POLYCOMB ROLE IN CELLULAR PROLIFERATION AND TRANSFORMATION

The Polycomb Group proteins (PcGs) are present in cells nuclei as two main repressive complexes named Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). Both have been involved in several cellular functions among which the ability to promote cellular proliferation is the main PcG feature that links their activity to cancer development. Both complexes are directly involved in repressing the transcription of the Ink4aArf locus that encodes for the tumor suppressive proteins p16 and p19/Arf (p14/Arf in humans), potent inhibitors of cell growth via the positive regulation of pRb and p53 functions. Thus, since the activity of both PRC1 and PRC2 complexes is frequently enhanced in different type of human tumors, inhibition of PcG function has been proposed for many years as a potential strategy for cancer treatment. Yet, the fact that the pro-proliferative role of PcG proteins depends on the repression of the pRb and p53 pathways, of which most if not all tumors are defective, generates a scientific paradox for the effectiveness of PcG inhibition in cancer treatment. In this thesis, with the help of my colleagues, (from now on referred as we) I will present data showing how PcGs genetic depletion dramatically impairs cellular proliferation independently on the expression of the Ink4a/Arf locus or p53 and pRb activities. We also genetically demonstrate, in cell culture and in vivo, that PcGs activity is required for both the transformation and the maintenance of the transformed phenotype obtained by expression of potent oncogenes such as H-RASV12 or c-MYC in cells defective for the pathways of p53 and pRb. Finally we suggest a potential mechanism to explain the reduced proliferation/tumorigenic potential involving DNA replication control by PcG proteins. We show defects both in fork progression and fork symmetry along with increased replication origin numbers in PcGs knockout transformed cells. Collectively these data strongly support PcGs as master regulators of cellular proliferation and transformation independently on the impairment of main tumor suppressive pathways and introduce a novel general mechanism through which PcGs regulate these processes. Overall this work supports PcGs as druggable targets in tumors where oncosuppressive pathways are de-regulated and proliferation, ergo DNA replication, is enhanced

Maintenance of leukemic cell identity by the activity of the Polycomb complex PRC1 in mice

Leukemia is a complex heterogeneous disease often driven by the expression of oncogenic fusion proteins with different molecular and biochemical properties. Whereas several fusion proteins induce leukemogenesis by activating Hox gene expression (Hox-activating fusions), others impinge on different pathways that do not involve the activation of Hox genes (non-Hox-activating fusions). It has been postulated that one of the main oncogenic properties of the HOXA9 transcription factor is its ability to control the expression of the p16/p19 tumor suppressor locus (Cdkn2a), thereby compensating Polycomb-mediated repression, which is dispensable for leukemias induced by Hox-activating fusions. We show, by genetically depleting the H2A ubiquitin ligase subunits of the Polycomb repressive complex 1 (PRC1), Ring1a and Ring1b, that Hoxa9 activation cannot repress Cdkn2a expression in the absence of PRC1 and its dependent deposition of H2AK119 monoubiquitination (H2AK119Ub). This demonstrates the essential role of PRC1 activity in supporting the oncogenic potential of Hox-activating fusion proteins. By combining genetic tools with genome-wide location and transcription analyses, we further show that PRC1 activity is required for the leukemogenic potential of both Hox-activating and non-Hox-activating fusions, thus preventing the differentiation of leukemic cells independently of the expression of the Cdkn2a locus. Overall, our results genetically demonstrate that PRC1 activity and the deposition of H2AK119Ub are critical factors that maintain the undifferentiated identity of cancer cells, positively sustaining the progression of different types of leukemia

Isolation of chromatin from dysfunctional telomeres reveals an important role for Ring1b in NHEJ-mediated chromosome fusions

When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh) in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ)-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation

Selective autophagy of mitochondria on a ubiquitin-endoplasmic reticulum platform

Correction: Developmental Cell, Volume 55, Issue 2 https://doi.org/10.1016/j.devcel.2020.10.002The dynamics and co-ordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes, or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required earliest followed by autophosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps whereas ULK1/2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way suggesting multiple initiation events. Targeted ubiquitinated mitochondrial are cradled by endoplasmic reticulum strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands providing platforms for formation of the mitophagosomes.Peer reviewe

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors

CpG binding protein (CFP1) occupies open chromatin regions of active genes, including enhancers and non-CpG islands

Additional file 1. Fig. S1: Analysis of CFP1 binding at individual loci and CpG islands (CGIs). (A-B) Analysis of CFP1 binding at the human α-globin locus in expressing and non-expressing cells. (A) Real-Time PCR analysis of immunoprecipitated chromatin using CFP1 antibody in human erythroblasts (red) and B-lymphocytes (blue). The y-axis represents enrichment over the input DNA, normalised to a control sequence in the human 18S gene. The x-axis represents the positions of Taqman probes used. The coding sequence is represented by the three exons (Promoter/Ex1, Ex2, Ex3) of the α-globin genes. 218 and hBact denote control sequences adjacent to the CpG islands of the human LUC7L (218) and ACTB promoters. Error bars correspond to ± 1 SD from at least two independent ChIPs. (B) Real-Time PCR analysis of immunoprecipitated chromatin using the CFP1 antibody indicated in humanised erythroblasts (normal, +MCS-R2 (left) and mutant, MCS-R2 (right). The y-axis represents enrichment over the input DNA, normalised to a control sequence in the mouse GAPDH gene. CpG Act denotes additional control sequence at the CGI of the mouse ACTB gene. The amplicons highlighted in red represent deleted regions in the humanised mice, for which no PCR signal is observed. Error bars correspond to ± 1 SD from at least two independent ChIPs. (C) CFP1 ChIP signal intensity in the top 200 peaks, by antibody and by cell type. Abcam, ab56035 antibody. Roeder, main antibody used in this study. (D) Analysis of CGI (green) and non-CGI (blue) transcription start sites (1-kb window, centred on TSS). Gene symbols shown with CpG content of individual loci in parentheses. Greek letters represent individual globin genes. Fig. S2: Peak overlaps of CFP1 and marks of active and repressed chromatin in transcription start sites (TSSs). Peaks were detected by MACS2. Venn diagrams show that CFP1 peaks within 1-kb of TSSs are strongly associated with H3K4me3 histone mark and poorly associated with H3K27me3 repressive histone mark. Cell types are (A) ERY and (B) EBV. Public data sets: * NCBI GEO GSE36985, ** NCBI GEO GSE50893. Fig. S3: UCSC tracks showing CFP1 and other ChIP signals in gene loci in erythroblasts (ERY) and EBV-transformed B-lymphoblasts (EBV). Hg38 coordinates for multiple genes, CpG islands (CGI, green boxes), and putative regulatory regions (blue boxes) are shown. CFP1 signals are shown in dark reds, inputs in grey, histone H3 signals in blues and open chromatin marks in greens. All ChIP pileups are scaled to 1x coverage genome-wide and shown in a range 0–50, except CFP1 (Roeder) is shown with extended range and H3K27me3 graphs scaled by 2x. (A) Tissue-specific binding of CFP1 to CGI promoters of tissue-specifically expressed genes. Left (chr16), CGI promoters of active genes in alpha globin locus are CFP1-bound in ERY, and unbound in EBV. Flanking regions are included, with known tissue-specific enhancers. Right (chr6), first seven exons of IRF4 locus, active in EBV and inactive in ERY, with CFP1 binding to CGI promoter in EBV only. (B) CGI promoters of housekeeping genes are CFP1 bound and unmarked by H3K27me3. Left (chr7), ACTB locus. Right (chr16), LUC7L locus. (C) CGI promoter of RHBDF1 locus (chr16) has H3K27me3 mark and the absence of CFP1 binding in both ERY and EBV. Fig. S4: Western blot analysis of CGBP (CFP1) expression in mouse and human erythroid and human lymphoid cell types. Whole cell extracts (20 µg) were loaded in each lane (1) mouse ES, (2) U-MEL, (3) I-MEL, (4) mouse primary erythroblasts and (5) human primary T lymphocytes and (6) human primary erythroblasts and separated on a 10% SDS-polyacrylamide gel. CFP1 antibody was used at a 1:1000 dilution. Fig. S5: Similar cell type-specific CFP1 read depth at CGI TSS of HBA1 gene and non-CGI TSS of HBB gene. Upper two tracks use the main antibody, and second two tracks use the commercial antibody. Coordinates are from the hg38 human genome build. Read depths are averaged in 50 bp bins and normalised to 1x genome-wide coverage. Blue boxes, known regulatory regions; green box, CGI. Fig. S6: Distribution of TrxG components in erythroid cells. Green indicates CGI and blue indicates other putative regulatory regions. All loci transcribed right to left. Pileups are shown scaled to 1x genome coverage, with full scale 0–50x depth. (A) Housekeeping genes ACTB, left (chr7), and LUC7L, right (chr16). (B) β-globin locus (chr11), (C) Non-expressed RHBDF1 locus (chr16). Fig. S7: Overlap of TrxG subunit ChIP peaks in a high-confidence subset of regions. SET1A complexes are represented by CFP1-SET1A colocalisation. MLL1/2 complexes are represented by Menin, and MLL3/4 complexes are represented by UTX, respectively. HCF1 is found in SET1A/B and MLL1/2 complexes, and RBBP5 is a member of SET1A/B and MLL1/2/3/4 complexes. Red outline (4220 peaks) shows strong colocalisation of Menin and CFP1-SET1A, accounting for the vast majority (99.5%) of 4242 CFP1-SET1A and half (50.0%) of 8432 Menin peak regions. Majority (87.0%, 2089/2400 peaks) of HCF1 (blue region) is accounted for by approximately half (49.5%, 2089/4220) of regions of Menin-SET1A-CFP1 colocalisation. Regions where either SET1A-CFP1 or Menin or both are colocalised with HCF1 (blue dashed line) accounts for nearly all (99.6%, 2390/2400) HCF1 regions, suggesting that HCF1 bound to DNA is primarily present as part of SET1A/B or MLL1/2 complexes. Fig. S8: Chromatin accessibility in TSSs and enhancers in erythroid cells as measured by ATAC-seq and DNase-seq. 1x-normalised, input-subtracted signals from ATAC-seq and DNase were averaged in a 2-kb window about TSSs and putative enhancers. Z-score transformed values for ATAC-seq and DNase-seq at a given locus were averaged. Fig. S9: Relationship of CFP1 signal to three predictive factors in top-decile open chromatin regions. A linear combination of CpG density and SET1A and H3K4me3 ChIP signals explains a substantial fraction of variation in CFP1 ChIP signal. Table S1: Bias of CFP1 for CGI TSSs in cell types and gene classes. Table S2: Bias of CFP1 for housekeeping gene TSSs. Table S3: Motifs associated with CFP1 peaks. Table S4: Dependence of CFP1 ChIP signal in erythroid cells on covariates putatively associated with its binding. Table S5: Analysis of variance of CFP1 signal in top-decile open chromatin regions surrounding TSSs and putative enhancers

Epigenetic factors in cancer development : polycomb group proteins

The role of chromatin-modifying factors in cancer biology emerged exponentially in the last 10 years, and increased attention has been focused on Polycomb group (PcG) proteins and their enzymatic activities. PcG proteins are repressive chromatin modifiers required for proliferation and development. The frequent deregulation of PcG activities in human tumors has direct oncogenic effects and results, essential for cancer cell proliferation. Here we will review the recent findings regarding PcG proteins in prospective tumor development, focusing on the molecular mechanisms that deregulate PcG expression in different tumors, at the downstream pathways to PcG expression (that contribute to cancer development) and at the mechanisms that regulate PcG recruitment to specific targets. Finally, we will speculate on the benefit of PcG inhibition for cancer treatment, reviewing potential pharmacological strategies