Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

Familial Alzheimer's mutations within APPTM increase Aß42 production by enhancing accessibility of ε-cleavage site.

The high Aß42/Aß40 production ratio is a hallmark of familial Alzheimer's disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aß is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ε-cleavage at either T48 or L49, resulting in subsequent production of Aß42 or Aß40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ε-cleavage, and consequently shift cleavage preference towards Aß42.

Host co-factors of the retrovirus-like transposon Ty1.

BACKGROUND: Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. RESULTS: To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. CONCLUSION: Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 cDNA include characterized RNA metabolism and modification genes, consistent with their having roles in early steps in retrotransposition such as expression, nuclear export, translation, localization, or packaging of Ty1 RNA. Our results suggest that Bud21, Hcr1, Loc1, and Puf6 promote efficient synthesis or stability of Ty1 Gag.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid ß peptide (Aß). Aß aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. Aß is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by ß- and γ-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of γ-secretase for Aß production. The interaction between APPTM and γ-secretase determines the production of different species of Aß. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP-APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D (15)N-(1)H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.

5' to 3' mRNA decay factors colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition.

The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the Saccharomyces cerevisiae Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5' to 3' mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5' to 3' mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5' to 3' decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5' to 3' exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5' to 3' mRNA decay is essential for the packaging of Ty1 RNA in VLPs.

S-phase checkpoint pathways stimulate the mobility of the retrovirus-like transposon Ty1.

The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101delta mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101delta mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms.

Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements.

Activation of a LTR-retrotransposon by telomere erosion.

Retrotransposons can facilitate repair of broken chromosomes, and therefore an important question is whether the host can activate retrotransposons in response to chromosomal lesions. Here we show that Ty1 elements, which are LTR-retrotransposons in Saccharomyces cerevisiae, are mobilized when DNA lesions are created by the loss of telomere function. Inactivation of telomerase in yeast results in progressive shortening of telomeric DNA, eventually triggering a DNA-damage checkpoint that arrests cells in G2/M. A fraction of cells, termed survivors, recover from arrest by forming alternative telomere structures. When telomerase is inactivated, Ty1 retrotransposition increases substantially in parallel with telomere erosion and then partially declines when survivors emerge. Retrotransposition is stimulated at the level of Ty1 cDNA synthesis, causing cDNA levels to increase 20-fold or more before survivors form. This response is elicited through a signaling pathway that includes Rad24, Rad17, and Rad9, three components of the DNA-damage checkpoint. Our findings indicate that Ty1 retrotransposons are activated as part of the cellular response to telomere dysfunction.