Copy Number-dependent Expression Research Articles

Copy number variation of TdDof controls solid-stemmed architecture in wheat

Stem solidness is an important agronomic trait of durum (Triticum turgidum L. var. durum) and bread (Triticum aestivum L.) wheat that provides resistance to the wheat stem sawfly. This dominant trait is conferred by the SSt1 locus on chromosome 3B. However, the molecular identity and mechanisms underpinning stem solidness have not been identified. Here, we demonstrate that copy number variation of TdDof, a gene encoding a putative DNA binding with one finger protein, controls the stem solidness trait in wheat. Using map-based cloning, we localized TdDof to within a physical interval of 2.1 Mb inside the SSt1 locus. Molecular analysis revealed that hollow-stemmed wheat cultivars such as Kronos carry a single copy of TdDof, whereas solid-stemmed cultivars such as CDC Fortitude carry multiple identical copies of the gene. Deletion of all TdDof copies from CDC Fortitude resulted in the loss of stem solidness, whereas the transgenic overexpression of TdDof restored stem solidness in the TdDof deletion mutant pithless1 and conferred stem solidness in Kronos. In solid-stemmed cultivars, increased TdDof expression was correlated with the down-regulation of genes whose orthologs have been implicated in programmed cell death (PCD) in other species. Anatomical and histochemical analyses revealed that hollow-stemmed lines had stronger PCD-associated signals in the pith cells compared to solid-stemmed lines, which suggests copy number-dependent expression of TdDof could be directly or indirectly involved in the negative regulation of PCD. These findings provide opportunities to manipulate stem development in wheat and other monocots for agricultural or industrial purposes.

Extensive genome heterogeneity leads to preferential allele expression and copy number-dependent expression in cultivated potato.

Relative to homozygous diploids, the presence of multiple homologs or homeologs in polyploids affords greater tolerance to mutations that can impact genome evolution. In this study, we describe sequence and structural variation in the genomes of six accessions of cultivated potato (Solanum tuberosum L.), a vegetatively propagated autotetraploid and their impact on the transcriptome. Sequence diversity was high with a mean single nucleotide polymorphisms (SNP) rate of approximately 1 per 50 bases suggestive of high levels of allelic diversity. Additive gene expression was observed in leaves (3605 genes) and tubers (6156 genes) that contrasted the preferential allele expression of between 2180 and 3502 and 3367 and 5270 genes in the leaf and tuber transcriptome, respectively. Preferential allele expression was significantly associated with evolutionarily conserved genes suggesting selection of specific alleles of genes responsible for biological processes common to angiosperms during the breeding selection process. Copy number variation was rampant with between 16098 and 18921 genes in each cultivar exhibiting duplication or deletion. Copy number variable genes tended to be evolutionarily recent, lowly expressed, and enriched in genes that show increased expression in response to biotic and abiotic stress treatments suggestive of a role in adaptation. Gene copy number impacts on gene expression were detected with 528 genes having correlations between copy number and gene expression. Collectively, these data suggest that in addition to allelic variation of coding sequence, the heterogenous nature of the tetraploid potato genome contributes to a highly dynamic transcriptome impacted by allele preferential and copy number-dependent expression effects.

Downregulation of human IL4 and IL13 expression by CRISPR/Cas9-mediated deletion of DNase I hypersensitive site HS11/12 from a transgenic human Th2 cytokine locus

Abstract Production of the Th2 cytokines IL4 and IL13 is frequently dysregulated in allergy and asthma. Multiple genetic elements regulating expression of murine Th2 cytokines have been characterized by gene targeting. To study the regulation of human Th2 cytokines, we created BAC transgenic mice (BAC5TG) carrying a 160 kb fragment of the human Th2 locus encompassing RAD50, IL13 and IL4. This transgene includes all the regulatory elements required for physiological expression of mouse Th2 cytokines. BAC5TG mice appear normal, are born at the expected Mendelian ratio, and demonstrate tissue-specific and transgene copy number-dependent expression of human Th2 cytokines. DNA methylation profiles across the transgenic human Th2 locus are highly comparable if not identical to those observed in human cells. A DNase I hypersensitive site (HS) in the human IL13-IL4 intergenic region, previously identified in our lab and designated HS11/12, colocalizes with an area of extensive and profound CpG hypomethylation both in human and mouse transgenic T cells. However, unlike most Th2 regulatory elements identified to date, HS11/12 is not conserved between humans and mice. Using the CRISPR/Cas9 approach, we deleted HS11/12 from the human Th2 transgene in BAC5TG mice. Notably, production of both human IL13 and IL4 by in vitro differentiated Th2 cells from BAC5DHS11/12 TG mice was reduced by ~50%. Our results suggest that HS11/12 represents a novel cis-regulatory element required for optimal production of the human Th2 cytokines IL4 and IL13. The application of CRISPR/Cas9 technology to BAC transgenic mice may provide a powerful tool to study human gene regulation.

Comparative Analysis of Transgene Copy Numbers and Expression Characteristics across Multiple Transgenic Marine Medaka Oryzias dancena Strains carrying the β-Actin Promoter-Driven GFP Reporter

【Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$ -actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$ -actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.】

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells.

CHO cells are the most frequently used host for commercial production of therapeutic proteins, and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. In this study, we have examined the interaction between Ubiquitous Chromatin Opening Elements (UCOE) and DHFR-linked amplification in relation to cell expression stability. In summary, the inclusion of UCOE elements generated cells that (1) achieved higher cell densities and exhibited increased production of recombinant mRNA per cell and protein yield, (2) allowed isolation of greater numbers of high-producing clones, (3) resulted in greater mRNA recovery per recombinant gene copy, (4) retained stable mRNA and protein expression after amplification provided Methotrexate (MTX) was present (but not in the absence of MTX when instability was observed) and (5) conferred copy number-dependent expression to linked transgene, suggesting that they are resistant to positional gene-silencing effects. It was concluded that the inclusion of UCOEs within expression constructs offers significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR, could be achieved.

270. Nanoparticle-Mediated Rhodopsin cDNA But Not the Intron-Containing DNA Delivery Causes Transgene Silencing in a Rhodopsin Knockout Model

Gene expression is moderated by a multitude of patterns. The cross-talk between the native promoters and all the non-coding endogenous elements, such as transcription factors, enhancers and introns, are tightly controlled in a precise and real-time manner.The use of cDNA transgene often causes transgene silencing as over-expression or under-expression is known to affect protein function. In contrast, large genomic DNA transgene showed positionindependent, copy number-dependent expression of the transgene. Precise control of rhodopsin expression is critical in maintaining viable photoreceptors. In the present study, two forms of rhodopsin DNAs, an intron-containing short form genomic DNA and an intron-less cDNA, were cloned into an S/MAR vector driven by the 500 bp mouse opsin (MOP) promoter. The vectors were further compacted into nanoparticles (NPs) and subretinally injected into a rhodopsin knockout mouse (RKO) model at postnatal day 3. Following injection, DNA methylation and histone status of the specific regions (bacteria plasmid backbone, promoter, rhodopsin gene and S/MAR) in the vectors were evaluated at various time points. Persistent expression and phenotypic improvement were found in RKO mice up to 8 months after NP-mediated intron-containing delivery but not the NPs housing the intron-less cDNA delivery (data submitted for publication under review). In this study, our results further indicated that epigenetic silencing occurred in vector-mediated gene transfer, which were contributed by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene as evaluated by DNA methylation specific PCR followed by DNA sequencing and chromatin immunoprocipitation (ChIP) assay with highly specific antibodies for euchromatin and heterochromatin. No toxicity or inflammation was found in the treated eyes as detected by both PCR for pro-inflammatory cytokines and immunohistochemistry using macrophage marker, F4/80. These results suggest that cDNA of transgene and bacteria backbone interfere with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochratin-associated modification. While the benefits of including genomic elements may vary from gene to gene, our data suggest reconsider the current paradigm using cDNA as the standard in gene delivery cassettes. This paradigm shift has wide-ranging application for ocular gene therapy for retinal diseases or for which a genetic treatment is of interest. Further experiments are currently underway to determine whether inclusion of additional endogenous elements will provide added advantages in terms of physiologically relevant gene expression.

Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.

Highly stable maintenance of a mouse artificial chromosome in human cells and mice

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis.

Genome‐wide expression and copy number analysis identifies driver genes in gingivobuccal cancers

The molecular mechanisms contributing to the development and progression of gingivobuccal complex (GBC) cancers-a sub-site of oral cancer, comprising the buccal mucosa, the gingivobuccal sulcus, the lower gingival region, and the retromolar trigone-remain poorly understood. Identifying the GBC cancer-related gene expression signature and the driver genes residing on the altered chromosomal regions is critical for understanding the molecular basis of its pathogenesis. Genome-wide expression profiling of 27 GBC cancers with known chromosomal alterations was performed to reveal differentially expressed genes. Putative driver genes were identified by integrating copy number and gene expression data. A total of 315 genes were found differentially expressed (P ≤ 0.05, logFC > 2.0) of which 11 genes were validated by real-time quantitative reverse transcriptase-PCR (qRT-PCR) in tumors (n = 57) and normal GBC tissues (n = 18). Overexpression of LY6K, in chromosome band 8q24.3, was validated by immunohistochemical (IHC) analysis. We found that 78.5% (2,417/3,079) of the genes located in regions of recurrent chromosomal alterations show copy number dependent expression indicating that copy number alteration has a direct effect on global gene expression. The integrative analysis revealed BIRC3 in 11q22.2 as a candidate driver gene associated with poor clinical outcome. Our study identified previously unreported differentially expressed genes in a homogeneous subtype of oral cancer and the candidate driver genes that may contribute to the development and progression of the disease. © 2011 Wiley Periodicals, Inc.

RFP2 Modulates 20S Proteasome Activity, NF Kappa B Activation, and Tumor Cell Growth in Multiple Myeloma.

Abstract 1786Poster Board I-812 BackgroundMultiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells. Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO- GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting. ResultsAt day six post transduction, 75-95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2a and Bip, and the induction of Caspase-8, 9 and 3 cleavage. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. The above described presence of ER stress, combined with the previous reports that RFP2 has E3 ubiquitin ligase activity prompted us to assess total protein ubiquitination. Concordant with its effects on ER stress, RFP2 downregulation was associated with significantly higher levels of poly-ubiquitinated proteins. Subsequently, we were able to document a significant reduction (60% inhibition) in 20S proteasome activity in RFP2 down regulated cells. Proteasome inhibition by RFP2 down regulation was confirmed in other MM cell lines and was partially abrogated by restoring RFP2 levels by overexpression. Importantly, RFP2 down regulated cells were more sensitive to bortezomib; indeed proteasome inhibition was synergistic with RFP2 downregulation in MM cells. The above results prompted us to study the mechanism whereby RFP2 impacts survival and proliferation of MM cells. Inhibition of the NF-kappa-B (NFκB) pathway is a hallmark of proteasome-related growth retardation and apoptosis and is a key pathway in MM. We show that NFkB luciferase reporter assay was associated with significant activity reduction with RFP2 downregulation. To define the mechanism of this process, we examined the level of NFkB related proteins in nuclear and cytoplasmic fractions. Interestingly, the most prominent effect observed in RFP2 down regulated cells was increased levels of IkBá in the nucleus. Altogether, these results support our supposition that the effects of RFP2 downregulation are mediated through an inhibition of the NFkB pathway that is associated with increased nuclear IkBa as well as a decrease in 20S proteasome activity. ConclusionsRFP2 is a gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest, mediated by proteasome inhibition and reduced NFkB activity. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, targeting RFP2 may represent a novel therapeutic approach in MM and other lymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Integrative genomics reveals mechanisms of copy number alterations responsible for transcriptional deregulation in colorectal cancer

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.

RFP2 Knockdown Disrupts Cell Growth and Induces Apoptosis in Multiple Myeloma

Background: Multiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells.Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO-GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting.Results: At day six post transduction, 75–95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2α and Bip, and the induction of Caspase-8, 9 and 3 cleavage. These effects could be abrogated by the ZVAD-FMK pancaspase inhibitor and by overcoming the G2 phase arrest with caffeine. Similar results were observed in MM cell lines RPMI-8226, NCI-H929, MM1S, and SACHI, and were independent of presence of a monoallelic 13q deletion. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. In order to identify potential RFP2 target proteins, RFP2 was immunoprecipitated from MM cell lines. RFP2 protein complexes are currently being analyzed by mass-spectometry and results of these studies will be presented.Conclusions: RFP2 is a copy number sensitive gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, disrupting RFP2 function may represent a novel therapeutic target in MM and other lymphoid malignancies.

Inducible gene inactivation in neurons of the adult mouse forebrain

BackgroundThe analysis of the role of genes in important brain functions like learning, memory and synaptic plasticity requires gene inactivation at the adult stage to exclude developmental effects, adaptive changes or even lethality. In order to achieve temporally controlled somatic mutagenesis, the Cre/loxP-recombination system has been complemented with the tamoxifen-inducible fusion protein consisting of Cre recombinase and the mutated ligand binding domain of the human estrogen receptor (CreERT2). To induce recombination of conditional alleles in neurons of the adult forebrain, we generated a bacterial artificial chromosome-derived transgene expressing the CreERT2 fusion protein under control of the regulatory elements of the CaMKIIα gene (CaMKCreERT2 transgene).ResultsWe established three mouse lines harboring one, two and four copies of the CaMKCreERT2 transgene. The CaMKCreERT2 transgene displayed reliable and copy number-dependent expression of Cre recombinase specifically in neurons of the adult forebrain. Using Cre reporter mice we show very low background activity of the transgene in absence of the ligand and efficient induction of recombination upon tamoxifen treatment in all three lines. In addition, we demonstrate in mice harboring two conditional glucocorticoid receptor (GR) alleles and the CaMKCreERT2 transgene spatially restricted loss of GR protein expression in neurons of the adult forebrain upon tamoxifen treatment.ConclusionThis is to our knowledge the first approach allowing highly efficient inducible gene inactivation in neurons of the adult mouse forebrain. This new approach will be a useful tool to dissect the function of specific genes in the adult forebrain. Effects of gene inactivation on pre- and postnatal brain development and compensatory mechanisms elicited by an early onset of gene inactivation can now be excluded.

Expression patterns of promoters for NPY Y1 and Y5 receptors in Y5RitTA and Y1RVenus BAC‐transgenic mice

In the rat brain, neuropeptide Y (NPY) Y(1) and Y(5) receptors are coexpressed in various forebrain regions where they mediate several NPY-activated functions, including feeding behaviour, anxiety, neuronal excitability and hormone secretion. We studied the distribution pattern and cellular colocalization of the Y(1) and the Y(5) receptor gene expression in the mouse brain by using transgenic mice with genomically integrated BAC clones, where the coding regions of the Y(1) and Y(5) receptor genes were replaced by Venus and the synthetic transcription factor itTA reporter genes, respectively (Tg(Y5RitTA/Y1RVenus) mice). Analysis of Venus fluorescence and itTA-mediated activation of Cre recombinase revealed copy number-dependent expression levels, between the lines, but similar expression patterns. In three transgenic lines the BAC encoded Y(5) receptor promoter induced strong Cre expression in the olfactory system, cerebral cortex, hippocampus and basal ganglia. Weaker expression was found in most of the hypothalamic nuclei of line 25, the highest-expressing transgenic line. Activation of Cre was itTA-dependent and could be regulated by doxycycline. The Y(1) receptor promoter-induced Venus fluorescence was intense, widely present through the brain and colocalized with Cre immunostaining in neurons of distinct brain regions, including the cerebral cortex, basolateral amygdala, dentate gyrus and paraventricular nucleus. These data provide a detailed and comparative mapping of Y(1) and Y(5) receptor promoter activity within cells of the mouse brain. The Tg(Y5RitTA/Y1RVenus)-transgenic mice generated here also represent a genetic tool for conditional mutagenesis via the Cre lox system, particularly of genes involved in feeding behaviour, neuronal excitability and hormone secretion.

A Transgenic Mouse Assay To Compare the Barrier Activities of the Chicken β-Globin 5′HS4, Human β-Globin 5′HS5 and Mouse 3′HS1.

Insulator elements are found at the boundary between euchromatin and heterochromatin, and are responsible for maintaining the correct chromatin configuration for a locus. The best characterized vertebrate insulator element, 5′ Hypersensitive Site (HS) 4 from the chicken β-globin locus (ch5′HS4), has two separable activities: enhancer blocking, which requires binding of the transcription factor CTCF, and barrier, which prevents transgene silencing. We have previously reported that transgenic mice carrying a wild-type erythrocyte ankyrin promoter (ANK-1E)/γ-globin gene showed uniform (γ-globin in 100% of red cells), position-independent (32/32 lines express), copy number-dependent (p=0.0005) expression of γ-globin mRNA and protein. Mutations in the ANK-1E promoter at positions −108 and −153 cause ankyrin-deficient Hereditary Spherocytosis. Transgenic mice with the −108/−153 ANK-1E/γ-globin transgene showed variegated (γ-globin in 0–80% of red cells), position-dependent (8/14 lines express), copy number-independent (p=0.27) expression of γ-globin. Flanking the −108/−153 ANK-1E/γ-globin transgene with the ch5′HS4 insulator restored uniform, position-independent (9/9 lines), copy number-dependent expression (p=0.003) at levels identical to the wild-type ANK-1E promoter. We hypothesized that we could test sequences for barrier activity by assaying their ability to restore normal expression to the −108/−153 ANK-1E/γ-globin gene in transgenic mice. In mammalian β-globin loci, human 5′HS5 and mouse 3′HS1 have been proposed to be insulator elements, similar to chicken 5′HS4, based on their ability to block enhancer element function. To test barrier function, we generated transgenic mice containing the −108/−153 ANK-1E/γ-globin transgene flanked by human 5′HS5, mouse wild-type 3′HS1, or mouse 3′HS1 with mutations that disrupt the binding of CTCF (×CTCF). A total of 5 lines of transgenic mice were generated containing the 5′HS5/−108/−153 ANK-1E/γ-globin transgene. γ-globin mRNA and protein were undetectable in 3/5 lines, indicating that expression was position-dependent, and in the two positive lines, mRNA levels did not correlate with copy number. A total of 9 lines of 3′HS1-flanked transgenic mice were generated, 3 of which did not express γ-globin mRNA and protein, demonstrating position-dependent expression. Among the 6 expressing lines, two lines showed variegated expression and the correlation between γ-globin mRNA level and copy number was significant (p=0.0117). In contrast, 3′HS1×CTCF transgenic mice expressed γ-globin in a uniform, position-independent (7/7 lines express), copy number-dependent (p=0.0005) manner. The levels of γ-globin mRNA in both the 3′HS1 and 3′HS1×CTCF transgenic mice were 2-fold greater than the levels measured in transgenic mice with the wild-type ANK-1E promoter (p=0.019; 0.0003 respectively), suggesting that 3′HS1 may contain an enhancer element. Our results indicate that while human 5′HS5 and mouse 3′HS1 block the effects of enhancer elements, neither are barrier elements as defined by the ability to prevent gene silencing. We hypothesize that the mutation of the CTCF binding sites allows the ANK-1E promoter to take greater advantage of the 3′HS1 enhancer, leading to a more uniform, position-independent, and copy number-dependent pattern of expression, as has been described for other enhancer elements in the β-globin Locus Control Region.

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

The Expression of Intact and Mutant Human apoAI/CIII/AIV/AV Gene Cluster in Transgenic Mice

The apoAI/CIII/AIV gene cluster is involved in lipid metabolism and has a complex pattern of gene expression modulated by a common regulatory element, the apoCIII enhancer. A new member of this cluster, apolipoprotein (apo) AV, has recently been discovered as a novel modifier in triglyceride metabolism. To determine the expression of all four apo genes in combination and, most importantly, whether the transcription of apoAV is coregulated by the apoCIII enhancer in the cluster, we generated an intact transgenic line carrying the 116-kb human apoAI/CIII/AIV/AV gene cluster and a mutant transgenic line in which the apoCIII enhancer was deleted from the 116-kb structure. We demonstrated that the apoCIII enhancer regulated hepatic and intestinal apoAI, apoCIII, and apoAIV expression; however, it did not direct the newly identified apoAV in the cluster. Furthermore, human apo genes displayed integrated position-independent expression and a closer approximation of copy number-dependent expression in the intact transgenic mice. Because apoCIII and apoAV play opposite roles in triglyceride homeostasis, we analyzed the lipid profiles in our transgenic mice to assess the effects of human apoAI gene cluster expression on lipid metabolism. The triglyceride level was elevated in intact transgenic mice but decreased in mutant ones compared with nontransgenic mice. In addition, the expression of human apoAI and apoAIV elevated high density lipoprotein cholesterol in transgenic mice fed an atherogenic diet. In conclusion, our studies with human apoAI/CIII/AIV/AV gene cluster transgenic models showed that the apoCIII enhancer regulated expression of apoAI, apo-CIII, and apoAIV but not apoAV in vivo and showed the influences of expression of the entire cluster on lipid metabolism.

Th2-Specific Chromatin Remodeling and Enhancer Activity in the Th2 Cytokine Locus Control Region

We recently identified a 3′ region of the rad50 gene possessing strong enhancer activity as well as activity consistent with function as a locus control region (LCR) for the flanking Th2 cytokine genes. In this study, we identify several functional elements within this region by examining chromatin changes as well as activity in transgenic mice. We find within this region four DNase I hypersensitive clusters, three of which are highly conserved and predominantly expressed in Th2 cells. Histone acetylation of this region is elevated in Th2 cells. Further, one of the hypersensitive sites (RHS7) is rapidly demethylated in Th2, but not Th1, cells. In transgenic mice, these hypersensitive sites impart strong, Th2-specific enhancer activity as well as copy number-dependent expression of the reporter gene, recapitulating LCR function. We postulate that these sites function alone or in combination with other regulatory elements to coordinate gene expression in the Th2 cytokine locus.

Defining the Active Chromatin Domain of the Erythroid Ankyrin Promoter.

Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p<0.001; χ2=9.0, p<0.01 respectively), indicating boundary function. We have previously shown that mutations at positions −153 and −108 in 5′HS associated with Hereditary Spherocytosis cause position dependent and variegated expression of the γ-globin transgene in mice. When the −153/−108 sequence was used to flank the β-globin/EGFP transgene gene silencing was observed in 5/10 K562 cell lines (χ2=5.7,p<0.02). Because boundary elements are sites of high levels of histone acetylase (HA) activity we hypothesized that the −153/−108 mutations disrupted histone acetylation in ankR. EMSA analysis showed that the −153/−108 mutations disrupted the binding of a complex that includes both CTCF (a zinc finger protein associated with boundary elements in the β-globin cluster) and Brg-1 (the ATP dependent subunit of the HA complex). Chomatin Immune Precipitation analysis of transfected K562 cells showed that histone acetylation in sequences containing the −153/−108 mutations was 4-fold less than in wild type sequences (p<0.02). These studies have defined the active chromatin domain of the ankR and have demonstrated that the −153/−108 mutations cause Hereditary Spherocytosis by inhibiting histone acetylation in ankR.

Analysis of the flanking regions of the human α-lactalbumin gene responsible for position-effect independent expression

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the α-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The −6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the −3.4 to −0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at −1.0 and −2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human α-lactalbumin gene.