Faculty Bibliography

Humans and yeast are separated by a billion years of evolution, yet their conserved histones retain central roles in gene regulation. Here, we "reset" yeast to use core human nucleosomes in lieu of their own (a rare event taking 20 days), which initially only worked with variant H3.1. The cells adapt by acquiring suppressor mutations in cell-division genes or by acquiring certain aneuploid states. Converting five histone residues to their yeast counterparts restored robust growth. We reveal that humanized nucleosomes are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy, globally reduce RNA content, and slow adaptation to new conditions by delaying chromatin remodeling. These humanized yeasts (including H3.3) pose fundamental new questions about how chromatin is linked to many cell processes and provide a platform to study histone variants via yeast epigenome reprogramming.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing beta-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg(2+) concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg(2+) transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg(2+) concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg(2+) concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg(2+) binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg(2+)-dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg(2+) concentrations, with implications for evolution and potential applications in gene targeting.

Mobile group II introns retrohome by an RNP-based mechanism in which the intron RNA reverse splices into a DNA site and is reverse transcribed by the associated intron-encoded protein. The resulting intron cDNA is then integrated into the genome by cellular mechanisms that have remained unclear. Here, we used an Escherichia coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify host factors that function in retrohoming. We then analyzed mutants identified in these and previous genetic screens by using a new biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro. The genetic and biochemical analyses indicate a retrohoming pathway involving degradation of the intron RNA template by a host RNase H and second-strand DNA synthesis by the host replicative DNA polymerase. Our results reveal ATP-dependent steps in both cDNA and second-strand synthesis and a surprising role for replication restart proteins in initiating second-strand synthesis in the absence of DNA replication. We also find an unsuspected requirement for host factors in initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming. Key features of the retrohoming mechanism may be used by human LINEs and other non-LTR-retrotransposons, which are related evolutionarily to mobile group II introns. Our findings highlight a new role for replication restart proteins, which function not only to repair DNA damage caused by mobile element insertion, but have also been co-opted to become an integral part of the group II intron retrohoming mechanism.

The organic anion transporters OAT1 (SLC22A6, originally identified by us as NKT) and OAT3 (SLC22A8) are critical for handling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Khandelwal, A., Swaan, P. W., and Nigam, S. K. (2008) J. Biol. Chem. 283, 8654-8663). Although microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, the individual contributions of these transporters in the three-dimensional context of the tissues in which they normally function remain unclear. Here, handling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed with three-dimensional ex vivo functional assays using knock-out tissue. To investigate the contribution of OAT1 and OAT3 in various nephron segments, the OAT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Although OAT1 function (uptake in oat3(-/-) tissue) was confined to portions of the cortex, consistent with a proximal tubular localization, OAT3 function (uptake in oat1(-/-) tissue) was apparent throughout the cortex, indicating localization in the distal as well as proximal nephron. This functional localization indicates a complex three-dimensional context, which needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) handled by both transporters. These results also raise the possibility of functional differences in the relative importance of OAT1 and OAT3 in antiviral handling in developing and mature tissue. Because the HIV antivirals are used in pregnant women, the results may also help in understanding how these drugs are handled by developing organs.

Deletion of integrin-beta1 (Itgb1) in the kidney collecting system led to progressive renal dysfunction and polyuria. The defect in the concentrating ability of the kidney was concomitant with decreased medullary collecting duct expression of aquaporin-2 and arginine vasopressin receptor 2, while histological examination revealed hypoplastic renal medullary collecting ducts characterized by increased apoptosis, ectasia and cyst formation. In addition, a range of defects from small kidneys with cysts and dilated tubules to bilateral renal agenesis was observed. This was likely due to altered growth and branching morphogenesis of the ureteric bud (the progenitor tissue of the renal collecting system), despite the apparent ability of the ureteric bud-derived cells to induce differentiation of the metanephric mesenchyme. These data not only support a role for Itgb1 in the development of the renal collecting system but also raise the possibility that Itgb1 links morphogenesis to terminal differentiation and ultimately collecting duct function and/or maintenance.

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues. Organic anion transporters (OATs) are believed to mediate the cellular uptake, and hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various OAT isoforms has been lacking. To characterize substrate interactions with mOat1, mOat3, and mOat6, a fluorescence-based competition assay in Xenopus oocytes as well as wild-type and knock-out whole embryonic kidney (WEK) organ culture systems was developed using 6-carboxyfluorescein, 5-carboxyfluorescein, and fluorescein. Of nine common antiviral drugs assessed in oocytes, many manifested higher affinity for SLC22a6 (mOat1), originally identified as NKT (e.g. adefovir and cidofovir), two (ddC and ddI) manifested significantly higher affinity for mOat3, while mOat6 had comparatively low but measurable affinity for certain antivirals. A live organ staining approach combined with fluorescent uptake in WEK cultures allowed the visualization of OAT-mediated uptake ex vivo into developing proximal tubule-like structures, as well as quantification of substrate interactions of individual OAT isoforms. In general, antiviral specificity of SLC22a6 (Oat1) (in Oat3(-/-) WEK culture) and SLC22a8 (Oat3) (in Oat1(-/-) WEK culture) was consistent with the Xenopus oocyte data. The combined observations suggest SLC22a8 (Oat3) is the major transporter interacting with ddC and ddI. Finally, quantitative structure-activity relationship analysis of the nine antivirals' physicochemical descriptors with their OAT affinity indicates that antiviral preferences of mOat1 are explained by high polar surface areas (e.g. phosphate groups), whereas mOat3 prefers hydrogen bond acceptors (e.g. amines, ketones) and low rotatable bond numbers. In contrast, hydrogen bond donors (e.g. amides, alcohols) diminish binding to mOat6. This suggests that, despite sharing close overall sequence homology, Oat1, Oat3, and Oat6 have signficantly different binding pockets. Taken together, the data provide a basis for understanding potential drug interactions in combination antiviral therapy, as well as suggesting structural mdifications for drug design, especially in the context of targeting toward or away from specific tissues.

Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions.