Characterization Of Cis-regulatory Elements Research Articles

A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Identification and Characterization of Cis-Regulatory Elements in the Human Genome

Identification and Characterization of Cis-Regulatory Elements in the Human Genome

Identification and characterization of cis-regulatory elements 'insulator and repressor' in PPARD gene.

Identification and functional characterization of cis-regulatory elements in human PPARD gene. We used various bioinformatic tools on the publicly available human genome and Encyclopedia of DNA Elements databases to explore potential cis-regulatory elements in PPARD gene region. We predicted an insulator and an enhancer element in intron 2 of PPARD gene. Functional characterization using transient transfection, reporter assay and CTCF binding confirmed the insulator status. However, the predicted enhancer element showed repressor/silencer activity. Finally, we observed a potential interaction between these two cis-regulatory elements which is in agreement with 5C-Encyclopedia of DNA Elements data. We report two functionally validated cis-regulatory elements in PPARD gene which will aid in understanding its regulation and role in metabolic functions.

Systems-wide Identification of cis-Regulatory Elements in Proteins

Protein interactions in cis that can activate or autoinhibit protein function play an important role in the fine-tuning of regulatory and signaling processes in the cell, but thus far cis-regulatory elements (CREs) in proteins have not been systematically identified and studied. Here, we introduce a computational tool that identifies intrinsically disordered protein segments that contribute to protein function regulation via interactions in cis. We apply this tool to estimate the prevalence of CREs in the human proteome and reveal that cis regulation is enriched in several signaling pathways, including the MAP kinase pathway, for which we provide a detailed map of its "cis regulome." We also show that disease-causing mutations are highly enriched in CREs, but not in motifs that classically mediate protein-protein interactions of disordered protein segments. Our approach should facilitate the discovery and characterization of CREs in proteins and the identification of disease-causing mutations that disrupt protein regulation in cis.

Non-coding transcription at cis-regulatory elements: Computational and experimental approaches

Mammalian genomes are pervasively transcribed, generating mostly RNAs with no coding potential that display different size, structure and interspecies sequence conservation. A prominent contribution to the ncRNA pool comes from the transcription of cis-regulatory elements, namely promoters, enhancers and locus control regions. While this phenomenon has been extensively documented, possible roles of such ncRNAs in gene regulation are still unclear. Addressing this issue will require experimental strategies dealing with the low abundance of enhancer-templated ncRNAs and aimed at specifically dissecting the relative role of transcription per se vs. RNA products.In this review, we first focus on the identification and characterization of cis-regulatory elements, highlighting the differences between emerging classes of ncRNAs associated to specific chromatin signatures. We then discuss current experimental strategies to dissect the function of nc transcription and computational approaches to the analysis and classification of regulatory sequences identified in next-generation sequencing experiments.

Characterization of cis-regulatory elements that control Pax7 expression during neural crest cell development

Characterization of cis-regulatory elements that control Pax7 expression during neural crest cell development

Characterization of human Collagen XVIII promoter 2: Interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides -186 and -21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position -700 (T>G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele -700G increases transcription and binding for a still unknown transcription factor. SNP -700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide -700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.

Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii

The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5′-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides −1245 to −625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides −1929 to −1067 and −456 to −222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii.

Definition of Regulatory Sequence Elements in the Promoter Region and the First Intron of the Myotonic Dystrophy Protein Kinase Gene

Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3'-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5' to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Co-transfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10-20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to up-regulate transcription of this gene in myoblasts.