Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR ...screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.
Recent advances in sample preparation and sequencing technology have made it possible to profile the transcriptomes of individual cells using single-cell RNA sequencing (scRNA-Seq). Compared to bulk ...RNA-Seq data, single-cell data often contain a higher percentage of zero reads, mainly due to lower sequencing depth per cell, which affects mostly measurements of low-expression genes. However, discrepancies between platforms are observed regardless of expression level. Using four paired datasets with multiple samples each, we investigated technical and biological factors that can contribute to this expression shift. Using two separate machine learning models we found that, in addition to expression level, RNA integrity, gene or UTR3 length, and the number of transcripts potentially also influence the occurrence of zeros. These findings could enable the development of novel analytical methods for cross-platform expression shift correction. We also identified genes and biological pathways in our diverse datasets that consistently showed differences when assessed at the single cell versus bulk level to assist in interpreting analysis across transcriptomic platforms. At the gene level, 25 genes (0.12%) were found in all datasets as discordant, but at the pathway level, 7 pathways (2.02%) showed shared enrichment in discordant genes.
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V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and ...inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.
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•Synergistic contacts between the elements of a super-enhancer contribute to activity•Igk enhancers play diverse roles in lymphocyte development•The Igk enhancer, MiEκ, contributes to regulation of Tcrb recombination•Enhancer sharing can involve active as well as inactive regulatory elements
Proudhon et al. show that, in B cells, synergistic chromatin contacts between the individual elements of the Igk super-enhancer contribute to the transcriptional output of all partner enhancers. In T cells, one component of the Igk enhancer cluster cooperates with the Tcrb enhancer to exert control over Tcrb recombination.
The Spectral Underpinning of word2vec Jaffe, Ariel; Kluger, Yuval; Lindenbaum, Ofir ...
Frontiers in applied mathematics and statistics,
12/2020, Letnik:
6
Journal Article
Recenzirano
Odprti dostop
Word2vec introduced by Mikolov et al. is a word embedding method that is widely used in natural language processing. Despite its success and frequent use, a strong theoretical justification is still ...lacking. The main contribution of our paper is to propose a rigorous analysis of the highly nonlinear functional of word2vec. Our results suggest that word2vec may be primarily driven by an underlying spectral method. This insight may open the door to obtaining provable guarantees for word2vec. We support these findings by numerical simulations. One fascinating open question is whether the nonlinear properties of word2vec that are not captured by the spectral method are beneficial and, if so, by what mechanism.
Purpose: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and
survival, and are targets of drugs in clinical development. We ...assessed expression of PI3K in melanomas and nevi, and studied
associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002.
Experimental Design: Using Automated Quantitative Analysis, we quantified expression of p85 and p110α subunits in 540 nevi and 523 melanomas.
We determined the IC 50 for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels
of PI3K pathway members and sensitivity to LY294002.
Results: p85 and p110α tend to be coexpressed ( P < 0.0001); expression was higher in melanomas than nevi ( P < 0.0001 ) for both subunits, and higher in metastatic than primary melanomas for p85 ( P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable.
We found no association by t tests between baseline p85, p110α, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser 235 and Ser 240 were lower in the more resistant cell lines ( P = 0.01 and P = 0.004, respectively).
Conclusions: Expression of p85 and p110α subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment
pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full
activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response
in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.
CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the ...role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.
Both pluripotent embryonic stem cells (ESCs), established from preimplantation murine blastocysts, and epiblast stem cells (EpiSCs), established from postimplantation embryos, can self‐renew in ...culture or differentiate into each of the primary germ layers. While the core transcription factors (TFs) OCT4, SOX2, and NANOG are expressed in both cell types, the gene expression profiles and other features suggest that ESCs and EpiSCs reflect distinct developmental maturation stages of the epiblast in vivo. Accordingly, “naïve” or “ground state” ESCs resemble cells of the inner cell mass, whereas “primed” EpiSCs resemble cells of the postimplantation egg cylinder. To gain insight into the relationship between naïve and primed pluripotent cells, and of each of these pluripotent states to that of nonpluripotent cells, we have used FAIRE‐seq to generate a comparative atlas of the accessible chromatin regions within ESCs, EpiSCs, multipotent neural stem cells, and mouse embryonic fibroblasts. We find a distinction between the accessible chromatin patterns of pluripotent and somatic cells that is consistent with the highly related phenotype of ESCs and EpiSCs. However, by defining cell‐specific and shared regions of open chromatin, and integrating these data with published gene expression and ChIP analyses, we also illustrate unique features of the chromatin of naïve and primed cells. Functional studies suggest that multiple stage‐specific enhancers regulate ESC‐ or EpiSC‐specific gene expression, and implicate auxiliary TFs as important modulators for stage‐specific activation by the core TFs. Together these observations provide insights into the chromatin structure dynamics accompanying transitions between these pluripotent states. Stem Cells 2015;33:378–391
Opioid use disorder (OUD) negatively impacts the HIV continuum of care for persons living with HIV (PLH). Medication treatment for OUD (MOUD) may have differential biological effects in individuals ...with HIV and OUD. To understand the role of MOUD – opioid agonist methadone, partial agonist buprenorphine and antagonist naltrexone – in HIV-1 persistence and reactivation, we will use molecular virology approaches to carry out the first prospective, longitudinal studies of adults living with HIV with OUD initiating MOUD. One of the major challenges to studying the impact of MOUD on HIV persistence is the low retention rate of study participants and the requirement of large-volume blood sampling to study the HIV proviral landscape and expression profiles.
A prospective cohort study is underway to study the HIV-1 expression, proviral landscape, and clonal expansion dynamics using limited blood sampling from persons with DSM-5 diagnosed OUD who are living with HIV infection and initiating treatment with methadone, buprenorphine, or extended-release naltrexone.
We describe the recruitment, laboratory, and statistical methods of this study as well as the protocol details of this on-going study. Out of the 510 screened for enrollment into the study, 35 (7%) were eligible and 27 were enrolled thus far. Retention through month 3 has been high at 95%.
This on-going study is evaluating the impact of MOUD on HIV persistence at the molecular virology level using limited blood sampling via a prospective, longitudinal study of people living with HIV DSM-5 OUD initiating treatment with MOUD.
Genomic aberrations can be used to determine cancer diagnosis and prognosis. Clinically relevant novel aberrations can be discovered using high-throughput assays such as Single Nucleotide ...Polymorphism (SNP) arrays and next-generation sequencing, which typically provide aggregate signals of many cells at once. However, heterogeneity of tumor subclones dramatically complicates the task of detecting aberrations.
The aggregate signal of a population of subclones can be described as a linear system of equations. We employed a measure of allelic imbalance and total amount of DNA to characterize each locus by the copy number status (gain, loss or neither) of the strongest subclonal component. We designed simulated data to compare our measure to existing approaches and we analyzed SNP-arrays from 30 melanoma samples and transcriptome sequencing (RNA-Seq) from one melanoma sample.We showed that any system describing aggregate subclonal signals is underdetermined, leading to non-unique solutions for the exact copy number profile of subclones. For this reason, our illustrative measure was more robust than existing Hidden Markov Model (HMM) based tools in inferring the aberration status, as indicated by tests on simulated data. This higher robustness contributed in identifying numerous aberrations in several loci of melanoma samples. We validated the heterogeneity and aberration status within single biopsies by fluorescent in situ hybridization of four affected and transcriptionally up-regulated genes E2F8, ETV4, EZH2 and FAM84B in 11 melanoma cell lines. Heterogeneity was further demonstrated in the analysis of allelic imbalance changes along single exons from melanoma RNA-Seq.
These studies demonstrate how subclonal heterogeneity, prevalent in tumor samples, is reflected in aggregate signals measured by high-throughput techniques. Our proposed approach yields high robustness in detecting copy number alterations using high-throughput technologies and has the potential to identify specific subclonal markers from next-generation sequencing data.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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