T had putative binding sites for the androgen receptor which plays a significant role in the signaling pathway involved in increase in ovulation rate; which consequently can have a tremendous effect on average litter size.

Effect of BsaA I genotyped intronic SNP of leptin gene on production and reproduction traits in Indian dairy cattle

In the present study, candidate single nucleotide polymorphism (SNP) g.92450765 G > A of leptin gene was explored for Bos indicus cattle with an aim to explore its possible effect on production and reproduction traits. The genotypic and allelic frequencies of BsaA 1 genotyped SNP g.92450765 G > A indicated the predominance of AG (0.65) genotyped Sahiwal cows in our studied population. The least-squares analysis showed a significant association (p < 0.05) of identified genetic variants with total milk yield (TMY) and 305 days milk yield (MY) (p < 0.05). The GG genotyped cows were found to be associated with higher milk yields. However, for reproduction traits under study AA genotype was found to be more favorable with respect to service period and calving interval (p < 0.05). Computational analysis was also performed to predict changes if any in the transcription factor binding sites (TFBS) caused due to the identified SNP in the intronic region of the leptin gene. TFBS analysis predicted that the targeted SNP at g.92450765G > A may lead to the disappearance of TFBS such as Hypermethylated in cancer 2 protein (HIC2), Max-binding protein MNT (MNT), Cyclic AMP-dependent transcription factor −3 (ATF3), Myc-associated factor X (MAX) and Microphthalmia-associated transcription factor (MITF). It may lead to changes in transcriptional regulation.

Transcriptome analysis of low-temperature-affected ripening revealed MYB transcription factors-mediated regulatory network in banana fruit

Low temperature leads to abnormal ripening and poor quality of the harvested banana fruit, which is an urgent problem limiting the development of industry in China. To comprehensively understand the mechanism underlying low-temperature-affected ripening, we performed comparative RNA-Seq analysis of ethylene-induced ripening of banana fruit after 3 days of pre-storage at 7 °C and 22 °C. A total of 986 differentially expressed genes (DEGs) were identified in both RT-0 d versus RT-3 d, LT-0 d versus LT-3 d, RT-0 d versus LT-0 d and RT-3 d versus LT-3 d, and the RNA-Seq outputs of 15 randomly selective DEGs were verified using qRT-PCR. Among the 986 DEGs obtained in the four groups, 9 MYB genes (MaMYB75/281/219/4/151/156/3/37 and MaMYB3R1) and 32 genes related to carotenoid biosynthesis (MaPSY1/2a), flavor formation (MaLOX6, MaADH7, MaAAT1), sucrose transport (MaSUS2/4), ethylene production (MaSAM1, MaACO9/10/12, MaACS1/12), starch degradation (MaAMY1A/1B, MaPHS1/2, MaMEX2, MapGlcT1) and cell wall degradation (MaPG3/X1, MaPME25/41, MaXTH5/7/22/23/25, MaEXP2/20/A1/A15) were characterized. Combining transcription factor binding site (TFBS) analysis as well as cis-acting element analysis, the regulatory network of low-temperature-affected ripening mediated by MYBs were constructed. The data generated in this study may unravel the transcriptional regulatory network of MYBs associated with low-temperature-affected ripening and provide a solid foundation for future studies.

STAT3 Partly Inhibits Cell Proliferation via Direct Negative Regulation of FST Gene Expression.

Follistatin (FST) is a secretory glycoprotein and belongs to the TGF-β superfamily. Previously, we found that two single nucleotide polymorphisms (SNPs) of sheep FST gene were significantly associated with wool quality traits in Chinese Merino sheep (Junken type), indicating that FST is involved in the regulation of hair follicle development and hair trait formation. The transcription regulation of human and mouse FST genes has been widely investigated, and many transcription factors have been identified to regulate FST gene. However, to date, the transcriptional regulation of sheep FST is largely unknown. In the present study, genome walking was used to close the genomic gap upstream of the sheep genomic FST gene and to obtain the FST gene promoter sequence. Transcription factor binding site analysis showed sheep FST promoter region contained a conserved putative binding site for signal transducer and activator of transcription 3 (STAT3), located at nucleotides −423 to −416 relative to the first nucleotide (A, +1) of the initiation codon (ATG) of sheep FST gene. The dual-luciferase reporter assay demonstrated that STAT3 inhibited the FST promoter activity and that the mutation of the putative STAT3 binding site attenuated the inhibitory effect of STAT3 on the FST promoter activity. Additionally, chromatin immunoprecipitation assay (ChIP) exhibited that STAT3 is directly bound to the FST promoter. Cell proliferation assay displayed that FST and STAT3 played opposite roles in cell proliferation. Overexpression of sheep FST significantly promoted the proliferation of sheep fetal fibroblasts (SFFs) and human keratinocyte (HaCaT) cells, and overexpression of sheep STAT3 displayed opposite results, which was accompanied by a significantly reduced expression of FST gene (P < 0.05). Taken together, STAT3 directly negatively regulates sheep FST gene and depresses cell proliferation. Our findings may contribute to understanding molecular mechanisms that underlie hair follicle development and morphogenesis.

Expression Quantitative Trait Locus Analysis in Systemic Sclerosis Identifies New Candidate Genes Associated With Multiple Aspects of Disease Pathology.

To identify the genetic variants that affect gene expression (expression quantitative trait loci [eQTLs]) in systemic sclerosis (SSc) and to investigate their role in the pathogenesis of the disease. We performed an eQTL analysis using whole-blood sequencing data from 333 SSc patients and 524 controls and integrated them with SSc genome-wide association study (GWAS) data. We integrated our findings from expression modeling, differential expression analysis, and transcription factor binding site enrichment with key clinical features of SSc. We detected 49,123 validated cis-eQTLs from 4,539 SSc-associated single-nucleotide polymorphisms (SNPs) (PGWAS < 10-5 ). A total of 1,436 genes were within 1 Mb of the 4,539 SSc-associated SNPs. Of those 1,436 genes, 565 were detected as having ≥1 eQTL with an SSc-associated SNP. We developed a strategy to prioritize disease-associated genes based on their expression variance explained by SSc eQTLs (r2 > 0.05). As a result, 233 candidates were identified, 134 (58%) of them associated with hallmarks of SSc and 105 (45%) of them differentially expressed in the blood cells, skin, or lung tissue of SSc patients. Transcription factor binding site analysis revealed enriched motifs of 24 transcription factors (5%) among SSc eQTLs, 5 of which were found to be differentially regulated in the blood cells (ELF1 and MGA), skin (KLF4 and ID4), and lungs (TBX4) of SSc patients. Ten candidate genes (4%) can be targeted by approved medications for immune-mediated diseases, of which only 3 have been tested in clinical trials in patients with SSc. The findings of the present study indicate a new layer to the molecular complexity of SSc, contributing to a better understanding of the pathogenesis of the disease.

A functional mutation in the AMPD1 promoter region affects promoter activity and breast meat freshness in chicken.

Freshness is an important index to determine the quality deterioration (protein degradation and changes in appearance) of chilled chicken meat and is a primary consideration of consumers. Adenosine monophosphate deaminase 1 (AMPD1) catalyzes the deamination of adenosine monophosphate to inosine monophosphate in skeletal muscle and is the rate-limiting step in the purine nucleotide cycle. Inosine monophosphate is regarded as an important indicator of meat freshness in chicken. This study investigated the association of polymorphisms in the chicken AMPD1 promoter region with meat freshness during freezing storage. An SNP (c. -905G>A) was found to be associated with the freshness (K-value) of chicken breast meat. Chickens with the AA genotype had significantly lower K-values than those with GG and AG genotypes (P<0.01). Individuals with the AA genotype also had higher breast meat AMPD1 mRNA levels than did those with the GG and AG genotypes (P<0.01, P<0.05). A luciferase assay revealed that genotype AA had greater transcriptional activity than genotype GG. Transcription factor binding site analysis identified distinct putative transcription factor binding sites in the two alleles of mutation site c. -905. In summary, we identified an SNP (c. -905G>A) in the promoter region of the AMPD1 gene that may modulate the binding affinity of different transcription factors to control AMPD1 expression and affect the freshness K-value of chicken meat.

Tnni1b-ECR183-d2, an 87 bp cardiac enhancer of zebrafish.

BackgroundSeveral heart malformations are associated with mutations in the regulatory regions of cardiac genes. Troponin I type 1b (tnni1b) is important for the formation of the atrioventricular canal in zebrafish hearts; however, the regulation of tnni1b is poorly understand. We aimed to identify a small but functional enhancer that is distal to tnni1b.MethodsEvolutionary Conserved Region (ECR) Browser was used to analyze the 219 kb zebrafish and human genomes covering the tnni1b gene as well as the 100 kb regions upstream and downstream of tnni1b. Putative transcription factor binding sites (TFBSs) were analyzed using JASPAR and PROMO, and the enhancer activity was identified using zebrafish embryos and the luciferase reporter assay. A correlation analysis between the enhancer and transcription factors (TFs) was performed via TF overexpression and TFBS mutation experiments and the electrophoretic mobility shift assay (EMSA). To analyze the conservation between zebrafish and human enhancers, human DNA fragments were functionally verified. Images were captured and analyzed by fluorescence microscopy or confocal microscopy.ResultsCombined with comparative analysis and functional validation, we identified a 183 bp ECR (termed tnni1b-ECR183) that was located approximately 84 kb upstream of tnni1b that had the heart-specific enhancer activity in zebrafish. TFBS analysis and the enhancer activity detection assay data showed that the 87 bp core region (termed tnni1b-ECR183-d2) was capable of driving specific GFP expression near the atrioventricular junction and increased luciferase expression in HEK293 and HL1 cell lines. The GFP pattern in zebrafish embryos was similar to the expression profiles of tnni1b. A correlation analysis showed that the enhancer activity of tnni1b-ECR183-d2 was increased when NKX2.5 (p = 0.0006) or JUN (p < 0.0001) was overexpressed and was decreased when the TFBSs of NKX2.5 (p < 0.0001) or JUN (p = 0.0018) were mutated. In addition, DNA-protein interactions were not observed between these TFs and tnni1b-ECR183-d2 in the EMSA experiment. The conservation analysis showed that tnni1b-ECR183-h179 (aligned from tnni1b-ECR183) drove GFP expression in the heart and skeletal muscles and increased the luciferase expression after NKX2.5 (p < 0.0001), JUN (p < 0.0001) or ETS1 (p < 0.0001) was overexpressed. Interestingly, the truncated fragment tnni1b-ECR183-h84 mainly drove GFP expression in the skeletal muscles of zebrafish and the enhancer activity decreased when NKX2.5 (p = 0.0028), ETS1 (p = 0.0001) or GATA4 (p < 0.0001) was overexpressed.ConclusionsAn 87 bp cardiac-specific enhancer located 84 kb upstream of tnni1b in zebrafish was positively correlated with NKX2.5 or JUN. The zebrafish and human enhancers in this study target different tissues. The GFP expression mediated by tnni1b-ECR183-d2 is a valuable tool for marking the domain around the atrioventricular junction.