The rockefeller university

Self-renewal is a hallmark of both hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs); therefore, the identification of mechanisms that are required for LSC, but not HSC, function could provide therapeutic opportunities that are more effective and less toxic than current treatments. Here, we employed an in vivo shRNA screen and identified jumonji domain-containing protein JMJD1C as an important driver of MLL-AF9 leukemia. Using a conditional mouse model, we showed that loss of JMJD1C substantially decreased LSC frequency and caused differentiation of MLL-AF9- and homeobox A9-driven (HOXA9-driven) leukemias. We determined that JMJU1C directly interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program. In contrast, loss of JMJD1C led to only minor defects in blood homeostasis and modest effects on HSC self-renewal. Together, these data establish JMJD1C as an important mediator of MLL-AF9- and HOXA9-driven LSC function that is largely dispensable for HSC function.

Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61 alpha, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid(1-10). The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation(11). How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (similar to 10 angstrom) or are of insufficient quality(6-8). Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

Background: A prophylactic HIV-1 vaccine is a global health priority. Objective: To assess a novel vaccine platform as a prophylactic HIV-1 regimen. Design: Randomized, double-blind, placebo-controlled trial. Both participants and study personnel were blinded to treatment allocation. (ClinicalTrials.gov: NCT01215149) Setting: United States, East Africa, and South Africa. Patients: Healthy adults without HIV infection. Intervention: 2 HIV-1 vaccines (adenovirus serotype 26 with an HIV-1 envelope A insert [Ad26.EnvA] and adenovirus serotype 35 with an HIV-1 envelope A insert [Ad35.Env], both administered at a dose of 5 x 10(10) viral particles) in homologous and heterologous combinations. Measurements: Safety and immunogenicity and the effect of baseline vector immunity. Results: 217 participants received at least 1 vaccination, and 210 (>96%) completed follow-up. No vaccine-associated serious adverse events occurred. All regimens were generally well-tolerated. All regimens elicited humoral and cellular immune responses in nearly all participants. Preexisting Ad26- or Ad35-neutralizing antibody titers had no effect on vaccine safety and little effect on immunogenicity. In both homologous and heterologous regimens, the second vaccination significantly increased EnvA antibody titers (approximately 20-fold from the median enzyme-linked immunosorbent assay titers of 30-300 to 3000). The heterologous regimen of Ad26-Ad35 elicited significantly higher EnvA antibody titers than Ad35-Ad26. T-cell responses were modest and lower in East Africa than in South Africa and the United States. Limitations: Because the 2 envelope inserts were not identical, the boosting responses were complex to interpret. Durability of the immune responses elicited beyond 1 year is unknown. Conclusion: Both vaccines elicited significant immune responses in all populations. Baseline vector immunity did not significantly affect responses. Second vaccinations in all regimens significantly boosted EnvA antibody titers, although vaccine order in the heterologous regimen had a modest effect on the immune response. Primary Funding Source: International AIDS Vaccine Initiative, National Institutes of Health, Ragon Institute, Crucell Holland.

Kaposi sarcoma (KS) is an endothelial malignancy caused by human herpes virus-8 (HHV-8) infection. The epidemic and iatrogenic forms of childhood KS result from a profound and acquired T cell deficiency. Recent studies have shown that classic KS of childhood can result from rare single-gene inborn errors of immunity, with mutations in WAS, IFNGR1, STIM1, and TNFRSF4. The pathogenesis of the endemic form of childhood KS has remained elusive. We review childhood KS pathogenesis and its relationship to inherited and acquired immunodeficiency to oncogenic HHV-8.

The transcription factor Blimp-1 is necessary for the generation of plasma cells. Here we studied its functions in plasmablast differentiation by identifying regulated Blimp-1 target genes. Blimp-1 promoted the migration and adhesion of plasmablasts. It directly repressed genes encoding several transcription factors and Aicda (which encodes the cytidine deaminase AID) and thus silenced B cell specific gene expression, antigen presentation and class-switch recombination in plasmablasts. It directly activated genes, which led to increased expression of the plasma cell regulator IRF4 and proteins involved in immunoglobulin secretion. Blimp-1 induced the transcription of immunoglobulin genes by controlling the 3' enhancers of the loci encoding the immunoglobulin heavy chain (Igh) and K-light chain (lgk) and, furthermore, regulated the post-transcriptional expression switch from the membrane-bound form of the immunoglobulin heavy chain to its secreted form by activating EII2 (which encodes the transcription-elongation factor ELL2). Notably, Blimp-1 recruited chromatin-remodeling and histone-modifying complexes to regulate its target genes. Hence, many essential functions of plasma cells are under the control of Blimp-1.

Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5'-3' exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit(+) cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.

The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 angstrom. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

Faces contain a variety of information such as one's identity and expression. One prevailing model suggests a functional division of labor in processing faces that different aspects of facial information are processed in anatomically separated and functionally encapsulated brain regions. Here, we demonstrate that facial identity and expression can be processed in the same region, yet with different neural coding strategies. To this end, we employed functional magnetic resonance imaging to examine two types of coding schemes, namely univariate activity and multivariate pattern, in the posterior superior temporal cortex (pSTS) - a face-selective region that is traditionally viewed as being specialized for processing facial expression. With the individual difference approach, we found that participants with higher overall face selectivity in the right pSTS were better at differentiating facial expressions measured outside of the scanner. In contrast, individuals whose spatial pattern for faces in the right pSTS was less similar to that for objects were more accurate in identifying previously presented faces. The double dissociation of behavioral relevance between overall neural activity and spatial neural pattern suggests that the functional-division-of-labor model on face processing is over-simplified, and that coding strategies shall be incorporated in a revised model.

Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.