Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-γ and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19 + B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.
Infection with human immunodeficiency virus type 1 (HIV-1) causes an inexorable depletion of CD4؉ T cells. The loss of these cells is particularly pronounced in the mucosal immune system during acute infection, and the data suggest that direct viral cytopathicity is a major factor. Cell cycle arrest caused by the HIV-1 accessory protein Vpr is strongly correlated with virus-induced cell death, and phosphorylation of Vpr serine 79 (S79) is required to activate G 2 /M cell cycle blockade. However, the kinase responsible for phosphorylating Vpr remains unknown. Our bioinformatic analyses revealed that S79 is part of a putative phosphorylation site recognized by protein kinase A (PKA). We show here that PKA interacts with Vpr and directly phosphorylates S79. Inhibition of PKA activity during HIV-1 infection abrogates Vpr cell cycle arrest. These findings provide new insight into the signaling event that activates Vpr cell cycle arrest, ultimately leading to the death of infected T cells.
Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins—including histones, transcription factors (TFs), and polymerases—interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.
Although the molecular events required for the repair of double-strand breaks (DSB) have been well characterized, the role of epigenetic processes in the recognition and repair of DSBs has only been investigated at low resolution. We rapidly and synchronously induced a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus, which has well-positioned nucleosomes. Utilizing MNase-seq epigenome mapping we interrogated the order of chromatin changes that occur immediately following a DSB by generating a base-pair resolution map of the chromatin landscape.In wild type cells, the first nucleosome left of the break was rapidly evicted. The eviction of this flanking nucleosome was dynamic and proceeded through an early intermediate chromatin structure where the nucleosome was repositioned in the adjacent linker DNA.Other nucleosomes bordering both sides of the break were also shifted away from the break; however, their loss was more gradual. These local changes preceded a broader loss of chromatin organization and nucleosome eviction that was marked by increased MNase sensitivity in the regions ~8 kb on each side of the break. While the broad loss of chromatin organization was dependent on the end-processing complex, Mre11-Rad50-Xrs2 (MRX), the early remodeling and repositioning of the nucleosome adjacent to the break was independent of the MRX and YKU70/80 complexes. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population, where 5' to 3' end-resection of DSB ends is blocked. Concomitant with DSB repair, we Tripuraneni December 6, 2019 2 observed the re-deposition and precise re-positioning of nucleosomes at the originally-occupied positions. This re-establishment of the pre-lesion chromatin landscape suggests that a DNA replication-independent mechanism exists in G1 cells to preserve epigenome organization following DSB repair.
Although the molecular events required for the repair of double-strand breaks (DSB) have been well characterized, the role of epigenetic processes in the recognition and repair of DSBs has only been investigated at low resolution. We rapidly and synchronously induced a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus, which has well-positioned nucleosomes. Utilizing MNase-seq epigenome mapping we interrogated the order of chromatin changes that occur immediately following a DSB by generating a base-pair resolution map of the chromatin landscape.In wild type cells, the first nucleosome left of the break was rapidly evicted. The eviction of this flanking nucleosome was dynamic and proceeded through an early intermediate chromatin structure where the nucleosome was repositioned in the adjacent linker DNA.Other nucleosomes bordering both sides of the break were also shifted away from the break; however, their loss was more gradual. These local changes preceded a broader loss of chromatin organization and nucleosome eviction that was marked by increased MNase sensitivity in the regions ~8 kb on each side of the break. While the broad loss of chromatin organization was dependent on the end-processing complex, Mre11-Rad50-Xrs2 (MRX), the early remodeling and repositioning of the nucleosome adjacent to the break was independent of the MRX and YKU70/80 complexes. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population, where 5' to 3' end-resection of DSB ends is blocked. Concomitant with DSB repair, we
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