Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function

Plasma cell survival and the consequent duration of immunity vary widely with infection or vaccination. Using fluorescent glucose analog uptake, we defined multiple developmentally independent mouse plasma cell populations with varying life- spans. Long-lived plasma cells imported more fluo- rescent glucose analog, expressed higher surface levels of the amino acid transporter CD98, and had more autophagosome mass than did short-lived cells. Low amino acid concentrations triggered re- ductions in both antibody secretion and mitochon- drial respiration, especially by short-lived plasma cells. To explain these observations, we found that glutamine was used for both mitochondrial respira- tion and anaplerotic reactions, yielding glutamate and aspartate for antibody synthesis. Endoplasmic reticulum (ER) stress responses, which link meta- bolism to transcriptional outcomes, were similar between long- and short-lived subsets. Accordingly, population and single-cell transcriptional compari- sons across mouse and human plasma cell subsets revealed few consistent and conserved dif- ferences. Thus, plasma cell antibody secretion and lifespan are primarily defined by non-transcriptional metabolic traits

ZBTB32 restrains antibody responses to murine cytomegalovirus infections, but not other repetitive challenges

ZBTB32 is a transcription factor that is highly expressed by a subset of memory B cells and restrains the magnitude and duration of recall responses against hapten-protein conjugates. To define physiological contexts in which ZBTB32 acts, we assessed responses by Zbtb32-/- mice or bone marrow chimeras against a panel of chronic and acute challenges. Mixed bone marrow chimeras were established in which all B cells were derived from either Zbtb32-/- mice or control littermates. Chronic infection of Zbtb32-/- chimeras with murine cytomegalovirus led to nearly 20-fold higher antigen-specific IgG2b levels relative to controls by week 9 post-infection, despite similar viral loads. In contrast, IgA responses and specificities in the intestine, where memory B cells are repeatedly stimulated by commensal bacteria, were similar between Zbtb32-/- mice and control littermates. Finally, an infection and heterologous booster vaccination model revealed no role for ZBTB32 in restraining primary or recall antibody responses against influenza viruses. Thus, ZBTB32 does not limit recall responses to a number of physiological acute challenges, but does restrict antibody levels during chronic viral infections that periodically engage memory B cells. This restriction might selectively prevent recall responses against chronic infections from progressively overwhelming other antibody specificities.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01AI99109, R01AI131680, U01AI131349, K08AI04991]; New York Stem Cell FoundationOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Defining phenotypic and functional heterogeneity of glioblastoma stem cells by mass cytometry

Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker-defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings

Looping Mediated Interaction between the Promoter and 3′ UTR Regulates Type II Collagen Expression in Chondrocytes

<div><p>Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (<em>Col2a1</em>) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of <em>Col2a1</em> expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigated the crosstalk between <em>cis</em>-acting DNA element and transcription factor on <em>Col2a1</em> expression in primary chondrocytes. Bioinformatic analysis revealed the potential regulatory regions in the <em>Col2a1</em> genomic locus. Among them, promoter and 3′ untranslated region (UTR) showed highly accessible chromatin architecture with enriched recruitment of active chromatin markers in primary chondrocytes. 3′ UTR has a potent enhancer function which recruits Lef1 (Lymphoid enhancer binding factor 1) transcription factor, leading to juxtaposition of the 3′ UTR with the promoter through gene looping resulting in up-regulation of <em>Col2a1</em> gene transcription. Knock-down of endogenous Lef1 level significantly reduced the gene looping and subsequently down-regulated <em>Col2a1</em> expression. However, these regulatory loci become inaccessible due to condensed chromatin architecture as chondrocytes dedifferentiate which was accompanied by a reduction of gene looping and down-regulation of <em>Col2a1</em> expression. Our results indicate that Lef1 mediated looping between promoter and 3′ UTR under the permissive chromatin architecture upregulates <em>Col2a1</em> expression in primary chondrocytes.</p> </div

Juxtaposition of 3′ UTR with the promoter through gene looping.

<p>(A) The relative position of the primers and <i>Pst1</i> cleavage sites designed to detect gene looping between the Col2a1 promoter and 3′ UTR are denoted as described in the main text and Fig. 1A. Figure was not drawn in scale. The filled black boxes indicate the promoter and 3′ UTR. X and Y denote primer pairs that do not flank any <i>Pst1</i> site. The empty horizontal arrows denote the 3C primers and the vertical black arrow indicates the Lef1 binding site (BS) in the 3′ UTR. (B, C) 3C assay was performed. Nuclei prepared from uncrosslinked (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828.s002" target="_blank">Fig. S2</a>), formaldehyde crosslinked or Lef1 siRNA transfected primary chondrocytes were subjected to <i>Pst1</i> digestion. Digestion efficiency was confirmed by RT-PCR using primers flanking the <i>Pst1</i> sites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828.s002" target="_blank">Fig. S2</a>). The formation of 3C product was detected by qRT-PCR using digested (cut), digested and ligated (cut + ligated) or Lef1 siRNA treated, digested and ligated (cut + ligated + siRNA) DNA samples as templates. The results are shown as EtBr stained gel images (B) or quantitative representation (C) of the product obtained with the indicated primer pairs relative to that obtained with control primer pairs X+Y. The data shown are expressed as mean ± SEM, n = 3 and *P<0.05.</p

Primer sequences for CHART-PCR and ChIP assay.

<p>Region encompassed by the analyzed CNSs are:</p><p>Promoter;−57/+166, CNS 1; +850/+3334, CNS 2; +5755/+9165, CNS 3; +14013/+14613, 3′ UTR; +28700/+29243.</p

Identification of regulatory elements in the <i>Col2a1</i> locus.

<p>(A) Conserved noncoding sequences (CNSs) in the <i>Col2a1</i> loci of mouse and human are shown. rVISTA 2.0 analysis depicting % conservation between mouse (as base) and human <i>Col2a1</i> loci. Peaks in pink color indicate the intronic regions, violet peaks indicate the exons and sky blue peaks indicate the 5′ and 3′ UTRs, respectively. The relative positions of CNS regions are named according to their distance in kb from the transcription start site. (B) HTB-94 human chondrosarcoma cells were transfected with the luciferase reporter alone (mock), construct containing the <i>Col2a1</i> minimal promoter only (mP) or with the constructs containing the individual CNS regions with the minimal promoter, and luciferase activity was measured 24 hrs post transfection. (C) Primary mouse chondrocytes were transfected with indicated luciferase reporter plasmids and luciferase activity was measured 24 hrs post transfection. In each case, plasmid encoding Renilla luciferase (pRLTK) was used as normalization control and data was expressed relative to the activity of Renilla luciferase as Relative Luciferase Unit (RLU). The data shown are expressed as mean ± SEM, n = 3 and **P<0.01, ***P<0.001.</p

Lef1 dependent <i>Col2a1</i> expression in primary chondrocytes.

<p>(A, B). Primary chondrocytes were transfected with Lef1 expression plasmid or empty vector control (mock). Lef1 over-expression was confirmed by immunoblotting with antibodies against Lef1 and β-actin (control) and relative level <i>of Col2a1</i> transcript was detected by qRT-PCR and expressed relative to the level of housekeeping control HPRT. (C, D) Primary chondrocytes were transfected with scrambled siRNA (mock) or Lef1 siRNA and Lef1 knock-down was confirmed by immunoblotting and relative level of <i>Col2a1</i> transcript was detected by qRT-PCR and expressed relative to the level of housekeeping control HPRT. Knock-down efficiency was expressed as percentage of scrambled siRNA (control) transfected sample. (E) Physiological binding of Lef1 to the predicted conserved Lef1-binding site in the 3′ UTR was assessed by ChIP assay. Crosslinked and fragmented DNA from primary chondrocytes were immunoprecipitated with Lef1 antibody and IgG (control). PCR analysis was performed using the primers for the 3′ UTR region of <i>Col2a1</i> locus. The same precipitate was also probed with primers specific for the 3′ UTR regions of Cox2 and MMP13 (positive controls) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828-Giese1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828-Kadauke1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828-OSullivan1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040828#pone.0040828-Yun1" target="_blank">[30]</a> or primers specific for the non-conserved region (+16801/+17024) in the <i>Col2a1</i> locus (negative control). All data are representative of three independent experiments. *P<0.05, **P<0.01. (F) EMSA was performed by incubating nuclear extract prepared from P0 stage chondrocyte with the indicated labeled probes, (hot-wild type (hot-WT) and hot-consensus (hot-Cons) and non-labeled competitor oligonucleotides (cold-wild type (cold-WT) and cold-Consensus (cold-Cons) or α-Lef1 antibody.</p

Primer sequences for Chromosome Conformation capture Assay.

<p>Primer sequences for Chromosome Conformation capture Assay.</p