Search Results (15)

Background: Busulfan is an FDA-approved alkylating drug used in the chemotherapy of advanced acute myeloid leukemia. The precise mechanisms by which Busulfan kills spermatogonia stem cells (SSCs) are not yet completely understood. Methods: Using a murine model, we evaluated Busulfan-induced apoptosis and DNA damage signaling between testis and ovary tissues. We executed RT-qPCR, analyzed single-nuclei RNA sequencing data and performed in situ hybridization for the localization of the gene expression in the tissues. Results: The results indicate that, in contrast to female germ cells, haploid male germ cells undergo significant apoptosis following Busulfan chemotherapy. Moreover, a gene enrichment analysis revealed that reactive oxygen species may activate the inflammatory response in part through the TNF-α/NF-κB signaling pathway. Interestingly, in the testis, the mRNA levels of TNF-α and TAF7 (TATA box-binding protein-associated factor 7) are downregulated, and testosterone levels suppressed. Mechanistically, the promoter of TNF-α has a conserved motif for binding TAF7, which is necessary for its transcriptional activation and may require further in-depth study. We next analyzed the tumorigenic function of TAF7 and revealed that it is highly overexpressed in several types of human cancers, particularly testicular germ cell tumors, and associated with poor patient survival. Therefore, we executed in situ hybridization and single-nuclei RNA sequencing, finding that less TAF7 mRNA is present in SSCs after chemotherapy. Conclusions: Thus, our data indicate a possible function of TAF7 in the regulation of SSCs and spermatogenesis following downregulation by Busulfan. These findings may account for the therapeutic effects of Busulfan and underlie its potential impact on cancer chemotherapy prognosis. Full article

In this study, we analyzed the regulatory properties of 26 (twenty-six) genes associated with nonsyndromic male infertility. We applied an in silico analysis in order to determine the number and distribution of promoters and identify relevant promoter consensus sequences and potential transcription factors. Underlining the concept of alternative transcriptional initiation (ATI), we have found that 65.4% of genes associated with nonsyndromic male infertility have 1 (one) to 6 (six) promoters, located in the region 1 kb upstream of the TSS, and 41% of them are located at a position below −500 bp. Although the TATA box consensus sequence TAWAAA, such as W is A or T, appears at a common location in all genes, it is shifted for at least 10 bp in the EFCAB9 gene. The C2H2 zinc finger is found to be the most significant common transcription factor, binding genes’ promoters GLIS1, ZSCAN21, GLIS3, GLIS1, ZNF770, ZNF780A, ZNF81, and ZNF264. On the other hand, basic leucine zipper factors (bZIPs) bind the JUNB gene promoter specifically, exhibiting unique regulatory properties of all genes associated with nonsyndromic male infertility. Two genes, NANOS1 and ZMYND15, are expected to be less susceptible to DNA methylation, due to the high density of CpG content found in their promoter regions. Full article

Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with the enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction in RPGs mRNAs, pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and a smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals. Full article

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64–10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor’s binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings. Full article

Transcription factors (TFs) are proteins that recognize specific DNA fragments in order to decode the genome and ensure its optimal functioning. TFs work at the local and global scales by specifying cell type, cell growth and death, cell migration, organization and timely tasks. We investigate the structure of DNA-binding motifs with the theory of finitely generated groups. The DNA ‘word’ in the binding domain—the motif—may be seen as the generator of a finitely generated group $F_{dna}$ on four letters, the bases A, T, G and C. It is shown that, most of the time, the DNA-binding motifs have subgroup structures close to free groups of rank three or less, a property that we call ‘syntactical freedom’. Such a property is associated with the aperiodicity of the motif when it is seen as a substitution sequence. Examples are provided for the major families of TFs, such as leucine zipper factors, zinc finger factors, homeo-domain factors, etc. We also discuss the exceptions to the existence of such DNA syntactical rules and their functional roles. This includes the TATA box in the promoter region of some genes, the single-nucleotide markers (SNP) and the motifs of some genes of ubiquitous roles in transcription and regulation. Full article

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts. Full article

Background: The therapeutically important DNA repair gene O⁶-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci.2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types. Full article

The Negative on TATA-less (NOT) 2_3_5 domain proteins play key roles in mRNA metabolism and transcription regulation, but few comprehensive studies have focused on this protein family in plants. In our study, a total of 30 NOT2_3_5 genes were identified in four cotton genomes: Gossypium. arboretum, G. raimondii, G. hirsutum and G. barbadense. Phylogenetic analysis showed that all the NOT2_3_5 domain proteins were divided into two classes. The NOT2_3_5 genes were expanded frequently, and segmental duplication had significant effects in their expansion process. The cis-regulatory elements analysis of NOT2_3_5 promoter regions indicated that NOT2_3_5 domain proteins might participate in plant growth and development processes and responds to exogenous stimuli. Expression patterns demonstrated that all of the GhNOT2_3_5 genes were expressed in the majority of tissues and fiber development stages, and that these genes were induced by multiple stresses. Quantitative real-time PCR showed that GbNOT2_3_5 genes were up-regulated in response to verticillium wilt and the silencing of GbNOT2_3_5-3/8 and GbNOT2_3_5-4/9 led to more susceptibility to verticillium wilt than controls. Identification and analysis of the NOT2_3_5 protein family will be beneficial for further research on their biological functions. Full article

The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC. Full article

Elastic fibers are one of the major structural components of the extracellular matrix (ECM) in human connective tissues. Among these fibers, microfibrillar-associated protein 4 (MFAP4) is one of the most important microfibril-associated glycoproteins. MFAP4 has been found to bind with elastin microfibrils and interact directly with fibrillin-1, and then aid in elastic fiber formation. However, the regulations of the human MFAP4 gene are not so clear. Therefore, in this study, we firstly aimed to analyze and identify the promoter region of the human MFAP4 gene. The results indicate that the human MFAP4 promoter is a TATA-less promoter with tissue- and species-specific properties. Moreover, the promoter can be up-regulated by retinol and coenzyme Q10 (coQ10) in Detroit 551 cells. Full article

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression of Abcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs. Full article

The EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements’ identification and cloning, the cis-regulatory elements’ mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/−1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes. Full article

The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS). Full article

Chlorella has great potential as a bio-factory for production of value-added compounds. To produce the desired chemicals more efficiently in Chlorella, genetic tools for modification of Chlorella need to be developed, especially an endogenous promoter. In this study, the promoter of photosystem I protein D (psaD) from Chlorella vulgaris UTEX395 was identified. Computational analysis revealed the presence of several putative cis-acting elements, including a potential core element, and light-responsive or stress-responsive elements. Gene expression analysis in heterologous expression system in Chlamydomonasreinhardtii and Nicotianabenthamiana showed that CvpsaD promoter can be used to drive the expression of genes. Functional analysis of this promoter suggested that the initiator element (Inr) is important for its function (i.e., TATA-less promoter) and that an additional factor (e.g., downstream of the transcriptional start site) might be needed for light response. We have shown that the CvpsaD promoter is functional, but not sufficiently strong, both in microalgae and higher plant. Full article

Carnitine palmitoyltransferase I (CPT I) is a key enzyme involved in the regulation of lipid metabolism and fatty acid β-oxidation. To understand the transcriptional mechanism of CPT Iα1b and CPT Iα2a genes, we cloned the 2695-bp and 2631-bp regions of CPT Iα1b and CPT Iα2a promoters of grass carp (Ctenopharyngodon idella), respectively, and explored the structure and functional characteristics of these promoters. CPT Iα1b had two transcription start sites (TSSs), while CPT Iα2a had only one TSS. DNase I foot printing showed that the CPT Iα1b promoter was AT-rich and TATA-less, and mediated basal transcription through an initiator (INR)-independent mechanism. Bioinformatics analysis indicated that specificity protein 1 (Sp1) and nuclear factor Y (NF-Y) played potential important roles in driving basal expression of CPT Iα2a gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the CPT Iα1b and CPT Iα2a genes. Moreover, some transcription factors, such as thyroid hormone receptor (TR), hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor (PPAR) family, were all identified on the CPT Iα1b and CPT Iα2a promoters. The TRα binding sites were only identified on CPT Iα1b promoter, while TRβ binding sites were only identified on CPT Iα2a promoter, suggesting that the transcription of CPT Iα1b and CPT Iα2a was regulated by a different mechanism. Site-mutation and electrophoretic mobility-shift assay (EMSA) revealed that fenofibrate-induced PPARα activation did not bind with predicted PPARα binding sites of CPT I promoters. Additionally, PPARα was not the only member of PPAR family regulating CPT I expression, and PPARγ also regulated the CPT I expression. All of these results provided new insights into the mechanisms for transcriptional regulation of CPT I genes in fish. Full article