Analysis of the Drosophila and human DPR elements reveals a distinct human variant whose specificity can be enhanced by machine learning.

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.

Designer genes courtesy of artificial intelligence.

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.

Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements.

Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. Here we show that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex, localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp ß2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. Our results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation.

Getting started: altering promoter choice as a mechanism for cell type differentiation.

In this issue of Genes & Development, Lu and colleagues (pp. 663-677) have discovered a key new mechanism of alternative promoter choice that is involved in differentiation of spermatocytes. Promoter choice has strong potential as mechanism for differentiation of many different cell types.

Transcription factor IID parks and drives preinitiation complexes at sharp or broad promoters.

Core promoters are sites where transcriptional regulatory inputs of a gene are integrated to direct the assembly of the preinitiation complex (PIC) and RNA polymerase II (Pol II) transcription output. Until now, core promoter functions have been investigated by distinct methods, including Pol II transcription initiation site mappings and structural characterization of PICs on distinct promoters. Here, we bring together these previously unconnected observations and hypothesize how, on metazoan TATA promoters, the precisely structured building up of transcription factor (TF) IID-based PICs results in sharp transcription start site (TSS) selection; or, in contrast, how the less strictly controlled positioning of the TATA-less promoter DNA relative to TFIID-core PIC components results in alternative broad TSS selections by Pol II.

From promoter motif to cardiac function: a single DPE motif affects transcription regulation and organ function in vivo.

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 promoter-dependent 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.

The punctilious RNA polymerase II core promoter.

The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter.

The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters.

DNA sequence signals in the core promoter, such as the initiator (Inr), direct transcription initiation by RNA polymerase II. Here we show that the human Inr has the consensus of BBCA+1BW at focused promoters in which transcription initiates at a single site or a narrow cluster of sites. The analysis of 7678 focused transcription start sites revealed 40% with a perfect match to the Inr and 16% with a single mismatch outside of the CA+1 core. TATA-like sequences are underrepresented in Inr promoters. This consensus is a key component of the DNA sequence rules that specify transcription initiation in humans.

Finding the start site: redefining the human initiator element.

Transcription by RNA polymerase II (Pol II) is dictated in part by core promoter elements, which are DNA sequences flanking the transcription start site (TSS) that help direct the proper initiation of transcription. Taking advantage of recent advances in genome-wide sequencing approaches, Vo ngoc and colleagues (pp. 6-11) identified transcripts with focused sites of initiation and found that many were transcribed from promoters containing a new consensus sequence for the human initiator (Inr) core promoter element.

Physical Peculiarity of Two Sites in Human Promoters: Universality and Diverse Usage in Gene Function.

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.

Establishment and Characterization of an HBV Viral Spread and Infectious System following Long-Term Passage of an HBV Clinical Isolate in the Primary Human Hepatocyte and Fibroblast Coculture System.

The existing cell culture-based methods to study hepatitis B virus (HBV) have limitations and do not allow for viral long-term passage. The aim of this study was to develop a robust in vitro long-term viral passage system with optimized cell culture conditions and a viral isolate with the ability to spread and passage. An HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized primary human hepatocyte (PHH)/human fibroblast coculture system. The passaged UV-treated virus was sequenced and further characterized. In addition, a panel of mutant viruses containing different combinations of mutations observed in this virus was investigated. The clinical isolate was passaged for 20 rounds with 21 days per round in an optimized PHH/human fibroblast coculture system while subject to UV mutagenesis. This passaged UV-mutated isolate harbored four mutations: G225A (sR24K) in the S gene, A2062T in the core gene, and two mutations G1764A and C1766T (xV131I) in the basal core promoter (BCP) region. In vitro characterization of the four mutations suggested that the two BCP mutations G1764A and C1766T contributed to the increased viral replication and viral infectivity. A robust in vitro long-term HBV viral passage system has been established by passaging a UV-treated clinical isolate in an optimized PHH/fibroblast coculture system. The two BCP mutations played a key role in the virus's ability to passage. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization. IMPORTANCE The existing cell culture-based methods to study HBV have limitations and do not allow for viral long-term passage. In this study, an HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized PHH/human fibroblast coculture system. This passaged UV-mutated isolate carried four mutations across the HBV genome, and in vitro characterization of the four mutations suggested that the two basal core promoter (BCP) mutations G1764A and C1766T played a key role in the virus's ability to passage. In summary, we have developed a robust in vitro long-term HBV viral passage system by passaging an UV-treated HBV genotype A clinical isolate in an optimized PHH/human fibroblast coculture system. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization.

Different mutation rates in the basal core promoter and precore regions between hepatitis B virus subgenotype F1b clusters.

Hepatitis B virus (HBV) is the main etiological agent of hepatocellular carcinoma (HCC) worldwide. It has been classified into nine genotypes and several subgenotypes, with uneven global distribution. There is growing evidence that the viral genotype influences the course and outcome of chronic hepatitis B infection. Two evolutionarily different clusters of the subgenotype F1b, called basal and cosmopolitan, have been described. The two clusters have constrained geographical distribution, with the particular feature that the basal cluster is present in regions of high HCC incidence, while the Cosmopolitan cluster is found in regions of low HCC incidence. The BCP/pC region was sequenced in 68 cases chronically infected with the F1b subgenotype to determine if there was a differential pattern of pathogenic-associated mutations between both clusters. Twenty-two of the 68 cases belonged to the subgenotype F1b basal cluster and 46 to the cosmopolitan cluster. Among the HBeAg-negative patients the A1762T/G1764A and G1896A mutations were more frequently found in the basal samples (85.7 and 92.9%) compared to the cosmopolitan ones (50 and 18.2%). Interestingly, no HBeAg loss-associated mutations were observed in 7.1 and 36.4% of the basal and cosmopolitan cases, respectively. The different rate of mutations associated with a more severe course of chronic hepatitis in the basal cluster would support the difference in the HCC incidence rate in the geographical regions where the basal cluster is restricted.

The A1762T/G1764A mutations enhance HBV replication by alternating viral transcriptome.

The A1762T/G1764A mutations, one of the most common mutations in the hepatitis B virus basal core promoter, are associated with the progression of chronic HBV infection. However, effects of these mutations on HBV replication remains controversial. This study aimed to systematically investigate the effect of the mutations on HBV replication and its underlying mechanisms. Using the prcccDNA/pCMV-Cre recombinant plasmid system, a prcccDNA-A1762T/G1764A mutant plasmid was constructed. Compared with wild-type HBV, A1762T/G1764A mutant HBV showed enhanced replication ability with higher secreted HBV DNA and RNA levels, while Southern and Northern blot indicated higher intracellular levels of relaxed circular DNA, single-stranded DNA, and 3.5 kb RNA. Meanwhile, the mutations increased expression of intracellular core protein and decreased the production of HBeAg and HBsAg. In vitro infection based on HepG2-NTCP cells and mice hydrodynamic injection experiment also proved that these mutations promote HBV replication. 5'-RACE assays showed that these mutations upregulated transcription of pregenomic RNA (pgRNA) while downregulating that of preC RNA, which was further confirmed by full-length transcriptome sequencing. Moreover, a proportion of sub-pgRNAs with the potential to express polymerase were also upregulated by these mutations. The ChIP-qPCR assay showed that A1762T/G1764A mutations created a functional HNF1α binding site in the BCP region, and its overexpression enhanced the effect of A1762T/G1764A mutations on HBV. Our findings revealed the mechanism and importance of A1762T/G1764A mutations as an indicator for management of CHB patients, and provided HNF1α as a new target for curing HBV-infected patients.

Core Promoter Regions of Antisense and Long Intergenic Non-Coding RNAs.

RNA polymerase II (POL II) is responsible for the transcription of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs). Previously, we have shown the evolutionary invariance of the structural features of DNA in the POL II core promoters of the precursors of mRNAs. In this work, we have analyzed the POL II core promoters of the precursors of lncRNAs in Homo sapiens and Mus musculus genomes. Structural analysis of nucleotide sequences in positions -50, +30 bp in relation to the TSS have shown the extremely heterogeneous 3D structure that includes two singular regions - hexanucleotide "INR" around the TSS and octanucleotide "TATA-box" at around ~-28 bp upstream. Thus, the 3D structure of core promoters of lncRNA resembles the architecture of the core promoters of mRNAs; however, textual analysis revealed differences between promoters of lncRNAs and promoters of mRNAs, which lies in their textual characteristics; namely, the informational entropy at each position of the nucleotide text of lncRNA core promoters (by the exception of singular regions) is significantly higher than that of the mRNA core promoters. Another distinguishing feature of lncRNA is the extremely rare occurrence in the TATA box of octanucleotides with the consensus sequence. These textual differences can significantly affect the efficiency of the transcription of lncRNAs.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Vitamin D signaling inhibits HBV activity by directly targeting the HBV core promoter.

Clinical and epidemiological studies support a role for vitamin D in suppressing hepatitis B virus (HBV). This antiviral role of vitamin D is widely attributed to vitamin D receptor (VDR)/retinoid X receptor-mediated regulation of host immunomodulatory genes through vitamin D response elements (VDREs) in their promoters. Here, we investigated the ability of calcitriol (1α,25-dihydroxyvitamin D3, metabolically activated vitamin D) to directly regulate HBV activity through this signaling pathway. We observed that calcitriol selectively inhibited only the HBV core promoter without affecting the HBV-PreS1, HBV-PreS2/S, or HBx promoters. We then identified a VDRE cluster in the HBV core promoter that is highly conserved across most HBV genotypes. Disruption of this VDRE cluster abrogated calcitriol-mediated suppression of the HBV core promoter. Furthermore, we showed that VDR interacts directly with the VDRE cluster in the HBV core promoter independent of retinoid X receptor. This demonstrates that calcitriol inhibits HBV core promoter activity through a noncanonical calcitriol-activated VDR pathway. Finally, we observed that calcitriol suppressed expression of the canonical HBV core promoter transcripts, pregenomic RNA, and precore RNA in multiple HBV cell culture models. In addition, calcitriol inhibited the secretion of hepatitis B "e" antigen and hepatitis B surface antigen (HBV-encoded proteins linked to poor disease prognosis), without affecting virion secretion. Our findings identify VDR as a novel regulator of HBV core promoter activity and also explain at least in part the correlation of vitamin D levels to HBV activity observed in clinical studies. Furthermore, this study has implications on the potential use of vitamin D along with anti-HBV therapies, and lays the groundwork for studies on vitamin D-mediated regulation of viruses through VDREs in virus promoters.

Core promoter mutation contributes to abnormal gene expression in bladder cancer.

BACKGROUND: Bladder cancer is one of the most mortal cancers. Bladder cancer has distinct gene expression signature, highlighting altered gene expression plays important roles in bladder cancer etiology. However, the mechanism for how the regulatory disorder causes the altered expression in bladder cancer remains elusive. Core promoter controls transcriptional initiation. We hypothesized that mutation in core promoter abnormality could cause abnormal transcriptional initiation thereby the altered gene expression in bladder cancer. METHODS: In this study, we performed a genome-wide characterization of core promoter mutation in 77 Spanish bladder cancer cases. RESULTS: We identified 69 recurrent somatic mutations in 61 core promoters of 62 genes and 28 recurrent germline mutations in 20 core promoters of 21 genes, including TERT, the only gene known with core promoter mutation in bladder cancer, and many oncogenes and tumor suppressors. From the RNA-seq data from bladder cancer, we observed  altered expression of the core promoter-mutated genes. We further validated the effects of core promoter mutation on gene expression by using luciferase reporter gene assay. We also identified potential drugs targeting the core promoter-mutated genes. CONCLUSIONS: Data from our study highlights that core promoter mutation contributes to bladder cancer development through altering gene expression.

Molecular evaluation of hepatitis B virus infection and predominant mutations of pre-core, basal core promoter and S regions in an Iranian population with type 2 diabetes mellitus: a case-control study.

BACKGROUND: This study was designed to evaluate the prevalence, genotypic patterns, and predominant mutations of hepatitis B virus (HBV) infection among diabetic patients. METHODS: Serum samples were obtained from 733 patients with type 2 diabetes mellitus and 782 non-diabetic controls. The presence of HBsAg and HBcAb was determined by ELISA. Nested PCR, targeting S and pre-core regions of the HBV genome, followed by sequencing was carried out to determine HBV genotypes and predominant mutations in the S, basal core promoter (BCP), and pre-core regions of the HBV genome. RESULTS: Of 733 diabetic patients, 94 cases (12.82%) were positive for HBcAb, 28 cases (3.82%) were positive for HBsAg, and 19 cases (2.59%) had HBV-DNA with genotype D, sub-genotype D1/D3 and subtype ayw2. An occult HBV infection was found in one of the HBV DNA-positive samples, which was positive for HBcAb but negative for HBsAg. P120T/G145R, G1896A/G1899A, and A1762T/G1764T were the most frequent point substitution mutations detected in the S, pre-core, and BCP regions of the HBV genome, respectively. P120T and G145R mutations were associated with low levels or undetectable levels of HBsAg in serum. Therefore, routine tests based on HBsAg detection cannot detect HBsAg-negative infected patients. CONCLUSIONS: Relatively high prevalence of HBV infection was found in diabetic patients, while all of the HBV-infected patients were unaware of their infection. Therefore, screening for HBV infection should be included in the management program of diabetes for timely diagnosis and treatment of infected but asymptomatic patients.

Involvement of TFAP2A in the activation of GSDMD gene promoter in hyperoxia-induced ALI.

Oxygen therapy is a common treatment in neonatal intensive care units, but long-term continuous hyperoxia ventilation may induce acute lung injury (ALI). Gasdermin D (GSDMD)-mediated pyroptosis participates in various diseases including ALI, but the role of GSDMD in hyperoxia-induced ALI is yet understood. Here, we showed a significant increase in GSDMD after exposure to high oxygen. To elucidate the molecular mechanisms involved in GSDMD regulation, we identified the core promoter of GSDMD, -98 ~ -12 bp relative to the transcriptional start site (TSS). The results of mutational analysis, overexpression or siRNA interference, EMSA and ChIP demonstrated that E2F4 and TFAP2A positively regulate the transcriptional activity of the GSDMD by binding to its promoter. However, only TFAP2A showed a regulatory effect on the expression of GSDMD. Moreover, TFAP2A was increased in the lung tissues of rats exposed to hyperoxia and showed a strong linear correlation with GSDMD. Our results indicated that TFAP2A positively regulates the GSDMD expression via binding to the promoter region of GSDMD.

Transcription initiation of distant core promoters in a large-sized genome of an insect.

BACKGROUND: Core promoters have a substantial influence on various steps of transcription, including initiation, elongation, termination, polyadenylation, and finally, translation. The characterization of core promoters is crucial for exploring the regulatory code of transcription initiation. However, the current understanding of insect core promoters is focused on those of Diptera (especially Drosophila) species with small genome sizes. RESULTS: Here, we present an analysis of the transcription start sites (TSSs) in the migratory locust, Locusta migratoria, which has a genome size of 6.5 Gb. The genomic differences, including lower precision of transcription initiation and fewer constraints on the distance from transcription factor binding sites or regulatory elements to TSSs, were revealed in locusts compared with Drosophila insects. Furthermore, we found a distinct bimodal log distribution of the distances from the start codons to the core promoters of locust genes. We found stricter constraints on the exon length of mRNA leaders and widespread expression activity of the distant core promoters in locusts compared with fruit flies. We further compared core promoters in seven arthropod species across a broad range of genome sizes to reinforce our results on the emergence of distant core promoters in large-sized genomes. CONCLUSIONS: In summary, our results provide novel insights into the effects of genome size expansion on distant transcription initiation.