Putative Transcriptional Regulatory Elements Research Articles

De Novo Genome Assemblies From Two Indigenous Americans from Arizona Identify New Polymorphisms in Non-Reference Sequences.

There is a collective push to diversify human genetic studies by including underrepresented populations. However, analyzing DNA sequence reads involves the initial step of aligning the reads to the GRCh38/hg38 reference genome which is inadequate for non-European ancestries. In this study, using long-read sequencing technology, we constructed de novo genome assemblies from two indigenous Americans from Arizona (IAZ). Each assembly included ∼17 Mb of DNA sequence not present [nonreference sequence (NRS)] in hg38, which consists mostly of repeat elements. Forty NRSs totaling 240 kb were uniquely anchored to the hg38 primary assembly generating a modified hg38-NRS reference genome. DNA sequence alignment and variant calling were then conducted with whole-genome sequencing (WGS) sequencing data from 387 IAZ using both the hg38 and modified hg38-NRS reference maps. Variant calling with the hg38-NRS map identified ∼50,000 single-nucleotide variants present in at least 5% of the WGS samples which were not detected with the hg38 reference map. We also directly assessed the NRSs positioned within genes. Seventeen NRSs anchored to regions including an identical 187 bp NRS found in both de novo assemblies. The NRS is located in HCN2 79 bp downstream of Exon 3 and contains several putative transcriptional regulatory elements. Genotyping of the HCN2-NRS revealed that the insertion is enriched in IAZ (minor allele frequency = 0.45) compared to other reference populations tested. This study shows that inclusion of population-specific NRSs can dramatically change the variant profile in an underrepresented ethnic groups and thereby lead to the discovery of previously missed common variations.

Mapping Transcription Regulation with Run-on and Sequencing Data Using the Web-Based tfTarget Gateway.

Run-on and sequencing assays like GRO-seq, PRO-seq, and ChRO-seq allow for joint profiling of transcription activity of transcriptional regulatory elements (TREs), i.e., promoters and active enhancers, and target genes. Variation in biological conditions, such as treated vs. control, results in changes in the activity of transcription factors (TFs), which induces concerted changes in TREs and target genes. By modeling the differences between two biological conditions, we developed the computational pipeline known as tfTarget that predicts a set of putative TREs and target genes responding to each TF under the biological condition of interest. In this chapter, we demonstrate the use of the new web-based tfTarget in mapping transcription regulation using run-on sequencing data.

Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

Coordinated transcriptional control of adipocyte triglyceride lipase (Atgl) by transcription factors Sp1 and peroxisome proliferator–activated receptor γ (PPARγ) during adipocyte differentiation

The breakdown of stored fat deposits into its components is a highly regulated process that maintains plasma levels of free fatty acids to supply energy to cells. Insulin-mediated transcription of Atgl, the enzyme that mediates the rate-limiting step in lipolysis, is a key point of this regulation. Under conditions such as obesity or insulin resistance, Atgl transcription is often misregulated, which can contribute to overall disease progression. The mechanisms by which Atgl is induced during adipogenesis are not fully understood. We utilized computational approaches to identify putative transcriptional regulatory elements in Atgl and then tested the effect of these elements and the transcription factors that bind to them in cultured preadipocytes and mature adipocytes. Here we report that Atgl is down-regulated by the basal transcription factor Sp1 in preadipocytes and that the magnitude of down-regulation depends on interactions between Sp1 and peroxisome proliferator-activated receptor γ (PPARγ). In mature adipocytes, when PPARγ is abundant, PPARγ abrogated transcriptional repression by Sp1 at the Atgl promoter and up-regulated Atgl mRNA expression. Targeting the PPARγ-Sp1 interaction could be a potential therapeutic strategy to restore insulin sensitivity by modulating Atgl levels in adipocytes.

Isolation, expression analysis and characterization of NEFA-interacting nuclear protein 30 and RING finger and SPRY domain containing 1 in skeletal muscle

Muscle atrophy results from a range of physiological conditions, including immobilization, spinal cord damage, inflammation and aging. In this study we describe two genes, NEFA-interacting nuclear protein 30 (Nip30) and RING Finger and SPRY domain containing 1 (Rspry1), which have not previously been characterized or shown to be expressed in skeletal muscle. Furthermore, Nip30 and Rspry1 were transcriptionally induced in response to neurogenic muscle wasting in mice and were also found to be expressed endogenously at the RNA and protein level in C2C12 mouse muscle cells. Interestingly, during analysis of Nip30 and Rspry1 it was observed that these genes share a 230 base pair common regulatory region that contains several putative transcription regulatory elements. In order to assess the transcriptional activity of the Nip30 and Rspry1 regulatory regions, a fragment of the promoter of each gene was cloned, fused to a reporter gene, and transfected into cells. The Nip30 and Rspry1 reporters were both found to have significant transcriptional activity in cultured cells. Furthermore, the Nip30-Rspry1 common regulatory region contains a conserved E-box enhancer, which is an element bound by myogenic regulatory factors that function in the regulation of muscle-specific gene expression. Therefore, in order to determine if the predicted E-box was functional, Nip30 and Rspry1 reporters were transfected into cells ectopically expressing the myogenic regulatory factor, MyoD1, resulting in significant induction of both reporter genes. In addition, mutation of the conserved E-box element eliminated MyoD1 activation of the Nip30 and Rspry1 reporters. Finally, GFP-tagged Nip30 was found to localize to the nucleus, while GFP-tagged Rspry1 was found to localize to the cytoplasm of muscle cells.

Functional analysis of 3’UTR polymorphisms in the caprine SLC11A1 gene and its association with the Mycobacterium avium subsp. paratuberculosis infection

The study aimed to investigate whether the genetic polymorphisms in the 3′UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3’end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3’UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level.

Bioinformatic analysis reveals an evolutional selection for DNA:RNA hybrid G-quadruplex structures as putative transcription regulatory elements in warm-blooded animals

Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both in vitro and in vivo conditions. Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand.

Genome-wide changes in expression profile of murine endogenous retroviruses (MuERVs) in distant organs after burn injury

BackgroundPrevious studies have shown that burn-elicited stress signals alter expression of certain murine endogenous retroviruses (MuERVs) in distant organs of mice. These findings suggest that MuERVs may participate in a network of pathophysiologic events during post-burn systemic response. To gain a better understanding of the biological roles of MuERVs in post-burn systemic response, we examined the genome-wide changes in the MuERV expression profiles in distant organs and the biological properties of the putative-burn related MuERVs were characterized.ResultsFemale C57BL/6J mice were subjected to an approximately 18 % total body surface area flame burn and tissues (liver, lung, and kidney) were harvested at 3 hours and 24 hours after injury. The changes in the MuERV expression profiles in these tissues were examined by RT-PCR using a primer set flanking the non-ecotropic MuERV U3 promoter region within the 3' long terminal repeat. There were differential changes in the expression profiles of MuERV U3 regions after injury in all three tissues examined. Subsequently, a total of 31 unique U3 promoter sequences were identified from the tissues of both burn and no burn mice. An analysis of viral tropisms revealed that putative MuERVs harboring these U3 promoter sequences were presumed to be either xenotropic or polytropic. Some putative transcription regulatory elements were present predominantly in U3 promoter sequences isolated from burn and no burn mice, respectively. In addition, in silico mapping using these U3 sequences as a probe against the mouse genome database identified 59 putative MuERVs. The biological properties (coding potentials for retroviral polypeptides, primer binding sites, tropisms, branching ages, recombination events, and neighboring host genes) of each putative MuERV were characterized. In particular, 16 putative MuERVs identified in this study retained intact coding potentials for all three retroviral polypeptides (gag, pol, and env). None of the putative MuERVs identified in this study were mapped to the coding sequences of host genes.ConclusionIn this study, we identified and characterized putative MuERVs whose expression might be altered in response to burn-elicited systemic stress signals. Further investigation is needed to understand the role of these MuERVs in post-burn systemic pathogenesis, in particular, via characterization of their interaction with host genes, MuERV gene products, and viral activities.

Molecular characterization of three forms of putative membrane-bound progestin receptors and their tissue-distribution in channel catfish, Ictalurus punctatus

Membrane-bound progestin receptors (mPR) were recently cloned and characterized as a new class of steroid receptors that transduce cell-signals through alteration of MAP kinase- and cAMP-dependent pathways. To further develop our understanding of this new class of steroid receptors, we characterized the cDNAs and genes of thealpha, beta and gamma forms of the channel catfish mPRs (IpmPR). The predicted alpha and beta proteins have 49% sequence identity, whereas they only have 30% and 27% identities, respectively, with the gamma form. Furthermore, IpmPRalpha and IpmPRbeta genes have similar structures featuring intronless coding regions, while IpmPRgamma gene is composed of 8 exons and 7 introns. The 5'-flanking region of each IpmPR gene differs, but each contains putative transcriptional regulatory elements of factors known to influence reproductive physiology and endocrine disruption, for example, responsive elements for cAMP and steroids and the recognition sites for steroidogenic factor-1 and for the aryl hydrocarbon receptor. The IpmPRalpha gene was detected in all the tissues tested with relatively greater expression in brain, pituitary, muscle and testis. The expression of IpmPRbeta was much lower than that of IpmPRalpha and the transcript was predominantly observed in brain, pituitary, ovary and testis. In contrast, the IpmPRgamma transcript was mainly detected in gill, ventral aorta, intestine, and trunk kidney. In conclusion, all the structural features of the IpmPRs strongly suggest that the closely related alpha and beta forms are distantly related to the gamma form. Additionally, regulatory features of the 5'-flanking regions and the differences in tissue-specific expression of each IpmPR gene suggest that they are involved in different endocrine functions in catfish.

Genomic organization and characterization of the promoter of rat malonyl-CoA decarboxylase gene

Malonyl-CoA decarboxylase (MCD) catalyzes the decarboxylation of malonyl-CoA, an elongating agent for fatty acid synthesis and also known as a fuel-sensing mediator. In order to elucidate the genome organization, we isolated a 2020 bp rat MCD cDNA from rat brain cDNA library and isolated the corresponding rat genomic clones from the rat genomic PAC library. Sequencing and comparison of these clones showed that the MCD genome consists of five exons and four introns spanning approximately 17 kb. The proximal upstream region is GC-rich, lacks a TATA box, and contains a variety of putative transcriptional regulatory elements within 2 kb. A major transcriptional initiation site was identified by a primer extension at a site 157 nucleotides upstream of the translational initiation site. To investigate the transcriptional regulation of MCD, a series of 5′-deletion constructs of the 5′-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in CV-1 cells, we suggest that an area of −15 bp 5′ from the first exon acted as a basal promoter for MCD and that there are positive cis-regulatory elements in the region from −55 to −325 bp and negative regulator elements in the region −1380 to −2240 bp.

Characterization of mouse Atp6i gene, the gene promoter, and the gene expression.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-type ATPase. We previously cloned a gene encoding a putative osteoclast-specific proton pump subunit, termed OC-116 kDa, approved mouse Atp6i (ATPase, H+ transporting, [vacuolar proton pump] member I). The function of Atp6i as osteoclast-specific proton pump subunit was confirmed in our mouse knockout study. However, the transcription regulation of Atp6i remains largely unknown. In this study, the gene encoding mouse Atp6i and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of Atp6i have been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning approximately 11 kilobases (kb) of genomic DNA. Alignment of the mouse Atp6i gene exon sequence and predicted amino acid sequence to that of the human reveals a strong homology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Met codon. Analysis of 4 kb of the putative promoter region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements. Northern blot analysis of RNAs from a number of mouse tissues reveals that Atp6i is expressed predominantly in osteoclasts, and this predominant expression was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical analysis. Whole-mount in situ hybridization shows that Atp6i expression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic development (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5. The expression level of Atp6i is largely reduced after E10.5. This is the first report of the characterizations of Atp6i gene, its promoter, and its gene expression patterns during mouse development. This study may provide valuable insights into the function of Atp6i, its osteoclast-selective expression, regulation, and the molecular mechanisms responsible for osteoclast activation.

Sequence and structural analysis of feline interleukin-5 cDNA

Abstract Objective To clone and characterize the cDNA encoding feline interleukin-5 (IL-5) cDNA and the 170 basepairs (bp) of the 5' flanking region of the feline IL-5 gene. Sample Population Blood mononuclear cells from a healthy cat. Procedures Cells were cultured, stimulated for 48 hours with concanavalin A, and harvested for RNA and DNA isolation. Recovered RNA was used in northern blot and reverse transcription-polymerase chain reaction analyses. Resulting cDNA was used for rapid amplification of 3' cDNA ends, dideoxy chain termination sequencing, and primer extension analysis. Results Full length cDNA was 838 bp, including a 402-bp open reading frame that encoded a precursor protein of 134 amino acids including a putative peptide signal of 19 residues. Homologies of the nucleotide and derived protein sequences between feline and human IL-5 cDNA were 72 and 71%, respectively. There also was homology between the human and predicted feline cytokines at amino acid positions that are critical for IL-5 receptor binding and signal transduction. The 5' flanking region of the feline gene was homologous to corresponding regions of the human (88%) and murine (72%) genes, and included putative transcriptional regulatory elements. Conclusions and Clinical Relevance Identification of feline IL-5 cDNA is an important step toward a detailed, fully comprehensive characterization of the mechanisms that may be operative in the pathogenesis of eosinophilic disorders in cats. The striking homology between the human and feline IL-5 genes suggests that cats can be used as animal models for human diseases characterized by eosinophil infiltration of tissues. (Am J Vet Res 1998;59:1263–1269)

Analysis of the 5′ Sequence, Genomic Structure, and Alternative Splicing of thepresenilin-1Gene (PSEN1) Associated with Early Onset Alzheimer Disease

Mutations in the human presenilin genes (PSEN1andPSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5′ upstream sequence and the structure of thePSEN1gene reveals that the 5′ sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5′UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem–loop structure with stabilities greater than −16 kcal/mol.

Isolation of a promoter region in mouse cytochrome P450 3A ( Cyp3A16) gene and its transcriptional control

An 11.5 kb fragment of the mouse Cyp3a16 gene containing the 5′ flanking region was isolated from the λDASHII mouse genomic library. A part of the 5′ flanking region and the first exon of Cyp3a16 gene were sequenced. S1 mapping analysis showed the presence of two transcriptional initiation sites. The first exon was completely identical to Cyp3a16 cDNA. The identity of 5′ flanking sequences between Cyp3a16 and Cyp3a11 genes was about 69%. A typical TATA box and a basic transcription element (BTE) were found as seen with other CYP3A genes from various animal species. Moreover, some putative transcriptional regulatory elements were also found in addition to the sequence motif seen for the formation of Z-type DNA. To examine the transcriptional activity of Cyp3a11 gene, DNA fragments in the 5′-flanking region of the gene were inserted front of the luciferase structural gene, and the constructs were transfected in primary hepatocytes. The analysis of the luciferase activity indicated that the region between −146 and −56 was necessary for the transcription of CYP3a16 gene.

Molecular cloning and sequence analysis of a seed-expressed acyl carrier protein (ACP) gene from Brassica campestris (Agrani)

We describe here the nucleotide sequence of the Brassica campestris ACPSF1 gene which encodes a seed-expressed acyl carrier protein (ACP). The 3600 by sequence consists of 1740 by upstream of the translation start codon, 828 by spanning the coding region which is interrupted by three introns and 1032 by downstream of the stop codon. Using a ACPSF1 gene-specific probe, transcripts could be detected in developing seeds, but not in leaves. This gene is now the only known member that represents group I seed-expressed ACP multigene family of Brassica species. The 5′ flanking sequence of the ACPSF1 gene was examined for putative transcriptional regulatory elements. A sequence alignment of the 5′ flanking regions of the available seed-expressed ACP genes of Brassica species showed some conserved regions which might have some common regulatory significance.

Nucleotide sequence and analysis of the mouse SPC3 promoter region

Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in β cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5′-upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non-functional TATA-like sequences and several putative transcriptional regulatory elements, including AP-1, Spl and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In βTC3 insulinoma cells, the sequence between the EcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).

Isolation and sequence of a rat glucose-6-phosphate dehydrogenase promoter

A 935 bp fragment of the rat glucose-6-phosphate dehydrogenase (G6PDH) gene containing promoter activity was isolated using the polymerase chain reaction (PCR). This fragment was sequenced and primer extension analysis showed a transcription initiation site in agreement with the human and mouse genes. Computer analysis of the sequence showed a 60% and 78% similarity to the human and mouse G6PDH sequences, respectively. A TATA box element, TTAAAT, was found and shown to be 100% similar to the human and mouse TATA box elements. Based on sequence comparison, some putative transcriptional regulatory elements were also found.

Structure of the human apolipoprotein D gene promoter region

A human genomic clone of 18.2 kbp encompassing the apolipoprotein D (apoD) exon-1 and 2 and 10 kbp of upstream sequence was isolated and characterized. DNA sequencing and primer extension analysis revealed a transcriptional initiation site located 27 bp downstream of a consensus TATA box sequence. The exon 1 was 66 bp long. Computer analysis of DNA sequence from positions −557 to +129 revealed some putative transcriptional regulatory elements including a stretch of (pyrimidine/purine) 26 located from nucleotide −263 to nucleotide −212, which could potentially form Z-DNA. Steroid hormone regulatory elements were identified which may be related to the modulation of apoD gene expression by androgens and estrogens in vitro.

A matched set of cat vectors for rapid mutational analysis of eukaryotic promoters and enhancers

The eukaryotic cat expression vectors, pBRAMS cat1 and pBRAMS cat2, were constructed to simplify the analysis of genomic fragments containing putative transcriptional regulatory elements. These vectors contain the f1 filamentous phage origin of replication for single-stranded DNA rescue, and permit site-directed mutagenesis, and dideoxy sequencing of nested deletion mutants using commercial T3, T7 and M13 universal forward/reverse primers. The above features eliminate the need to shuttle back and forth between a conventional cloning vector and the cat expression vector during the analysis of putative eukaryotic gene regulatory elements. Plasmid pBRAMS cat1 contains the bacterial chloramphenicol acetyltransferaseencoding gene ( cat) and no eukaryotic promoter and was designed for the analysis of eukaryotic promoters. Plasmid pBRAMS cat2 contains the cat gene under the control of the Herpes simplex virus thymidine kinase promoter and was designed for the analysis of eukaryotic enhancers.