Chromatin Composition Is Changed by Poly(ADP-ribosyl)ation during Chromatin Immunoprecipitation

Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition

A proteomic atlas of senescence-associated secretomes for aging biomarker development.

The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo

Correction to: EGFR/Ras-induced CCL20 production modulates the tumour microenvironment

The article ‘EGFR/Ras-induced CCL20 production modulates the tumour microenvironment’, written by Andreas Hippe, Stephan Alexander Braun, Péter Oláh, Peter Arne Gerber, Anne Schorr, Stephan Seeliger, Stephanie Holtz, Katharina Jannasch, Andor Pivarcsi, Bettina Buhren, Holger Schrumpf, Andreas Kislat, Erich Bünemann, Martin Steinhoff, Jens Fischer, Sérgio A. Lira, Petra Boukamp, Peter Hevezi, Nikolas Hendrik Stoecklein, Thomas Hoffmann, Frauke Alves, Jonathan Sleeman, Thomas Bauer, Jörg Klufa, Nicole Amberg, Maria Sibilia, Albert Zlotnik, Anja Müller- Homey and Bernhard Homey, was originally published electronically on the publisher’s internet portal on 30 June 2020 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 16 September 2021 to © The Author(s) 2021 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Open Access funding enabled and organized by Projekt DEAL

Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection

Summary: Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species.For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020)

PARylation affects binding of transcription factors and histone H1 differently dependent on the binding site.

<p>Preparation of chromatin and ChIP was done by JLI and BMB protocol, respectively. Three independent chromatin preparations were analyzed by three independent PCRs each (panels A, C), or by only one PCR each (panel B) due to lack of material. (<b>A</b>) Suppression of PARylation improves ChIP efficiency in general, but with some specificity. Column color code: blue: ChIP with anti-CTCF antibody; white: ChIP with anti-E2F1 antibody; green: ChIP with anti-p65/RELA (NFκB) antibody; black: ChIP with anti-NFYB antibody. Respective binding sites and promoters are indicated on Y-axis of panel C. J = JLI protocol; B = BMB protocol. Error bars represent mean±s.e.m. (N = 3), exact P-values are indicated; data were analyzed by unpaired t-test. Note that CTCF binding is affected by PARylation at the <i>H19_ICR</i> locus, but not at the <i>BRCA1</i> promoter. (<b>B</b>) PARylation impacts on histone H1 binding independent of transcription factors. ChIP was performed with anti-H1 antibody and analyzed for binding at the same positions as in (A). Respective binding sites and promoters are indicated on Y-axis of panel C. Coding was maintained to simplify comparison. Error bars represent mean±s.e.m. (N = 3), exact P-values are indicated; data were analyzed by unpaired t-test. Note that H1 binding at the CTCF sites is oppositely affected by PARylation. (<b>C</b>) Increased formaldehyde concentration during fixation does not impact on PCR efficiency. Product signal intensities from input PCRs were compared. Respective binding sites and promoters are indicated on Y-axis. J = JLI protocol, B = BMB protocol. Coding was maintained to simplify comparison between panels. Only NFκB/RELA binding to <i>HIF1A</i> promoter displayed border-line significance in PCR efficiency. Error bars represent mean±s.e.m. (N = 3), *P = 0.045; data were analyzed by unpaired t-test.</p

Low-dose formaldehyde induces γH2AX formation.

<p>Cells were fixed with 1% or 4% paraformaldehyde and analyzed by confocal microscopy. (<b>A</b>) γH2AX foci were counted using one confocal slice after reducing background staining by ImageJ software. Reduction parameters were identical for respective pictures. Cells were split into three groups with (i) less than 5, (ii) between 5 and 20, (iii) more than 20 foci, and percent of total cells was calculated. 10 min 1% paraformaldehyde (1% FA) induces more than sevenfold increase in cells with more than 20 foci, and a decrease in cells with less than 5 foci compared to 4% paraformaldehyde (4% FA). Error bars represent mean±s.e.m. (N = 3), ***P<0.001; data were analyzed with one-way ANOVA and Dunnett's Multiple Comparison Test. (<b>B</b>) All pictures from a z-stack were analyzed for γH2AX foci intensity and normalized to cells fixed for 20 min with 4% paraformaldehyde. Intensity increases eightfold with 1% FA compared to 4% FA. Error bars represent mean±s.e.m. (N = 3), *P = 0.016; data were analyzed by unpaired t-test.</p

PARP inhibition suppresses PAR formation only if present in every step.

<p>(<b>A</b>) Detection of PAR by immunofluorescence after different fixation strategies. H₂O₂ in combination with methanol fixation induces an even distribution of PAR-staining within the nucleus (I). Pretreatment of cells with 2 µM PJ34 6 h in advance of damage induction and methanol fixation completely suppresses PAR formation (II). Fixation of cells with JLI protocol induces polymer synthesis without H₂O₂ (III), but in contrast to (II), PJ34 is not able to block PARP activity completely (IV). Methanol fixation directly after the formaldehyde step reduces PAR staining (V), but not if the cells were fixed after PBS washing (VI). PJ34 is able to suppress PAR formation only if present in all steps until lysis (VII). Scale bars represent 10 µM. (<b>B</b>) Flow chart of the different fixation strategies. Standard JLI fixation (III) encompasses all steps until lysis/permeabilization for immunofluorescence detection. Preincubation with 2 µM PARP inhibitor PJ34 (IV) is otherwise identical to (III). Methanol is used to fix cells either directly after formaldehyde treatment (V), or after PBS washes (VI). (VII) 2 µM PJ34 is used for preincubation and continuous treatment of cells during all steps until lysis/permeabilization for immunofluorescence detection.</p

Model of PAR-dependent chromatin remodeling during ChIP fixation.

<p>On the left side, standard ChIP protocol leads to PARP (brown) activation and subsequent poly(ADP-ribose) formation (orange lines) by damaging DNA (red asterisk). This either dislodges proteins (blue, X) from DNA or attracts proteins (green, Y) to DNA with subsequent crosslinking (red arc). Therefore, after lysis and sonication, immunoprecipitated proteins can be present either in wrong amounts (reduced efficiency), or proteins are crosslinked that are not present on DNA in physiological conditions (false positive). On the right side, using 3.7% formaldehyde for 10 min as fixation protocol or treatment with the PARP inhibitor PJ34 throughout the experiment until lysis abolishes PAR formation completely. Thus, chromatin composition is unaltered and reflects in vivo situation.</p

Suppression of PARylation impacts on ChIP efficiency.

<p>Evaluation of ChIP efficiency of PARP1 bound to <i>PARP1</i> promoter. Both modifications improve significantly ChIP efficiency. Error bars represent mean±s.e.m. (N = 3), *P<0.05, **P<0.01; data were analyzed with one-way ANOVA and Bonferroni's Multiple Comparison Test.</p