Enhancer-promoter competition between homologous alleles leads to reduced transcription in early Drosophila embryos

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. Here, we ask if inter-allelic interactions between two homologous alleles can affect gene regulation. Using MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcription of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the hemizygous allele, suggesting the possibility of allelic competition in homozygotes. Moreover, the MS2-yellow reporter gene showed reduced transcriptional activity when a partial transcription unit (enhancer or promoter only) was in the homologous position. We propose that the transcriptional machinery that binds to both the enhancer and promoter region, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. To support this idea, we showed that the homologous alleles did not interfere with each other in earlier nuclear cycles when Pol II is in excess, while the degree of interference gradually increased in nuclear cycle 14. Such allelic competition was observed for endogenous snail as well. Our study provides new insights into the role of 3D inter-allelic interactions in gene regulation.

LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology

Swelling-activated VRACs are heterohexameric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryo-electron microscopy (EM) structures of non-native LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM DCPIB in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, a LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ-dependent. At normal intracellular µ, LRRC8A+LRRC8C heterohexamers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage-dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heterohexameric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homohexameric channels.

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+–Cl−–glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

ABSTRACT The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.

Phenotypic variability and origins of mutations in the gene encoding 3beta-hydroxysteroid dehydrogenase type II

Mutations in HSD3B2, the gene for 3beta-hydroxysteroid dehydrogenase type II (3beta-HSD II) have been detected and activities analysed through the in vitro expression of mutant cDNAs. Two full sibs with male pseudohermaphroditism were found to be double heterozygotes: N100S/266DeltaA. This genotype leads to the most profound loss of 3beta-HSD II enzyme activity (1.3% of normal) described to date in cases without severe salt-loss. One sib (N100S/266DeltaA) is the first reported male case of type II deficiency affected with premature adrenarche. Three apparently independent kindreds had propositi affected with the HSD3B2 mutation A82T/A82T, which is associated with a non salt-losing phenotype with variable expressivity in females. These three families had the same extended HSD3B haplotype and are likely to have inherited the same ancestral mutation. The significance of this finding is discussed in the light of the presence of A82T mutation at a homologous position in pseudogene varphi5 that is present in the HSD3B cluster.

Transgene Repeat Arrays Interact With Distant Heterochromatin and Cause Silencing in cis and trans

Tandem repeats of Drosophila transgenes can cause heterochromatic variegation for transgene expression in a copy-number and orientation-dependent manner. Here, we demonstrate different ways in which these transgene repeat arrays interact with other sequences at a distance, displaying properties identical to those of a naturally occurring block of interstitial heterochromatin. Arrays consisting of tandemly repeated white transgenes are strongly affected by proximity to constitutive heterochromatin. Moving an array closer to heterochromatin enhanced variegation, and enhancement was reverted by recombination of the array onto a normal sequence chromosome. Rearrangements that lack the array enhanced variegation of white on a homologue bearing the array. Therefore, silencing of white genes within a repeat array depends on its distance from heterochromatin of the same chromosome or of its paired homologue. In addition, white transgene arrays cause variegation of a nearby gene in cis, a hallmark of classical position-effect variegation. Such spreading of heterochromatic silencing correlates with array size. Finally, white transgene arrays cause pairing-dependent silencing of a non-variegating white insertion at the homologous position.

The enhancer-like sequence 3′ to the A gamma gene is polymorphic in human populations

Cloning and sequencing of the enhancer 3′ of the A gamma globin gene of a particularly low G gamma and HbF sickle cell anemia (SCA) patient unexpectedly revealed three base changes (T----C, C----A, and A----G at sites +2285, +2460, and +2676) previously associated with the Seattle- type HPFH, thus leading the authors to suspect that the three mutations were polymorphic. The determination of the incidence of the mutations among various ethnic groups allowed the authors to conclude that this is a widely spread polymorphism, thus excluding any role of these base changes in the determination of the hereditary persistence of fetal hemoglobin (HPFH) phenotype. The origin of these three mutations is not clear because they appear linked, and the same bases (C, A, G) are found in homologous position in the 3′ of the normal G gamma gene. As C, A, G at positions +2285, +2460, and +2676 are found with a 100% frequency in African SS patients and presumably among normal Africans (to explain the extremely high frequency among normal American blacks), it is likely that this was the sequence preceding the division of races. The presence of T, C, and A at the same positions apparently occurred after the divergence between blacks and the other races, that is, within the last 1 million years.

The enhancer-like sequence 3′ to the A gamma gene is polymorphic in human populations

Cloning and sequencing of the enhancer 3′ of the A gamma globin gene of a particularly low G gamma and HbF sickle cell anemia (SCA) patient unexpectedly revealed three base changes (T----C, C----A, and A----G at sites +2285, +2460, and +2676) previously associated with the Seattle- type HPFH, thus leading the authors to suspect that the three mutations were polymorphic. The determination of the incidence of the mutations among various ethnic groups allowed the authors to conclude that this is a widely spread polymorphism, thus excluding any role of these base changes in the determination of the hereditary persistence of fetal hemoglobin (HPFH) phenotype. The origin of these three mutations is not clear because they appear linked, and the same bases (C, A, G) are found in homologous position in the 3′ of the normal G gamma gene. As C, A, G at positions +2285, +2460, and +2676 are found with a 100% frequency in African SS patients and presumably among normal Africans (to explain the extremely high frequency among normal American blacks), it is likely that this was the sequence preceding the division of races. The presence of T, C, and A at the same positions apparently occurred after the divergence between blacks and the other races, that is, within the last 1 million years.

Molecular characterization of the C3HfB/HeN H-2Kkm2 mutation. Implications for the molecular basis of alloreactivity.

The C3HfB/HeN (C3Hf) mouse strain expresses an H-2Kk molecule, previously denoted H-2Kkv1, that is structurally and functionally distinct from H-2Kk of the parental C3H strain. By molecular genetic analysis, we demonstrate that the C3Hf H-2K gene carries a homozygous coding region mutation relative to the C3H allele, revealing that C3Hf meets the requirements for assignment of a mutant haplotype, H-2km2. C3Hf H-2Kkm2 bears a single clustered substitution of four nucleotides within 14 contiguous nucleotides in exon 3. Since this sequence also is present intact at the homologous position in H-2Dk of both C3H and C3Hf, the origin of the H-2Kkm2 mutation is consistent with a nonreciprocal sequence transfer from the H-2Dk donor gene, analogous to the mechanism proposed for generation of the H-2Kb mutations. The H-2Kkm2 mutation encodes three clustered amino acid substitutions, at positions 95, 98, and 99, that map to one of the large beta strands at the bottom of the peptide antigen binding cleft of the H-2Kkm2 molecule. The nature and location of these amino acid substitutions are unique relative to any other known H-2 mutant or HLA variant, and underscore the importance of the beta-pleated sheet in influencing CTL recognition. These results indicate that H-2Kkm2 alloantigenicity may derive largely from altered presentation of self cellular peptides.