Critical Cis-regulatory Elements Research Articles

Genome-wide enhancer identification by massively parallel reporter assay in Arabidopsis.

Enhancers are critical cis-regulatory elements controlling gene expression during cell development and differentiation. However, genome-wide enhancer characterization has been challenging due to the lack of a well-defined relationship between enhancers and genes. Function-based methods are the gold standard for determining the biological function of cis-regulatory elements, however, these methods have not been widely applied to plants. Here, we applied a massively parallel reporter assay on Arabidopsis to measure enhancer activities across the genome. We identified 4327 enhancers with various combinations of epigenetic modifications distinctively different from animal enhancers. Furthermore, we showed that enhancers differ from promoters in their preference for transcription factors. Although some enhancers are not conserved and overlap with transposable elements forming clusters, enhancers are generally conserved across thousand Arabidopsis accessions, suggesting they are selected under evolution pressure and could play critical roles in the regulation of important genes. Moreover, comparison analysis reveals that enhancers identified by different strategies do not overlap, suggesting these methods are complementary in nature. In sum, we systematically investigated enhancer features in A. thaliana with enhancers identified by functional assay, which lays the foundation for further investigation into enhancers' functional mechanisms in plants.

Single-cell chromatin accessibility profiling of cell-state-specific gene regulatory programs during mouse organogenesis.

In mammals, early organogenesis begins soon after gastrulation, accompanied by specification of various type of progenitor/precusor cells. In order to reveal dynamic chromatin landscape of precursor cells and decipher the underlying molecular mechanism driving early mouse organogenesis, we performed single-cell ATAC-seq of E8.5-E10.5 mouse embryos. We profiled a total of 101,599 single cells and identified 41 specific cell types at these stages. Besides, by performing integrated analysis of scATAC-seq and public scRNA-seq data, we identified the critical cis-regulatory elements and key transcription factors which drving development of spinal cord and somitogenesis. Furthermore, we intersected accessible peaks with human diseases/traits-related loci and found potential clinical associated single nucleotide variants (SNPs). Overall, our work provides a fundamental source for understanding cell fate determination and revealing the underlying mechanism during postimplantation embryonic development, and expand our knowledge of pathology for human developmental malformations.

Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.

A Genome-Wide Analysis of Pathogenesis-Related Protein-1 (PR-1) Genes from Piper nigrum Reveals Its Critical Role during Phytophthora capsici Infection.

Black pepper (Piper nigrum L.) is a prominent spice that is an indispensable ingredient in cuisine and traditional medicine. Phytophthora capsici, the causative agent of footrot disease, causes a drastic constraint in P. nigrum cultivation and productivity. To counterattack various biotic and abiotic stresses, plants employ a broad array of mechanisms that includes the accumulation of pathogenesis-related (PR) proteins. Through a genome-wide survey, eleven PR-1 genes that belong to a CAP superfamily protein with a caveolin-binding motif (CBM) and a CAP-derived peptide (CAPE) were identified from P. nigrum. Despite the critical functional domains, PnPR-1 homologs differ in their signal peptide motifs and core amino acid composition in the functional protein domains. The conserved motifs of PnPR-1 proteins were identified using MEME. Most of the PnPR-1 proteins were basic in nature. Secondary and 3D structure analyses of the PnPR-1 proteins were also predicted, which may be linked to a functional role in P. nigrum. The GO and KEGG functional annotations predicted their function in the defense responses of plant-pathogen interactions. Furthermore, a transcriptome-assisted FPKM analysis revealed PnPR-1 genes mapped to the P. nigrum-P. capsici interaction pathway. An altered expression pattern was detected for PnPR-1 transcripts among which a significant upregulation was noted for basic PnPR-1 genes such as CL10113.C1 and Unigene17664. The drastic variation in the transcript levels of CL10113.C1 was further validated through qRT-PCR and it showed a significant upregulation in infected leaf samples compared with the control. A subsequent analysis revealed the structural details, phylogenetic relationships, conserved sequence motifs and critical cis-regulatory elements of PnPR-1 genes. This is the first genome-wide study that identified the role of PR-1 genes during P. nigrum-P. capsici interactions. The detailed in silico experimental analysis revealed the vital role of PnPR-1 genes in regulating the first layer of defense towards a P. capsici infection in Panniyur-1 plants.

Adenine base editing of the DUX4 polyadenylation signal for targeted genetic therapy in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation of the D4Z4 repeat resulting in misexpression of the D4Z4-encoded DUX4 gene in skeletal muscle. One of the key genetic requirements for the stable production of full-length DUX4 mRNA in skeletal muscle is a functional polyadenylation signal (ATTAAA) in exon three of DUX4 that is used in somatic cells. Base editors hold great promise to treat DNA lesions underlying genetic diseases through their ability to carry out specific and rapid nucleotide mutagenesis even in postmitotic cells such as skeletal muscle. In this study, we present a simple and straightforward strategy for mutagenesis of the somatic DUX4 polyadenylation signal by adenine base editing in immortalized myoblasts derived from independent FSHD-affected individuals. We show that mutating this critical cis-regulatory element results in downregulation of DUX4 mRNA and its direct transcriptional target genes. Our findings identify the somatic DUX4 polyadenylation signal as a therapeutic target and represent the first step toward clinical application of the CRISPR-Cas9 base editing platform for FSHD gene therapy.

Multifaceted genome-wide study identifies novel regulatory loci in SLC22A11 and ZNF45 for body mass index in Indians.

Obesity, a risk factor for multiple diseases (e.g. diabetes, hypertension, cancers) originates through complex interactions between genes and prevailing environment (food habit and lifestyle) that varies across populations. Indians exhibit a unique obesity phenotype with high abdominal adiposity for a given body weight compared to matched white populations suggesting presence of population-specific genetic and environmental factors influencing obesity. However, Indian population-specific genetic contributors for obesity have not been explored yet. Therefore, to identify potential genetic contributors, we performed a two-staged genome-wide association study (GWAS) for body mass index (BMI), a common measure to evaluate obesity in 5973 Indian adults and the lead findings were further replicated in 1286 Indian adolescents. Our study revealed novel association of variants-rs6913677 in BAI3 gene (p = 1.08 × 10-8) and rs2078267 in SLC22A11 gene (p = 4.62 × 10-8) at GWAS significance, and of rs8100011 in ZNF45 gene (p = 1.04 × 10-7) with near GWAS significance. As genetic loci may dictate the phenotype through modulation of epigenetic processes, we overlapped genetic data of identified signals with their DNA methylation patterns in 236 Indian individuals and performed methylation quantitative trait loci (meth-QTL) analysis. Further, functional roles of discovered variants and underlying genes were speculated using publicly available gene regulatory databases (ENCODE, JASPAR, GeneHancer, GTEx). The identified variants in BAI3 and SLC22A11 genes were found to dictate methylation patterns at unique CpGs harboring critical cis-regulatory elements. Further, BAI3, SLC22A11 and ZNF45 variants were located in repressive chromatin, active enhancer, and active chromatin regions, respectively, in human subcutaneous adipose tissue in ENCODE database. Additionally, these genomic regions represented potential binding sites for key transcription factors implicated in obesity and/or metabolic disorders. Interestingly, GTEx portal identify rs8100011 as a robust cis-expression quantitative trait locus (cis-eQTL) in subcutaneous adipose tissue (p = 1.6 × 10-7), and ZNF45 gene expression in skeletal muscle of Indian subjects showed an inverse correlation with BMI indicating its possible role in obesity. In conclusion, our study discovered 3 novel population-specific functional genetic variants (rs6913677, rs2078267, rs8100011) in 2 novel (SLC22A11 and ZNF45) and 1 earlier reported gene (BAI3) for BMI in Indians. Our study decodes key genomic loci underlying obesity phenotype in Indians that may serve as prospective drug targets in future.

CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.

The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1–2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5′UTR sequences crucial for chinmo's post-transcriptional regulation.

Abstract 17134: Variation Within a Left Ventricle-Specific Hand1 Enhancer Impairs Gata Transcription Factor Binding and Disrupts Ventricular Conduction System Development

The ventricular conduction system (VCS), composed of the His bundle, the left and right bundle branches, and the Purkinje fiber network, rapidly propagates electrical impulses through the ventricles to coordinate chamber contraction. An ECG depicts VCS-mediated ventricular depolarization as the QRS interval. Disorders of the VCS may manifest as arrhythmias, and are associated with increased risk of sudden cardiac death and overall mortality. The genetic and developmental mechanisms underlying VCS dysfunction are poorly understood. Genome-wide association studies have identified multiple single nucleotide polymorphisms (SNPs) associated with VCS arrhythmias. A majority of these SNPs occur outside of coding domains, within intergenic or intronic regions. We hypothesize that such pathogenic SNPs may impact VCS development and function by altering the expression of critical genes. Two intergenic SNPs associated with QRS prolongation occur near the bHLH cardiac transcription factor HAND1 . We have identified a left ventricle (LV) enhancer, located between these two SNPs, that is necessary and sufficient for LV cis -regulatory activity. Two evolutionarily conserved GATA transcription factor consensus-binding sites within this enhancer are bound by GATA4 and necessary for cis -regulatory activity. CRISPR-mediated deletion of this enhancer dramatically reduced Hand1 expression solely within the LV. Mice homozygous for this deleted enhancer displayed dysregulated LV gene expression, morphologically abnormal His bundles and left bundle branches, and a VCS phenotype consistent with right bundle branch block. Genome-wide association analyses of human patients with QRS prolongation revealed additional, linked SNPs, one of which overlaps with a critical GATA cis -regulatory element within the LV-specific Hand1 enhancer. This SNP disrupts GATA4 binding. Ongoing studies of mice that genetically mimic these minor SNP variants will assess reduced Hand1 expression and impaired VCS function. We conclude that a LV-specific Hand1 enhancer is necessary for VCS development and a SNP associated with QRS prolongation directly influences GATA4 binding to a critical cis -regulatory element within this enhancer.

C/ebpα controls osteoclast terminal differentiation, activation, function, and postnatal bone homeostasis through direct regulation of Nfatc1.

Osteoclast lineage commitment and differentiation have been studied extensively, although the mechanism by which transcription factor(s) control osteoclast terminal differentiation, activation, and function remains unclear. CCAAT/enhancer-binding protein α (C/ebpα) has been reported to be a key regulator of osteoclast cell lineage commitment, yet C/ebpα's roles in osteoclast terminal differentiation, activation and function, and bone homeostasis, under physiological or pathological conditions, have not been studied because newborn C/ebpα-null mice die within several hours after birth. Furthermore, the function of C/ebpα in osteoclast terminal differentiation, activation, and function is largely unknown. Herein, we generated and analyzed an osteoclast-specific C/ebpα conditional knockout (CKO) mouse model via Ctsk-Cre mice and found that C/ebpα-deficient mice exhibited a severe osteopetrosis phenotype due to impaired osteoclast terminal differentiation, activation, and function, including mildly reduced osteoclast number, impaired osteoclast polarization, actin formation, and bone resorption, which demonstrated the novel function of C/ebpα in cell function and terminal differentiation. Interestingly, C/ebpα deficiency did not affect bone formation or monocyte/macrophage development. Our results further demonstrated that C/ebpα deficiency suppressed the expression of osteoclast functional genes, e.g. encoding cathepsin K (Ctsk), Atp6i (Tcirg1), and osteoclast regulator genes, e.g. encoding c-fos (Fos), and nuclear factor of activated T-cells 1 (Nfatc1), while having no effect on Pu.1 (Spi1) expression. Promoter activity mapping and ChIP assay defined the critical cis-regulatory element (CCRE) in the promoter region of Nfatc1, and also showed that the CCREs were directly associated with C/ebpα, which enhanced the promoter's activity. The deficiency of C/ebpα in osteoclasts completely blocked ovariectomy-induced bone loss, indicating that C/ebpα is a promising new target for the treatment of osteolytic diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Monocyte-Specific Knockout of C/ebpα Results in Osteopetrosis Phenotype, Blocks Bone Loss in Ovariectomized Mice, and Reveals an Important Function of C/ebpα in Osteoclast Differentiation and Function.

CCAAT/enhancer-binding protein α (C/ebpα) is critical for osteoclastogenesis by regulating osteoclast (OC) lineage commitment and is also important for OC differentiation and function in vitro. However, the role of C/ebpα in postnatal skeletal development has not been reported owing to lethality in C/ebpα-/- mice from hypoglycemia within 8 hours after birth. Herein, we generated conditional knockout mice by deleting the C/ebpα gene in monocyte via LysM-Cre to examine its role in OC differentiation and function. C/ebpαf/f LysM-Cre mice exhibited postnatal osteopetrosis due to impaired osteoclastogenesis, OC lineage priming defects, as well as defective OC differentiation and activity. Furthermore, our ex vivo analysis demonstrated that C/ebpα conditional deletion significantly reduced OC differentiation, maturation, and activity while mildly repressing macrophage development. At the molecular level, C/ebpα deficiency significantly suppresses the expressions of OC genes associated with early stages of osteoclastogenesis as well as genes associated with OC differentiation and activity. We also identified numerous C/ebpα critical cis-regulatory elements on the Cathepsin K promoter that allow C/ebpα to significantly upregulate Cathepsin K expression during OC differentiation and activity. In pathologically induced mouse model of osteoporosis, C/ebpα deficiency can protect mice against ovariectomy-induced bone loss, uncovering a central role for C/ebpα in osteolytic diseases. Collectively, our findings have further established C/ebpα as a promising therapeutic target for bone loss by concurrently targeting OC lineage priming, differentiation, and activity. © 2017 American Society for Bone and Mineral Research.

Genome-wide methylome and chromatin interactome identify abnormal enhancer to be risk factor of breast cancer.

Enhancer is critical cis regulatory elements in gene expression. To understand whether and how the aberrant enhancer activation may contribute to cancer risk, the differentially methylated enhancers (eDMRs) in normal and malignant breast tissues were identified and analyzed. By incorporating genome-wide chromatin interaction, integrated analysis of eDMRs and target gene expression identified 1,272 enhancer-promoter pairs. Surprisingly, two functionally distinct groups of genes were identified in these pairs, one showing better correlation to enhancer methylation (eRGs) and the other showing better correlation to promoter methylation (pRGs), and the former group is functionally enriched with cancer related genes. Moreover, enhancer methylation based clustering of breast cancer samples is capable of discriminating basal breast cancer from other subtypes. By correlating enhancer methylation status to patient survival, 345 enhancers show the impact on the disease outcome and the majority of their target genes are important regulators of cell survival pathways including known cancer related genes. Together, these results suggest reactivation of enhancers in cancer cells has the add-on effect and contributes to cancer risk in combination.

Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells

Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.

RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes

A broad range of human leukemias carries RUNX1 and MLL genetic alterations. Despite such widespread involvements, the relationship between RUNX1 and MLL has never been appreciated. Recently, we showed that RUNX1 physically and functionally interacts with MLL, thereby regulating the epigenetic status of critical cis-regulatory elements for hematopoietic genes. This newly unveiled interaction between the two most prevalent leukemia genes has solved a long-standing conundrum: leukemia-associated RUNX1 N-terminal point mutants that exhibit no obvious functional abnormalities in classical assays for the assessment of transcriptional activities. These mutants turned out to be defective in MLL interaction and subsequent epigenetic modifications that can be examined by the histone-modification status of cis-regulatory elements in the target genes. RUNX1/MLL binding confirms the importance of RUNX1 function as an epigenetic regulator. Recent studies employing next-generation sequencing on human hematological malignancies identified a plethora of mutations in epigenetic regulator genes. These new findings would enhance our understanding on the mechanistic basis for leukemia development and may provide a novel direction for therapeutic applications. This review summarizes the current knowledge about the epigenetic regulation of normal and malignant hematopoiesis by RUNX1 and MLL.

Transcriptional networks driving enhancer function in the CFTR gene

A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.

Abstract 2922: Androgen receptor driven transcription in a subtype of estrogen receptor negative breast cancer is controlled by FoxA1

Abstract Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumors were recently identified as an additional subgroup, characterised as ERα- AR+, but with an expression profile that resembles ERα+ luminal breast cancer. Previously, we mapped global AR binding events in a cell line model of molecular apocrine breast cancer (termed MDA-MB-453 cells) using ChIP-seq. Our results suggested that these tumours are driven by AR through its interaction with critical ER cis-regulatory elements, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers. Additionally, we found that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a Nuclear Receptor. By silencing FoxA1 using specific siRNA, we observe dramatic changes in the gene expression profile of the cells (730 genes are differentially expressed) including many of the signature molecular apocrine genes. Furthermore, silencing of FoxA1 in these breast cancer cells results in altered AR binding to the chromatin (lost, conserved and de novo binding events are found) as has been previously seen in the prostate cancer cell line, LNCaP. We are investigating this phenomenon further using proteomics to discover new AR interacting proteins and histone mark ChIP-seq to understand the proteins and epigenetic events that may mediate the reprogrammed AR binding in the absence of FoxA1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2922. doi:1538-7445.AM2012-2922

ATF2 Interacts with β-Cell-enriched Transcription Factors, MafA, Pdx1, and Beta2, and Activates Insulin Gene Transcription

Pancreatic β-cell-restricted expression of insulin is established through several critical cis-regulatory elements located in the insulin gene promoter region. The principal cis elements are A-boxes, E1, and C1/RIPE3b. The β-cell-enriched transcription factors Pdx1 and Beta2 bind to the A-boxes and E1 element, respectively. A β-cell-specific trans-acting factor binding to C1/RIPE3b (termed RIPE3b1 activator) was detected by electrophoretic mobility shift assay and has been identified as MafA, a member of the Maf family of basic leucine zipper (bZip) proteins. Here, ATF2, a member of the ATF/CREB family of basic leucine zipper proteins, was identified as a component of the RIPE3b1 activator. ATF2 alone was unable to bind to the C1/RIPE3b element but acquired binding capacity upon complex formation with MafA. ATF2 also interacted with Pdx1 and Beta2, and co-expression of ATF2, MafA, Pdx1, and Beta2 resulted in a synergistic activation of the insulin promoter. Immunohistochemical analysis of mouse pancreas tissue sections showed that ATF2 is enriched in islet endocrine cells, including β-cells. RNAi-mediated knockdown of MafA or ATF2 in the MIN6 β-cell line resulted in a significant decrease in endogenous levels of insulin mRNA. These data indicate that ATF2 is an essential component of the positive regulators of the insulin gene expression.

Identification of a Spatio-Temporal Enhancer Element for the Alzheimer’sAmyloid Precursor Protein-Like-1Gene in the NematodeCaenorhabditis elegans

During the larva-to-adult transition of the nematode Caenorhabditis elegans, expression of the Alzheimer's amyloid precursor protein-like gene, apl-1, is temporally regulated in seam cells. This regulation requires several transcription factors, including nuclear receptor NHR-25. Here we describe a critical cis-regulatory element of apl-1 transcription. The element is conserved among Caenorhabditis sister species and is similar to nuclear receptor binding consensus.

Mammalian origins of replication: Paradigms lost.

The Chinese hamster dihydrofolate reductase (DHFR) origin consists of more than 30 potential initiation sites distributed throughout the 55 kb spacer between the convergently transcribed DHFR and 2BE2121 genes. Some of these sites are clearly more active than others, but no critical cis-regulatory elements appear to reside within the spacer (origin) itself. In contrast, deletion of either the promoter or the 3′ processing signals of the DHFR gene eliminates initiation in the downstream origin. To determine whether the DHFR initiation zone represents an exception rather than the rule, we have devised a gel trapping method that will allow us to isolate all of the active origins from genomes ranging in complexity from yeast to mammals. To date, we have made origin libraries from both hamster and human genomes. Surprisingly, fourteen of the fifteen anonymous origins that we have so far analyzed by a stringent 2-D gel replicon mapping procedure correspond to a zone of inefficient sites rather than to a fixed replicator such as those that characterize bacterial and viral origins. We propose that a hierarchy of inefficient initiation sites is distributed at intervals of a few kilobases or less throughout higher eukaryotic genomes, the activities of which are controlled largely by epigenetic effects such as local transcription and chromatin architecture. While some of these sites may have evolved into high efficiency replicators in order to satisfy certain developmental constraints, most origins would appear to coincide simply with non-transcribed intergenic spacers in permissive chromatin domains.

3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.

CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.

Involvement of upstream stimulatory factor in regulation of the mouse Prnd gene coding for Doppel protein

Promoter of the Prnd gene coding for the Prion-like protein Doppel contains two critical cis-regulatory elements, NF-Y consensus motif and canonical E-box. Here, we studied a role of the upstream stimulatory factor (USF) in the E-box-mediated activation of Prnd transcription. Co-expression of USF-1 with the luciferase reporter gene driven by the −185/+27 Prnd promoter fragment resulted in several fold increase of the luciferase activity. Conversely, mutations within the E-box led to a significantly reduced Prnd promoter activation. USF-1 binding was supported by the gel shift assay, supershift with USF-1 antibody and UV cross-linking. The activation capacity of the related USF-2, c-Myc and HIF-2α proteins was lower compared to USF-1 suggesting that USF-1 is the major E-box-binding transcription factor regulating the Prnd promoter.