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Abstract The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD 5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD 5 regulation, we cloned and characterized the promoter region of ABHD 5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD 5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of –109/+307 was identified as proximal minimum core promoter region of ABHD 5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD 5 gene. Taken together these findings we can conclude that ABHD 5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits. Highlights • The mRNA expression level of ABHD 5 was quantified in different tissue at different age groups of Qinchuan cattle. • The transcription factor binding sites of C/EBPα and Evi1 are conserved for common livestock, and their mutations lead to an increase in ABHD 5 gene promoter activity in bovine intramuscular preadipocytes. • Luciferase reporter assay, site directed mutation and EMSA were used to validate the role of Evi1 and C/EBPα transcription factors in the regulation of ABHD 5 gene. [ABSTRACT FROM AUTHOR]

The radioresistant D. radiodurans regulates its DNA damage regulon (DDR) through interaction between a 17 bp palindromic cis -regulatory element called the Radiation Desiccation Response Motif (RDRM), the DdrO repressor and a protease IrrE. The role of RDRM in regulation of DDR was dissected by constructing RDRM sequence-, position- or deletion-variants of Deinococcal gyrB gene (DR0906) promoter and by RDRM insertion in the non-RDRM groESL gene (DR0606) promoter, and monitoring the effect of such modifications on the basal as well as gamma radiation inducible promoter activity by quantifying fluorescence of a GFP reporter. RDRM sequence-variants revealed that the conservation of sequence at the 5th and 13th position and the ends of RDRM is essential for basal repression by interaction with DdrO. RDRM position-variants showed that the sequence acts as a negative regulatory element only when located around transcription start site (TSS) and within the span of RNA polymerase (RNAP) binding region. RDRM deletion-variants indicated that the 5′ sequence of RDRM possibly possesses an enhancer-like element responsible for higher expression yields upon repressor clearance post-irradiation. The results suggest that RDRM plays both a negative as well as a positive role in the regulation of DDR in D. radiodurans . [ABSTRACT FROM AUTHOR]

Recent reports of a link between human- and primate-specific genetic factors and human/primate-specific characteristics and diseases necessitate genome-wide identification of those factors. We have previously reported core promoter short tandem repeats (STRs) of extreme length (≥ 6-repeats) that have expanded exceptionally in primates vs. non-primates, and may have a function in adaptive evolution. In the study reported here, we extended our study to the human STRs of ≥ 3-repeats in the category of penta and hexaucleotide STRs, across the entire human protein coding gene core promoters, and analyzed their status in several superorders and orders of vertebrates, using the Ensembl database. The ConSite software was used to identify the transcription factor (TF) sets binding to those STRs. STR specificity was observed at different levels of human and non-human primate (NHP) evolution. 73% of the pentanucleotide STRs and 68% of the hexanucleotide STRs were found to be specific to human and NHPs. AP-2alpha, Sp1, and MZF were the predominantly selected TFs (90%) binding to the human-specific STRs. Furthermore, the number of TF sets binding to a given STR was found to be a selection factor for that STR. Our findings indicate that selected STRs, the cognate binding TFs, and the number of TF set binding to those STRs function as switch codes at different levels of human and NHP evolution and speciation. [ABSTRACT FROM AUTHOR]

Improvement of host plant resistance is one of the best methods to protect the yield from biotic stresses. Incorporation of major resistance genes or their variants into elite rice varieties will enhance the host plant resistance and its durability. Allele mining is a preferred choice to discover the novel allelic variants of major genes from wide range of germplasm. ‘True’ allele mining includes coding and noncoding regions, which are known to affect the plant phenotype, eventually. In this study, major blast resistance gene, Pita was analyzed by allele and promoter mining strategy and its different allelic variants were discovered from landraces and wild Oryza species. Polymorphisms at allelic sequences as well as transcription factor binding motif (TFBM) level were examined. At motif level, MYB1AT is present in Pita Tadukan and other resistance alleles, but was absent in the susceptible allele. Core promoter was demarked with 449 bp, employing serial promoter deletion strategy. Promoter with 1592 bp upstream region could express the gfp two fold higher than the core promoter. The identified Pita resistance allele ( Pita Konibora ) can be directly used in rice blast resistance breeding programs. Moreover, characterization of Pita core promoter led to deeper understanding of resistance gene's regulation and the identified core promoter can be utilized to express similar genes in rice. [ABSTRACT FROM AUTHOR]

Abstract: Protein complexes that bind to ‘GAGA’ DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1×10−8). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution. [Copyright &y& Elsevier]

Abstract: Short tandem repeats (STRs) are variable elements that play a significant role in genome evolution by creating and maintaining quantitative genetic variation. Because of their proximity to the +1 transcription start site (TSS) and polymorphic nature, core promoter STRs may be considered a novel source of variation across species. In a genome-scale analysis of the entire human protein-coding genes annotated in the GeneCards database (19,927), we analyze the prevalence and repeat numbers of different classes of core promoter STRs in the interval between −120 and +1 to the TSS. We also analyze the evolutionary trend of exceptionally long core promoter STRs of ≥6-repeats. 133 genes (~2%) had core promoter STRs of ≥6-repeats. In the majority of those genes, the STR motifs were found to be conserved across evolution. Di-nucleotide repeats had the highest representation in the human core promoter long STRs (72 genes). Tri- (52 genes), tetra-, penta-, and hexa-nucleotide STRs (9 genes) were also present in the descending prevalence. The majority of those genes (84 genes) revealed directional expansion of core promoter STRs from mouse to human. However, in a number of genes, the difference in average allele size across species was sufficiently small that there might be a constraint on the evolution of average allele size. Random drift of STRs from mouse to human was also observed in a minority of genes. Future work on the genes listed in the current study may further our knowledge into the potential importance of core promoter STRs in human evolution. [Copyright &y& Elsevier]

Abstract: In a genome-scale analysis of the composition of core promoter sequences, we have recently shown that approximately 25% of the human protein-coding genes have at least one short tandem repeat (STR) of 3-repeats in their core promoters (i.e. the interval between −120 to +1). Through their nucleosome processing effect, GA-repeats play a crucial role in the regulation of gene transcription. In this study, we chose the human SRY (sex determining region Y)-box 5 (SOX5) gene as a prototype of the GA-rich core promoters to investigate the role of core promoter GA-STRs in gene expression. The human SOX5 gene is indispensable for diverse embryonic developmental processes, ranging from oligodendrocyte development and corticogenesis to chondrogenesis, and regulation of the cell cycle. Whereas the absolute ratio of 99% of the genes range between 0.2 and 2, the composition of the core promoter of the two most ubiquitously expressed mRNAs of the human SOX5 gene (transcripts ID: ENST00000451604 and ENST00000309359) is exceptionally rich in purine nucleotides (purine/pyrimidine ratio: 61.5). Indeed, this core promoter is an island of four tandem GA-STRs, and lacks the known TATA and TATA-less elements for gene transcription. Evolutionary conservation of this region between human and mouse (75% homology) supports important functional role for this promoter. In this study, we show that this nucleotide composition is indeed a potent promoter (p <1 × 10−10), and different haplotypes across the region result in significant difference in gene expression (p <1 × 10−6). To our knowledge, this is the first report of functional STRs in a human gene core promoter. Based on our search on the core promoters of the entire human protein-coding genes annotated in the GeneCards database (19,927genes) for the presence of pure GA-STRs, 429 genes contain at least one GA(3)-repeat in their core promoter. Core promoters with pure GA-STRs of GA(4) and above were observed in 61 genes. Our data unravel a novel mechanism for inter-individual variation in gene expression and complex traits/phenotypes through core promoter GA-STRs. [Copyright &y& Elsevier]

Abstract: The proximal promoter sequences contain basic motifs for the expression of the downstream genes. We present genome-scale computational analyses of the 120-bp immediate upstream sequences to the +1 transcription start sites (TSSs) of 10,117 human protein-coding genes, and unravel exceptional genes in respect with the core promoter nucleotide composition. Our data reveal that while in 99% of the genes the absolute purine/pyrimidine ratio ranges between 0.2 and 2.5, certain genes show exceptional skew in this balance (e.g. ratios of 82.3 in VWA3A, 61.5 in Sox5, and 24.0 in BRWD3), and consist of islands of purines or pyrimidines. Furthermore, while over 95% of the genes lack more than one short tandem repeat (STR) in their core promoters, certain gene promoters are exceptionally rich in multiple STRs (e.g. eight consecutive STRs in UBE2QL1, and six STRs in GRIA2). We found sequence bias for the majority of those promoters across species, supporting functional roles for them in gene expression. Genes downstream to those promoters were also found to be of ontologic importance (i.e. we were able to track the majority of those genes to the lower species such as Saccharomyces cerevisiae and Caenorhabditis elegans). The exceptional promoters presented in this study lack the conventional motifs for the TATA, and TATA-less promoters, hence offering novel mechanisms for gene expression. They may also provide potential mechanisms for inter-individual variations in gene expression, and complex traits/disorders. [Copyright &y& Elsevier]

Abstract: hZip1 has been characterized as the major zinc uptake transporter regulating the accumulation of zinc in prostate cells. The mechanisms regulating expression of hZip1 have not been described. To explore the mechanisms of transcriptional regulation of the hZip1 gene, we determined the putative promoter sequence for hZip1 and identified the potential transcription start site within the predicted hZip1 promoter region. To further characterize the promoter region for basal hZip1 transcription, 3′ and 5′ deletion constructs and constructs with mutated binding sites for putative transcription factors were generated by PCR amplification and assessed for transcriptional activity with a luciferase reporter assay in PC-3 prostate cancer cells. The ability of the specific transcription factors to bind the hZip1 core promoter was confirmed by EMSA, GelSupershift and ChIP assays. Our experiments identified the core promoter region responsible for constitutive expression of hZip1 and demonstrated critical roles for SP1 and CREB1 in transcriptional regulation of the hZip1 gene in prostate cancer cells. [Copyright &y& Elsevier]

• ZNFO core promoter is located within the region −57 to +36 relative to the TSS. • ZNFO core promoter contains a USF1/USF2 binding site required for promoter activity. • USF1/USF2 complex activates ZNFO basal transcription through interaction with its binding site. ZNFO is a Krüppel-associated box (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocytes. Previous studies have demonstrated that ZNFO possesses an intrinsic transcriptional repressive activity and is essential for early embryonic development in cattle. However, the mechanisms regulating ZNFO transcription remain elusive. In the present study, the core promoter that controls the ZNFO basal transcription was identified. A 1.7 kb 5′ regulatory region of the ZNFO gene was cloned and its promoter activity was confirmed by a luciferase reporter assay. A series of 5′ deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core promoter region has to include the sequence located within 57 bp to 31 bp upstream of the transcription start site. Sequence analysis revealed that a putative USF1/USF2 binding site (GGTCACGTGACC) containing an E-box motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene. [ABSTRACT FROM AUTHOR]