Michael Antoniou

Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

The human beta‐globin dominant control region (DCR) was previously identified as a region from the 5′ end of the human beta‐globin locus which directs high level, site of integration‐independent, copy number‐dependent expression on a linked human beta‐globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta‐globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta‐globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3′ enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.

Inefficient processing impairs release of RNA from the site of transcription

We describe here for the first time the site of retention within the nucleus of pre‐mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human β‐globin genes that are either normal or defective in splicing or 3′ end formation. Nuclear transcripts of both wild‐type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, α‐amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild‐type human β‐globin RNA. In contrast, pre‐mRNA mutants defective in either splicing or 3′ end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3′ end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells

BackgroundThe hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing.ResultsIn this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression.ConclusionVectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

An interaction between U2AF 65 and CF Im links the splicing and 3′ end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3′ end processing of vertebrate pre‐mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre‐mRNA 3′ end processing. We identify an arginine/serine‐rich region of U2AF 65 that mediates an interaction with an RS‐like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF Im), an essential 3′ processing factor that functions at an early step in the recognition of the 3′ end processing signal. Tethered functional analysis shows that the U2AF 65/CF Im 59 interaction stimulates in vitro 3′ end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF Im 59 interaction in the functional coordination of splicing and 3′ end processing.

Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing.

The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein (TBP)-proteasome component-B1 (PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)-heterochromatin protein 1Hs-gamma (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression. These observations suggest that methylation-free CpG islands associated with dual, divergently transcribed promoters possess an independent dominant chromatin opening function and may therefore be major determinants in establishing and maintaining a region of open chromatin at housekeeping gene loci.

The human beta-globin promoter; nuclear protein factors and erythroid specific induction of transcription.

We have shown that the promoter of the human beta‐globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF‐E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT‐box factor (CP1). Deletion experiments suggest that only the combination of NF‐E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta‐globin promoter in mouse erythroleukaemia (MEL) cells.

Concerns over use of glyphosate-based herbicides and risks associated with exposures: A consensus statement

The broad-spectrum herbicide glyphosate (common trade name “Roundup”) was first sold to farmers in 1974. Since the late 1970s, the volume of glyphosate-based herbicides (GBHs) applied has increased approximately 100-fold. Further increases in the volume applied are likely due to more and higher rates of application in response to the widespread emergence of glyphosate-resistant weeds and new, pre-harvest, dessicant use patterns. GBHs were developed to replace or reduce reliance on herbicides causing well-documented problems associated with drift and crop damage, slipping efficacy, and human health risks. Initial industry toxicity testing suggested that GBHs posed relatively low risks to non-target species, including mammals, leading regulatory authorities worldwide to set high acceptable exposure limits. To accommodate changes in GBH use patterns associated with genetically engineered, herbicide-tolerant crops, regulators have dramatically increased tolerance levels in maize, oilseed (soybeans and canola), and alfalfa crops and related livestock feeds. Animal and epidemiology studies published in the last decade, however, point to the need for a fresh look at glyphosate toxicity. Furthermore, the World Health Organization’s International Agency for Research on Cancer recently concluded that glyphosate is “probably carcinogenic to humans.” In response to changing GBH use patterns and advances in scientific understanding of their potential hazards, we have produced a Statement of Concern drawing on emerging science relevant to the safety of GBHs. Our Statement of Concern considers current published literature describing GBH uses, mechanisms of action, toxicity in laboratory animals, and epidemiological studies. It also examines the derivation of current human safety standards. We conclude that: (1) GBHs are the most heavily applied herbicide in the world and usage continues to rise; (2) Worldwide, GBHs often contaminate drinking water sources, precipitation, and air, especially in agricultural regions; (3) The half-life of glyphosate in water and soil is longer than previously recognized; (4) Glyphosate and its metabolites are widely present in the global soybean supply; (5) Human exposures to GBHs are rising; (6) Glyphosate is now authoritatively classified as a probable human carcinogen; (7) Regulatory estimates of tolerable daily intakes for glyphosate in the United States and European Union are based on outdated science. We offer a series of recommendations related to the need for new investments in epidemiological studies, biomonitoring, and toxicology studies that draw on the principles of endocrinology to determine whether the effects of GBHs are due to endocrine disrupting activities. We suggest that common commercial formulations of GBHs should be prioritized for inclusion in government-led toxicology testing programs such as the U.S. National Toxicology Program, as well as for biomonitoring as conducted by the U.S. Centers for Disease Control and Prevention.

The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers.

We have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta‐globin gene and the murine H‐2Kb gene. S1 nuclease and transcriptional run‐off analysis showed that the human beta‐globin gene contains at least three globin specific transcriptional control elements. One is a promoter element and is located 160 bp upstream from the transcription initiation site; it increases the efficiency of the promoter after MEL cells are induced to differentiate. The other two elements are tissue‐specific transcriptional enhancers and are located downstream from the transcription initiation site, the first in the structural beta‐globin gene and the second in the 3′ flanking sequences.

A Ubiquitous Chromatin Opening Element (UCOE) Confers Resistance to DNA Methylation–mediated Silencing of Lentiviral Vectors

DNA methylation may restrict the activity of gene transfer vectors due to inadvertent silencing. In P19 embryonic carcinoma cells in vitro, we found that transgene expression regulated by the SFFV LTR and EF1 alpha promoter declined rapidly within 16 days, but for A2UCOE derived from the human HNRPA2B1-CBX3 housekeeping gene locus, remained completely stable. Silencing correlated with extensive epigenetic methylation of CpG sites, whereas the A2UCOE was almost completely resistant. Linking of the A2UCOE upstream of the SFFV LTR protected this element from both DNA methylation and silencing. Analysis of engrafted hematopoietic cells in vivo transduced with the same vectors revealed a similar pattern. The A2UCOE displayed little or no methylation in either primary or secondary graft recipients, and gene expression profiles were highly conserved between the two groups. These studies provide convincing evidence that DNA methylation plays a direct role in regulating self-inactivating (SIN) lentiviral transgene expression, and that the stability of expression from the A2UCOE is, at least in part, due to methylation resistance. The A2UCOE therefore has considerable utility for gene therapy applications where reliable and sustained gene expression is desirable.

In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of β-thalassemia

Gene therapy for β-thalassemia requires stable transfer of a β-globin gene into hematopoietic stem cells (HSCs) and high and regulated hemoglobin expression in the erythroblastic progeny. We developed an erythroid-specific lentiviral vector driving the expression of the human β-globin gene from a minimal promoter/enhancer element containing two hypersensitive sites from the β-globin locus control region. Transplantation of transduced HSCs into thalassemic mice leads to stable and long-term correction of anemia with all red blood cells expressing the transgene. A frequency of 30–50% of transduced HSCs, harboring an average vector copy number per cell of 1, was sufficient to fully correct the thalassemic phenotype. In the mouse model of Cooleys anemia transplantation of transduced cells rescues lethality, leading to either a normal or a thalassemia intermedia phenotype. We show that genetically corrected erythroblasts undergo in vivo selection with preferential survival of progenitors harboring proviral integrations in genome sites more favorable to high levels of vector-derived expression. These data provide a rationale for a gene therapy approach to β-thalassemia based on partially myeloablative transplantation protocols.