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Myeloid malignancy is a broad term encapsulating myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Initial studies into genomic profiles of these diseases have shown 2000 somatic mutations prevalent across the spectrum of myeloid blood disorders. Epigenetic mutations are emerging as critical components of disease progression, with mutations in genes controlling chromatin regulation and methylation/acetylation status. Genes such as DNA methyltransferase 3A (DNMT3A), ten eleven translocation methylcytosine dioxygenase 2 (TET2), additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2) and isocitrate dehydrogenase 1/2 (IDH1/2) show functional impact in disease pathogenesis. In this review we discuss how current knowledge relating to disease progression, mutational profile and therapeutic potential is progressing and increasing understanding of myeloid malignancies.

Introduction The molecular basis of the association between mutations in specific remodeler subunits and particular types of cancer is poorly understood. To understand how remodelers are targeted in a cell-type specific manner, we aim to explore the mechanisms of the Nucleosome Remodelling and Deacetylase (NURD) complex during cancer progression. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCCs leads to a reversal of Epithelial-mesenchymal transition (EMT). Material and methods We examined DOC1 expression in normal and cancerous tongue tissue with immunohistochemistry. Besides, 4 different human OSCC cell lines were examined using immunofluorescence and immunoblotting. To study its role in these cells, we re-expressed DOC1 and utilised shRNA-mediated knockdowns (remodelers, EMT regulators). Subsequently we carried out proteomics, genomics, cell-based and biochemistry assays. Results and discussions The loss of DOC1 in oral cancer cells leads to a failure of NURD to bind and repress master transcriptional regulators of EMT. Re-expression of DOC1 in OSCC cells restores NURD recruitment to key target genes, a switch from open to closed chromatin, transcriptional repression, and reversal of EMT (MET). We speculate that, during the development of oral cancer, DOC1 is lost after the inactivation of p53 and INK4a. Our genome-wide binding site analysis showed that DOC1 is crucial for NURD recruitment to a subset of target loci; in particular, promoter regions harbouring CpG islands. DOC1-mediated NURD binding to the Twist1/2 and Zeb2 promoters leads to eviction of SWI/SNF involving nucleosome repositioning, epigenetic reprogramming, and shutdown of transcription. Binding of either SWI/SNF or NURD determines opposite epigenetic states, thereby committing OSCC cells to either EMT or MET. Conclusion Re-expression of DOC1 in OSCC cells restores the switch from open- to closed chromatin and reversal of EMT. We suggest that subunit-dependent gene selection is a major cause of the association between the loss of specific remodeler subunits and particular types of cancer. Our results emphasise that gene control involves a dynamic equilibrium between opposing chromatin modulating enzymes rather than a static chromatin state. Disturbances in this balance can initiate a cascade of chromatin reprogramming events that drives oncogenesis.

Introduction: ATP-dependent chromatin remodelers are frequently mutated in cancers. Similar to the better studied SWI/SNF remodelers, the sequencing of cancer genomes has uncovered frequent mutations in genes encoding the Nucleosome remodeling and histone deacetylase (NuRD) subunits. Upon closer inspection, NuRD complex is highly amplified in breast cancer, deleted in prostate cancer, yet mutated in lung cancer. These observations suggest that (in)activation of NuRD might contribute to oncogenesis in a cell-type specific manner. Prompted by these findings, and to gain insights into the functions of the NuRD complex, we first need to understand the composition of this complex(es). Material & Methods: We carried out Immunoprecipitation (IP) with antibodies raised against NuRD members DOC1 and CHD4, followed by mass spectrometry (MS) with HeLa nuclear extracts. This was followed by ‘reverse’ IP-MS i.e. using antibodies raised against these novel NuRD-associated factors (AFs) on HeLa nuclear extracts and data mining for the NuRD-AFs (cBioPortal). Re-expression and knockdowns of DOC1, CHD4 and NuRD-AFs were executed in selected breast, prostate and lung carcinoma cell lines. High resolution MNase nucleosomal mapping followed by qPCR was performed at specific target loci. Results & Discussions: Proteomics data suggest that ZFHX3 (cancer determinant), ZBTB2 (developmental regulator) and SMAD4 (BMP/TGF-beta signalling pathway) as NuRD-AFs. Interestingly, DOC1 IP reveals interaction only with SMAD4 while CHD4 immunoprecipitation uncovered interaction only with ZFHX3. Besides, ZBTB2 associated with both DOC1 and CHD4-NuRD. It is worth noting that these novel NuRD-AFs were found to be frequently altered in specific cancers e.g. prostate, non-small cell lung and breast carcinomas. Re-expression and knockdowns of NuRD-AFs affected epithelial-mesenchymal transitions (EMT) and chromatin dynamics during cancer progression. Conclusion: Remodeling of chromatin is an essential step towards gene expression, therefore in order to study the critical role of NuRD-AFs during carcinogenesis, a higher resolution picture of the dynamic changes in chromatin and nucleosomal structure is pertinent. These mapping experiments revealed nucleosome occupancy as well as (in)accessibility of chromatin during carcinogenesis, in particular at specific target loci. Chromatin dynamics uncovered in specific carcinoma cell lines thereby generating a comprehensive picture of the various chromatin states during carcinogenesis and expanding our understanding of chromatin function. The impact will be protein/gene expression as well as chromatin (in)accessibility and structure could be used in (early) diagnostics and prediction of carcinogenesis or biomarker discovery, in combination with already available diagnostics/biomarkers; towards a more targeted and personalised therapy for cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Adone Mohd-Sarip, Diana Zatreanu, Jeroen Demmers. The mutually exclusive and diverse NuRD chromatin remodeling complex in cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5229.

Nucleotide biosynthesis is fundamental to normal cell proliferation as well as to oncogenesis. Tumor suppressor p53, which prevents aberrant cell proliferation, is destabilized through ubiquitylation by MDM2. Ubiquitin-specific protease 7 (USP7) plays a dualistic role in p53 regulation and has been proposed to deubiquitylate either p53 or MDM2. Here, we show that guanosine 5'-monophosphate synthase (GMPS) is required for USP7-mediated stabilization of p53. Normally, most GMPS is sequestered in the cytoplasm, separated from nuclear USP7 and p53. In response to genotoxic stress or nucleotide deprivation, GMPS becomes nuclear and facilitates p53 stabilization by promoting its transfer from MDM2 to a GMPS-USP7 deubiquitylation complex. Intriguingly, cytoplasmic sequestration of GMPS requires ubiquitylation by TRIM21, a ubiquitin ligase associated with autoimmune disease. These results implicate a classic nucleotide biosynthetic enzyme and a ubiquitin ligase, better known for its role in autoimmune disease, in p53 control.

ATP-dependent chromatin remodelers are frequently mutated in cancers. However, the molecular basis of the association between mutations in specific remodeler subunits and particular types of cancer is poorly understood. To understand how remodelers are targeted in a cell-type specific manner, we aim to explore the mechanisms of the Nucleosome Remodeling and Deacetylase (NURD) complex during cancer progression. We recently showed that DOC1-dependent recruitment of NuRD reveals antagonism with SWI/SNF during epithelial-mesenchymal transition in oral cancer cells (Mohd-Sarip et al., 2017). It is instructive to compare the function of DOC1 in oral squamous cell carcinomas (OSCCs) with that of the SWI/SNF subunit SMARCB1/hSNF5 in malignant rhabdoid tumors (MRTs). MRTs are an extremely aggressive pediatric cancer caused by the loss of SMARCB1 (Kia et al., 2008). Although the loss of DOC1 in OSCCs or that of SMARCB1 in MRTs generates opposite epigenetic states of their target genes, in both cases, this is caused by failed remodeler recruitment. The loss of a single subunit, such as DOC1 or SMARCB1, does not abrogate all other remodeler functions. We suggest that subunit-dependent gene selection is a major cause of the association between the loss of specific remodeler subunits and particular types of cancer. Our results emphasize that gene control involves a dynamic equilibrium between opposing chromatin modulating enzymes rather than a static chromatin state. Disturbances in this balance can initiate a cascade of chromatin reprogramming events that drives oncogenesis. Such an intertwined system of epigenetic regulation suggests therapeutic strategies aimed at restoring the balance between antagonistic activities. Prompted by these findings, we will present an inclusive view of the protein regulatory network in order to gain a wholesome understanding of the molecular transactions and dynamic composition of NuRD complexes in specific cancer types. These were identified by immunopurifications using in-house antibodies followed by mass spectrometry. Our proteomic studies of the NuRD complex in flies (Reddy et al., 2010) and HeLa nuclear extracts suggest that mammalian NuRD share the same subunits with flies. Upon closer inspection, we found unanticipated novel interacting partners of NuRD and that they are altered in specific cancers namely prostate, pancreatic and ovarian. This will be followed by confirmation of the interactomes using cell-based and biochemistry assays in combination with genome-wide approaches, as well as potentially leading to the characterization of novel targets of NuRD. These findings will represent the types of multiprotein NuRD-like complexes that can form during cancer progression and how they are targeted to chromatin. Citation Format: Adone Mohd-Sarip, Diana Zatreanu, Jeroen A. Demmers, C Peter Verrijzer. Gene-selective recruitment of NuRD drives chromatin reprogramming in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1478.

Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.

Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.

The hSNF5 chromatin-remodeling factor is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). A number of studies have shown that hSNF5 re-expression blocks MRT cell proliferation. However, the pathway through which hSNF5 acts remains unknown. To address this question, we generated MRT-derived cell lines in which restoration of hSNF5 expression leads to an accumulation in G0/G1, induces cellular senescence and increased apoptosis. Following hSNF5 expression, we observed transcriptional activation of the tumor suppressor p16INK4a but not of p14ARF, repression of several cyclins and CD44, a cell surface glycoprotein implicated in metastasis. Chromatin immunoprecipitations indicated that hSNF5 activates p16INK4a transcription and CD44 down-regulation by mediating recruitment of the SWI/SNF complex. Thus, hSNF5 acts as a dualistic co-regulator that, depending on the promoter context, can either mediate activation or repression. Three lines of evidence established that p16INK4a is an essential effector of hSNF5-induced cell cycle arrest. 1) Overexpression of p16INK4a mimics the effect of hSNF5 induction and leads to cellular senescence. 2) Expression of a p16INK4a-insensitive form of CDK4 obstructs hSNF5-induced cell cycle arrest. 3) Inhibition of p16INK4a activation by siRNA blocks hSNF5-mediated cellular senescence. Collectively, these results indicate that in human MRT cells, the p16INK4a/pRb, rather than the p14ARF/p53 pathway, mediates hSNF5-induced cellular senescence.

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.

Silence, Please Polycomb group (PcG) proteins play pivotal roles in epigenetic gene control, development, and disease. PcG proteins can control cell cycle progression, but the underlying mechanisms remain unclear. PcG proteins are thought to silence transcription of target genes through modulating chromatin structure. Mohd-Sarip et al. (p. 744 , published online 5 April) uncovered an unanticipated transcription-independent function for the canonical PcG protein “Posterior sex combs” in cell cycle control, through its direct regulation of cyclin B destruction.

Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (PHO) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of PHO and the multisubunit Polycomb core complex (PCC). Here, we analyzed the architecture of the PHO/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that PHO/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. We discuss the implications of these findings for models explaining PRE function.

The hSNF5 chromatin-remodeling factor is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). A number of studies have shown that hSNF5 re-expression blocks MRT cell proliferation. However, the pathway through which hSNF5 acts remains unknown. To address this question, we generated MRT-derived cell lines in which restoration of hSNF5 expression leads to an accumulation in G(0)/G(1), induces cellular senescence and increased apoptosis. Following hSNF5 expression, we observed transcriptional activation of the tumor suppressor p16(INK4a) but not of p14(ARF), repression of several cyclins and CD44, a cell surface glycoprotein implicated in metastasis. Chromatin immunoprecipitations indicated that hSNF5 activates p16(INK4a) transcription and CD44 down-regulation by mediating recruitment of the SWI/SNF complex. Thus, hSNF5 acts as a dualistic co-regulator that, depending on the promoter context, can either mediate activation or repression. Three lines of evidence established that p16(INK4a) is an essential effector of hSNF5-induced cell cycle arrest. 1) Overexpression of p16(INK4a) mimics the effect of hSNF5 induction and leads to cellular senescence. 2) Expression of a p16(INK4a)-insensitive form of CDK4 obstructs hSNF5-induced cell cycle arrest. 3) Inhibition of p16(INK4a) activation by siRNA blocks hSNF5-mediated cellular senescence. Collectively, these results indicate that in human MRT cells, the p16(INK4a)/pRb, rather than the p14(ARF)/p53 pathway, mediates hSNF5-induced cellular senescence.

We have cloned Af10, the murine homologue of the MLL partner gene AF10. The predicted open reading frame of Af10 contains 1069 aa which are 90% identical to those of AF10. Af10 contains an N-terminal cysteine-rich region with a LAP/PHD finger, a leucine zipper domain and a glutamine-rich region at the C-terminus, features also found in the human proteins AF10 and AF17. A single 5. 5-kb transcript was detected in murine tissues with the highest level of expression in the testes. A polyclonal antibody raised to the cysteine-rich region of AF10 was able to identify a double band of 140 kDa on Western analysis in mouse testicular extracts. After subcellular separation Af10 was identified in both the nuclear and cytoplasmic extracts, again as a double band of 140 kDa in size. In situ hybridisation studies were performed with sense and antisense digoxigenin-labelled oligonucleotides. High levels of expression were noted in postmeiotic germ cells, especially in spermatids from around stage VI to stage VIII. High levels of expression were also seen in the white matter of the cerebellum, extending into the granular layer. The expression in differentiated rather than in proliferating cells suggests that the role of Af10 may lie in the suppression of proliferation rather than in differentiation. Since the LAP/PHD finger domains are lost in the MLL-AF10 fusion, arguably such a function could be carried out by this domain.

It is well-established that compaction of chromatin affects gene expression by regulating the accessibility of DNA to factors that control gene expression. Although the three-dimensional structure of nucleosomes has been elucidated in exquisite detail, the next level of chromatin structure is still poorly understood. In their Perspective, [Mohd-Sarip and Verrijzer][1] discuss two new studies ([ Dorigo et al .][2] and [ Francis et al .][3]) that shed light on how compaction of the nucleosomes into a higher order structure--a 30-nm chromatin fiber--contributes to the regulation of gene expression. [1]: http://www.sciencemag.org/cgi/content/full/306/5701/1484 [2]: http://www.sciencemag.org/cgi/content/short/306/5701/1571 [3]: http://www.sciencemag.org/cgi/content/short/306/5701/1574