The promoter of the human interleukin-2 gene contains two octamer-binding sites and is partially activated by the expression of Oct-2

The gene encoding interleukin-2 (IL-2) contains a sequence 52 to 326 nucleotides upstream of its transcriptional initiation site that promotes transcription in T cells that have been activated by costimulation with tetradecanoyl phorbol myristyl acetate (TPA) and phytohemagglutinin (PHA). We found that the ubiquitous transcription factor, Oct-1, bound to two previously identified motifs within the human IL-2 enhancer, centered at nucleotides -74 and -251. Each site in the IL-2 enhancer that bound Oct-1 in vitro was also required to achieve a maximal transcriptional response to TPA plus PHA in vivo. Point mutations within either the proximal or distal octamer sequences reduced the response of the enhancer to activation by 54 and 34%, respectively. Because the murine T-cell line EL4 constitutively expresses Oct-2 and requires only TPA to induce transcription of the IL-2 gene, the effect of Oct-2 expression on activation of the IL-2 promoter in Jurkat T cells was determined. Expression of Oct-2 potentiated transcription 13-fold in response to TPA plus PHA and permitted the enhancer to respond to the single stimulus of TPA. Therefore, both the signal requirements and the magnitude of the transcription response of the IL-2 promoter can be modulated by Oct-2

Molecular cloning of an enhancer binding protein:Isolation by screening of an expression library with a recognition site DNA

A novel strategy has been used to isolate a cDNA clone that encodes a DNA binding domain whose recognition properties overlap those of the mammalian transcription factors H2TF1 and NF-KB. These two factors are distinguished by their cell type distributions and their relative affinities for related sequence elements in the enhancers of the major histocompatibility complex (MHC) class I and immunoglobulin K chain genes. The human cDNA clone was detected by screening a ~ phage expression library with a binding site probe derived from the MHC enhancer. The phage encoded fusion protein binds specifically to both the MHC and K gene enhancers. The cDNA hybridizes to a single copy gene that is expressed as a 10 kb mRNA in both B and non-B cells. The strategy used in this study may prove generally useful in the cloning and analysis of sequence-specific DNA binding proteins

Characterization of glycan substrates accumulating in GM1 Gangliosidosis

Introduction: GM1 gangliosidosis is a rare autosomal recessive genetic disorder caused by the disruption of the GLB1 gene that encodes β-galactosidase, a lysosomal hydrolase that removes β-linked galactose from the non-reducing end of glycans. Deficiency of this catabolic enzyme leads to the lysosomal accumulation of GM1 and its asialo derivative GA1 in β-galactosidase deficient patients and animal models. In addition to GM1 and GA1, there are other glycoconjugates that contain β-linked galactose whose metabolites are substrates for β-galactosidase. For example, a number of N-linked glycan structures that have galactose at their non-reducing end have been shown to accumulate in GM1 gangliosidosis patient tissues and biological fluids. Objective: In this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover. Results: Using tandem mass spectrometry and glycan reductive isotope labeling with data-dependent mass spectrometry, we have confirmed the accumulation of glycolipids (GM1 and GA1) and N-linked glycans with terminal beta-linked galactose. We have also discovered a novel set of core 1 and 2 O-linked glycan metabolites, many of which are part of structurally-related isobaric series that accumulate in disease. In the brain of GLB1 null mice, the levels of these glycan metabolites increased along with those of both GM1 and GA1 as a function of age. In addition to brain tissue, we found elevated levels of both N-linked and O-linked glycan metabolites in a number of peripheral tissues and in urine. Both brain and urine samples from human GM1 gangliosidosis patients exhibited large increases in steady state levels for the same glycan metabolites, demonstrating their correlation with this disease in humans as well. Conclusions: Our studies illustrate that GLB1 deficiency is not purely a ganglioside accumulation disorder, but instead a broad oligosaccharidosis that include representatives of many β-linked galactose containing glycans and glycoconjugates including glycolipids, N-linked glycans, and various O-linked glycans. Accounting for all β-galactosidase substrates that accumulate when this enzyme is deficient increases our understanding of this severe disorder by identifying metabolites that may drive certain aspects of the disease and may also serve as informative disease biomarkers to fully evaluate the efficacy of future therapies

A Negative Regulatory Element Controls mRNA Abundance of the Leishmania mexicana Paraflagellar Rod Gene PFR2†

The Leishmania mexicana PFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle. PFR2 mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in PFR2 mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3′ untranslated region (UTR) of PFR2C led to the identification of a regulatory element contained within 10 nucleotides of the 3′ UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of PFR2 mRNA levels. Comparison of the half-lives of PFR2 transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the PFR2 mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric PFR2 transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3′ UTR of all L. mexicana PFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes

Utilizing ExAC to assess the hidden contribution of variants of unknown significance to Sanfilippo Type B incidence

<div><p>Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB). Sanfilippo Type B is caused by mutations in the gene encoding <i>α</i>-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60,000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown. To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence.</p></div

The enzymatic activity of NAGLU variants.

<p><b>(A)</b> Variants are ordered by average %wt activity. Standard deviation in %wt activity in replicates is represented by vertical bars. Previously identified disease variants are shown in orange. A dashed line shows the 15%wt activity threshold below which variants are considered to be pathogenic. <b>(Insert B)</b> A box plot (y-axis log scale) of the Non-Finnish European allele frequency of variants with ≤15%wt activity (average allele frequency of 3.76 × 10⁻⁵ and those with >15%wt (average allele frequency of 0.0014). The difference in the average allele frequency between the two groups was not statistically significant (p-value = 0.372, t-test).</p

The statistical power of ExAC.

<p><b>(A)</b> Blue lines show the percent margin of error when calculating incidence using the overall sample size of ExAC given a 95% confidence interval. Red lines show the percent margin of error when calculating incidence using the sample size of the Non-Finnish European cohort in ExAC given a 95% confidence interval. Upper and lower limits of the confidence intervals are shown as dotted and solid lines respectively. The vertical grey dashed line represents the 1 in 321,128 Sanfilippo Type B incidence rate estimated by Heron et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200008#pone.0200008.ref009" target="_blank">9</a>]. <b>(B)</b> The number of individuals which should be sequenced such that the lower critical value will represent a 20% margin of error given a 95% confidence interval for a range of estimated incidence values.</p

Allele frequency based Sanfilippo Type B incidence estimates.

<p><b>(A)</b> The impact of each category of variants on our estimate of Sanfilippo Type B incidence. The contribution of HGMD variants, sorted by allele frequency, is shown in orange. The contribution of LoF variants which have yet to be documented in patients, also sorted by allele frequency, is shown in light blue. The contribution of VUS with %wt activity values below 15% is shown in grey. VUS are sorted by %wt values. Percentages in parenthesis represent the contribution of each category of variants to our final incidence estimate. <b>(B)</b> Confidence interval calculations. Grey bars represent the distribution of incidence rates observed through bootstrapping simulation. The orange solid line represents the distribution of incidence rates as modeled using the beta distribution. Using bootstrapping simulation we observed that 95% of simulated incidence values fell in the range of 1 in 558,306 and 1 in 241,749 for an equal tailed interval (vertical black lines). In comparison, using the beta-distribution we estimated that 95% of incidence values would fall in the range of 1 in 566,863 to 1 in 243,753 (vertical dashed orange lines). Using the normal approximation we estimated that 95% of incidence values would fall in the range of 1 in 610,093 to 1 in 250,830 (vertical dashed blue lines).</p

The structural characteristics of NAGLU variants.

<p>Mutations at N-linked glycosylation sites are shown in cyan, active-site pocket in red, disulfides in orange and trimer interface in blue. <b>(A)</b> Mutations (spheres) with ≤15%wt activity and DM variants in HGMD (left) and >15%wt activity (right) mapped onto a NAGLU monomeric structure (PDB ID 4XWH) <b>(B)</b> The relative solvent exposure in the trimer NAGLU structure of variants with ≤15%wt activity and DM variants in HGMD compared to those with >15%wt activity. ≤15%wt activity and DM variants were found to have an average percent solvent exposure of 5.96%. Variants with >15%wt activity had an average percent solvent exposure of 23.20%. The differences in the average solvent exposure between the two groups was statistically significant at the 5% significance level with a p-value of 1.09 × 10⁻¹². <b>(C)</b> The distance to the active site in the NAGLU structure of variants with ≤15%wt activity and DM variants in HGMD compared to those with >15%wt activity. ≤15%wt activity and DM variants were found to have an average distance to the active site of 24.03Å. Variants with >15%wt activity had an average distance to the active site of 27.23Å. The differences in the average distance to the active site between the two groups was statistically significant at the 5% significance level with a p-value of.001.</p