TBP binding and the rate of transcription initiation from the human β-globin gene.

DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription

Isolation of β-globin related genes from a human cosmid library.

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA

β-globin gene promoter generates 5' truncated transcripts in the embryonic foetal erythroid environment.

We report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly. Using hybrid constructs of these two genes we demonstrate that aberrant activity is conferred by sequences 5' of the beta-globin gene. Thus beta-globin promoter attached to the H2 coding sequence produces H2 transcripts with truncated 5' ends. By introducing a series of deletions in the beta-globin promoter we restrict these sequences to the -77/+28 base pair region spanning the CAAT element to the translation initiation site. These results are consistent with the lack of recognition of the beta-globin gene major cap site in Putko and K562 cells. We suggest that inactivity of the adult globin gene in the embryonic/fetal environment is at least in part conferred by sequences within the beta-globin gene promoter

Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes

Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes

Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH-VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique,soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs

Chromatin Dynamics of the mouse β-globin locus

Lately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors (TF) to TF binding sites also becomes an important factor to the gene’s activation status. Both such changes have been ascribed to the mouse ?- haemoglobin gene cluster as a trigger to activate globin expression in the erythroid cell lineage. Early models speculated a scanning, random activation or a looping mechanism to activate globin transcription. The chromatin conformation capture (3C) technique has shown that there is a molecular interaction between various DNAse I hypersensitive (HS) sites that are located up- and downstream of the ?-globin gene cluster, the HS sites of the Locus Control Region (LCR) and the promoter by means of a dynamic looping mechanism. The clustering of the HS sites of the LCR and the up- and down- stream HS sites results in the formation of a so called Active Chromatin Hub (ACH) which is depending on at least two erythoid TF: EKLF and GATA-1. The long range interactions between the outlying HS -84/-85, -62/-60 and the 3’HS1 are depending on the presence of CTCF, a TF that is thought to play an important role in long range chromatin interactions across the whole genome. Prior to gene activation, cells of the early erythroid lineage (progenitors) already show a presence of an ACH, which is not found in non-erythroid cells. The final chromatin 3D structure consist of four major loops sizing 25- 38Kb and two minor loops within the LCR sizing 4.5 and 12Kb. To confirm this looping hypothesis (based on 3C technology) we used an in situ hybridization approach to visualize and, after image restoration, quantitatively measure the 3D conformational changes that take place within the locus in erythroid cells before and after differentiation. Globin gene activation is depending on long distance looping of the up- and downstream HS sites and the ?-major promotor to the LCR, resulting in a complex 3D chromatin structure. By staining the m?- globin loci with fluorescence labeled sequence specific probes followed by high-resolution 3D imaging and 3D volume rendering of the deconvolved images, the loci reveal changes in the geometric size and shape properties when cells are differentiated into a active globin transcribing cell. An almost 2x decrease in volume was measured, which was mostly due to a reduction of the longest length measured. This can be explained by a change in loop formation. The almost 70Kb loop between the LCR and the 3’HS1 is folded into two loops of 34 and 35Kb upon interaction of the promotor to the ACH to activate transcription. The limited decrease in volume and length when the locus was probed with an additional 5’ and 3’ end region is surprising. The 5’ end is actively participating in the looping process that stabilizes the ACH. However, the 3’ end has (until now) not been seen to be participate in ACH formation or any complex looping mechanism for globin gene activation. As this part of the locus seems to be the most un-dynamic, it could be the dominating factor that influences the fluorescent signal emitting from the probed DNA region and therefore cloud subtle changes in the chromatin folding mechanism of gene activation. Next to the dynamic chromatin folding process that is occurring between the HS-85/84 and the 3’HS1, a stretch of “rigid” DNA can prevent a DNA region containing activated genes to stay at the edge of a chromosome territory and possibly prevent a close proximity to the silencing effect of (spreading) heterochromatin. An increase in lateral and axial resolution like the 3D Structural Illumination Microscope (SIM) provides, could help solve the problem of detecting subtle 3D changes in chromatin structure. And in the near future will reveal many more details of 3D chromatin organization of not only the m?-globin locus but of many other intra-cellular processes

The 3D chromatin structure of the mouse β-haemoglobin gene cluster

Here we show a 3D DNA-FISH method to visualizes the 3D structure of the β-globin locus. Geometric size and shape measurements of the 3D rendered signals (128Kb) show that the volume of the β-globin locus decreases almost two fold upon gene activation. A decrease in length and a distinctive change in shape and surface structure of the locus are also observed. Adding 5’ and 3’end regions to the probe (175Kb) showed a less prominent change in length, shape and structure. It was shown (data not on this poster) that the physical distance between the two flanking regions shift in a similar limited manner, indicating that the flanking regions do not participate in ACH formation and thus active chromatin folding is occurring only within the locus proper

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock