Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

CONCLUSIONS: ALDH7B4 promoter analysis in A. thaliana and E. salsugineum reveals that both genetic background and promoter architecture contribute to gene expression in response to stress in different species. Many genes are differentially regulated in a comparison of salinity-sensitive and salinity-tolerant plant species. The aldehyde dehydrogenase 7B4 (ALDH7B4) gene is turgor-responsive in A. thaliana and encodes a highly conserved detoxification enzyme in plants. This study compared the ALDH7B4 gene in A. thaliana (salinity-sensitive) and in the salinity-tolerant close relative Eutrema salsugineum. EsALDH7B4 in E. salsugineum is the ortholog of AtALDH7B4 and the expression is also salinity, drought, and wound responsive. However, E. salsugineum requires higher salinity stress to induce the EsALDH7B4 transcriptional response. The GUS expression driven either by the promoter AtALDH7B4 or EsALDH7B4 was induced under 300 mM NaCl treatment in A. thaliana while 600 mM NaCl treatment was required in E. salsugineum, suggesting that the genetic background plays a crucial role in regulation of gene expression. Promoter sequences of ALDH7B4 are less conserved than the protein coding region. A series of EsALDH7B4 promoter deletion fragments were fused to the GUS reporter gene and promoter activity was determined in A. thaliana. The promoter region that contains two conserved ACGT-containing motifs was identified to be essential for stress induction. Furthermore, a 38 bp \"TC\" rich motif in the EsALDH7B4 promoter, absent from the AtALDH7B4 promoter, negatively affects EsALDH7B4 expression. A MYB-like transcription factor was identified to bind the \"TC\" motif and to repress the EsALDH7B4 promoter activity. This study reveals that genetic background and cis-acting elements coordinately regulate gene expression.

Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Corynebacterium glutamicum. Here, 17 core promoters and 29 RBSs in C. glutamicum were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs\' strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to C. glutamicum, which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in C. glutamicum. Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanced, and the yield of a target metabolite can be further improved.

Background: Human β-defensin-2 is an antimicrobial peptide with antibiotic properties secreted by the oral cavity to protect the host against microbial attack. The inter-individual differences in defensin expression profiles due to genetic variation might be partly responsible for differences in disease susceptibility. Aims: The objective of this study was to examine whether variation in the human β-defensin-2 gene (DEFB4A) is associated with chronic periodontitis (CP). Materials and Methods: This case-control study used Sanger sequencing to analyze two promoter polymorphisms of the DEFB4A gene with potential functional consequences using DNA samples collected from 200 unrelated individuals. Results: The DEFB4A rs1339258595 promoter polymorphism is associated with CP risk and clinical attachment level (CAL) but the rs3762040 polymorphism is not. Carriers of the T allele (rs1339258595) were approximately three times less likely to develop periodontitis compared with noncarriers (p = 0.0004, odds ratio = 0.35). Consistent with a protective role, the carriers of T allele had a lower CAL compared with the wild-type (G) allele. Moreover, the wild-type diplotype (GGGG) had a significantly higher risk of tooth loss compared with other diplotypes (p = 0.016). Conclusion: This study demonstrates that genetic variation in the promoter region of DEFB4A likely affects resistance to periodontal infection and might be a potential marker for CP risk and severity.

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2⁻3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2⁻2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.

Objective: To clarify the characteristics of the A20 regulatory changes by analyzing mutations in the non-coding region of the A20 gene in patients with T-cell lymphoma leukemia (T-LCL) . Methods: PCR and nucleotide sequence analysis were used to detect mutations in the non-coding region of the A20 gene, and DNA samples from PBMCs of 52 cases of T-LCL and 99 healthy controls. Results: A missense mutation (c.-672T>G) was detected in the A20 gene promoter from one T-LCL patient, which has been registered as a SNP (rs139054966) in gene bank. Meanwhile, a new mutation was detected in the 3\' UTR mRNA (3916 (C>G) ) . These two mutations were absent in other T-LCL samples and controls. Conclusion: The rs139054966 (c.-672T>G) and 3916 (C>G) mutations in the A20 gene were detected in T-LCL patients for the first time. There was also rs139054966 located on the binding region of the transcription factor P53, and its significance remained to be further clarified.
目的： 分析T细胞淋巴瘤白血病患者A20基因非编码区（UTR）突变情况，了解A20基因调控改变特点。 方法： 利用PCR和核苷酸序列分析方法检测52例T细胞白血病患者和99名健康对照者外周血单个核细胞中A20基因启动子区和3′UTR基因突变。 结果： 在1例T细胞淋巴瘤白血病患者中，发现其A20基因启动子区存在错义突变（c.-672T>G），该突变在基因库中已作为单核苷酸多态性登记（rs139054966），同时发现在A20基因3′UTR mRNA的3916位点存在核苷酸替换（C>G），而在其他T细胞白血病样本和健康对照组中均未发现这两种改变。 结论： 首次发现T细胞肿瘤中存在A20基因的rs139054966（c.-672T>G）和3916（C>G）改变，前者位于转录因子P53的结合区域，其具体意义有待进一步明确。.

Essential hypertension (EH) is a multifactorial disease. Interferon-γ (IFN-γ) plays an important role in the onset of EH through cytokine-mediated systemic inflammatory responses. We aimed to determine whether the methylation status of the IFN-γ gene (IFNG) promoter is involved in the pathogenesis of EH. Six copies of CpG dinucleotides are distributed between 3,203 bp and 3,121 bp upstream from the transcription initiation site of IFNG, termed CpG1 to CpG6 in the 5\'-to-3\' direction. We recruited 96 patients with EH and 96 sex- and age-matched healthy subjects as controls. Using bisulfate pyrosequencing datasets, we analyzed the methylation status of the six CpG sites and thus found that CpG5 was consistently methylated in all of the 96 EH patients and 96 control subjects. Among the remaining five CpG sites, there was no significant difference in the methylation levels of CpG4 and CpG6 between the two groups. By contrast, CpG1 (P = 0.003) and CpG3 (P = 5.87 × 10-7) were highly methylated among the EH subjects compared with the controls, whereas CpG2 (P = 1.24 × 10-12) was significantly less methylated in among EH subjects. The methylation levels of CpG2 were still lower after adjustment with logistic regression (adjusted P = 0.032). The CpG2 methylation level was an effective marker of EH (area under curve = 0.384; P = 1.40 × 10-15). The present study shows that hypomethylation of the IFNG promoter is significantly related to the risk of EH, providing new insights into the pathogenesis of EH.

Mutations in the cardiac sodium channel gene SCN5A may result in various arrhythmia syndromes such as long QT syndrome type 3 (LQTS), Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction diseases (CCD) and possibly dilated cardiomyopathy (DCM). In most of these inherited cardiac arrhythmia syndromes the phenotypical expression may range from asymptomatic phenotypes to sudden cardiac death (SCD). A 16-year-old female died during sleep. Autopsy did not reveal any explanation for her death and a genetic analysis was performed. A variant in the SCN5A gene (E1053K) that was previously described as disease causing was detected. Family members are carriers of the same E1053K variant, some even in a homozygous state, but surprisingly did not exhibit any pathological cardiac phenotype. Due to the lack of genotype-phenotype correlation further genetic studies were performed. A novel deletion in the promoter region of SCN5A was identified in the sudden death victim but was absent in other family members. These findings demonstrate the difficulties in interpreting the results of a family-based genetic screening and underline the phenotypic variability of SCN5A mutations.

Accumulating evidence of population association studies support the hypothesis that the high heritability of major psychiatric disorders is a combination of relatively common alleles of modest effect, and rare alleles some with relatively larger effects. We have previously reported low frequency mutations in the proximal promoter of the human calreticulin (CALR) gene that co-occur with the spectrum of major psychiatric disorders. One of those mutations at -205C>T (rs556992558) was detected in an isolate case of schizoaffective disorder. In the current study, the functional implication of mutation -205T is studied in the human neuronal cell lines LAN-5, BE(2)-C and HEK-293. In contrast with other mutations in the promoter region which increase gene expression activity, the -205T mutation significantly decreased gene expression in those cell lines in comparison with the wild-type -205C nucleotide (p < 0.000001, p < 0.0005, and p < 0.017, respectively). Treatment of the cell lines with the mood-stabilizing drug, valproic acid (VPA) resulted in differential gene expression activity in the mutant -205T versus the wild-type -205C construct. VPA increased gene expression activity in both constructs, while a significantly higher expression activity was observed in the mutant construct (p < 0.01), indicative of the creation of a positive effector binding site for VPA as a result of the -205T mutation. We conclude that deviation from normalcy in the level of CALR in either direction is associated with major psychiatric disorders.

BACKGROUND: Polymorphisms of G174C and C572G in the interleukin-6 (IL-6) promoter gene can affect both transcription and secretion of IL-6 and may be involved in inflammation related to and pathogenesis of ischemic stroke (IS). Whether these IL-6 gene polymorphisms are risk factors for IS or not, remains controversial.
OBJECTIVE: The aim of this study was to determine the association between IL-6 G174C and C572G gene polymorphisms and susceptibility to ischemic stroke in North Indian Population.
METHODS: Two hundred and fifty IS patients and 250 age- and sex-matched controls were studied. Genotyping was performed using SNaPshot method. Stroke was classified using Trial of Org 10172 in acute stroke treatment classification. Conditional logistic regression analysis was used to calculate the strength of association between IL-6 (G174C and C572G) polymorphisms and risk of IS.
RESULTS: Hypertension, diabetes, dyslipidemia, alcohol, smoking, family history of stroke, sedentary life style and low socioeconomic status were found to be associated with the risk of IS. Conditional logistic regression analysis showed a significant association of IL-6 G174C with the risk of IS under dominant model (OR, 1.61; 95%CI, 1.0-2.4; P value 0.02) and allelic model (OR, 1.5; 95%CI, 1.0-2.1; P value 0.02). For IL-6 C572G, multivariate adjusted analysis showed a significant association with the risk of IS under dominant model for overall IS (OR, 1.81; 95%CI, 1.04-3.15; P value 0.03) and small vessel disease subtype of IS (OR, 2.8; 95%CI, 1.3-6.0; P value 0.006).
CONCLUSIONS: Our results suggest that IL-6 (G174C) polymorphism is significantly associated with the risk of IS in North Indian population. However, IL-6 (C572G) polymorphism is found significantly associated with the risk of IS after adjusting the demographic and risk factors variables. Prospective studies with large sample size are required for independent validation. Our findings could be helpful in identifying individuals at increased risk for developing IS.