Galectin-3 interacts with components of the nuclear ribonucleoprotein complex

Differentially spliced mRNAs following galectin‐3 depletion. (PDF 122 kb

YM155-Adapted Cancer Cell Lines Reveal Drug-Induced Heterogeneity and Enable the Identification of Biomarker Candidates for the Acquired Resistance Setting

Survivin is a drug target and its suppressant YM155 a drug candidate mainly investigated for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of 10 additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterized by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualized therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB

MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

<div><p>Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.</p></div

PRC1.6 binding sites partially overlap with cPRC1, PRC2 and ncPRC1.1 binding sites.

<p>(A) ChIP-seq heatmaps of Pcgf6, IgG control, Ring1b (GSM1041372) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Rybp (GSM1041375) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Cbx6-HA (GSM2610616) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Cbx7 (GSM2610619) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Pcgf2 (GSM1657387) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref056" target="_blank">56</a>], Suz12 (GSM1041374) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Kdm2b (GSM1003594) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref006" target="_blank">6</a>] and H3K27me3 (GSM1341951) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref010" target="_blank">10</a>] peaks in mESCs at +/- 2 kb regions centred over the Mga-L3mbtl2-Pcgf6 peaks. (B) Venn diagrams showing the overlap of high confidence Pcgf6 target genes (location of binding sites between -2.5 kb of TSS and TES) with those of Cbx7 (cPRC1), Suz12 and H3K27me3 (PRC2) and Kdm2b (ncPRC1.1). (C) Genome browser screenshots of ChIP-seq tracks at promoters of representative meiosis-related genes (<i>Dazl</i>, <i>Sycp3</i>, <i>Stk31 and Mei1</i>) (D) Genome browser screenshots of ChIP-seq tracks at cPRC1 target genes (<i>Nkx2-4</i> and <i>Hoxa7)</i>.</p

Commercially available antibodies used in this study.

<p>Commercially available antibodies used in this study.</p

MGA is essential for genomic binding of PRC1.6.

<p>(A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the <i>AEBP2</i>, <i>RPA2</i>, <i>RFC1</i> and <i>SPOP</i> promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) <i>L3MBTL2</i>-, <i>E2F6</i>- and <i>PCGF6</i> transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. <i>B2M</i> transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA<i>ko</i> cells, and diminished deposition of H2AK119ub1. The <i>CDC7</i> -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA<i>ko</i> cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 (<i>FUT9</i>, <i>MYT1</i> and <i>TSH2B</i>) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.</p

L3MBTL2 and E2F6 recruit PRC1.6 differentially in a promoter-specific manner.

<p>(A) Left panel, scatter plot comparing the extent of reduction (fold change of normalized tag counts) of MGA binding in L3MBTL2<i>ko</i> cells with the extent of reduction in E2F6<i>ko</i> cells. Right panel, scatter plot comparing the extent of reduction of E2F6 binding in L3MBTL2<i>ko</i> cells with the extent of reduction of L3MBTL2 in E2F6<i>ko</i> cells. The E2F6-dependent <i>RNF130</i> and the L3MBTL2-dependent <i>ZFR</i> promoters are indicated for clarity. (B) Genome browser screenshots of ChIP-seq tracks showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the <i>RNF130 and ZFR</i> promoters in wild type cells (WT), and in MGA<i>ko</i>, L3MBTL2<i>ko</i>, E2F6<i>ko</i> and PCGF6<i>ko</i> cells. (C) ChIP-qPCR analysis of MGA binding to selected promoters in two different L3MBTL2<i>ko</i> (L2ko cl10 and L2ko cl14, upper panel) and in two different E2F6<i>ko</i> (E2F6<i>ko</i> cl1 and E2F6<i>ko</i> cl11, lower panel) cell clones. The <i>CDC7</i> -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (D) Expression of L3MBTL2 in L3MBTL2<i>ko</i> cells rescues binding of PRC1.6. Left, Western blot for L3MBTL2. Right, ChIP-qPCR data showing binding of exogenous L3MBTL2 and of endogenous MGA, E2F6 and PCGF6 to representative PRC1.6 target promoters. Percent of input values represent the mean of at least three independent experiments +/- SD. (E) Expression of wild type E2F6 but not of a DNA binding-deficient E2F6 mutant (E2F6mut) in E2F6<i>ko</i> cells rescues binding of PRC1.6. Left, Western blot for E2F6. Right, ChIP-qPCR data showing binding of exogenous E2F6 (wild type or DNA-binding deficient mutant) and of endogenous MGA and L3MBTL2 to representative PRC1.6 target promoters. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) Venn diagram showing the overlap of E2F6 peaks in L3MBTL2<i>ko</i> cells and L3MBTL2 peaks in E2F6<i>ko</i> cells. Logos of the enriched sequence motifs were obtained by running MEME-ChIP with 300 bp summits of the ChIP-seq peaks. (G) GO analyses of biological functions of E2F6-dependent and of L3MBTL2-dependent PRC1.6 target genes. Enriched GO terms were retrieved using Enrichr. p values are plotted in -log2 scale.</p