Interplay of chromatin remodeling, transcriptional regulation, and nuclear organization

Transcription is regulated on different levels to ensure that genes are expressed at the correct time and in the amounts required. At the chromatin level, DNA is wound onto histone proteins, forming nucleosomes that influence accessibility of DNA elements. Modifications on those histones and interactions with other chromatin proteins can either encourage or inhibit recruitment of the transcription machinery. Genomic regions of similar character form chromatin domains, organizing the genome based on their transcription states. Within the nucleus, both individual loci and entire chromosomes assume non-random positions, based on their transcription levels and interactions with nuclear landmarks. This thesis examines the effects of the Fun30 chromatin remodeling enzymes on transcription regulation and nuclear organization, both on the local chromatin level as well as on a genome-wide scale. Using the fission yeast Schizosaccharomyces pombe as a model organism, we mapped the interactions between the genome and two inner nuclear membrane proteins, Ima1 and Man1. We observed a preference for lowly expressed genes to associate with the nuclear envelope, similar to what had been observed in mammalian and fruit fly cells. When comparing Ima1 and Man1 binding patterns, we found both common and separate target sites, suggesting a role for inner nuclear membrane proteins in organizing the fission yeast genome. Following up on these results, we went on to examine subtelomeric chromatin domains, which are regulated through the Fun30 remodeler Fft3. These domains contain repressed genes, whose transcription levels increase in cells carrying an fft3Δ deletion. While the subtelomeres associate with the nuclear envelope through Man1 in wild-type cells, this interaction is lost in fft3Δ cells. In these cells, we also observed changes in nucleosome occupancy at the subtelomeric borders. Interestingly, a strain carrying a catalytically inactive version of the Fft3 remodeler showed the same behavior as the deletion strain, with upregulation of subtelomeric genes and loss of Man1 interactions. Together, these results point to an active role of Fft3 in regulating subtelomeric chromatin, transcription, and nuclear periphery interactions. In addition to their role at subtelomeres, Fun30 remodelers also control transcription in other parts of the genome. When we examined a strain lacking Fft2, a paralog of Fft3, we found increased transcription of the fission yeast Tf2 retrotransposons. This increase is accompanied by a shift in transcription start site (TSS) further upstream and is especially pronounced when both fft2 and fft3 are deleted. By mapping nucleosome positioning, we were able to establish that Fft2 and Fft3 collaborate in stabilizing a nucleosome over the upstream TSS, resulting in transcription initiation further downstream and production of an mRNA incapable of transposition. Expression of both remodelers is downregulated in stress conditions, allowing for production of the longer transcript under these circumstances. We propose that the shift in TSS choice allows for bursts of transposition in cells under environmental stress. This can enable cells to adapt to changed conditions through favorable insertion events altering expression of nearby genes

Regulating retrotransposon activity through the use of alternative transcription start sites

International audienceRetrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome

IL-10 Receptor Signaling Is Essential for T R 1 Cell Function In Vivo

Interleukin-10 (IL-10) is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and being therefore currently examined in clinical trials as T-cell therapy in patients with inflammatory bowel disease (IBD). However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified in order to optimize the efficiency and to ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo

The induction and function of the anti-inflammatory fate of TH17 cells

TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes.Fil: Xu, Hao. University of Yale. School of Medicine; Estados UnidosFil: Agalioti, Theodora. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Zhao, Jun. University of Yale. School of Medicine; Estados UnidosFil: Steglich, Babett. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Wahib, Ramez. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Amezcua Vesely, Maria Carolina. University of Yale. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bielecki, Piotr. University of Yale. School of Medicine; Estados UnidosFil: Bailis, Will. University of Yale. School of Medicine; Estados UnidosFil: Jackson, Ruaidhri. University of Yale. School of Medicine; Estados UnidosFil: Perez, Daniel. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Izbicki, Jakob. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Licona-Limón, Paula. University of Yale. School of Medicine; Estados UnidosFil: Kaartinen, Vesa. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Geginat, Jens. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Esplugues, Enric. University of Yale. School of Medicine; Estados UnidosFil: Tolosa, Eva. University of Yale. School of Medicine; Estados UnidosFil: Huber, Samuel. University of Yale. School of Medicine; Estados UnidosFil: Flavell, Richard A.. University of Yale. School of Medicine; Estados UnidosFil: Gagliani, Nicola. University Medical Center Hamburg-Eppendorf; Alemani

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

Background &amp; Aims: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. Methods: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. Results: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Conclusions: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Impact and implications: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.</p

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

Background &amp; Aims: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. Methods: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. Results: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Conclusions: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Impact and implications: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.</p

The Fun30 Chromatin Remodeler Fft3 Controls Nuclear Organization and Chromatin Structure of Insulators and Subtelomeres in Fission Yeast

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3? cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope

Fft3 and Bqt4 anchor subtelomeres at the nuclear envelope.

<p><b>(A)</b> Live cell microscopy examples for wild type and <i>bqt4Δ fft3Δ</i> cells. The nuclear envelope is stained by Ccr1N-GFP (green) and the telomeres by Taz1-mCherry (red). Cells were scored as belonging to either of three equal volume zones depending on the distance between the nuclear envelope and the telomere signal. <b>(B-E)</b> Telomeres are released from the nuclear envelope in <i>bqt4Δ</i> cells, but move even further into the nuclear interior in <i>bqt4Δ fft3Δ</i> cells, where subtelomeres lose association with the nuclear envelope. Percentages of cells in each of the zones in different strains are shown, with number of cells measured in the legend. Significance testing was done using a two-sided Chi-square test.</p

Fft3 interacts with TFIIIC at Pol III transcribed loci and affects peripheral localization of tRNA genes.

<p><b>(A)</b> Fft3 is significantly enriched at tRNA genes. Fft3-enrichment over anti-myc control is shown as boxplot. Fft3-myc ChIP-chip data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005101#pgen.1005101.ref014" target="_blank">14</a>]. <b>(B)</b> Fft3-myc binds to loci also bound by the TFIIIC-subunit Sfc6 and the Pol III-component Rpc130. Shown are ChIP-enrichments over 2-fold over input on 1.5 Mb on the left arm of chromosome 2. The subtelomeric chromatin is marked in gray. Fft3-myc ChIP-chip data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005101#pgen.1005101.ref014" target="_blank">14</a>], Sfc6 and Rpc130 ChIP-chip data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005101#pgen.1005101.ref028" target="_blank">28</a>]. <b>(C)</b> Fft3 is significantly enriched at loci bound by TFIIIC and Pol III together and Pol III alone, but not at loci bound by TFIIIC alone. Fft3-enrichment over input is shown as boxplot. Targets were defined as probes with more than two-fold enrichment (both Sfc6 and Rpc130: 896 probes (53.8 kb), Rpc130 only: 1656 probes (99.3 kb), Sfc6 only: 750 probes (45 kb), neither: 37917 (2,275 kb)). <b>(D)</b> Fft3 and Sfc4 interact in a Yeast-2-hybrid screen. Bait fragments of Sfc4 are shown aligned to full-length Sfc4, with overlap between them marked in red. <b>(E)</b> Sfc4 is co-purified in a Fft3-TAP purification. Shown are input (diluted to 10%), final wash and eluate for the double-tagged strain (Sfc4-Myc Fft3-TAP) and the two single tagged strains (Sfc4-Myc and Fft3-TAP). <b>(F)</b> Nup85 is significantly enriched at tRNA genes. Nup85-Dam enrichment over Dam-only control is shown as boxplot. Nup85-Dam data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005101#pgen.1005101.ref024" target="_blank">24</a>]. <b>(G)</b> The INM protein Man1 is enriched at tRNA genes in wild type <i>S</i>. <i>pombe</i>. Man1-Dam enrichment over Dam-only control is shown as boxplot. <b>(H)</b> Man1 is no longer enriched at tRNA genes in <i>fft3Δ</i> cells. Man1-Dam enrichment over Dam-only control in <i>fft3Δ</i> cells is shown as boxplot. <b>(I)</b> Nucleosome occupancy at tRNA genes is reduced in <i>fft3Δ</i> cells. The average number of reads mapping to each tRNA is shown for wild type and <i>fft3Δ</i>. P-value was obtained using paired, two-sided Mann-Whitney U test.</p

Subtelomeric silencing requires remodeling activity of Fft3.

<p><b>(A)</b> Nucleosome occupancy at subtelomeric borders is affected by Fft3. Map shows nucleosome occupancy profiles over the border of the left subtelomere on chromosome I (top panel) and Fft3 enrichment (blue). Genes are shown in light grey, LTRs in dark grey. Asterisks mark positions were nucleosome occupancy is reduced. <b>(B)</b> Schematic representation of the Fft3 ATPase mutant containing a point mutation in the ATPase domain that replaces lysine at position 418 (AAA) by arginine (AGA) and a C-terminal Myc-tag. The split ATPase domain is highlighted in grey [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005101#pgen.1005101.ref016" target="_blank">16</a>]. <b>(C)</b> The Fft3 ATPase mutant (<i>fft3-K418R</i>) is expressed at similar levels as the wild type protein (Fft3-myc). Whole-cell lysate was immunoblotted with anti-myc antibodies. A non-tagged wild type strain was included as a control for the specificity of the antibody and actin blot confirms equal loading. <b>(D)</b> The Fft3 ATPase mutant is recruited to the LTR elements of the subtelomeric border. Data from duplicate ChIP-qPCR of non-tagged wild type, Fft3-myc and Fft3-K418R-myc is shown as fold difference in enrichment at LTR over <i>dg</i>, normalized to input. Error bars represent the standard deviation. <b>(E)</b> The Fft3 ATPase mutant shows an upregulation of subtelomeric genes. Total RNA was extracted in duplicate and reversely transcribed into cDNA. The expression levels of two subtelomeric genes were measured with qPCR. Error bars represent the standard deviation.</p