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Intramuscular fat (IMF) content is an important factor that affects the edible and processing quality of pork. Studying the transcriptional regulation mechanisms of genes affecting intramuscular fat deposition can provide theoretical support for genetic improvement in pigs. Long-chain fatty acyl-CoA synthase 3 (ACSL3), as a key enzyme in the process of lipid synthesis in mammals. However, no information about the core promoter of the ACSL3 gene and its transcriptional regulation has been reported so far. In this experiment, we successfully cloned 3112 bp of the porcine ACSL3 gene promoter region. In order to find out the core promoter of the ACSL3 gene. The results indicated that the core promoter region of the ACSL3 gene is located from −111 bp to −59 bp upstream of the transcription initiation site (TSS). To identify the interaction between SP1 and the ACSL3 gene promoter, we mutated the predicted binding sites of ACSL3 gene promoter. The results showed that the activity of the promoter was decreased by site-specific mutagenesis of the SP1 transcription factor binding site, while overexpression of SP1 increased the expression of the ACSL3 gene. In summary, our study identified a core promoter region of the porcine ACSL3 gene, and the SP1 binding site is responsible for the promoter activity. [ABSTRACT FROM AUTHOR]

Simple Summary: The HMGA2 gene has been known to regulate body size or muscle development in various animals, including mice. In this study, we explored the role of HMGA2 in sheep. We found that HMGA2 significantly promotes the proliferation of sheep muscle cells. Additionally, a specific genetic variation in the HMGA2 gene was identified, which is associated with important growth traits in sheep. These findings suggest that HMGA2 could be a valuable marker for breeding programs aimed at improving meat production in sheep. Uncovering genes associated with muscle growth and body size will benefit the molecular breeding of meat Hu sheep. HMGA2 has proven to be an important gene in mouse muscle growth and is associated with the body size of various species. However, its roles in sheep are still limited. Using sheep myoblast as a cell model, the overexpression of HMGA2 significantly promoted sheep myoblast proliferation, while interference with HMGA2 expression inhibited proliferation, indicated by qPCR, EdU, and CCK-8 assays. Furthermore, the dual-luciferase reporter system indicated that the region NC_056056.1: 154134300-154134882 (-618 to -1200 bp upstream of the HMGA2 transcription start site) was one of the habitats of the HMGA2 core promoter, given the observation that this fragment led to a ~3-fold increase in luciferase activity. Interestingly, SNP rs428001129 (NC_056056.1:g.154134315 C>A) was detected in this fragment by Sanger sequencing of the PCR product of pooled DNA from 458 crossbred sheep. This SNP was found to affect the promoter activity and was significantly associated with chest width at birth and two months old, as well as chest depth at two and six months old. The data obtained in this study demonstrated the phenotypic regulatory role of the HMGA2 gene in sheep production traits and the potential of rs428001129 in marker-assisted selection for sheep breeding in terms of chest width and chest depth. [ABSTRACT FROM AUTHOR]

The core promoter determines not only where gene transcription initiates but also the transcriptional activity in both basal and enhancer-induced conditions. Multiple short sequence elements within the core promoter have been identified in different species, but how they function together and to what extent they are truly species-specific has remained unclear. In this issue of Genes & Development, Vo ngoc and colleagues (pp. 377-382) report undertaking massively parallel measurements of synthetic core promoters to generate a large data set of their activities that informs a statistical learning model to identify the sequence differences of human and Drosophila core promoters. This machine learning model was then applied to design gene core promoters that are particularly specific for the human transcriptional machinery.

The RNA polymerase II core promoter is the site of convergence of the signals that lead to the initiation of transcription. Here, we performed a comparative analysis of the downstream core promoter region (DPR) in Drosophila and humans by using machine learning. These studies revealed a distinct human-specific version of the DPR and led to the use of machine learning models for the identification of synthetic extreme DPR motifs with specificity for human transcription factors relative to Drosophila factors and vice versa. More generally, machine learning models could similarly be used to design synthetic DNA elements with customized functional properties.

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoterdependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7 bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation. Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoterdependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7 bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation. [ABSTRACT FROM AUTHOR]

Numerous noncoding (nc)RNAs have been identified. Similar to the transcription of protein-coding (mRNA) genes, long noncoding (lnc)RNA genes and most of micro (mi)RNA genes are transcribed by RNA polymerase II (Pol II). In the transcription of mRNA genes, core promoters play an indispensable role; they support the assembly of the preinitiation complex (PIC). However, the structural and/or physical properties of the core promoters of lncRNA and miRNA genes remain largely unexplored, in contrast with those of mRNA genes. Using the core promoters of human genes, we analyzed the repertoire and population ratios of residing core promoter elements (CPEs) and calculated the following five DNA physical properties (DPPs): duplex DNA free energy, base stacking energy, protein-induced deformability, rigidity and stabilizing energy of Z-DNA. Here, we show that their CPE and DPP profiles are similar to those of mRNA gene promoters. Importantly, the core promoters of these three classes of genes have two highly distinctive sites in their DPP profiles around the TSS and position −27. Similar characteristics in DPPs are also found in the 5′-flanking regions of tRNA genes, indicating their common essential roles in transcription initiation over the kingdom of RNA polymerases. [ABSTRACT FROM AUTHOR]

Since the discovery of physical peculiarities around transcription start sites (TSSs) and a site corresponding to the TATA box, research has revealed only the average features of these sites. Unsettled enigmas include the individual genes with these features and whether they relate to gene function. Herein, using 10 physical properties of DNA, including duplex DNA free energy, base stacking energy, protein-induced deformability, and stabilizing energy of Z-DNA, we clarified for the first time that approximately 97% of the promoters of 21,056 human protein-coding genes have distinctive physical properties around the TSS and/or position −27; of these, nearly 65% exhibited such properties at both sites. Furthermore, about 55% of the 21,056 genes had a minimum value of regional duplex DNA free energy within TSS-centered ±300 bp regions. Notably, distinctive physical properties within the promoters and free energies of the surrounding regions separated human protein-coding genes into five groups; each contained specific gene ontology (GO) terms. The group represented by immune response genes differed distinctly from the other four regarding the parameter of the free energies of the surrounding regions. A vital suggestion from this study is that physical-feature-based analyses of genomes may reveal new aspects of the organization and regulation of genes. [ABSTRACT FROM AUTHOR]

Abnormal gene expression plays key role in cancer development. A core promoter is located around the transcriptional start site. Through interaction between core promoter sequences and transcriptional factors, core promoter controls transcriptional initiation. We hypothesized that in cancer, core promoter sequences could be mutated to interfere the interaction with transcriptional factors, resulting in altered transcriptional initiation and abnormal gene expression and cancer development. We used triple-negative breast cancer (TNBC) as a model to test our hypothesis. We collected genome-wide core promoter variants from 279 TNBC genomes. After extensive filtering of normal genomic polymorphism, we identified 19,427 recurrent somatic variants in 1,238 core promoters of 1,274 genes and 1,694 recurrent germline variants in 272 core promoters of 294 genes. Many of the affected genes were oncogenes and tumor suppressors. Analysis of RNA-seq data from the same patient cohort identified increased or decreased gene expression in 439 somatic and 85 germline variants-affected genes, and the results were validated by luciferase reporter assay. By comparing with the core promoter variation data from 610 unclassified breast cancer, we observed that core promoter variants in TNBC were highly TNBC-specific. We further identified the drugs targeting the genes with core promoter variation. Our study demonstrates that core promoter is highly mutable in cancer, and can play etiological roles in TNBC and other types of cancer through influencing transcriptional initiation.

ABSTRACT: Chicken is considered an ideal model species to study the synthesis of polyunsaturated fatty acids (PUFAs) due to its appropriate proportions of fatty acids and abundant content of PUFAs, suitable for human consumption. However, the molecular mechanisms regulating poultry PUFA synthesis remain unclear. Here, we systematically explored the transcriptional regulation activity of the gene family related to PUFA synthesis in chicken by carrying out the Dual-Luciferase Reporter Assay. We identified the core promoter regions of members of the chicken PUFA synthesis-related gene family, including ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, FADS1, FADS2, FADS6, SCD, and SCD5. Additionally, changes in relative fluorescence values of different truncated segments in the upstream regulatory region of these genes indicate the existence of regulatory regions. Furthermore, we predicted the transcription factors that bind to the identified core promoter regions of multiple genes, including Sp1, NF-1, C/EBPalpha, etc. These findings provide a basis for the molecular mechanisms regulating poultry PUFA synthesis and offer new scientific insight into the potential improvement of poultry meat quality in the future.

The core promoter plays an essential role in regulating transcription initiation by controlling the interaction between transcriptional factors and sequence motifs in the core promoter. Although mutation in core promoter sequences is expected to cause abnormal gene expression leading to pathogenic consequences, limited supporting evidence showed the involvement of core promoter mutation in diseases. Our previous study showed that the core promoter is highly polymorphic in worldwide human ethnic populations in reflecting human history and adaptation. Our recent characterization of the core promoter in triple-negative breast cancer (TNBC), a subtype of breast cancer, in a Chinese TNBC cohort revealed the wide presence of core promoter mutation in TNBC. In the current study, we analyzed the core promoter in a Thai TNBC cohort. We also observed rich core promoter mutation in the Thai TNBC patients. We compared the core promoter mutations between Chinese and Thai TNBC cohorts. We observed substantial differences of core promoter mutation in TNBC between the two cohorts, as reflected by the mutation spectrum, mutation-effected gene and functional category, and altered gene expression. Our study confirmed that the core promoter in TNBC is highly mutable, and is highly ethnic-specific. [ABSTRACT FROM AUTHOR]

COL1A1 encodes the type I collagen α1 chain, which shows the highest abundance among members of the collagen family and is widely expressed in different mammalian cells and tissues. However, its molecular characteristics are not completely elucidated. In this study, the molecular profiles of COL1A1 and characteristics of the COL1A1 protein were investigated using a promoter activity assay and multiple bioinformatics tools. The results showed that the 5′ flanking region of porcine COL1A1 contained two CpG islands, five core promoter sequences, and twenty-six transcription factor-binding sites. In the luciferase assay, the upstream 294 bp region of the initiation codon of COL1A1 showed the highest activity, confirming that this section is the core region of the porcine COL1A1 promoter. Bioinformatic analysis revealed that COL1A1 is a negatively charged, hydrophilic secreted protein. It does not contain a transmembrane domain and is highly conserved in humans, mice, sheep, and pigs. Protein interaction analysis demonstrated that the interaction coefficient of COL1A1 with COL1A2, COL3A1, ITGB1, and ITGA2 was greater than 0.9, suggesting that this protein plays a crucial role in collagen structure formation and cell adhesion. These results provide a theoretical basis for further investigation of the functions of porcine COL1A1. [ABSTRACT FROM AUTHOR]

Decreased expression of chicken cholecystokinin A receptor (CCKAR) attenuates satiety, which contributes to increased food intake and growth for modern broilers. The study aims to define the core promoter of CCKAR, and to identify variants associated with expression activity. A 21 kb region around the CCKAR was re-sequenced to detect sequence variants. A series of 5′-deleted promoter plasmids were constructed to define the core promoter of CCKAR. The effects of sequence variants located in promoter (PSNP) and conserved (CSNP) regions on promoter activity were analyzed by comparing luciferase activity between haplotypes. A total of 182 variants were found in the 21 kb region. There were no large structural variants around CCKAR. pNL−328/+183, the one with the shortest insertion, showed the highest activity among the six promoter constructs, implying that the key cis elements regulating CCKAR expression are mainly distributed 328 bp upstream. We detected significant activity differences between high- and low-growth associated haplotypes in four of the six promoter constructs. The high-growth haplotypes of constructs pNL−1646/+183, pNL−799/+183 and pNL−528/+183 showed lower activities than the low-growth haplotypes, which is consistent with decreased expression of CCKAR in high-growth chickens. Lower expression of the high-growth allele was also detected for the CSNP5-containing construct. The data suggest that the core promoter of CCKAR is located the 328 bp region upstream from the transcription start site. Lower expression activities shown by the high-growth haplotypes in the reporter assay suggest that CSNP5 and variants located between 328 bp and 1646 bp upstream form a promising molecular basis for decreased expression of CCKAR and increased growth in chickens. [ABSTRACT FROM AUTHOR]

Close Homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules. CHL1 gene is located on human chromosome 3 and has been linked to several pathologies, including 3p deletion syndrome, schizophrenia, and tumor growth and metastasis. The goal of the present study was to determine which region of the CHL1 promoter is most competent in driving CHL1 gene expression. Methods: Five candidate DNA fragments in the promoter regions were selected by screening across six species for evolutionary conserved sequences. The activity of these five promoter regions was quantitatively evaluated using a GFP reporter gene in transfection experiments, performed in C6 glioma cells. Of the five promoter regions tested, three drove reporter GFP expression, with the conserved region 6 (CR6, Gene ID AC066595.5, 25851-26850) being the most active for transcription. The identification of the CR6 activity provides a better understanding of the regulatory mechanisms underlying CHL1 expression. It may help future discovery of therapeutic strategies that involve influencing critical promoter regions to drive transcriptional regulation of the mammalian CHL1 gene. Conserved regions of CHL1 promoter sequences were identified by in-silico analysis. Five conserved regions were tested for gene regulatory activity using a reporter assay. Conserved regions CR5, CR6 and CR7 show gene regulatory function in a reporter assay. Co-transfection of CR5 and CR6 yielded the highest reporter activity. The core region of CR6 (CR6core) was identified as a cis-acting element. In-tandem promoter CR5core-CR6core was the best in a reporter assay. [ABSTRACT FROM AUTHOR]

As a member of the large tumor suppressor (LATS) gene family, LATS1 plays an important role in regulating muscle growth and development. In this study, we determined the distinct exhibit patterns of tissue expression of bovine LATS1. Further, we determined the functional proximal minimal promoter of bovine LATS1 and identified the key transcription factors in the core promoter region to elucidate its molecular regulation mechanism. The results showed that bovine LATS1 was highly expressed in the longissimus thoracis and upregulation in infancy muscle. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and small interfering RNA (siRNA) interference demonstrated that myogenic differentiation 1 (Myod1) and myocyte enhancer factor 2A (MEF2A) binding in the core promoter region (−298/−123 bp) play important roles in the transcriptional regulation of the bovine LATS1 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of LATS1 transcription in mediating skeletal muscle growth in cattle.

Despite the elucidation of the many processes by which a single eukaryotic cell develops into a complex mature organism, it is still puzzling to some biologists how it is that an unvarying, interconnected set of processes becomes coordinated and insulated from a stochastic universe. This article suggests that electromagnetic processes deriving from the chemistry of an organism may provide such coordination. Specifically, the author develops the pacemaker concept, the periodic, autonomous electrical signal to the entire embryo, the result of which, after each pulse, is to alter or enlarge the transcriptome to produce the next level of complexity and maturity of the organism., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

Background: Hepatitis B virus (HBV) infection is still a serious threat to global health and can lead to a variety of liver diseases, including acute and chronic hepatitis, liver cirrhosis, liver failure, hepatocellular carcinoma (HCC), and so on. At present, there are mainly two kinds of drugs for the treatment of hepatitis B at home and abroad: interferon (IFN) and nucleoside/nucleotide analogs (NAs). In recent years, natural compounds have been considered an important source for the development of new anti-HBV drugs due to their complex structure, diverse components, high efficiency, and low toxicity. Many studies have demonstrated that Solamargine has significant anticancer activity, but the antiviral effect is rarely studied. This study aimed to verify the anti-HBV effect of Solamargine and to explore the specific mechanism., Method: The relative expression of HBV pregenomic RNA (pgRNA) was detected by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR). Northern blot and western blot were used to detect the relative expression of HBV pgRNA and target protein. PCR was used in the construction of HBV pg-promoter, ENII/BCP, and a series of gene deletion mutant fluorescent reporter vectors. The fluorescence relative expression of each mutant was detected by Renilla luciferase assay., Results: By binding to MZF1 (Myeloid zinc finger protein 1, MZF1), Solamargine inhibits HBV core promoter activity, reduces pregenomic RNA level, and inhibits HBV, achieving antiviral effects.

Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman ( tin ) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation., Competing Interests: Competing Interest Statement The authors declare no competing interests.

Members of the tripartite motif (TRIM) protein family strongly induced by interferons (IFNs) are parts of the innate immune system with antiviral activity. However, it is still unclear which TRIMs could play important roles in hepatitis B virus (HBV) inhibition. Here, we identified that TRIM56 expression responded in IFN-treated HepG2-NTCP cells and HBV-infected liver tissues, which was a potent IFN-inducible inhibitor of HBV replication. Mechanistically, TRIM56 suppressed HBV replication via its Ring and C-terminal domain. C-terminal domain was essential for TRIM56 translocating from cytoplasm to nucleus during HBV infection. Further analysis revealed that TRIM56's Ring domain targeted IκBα for ubiquitination. This modification induced phosphorylation of p65, which subsequently inhibited HBV core promoter activity, resulting in the inhibition of HBV replication. The p65 was found to be necessary for NF-κB signal pathway to inhibit HBV replication. We verified our findings using HepG2-NTCP and primary human hepatocytes. Our findings reveal that TRIM56 is a critical antiviral immune effector and exerts an anti-HBV activity via NF-κB signal pathway, which is essential for inhibiting transcription of HBV covalently closed circular DNA. [Display omitted] • TRIM56 inhibits infection and replication of HBV. • TRIM56 restricts HBV core promoter activity. • TRIM56 Ring domain ubiquitinates IκBα to active NF-КB pathway. • TRIM56 inhibit HBV replication through phosphorylated p65 to suppress HBV transcription. [ABSTRACT FROM AUTHOR]