Nucleosome Remodeling And Histone Deacetylase Research Articles

Gatad2b, associated with the neurodevelopmental syndrome GAND, plays a critical role in neurodevelopment and cortical patterning

GATAD2B (GATA zinc finger domain containing 2B) variants are associated with the neurodevelopmental syndrome GAND, characterized by intellectual disability (ID), infantile hypotonia, apraxia of speech, epilepsy, macrocephaly and distinct facial features. GATAD2B encodes for a subunit of the Nucleosome Remodeling and Histone Deacetylase (NuRD) complex. NuRD controls transcriptional programs critical for proper neurodevelopment by coupling histone deacetylase with ATP-dependent chromatin remodeling activity. To study mechanisms of pathogenesis for GAND, we characterized a mouse model harboring an inactivating mutation in Gatad2b. Homozygous Gatad2b mutants die perinatally, while haploinsufficient Gatad2b mice exhibit behavioral abnormalities resembling the clinical features of GAND patients. We also observed abnormal cortical patterning, and cellular proportions and cell-specific alterations in the developmental transcriptome in these mice. scRNAseq of embryonic cortex indicated misexpression of genes key for corticogenesis and associated with neurodevelopmental syndromes such as Bcl11b, Nfia and H3f3b and Sox5. These data suggest a crucial role for Gatad2b in brain development.

BCL11B and the NuRD complex cooperatively guard T-cell fate and inhibit OPA1-mediated mitochondrial fusion in T cells.

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.

HOXA9 forms a repressive complex with nuclear matrix–associated protein SAFB to maintain acute myeloid leukemia

AbstractHOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)–chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ–associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.

Hemogen/BRG1 cooperativity modulates promoter and enhancer activation during erythropoiesis

AbstractHemogen is a hematopoietic tissue–specific gene that regulates the proliferation and differentiation of hematopoietic cells; however, the mechanism underlying its function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role for hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of corepressors nucleosome remodeling and histone deacetylase (NuRD) complex and facilitated LDB1 complex–mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a coactivator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To determine whether hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen-knockout/knockin mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in embryonic days 12.5 to 16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. Chromatin immunoprecipitation sequencing in wild-type and hemogen-knockout animals revealed that BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, the hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.

Abstract 1175: Identification of novel combination therapies with the LSD1 inhibitor SP-2577 (Seclidemstat) in Ewing's Sarcoma using a complete druggable CRISPR library approach

Abstract Introduction: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. It has been previously observed in ES patient samples have high levels of the histone lysine specific demethylase 1 (LSD1). LSD1 is recruited by EWS/FLI as a member of the nucleosome remodeling and histone deacetylase (NuRD) complex to repress the critical EWS/FLI targets LOX and TGFBR2. Treatment with the potent LSD1 reversible inhibitor, SP-2577 (Seclidemstat), causes de-repression of these genes and impaired tissue culture cell viability in multiple ES cell lines. In this study we investigated new targeted therapies to use in combination with SP-2577 to help increase the cytotoxicity of the drug within ES cell types. Methods: Synthego® arrayed CRISPR screening platform was used to knock down genes associated with known druggable interactions in the ES cell line SK-N-MC. Genes were knocked down singularly either in the presence/absence of SP-2577. Viability screening was used to determine genes that increased the cytotoxicity of SP-2577. Statistical analysis, z-score, and IPA analysis was performed to investigate correlations between loss of gene effect and LSD1 pathway. Hit-gene validation was performed using siRNA and/or sgRNA knockdown/knockout, and other methods such as qPCR, and western blot assay for protein expression. Results: Arrayed CRISPR screening identified 103 gene deletions that resulted in increased SP-2577 cytotoxicity in SK-N-MC cell line. Among those, the top 19 hits were selected by identifying a FDA approved drug-gene interaction. Two high- interest hits were seen to have significant interaction with the LSD1 pathway: ABL2 and SH2B3. Drug combination treatment of SP-2577 with either Ruxolitinib or Dasatinib in ES cell lines showed high synergy at low dose ratio. Downstream pathways analysis confirmed the efficacy of the drugs combination. Detailed data will be presented. Conclusion: Our data support the validity of the arrayed CRISPR screening to identify new therapeutic strategies in ES, and highlights the potential for Ruxolitinib or Dasatinib treatment in combination with SP-2577 in ES patients. Citation Format: Samuel Sampson, Trason Thode, Mohan Kaadige, Alexis Weston, Serina Ng, Kevin Drenner, Raffaella Soldi, Sunil Sharma. Identification of novel combination therapies with the LSD1 inhibitor SP-2577 (Seclidemstat) in Ewing's Sarcoma using a complete druggable CRISPR library approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1175.

Cancer metastasis-associated protein 1 localizes to the nucleolus and regulates pre-rRNA synthesis in cancer cells.

Metastasis-associated protein 1 (MTA1) is a critical component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA1 has several biological functions, and it is closely associated with the malignant properties of human cancers; however, the mechanisms and subcellular localization of MTA1 in cells remain unclear. Some initial studies indicated that MTA1 was absent from the nucleolus; however, several NuRD components were recently found to be present in the nucleolus, where they regulate preribosomal RNA (pre-rRNA) transcription. In this study, we demonstrated that MTA1 is definitely localized to the nucleolus and regulates pre-rRNA transcription, which is consistent with the recent reports on NuRD. To determine if MTA1 was present in the nucleolus, we utilized the following complementary molecular approaches: immunofluorescence, GFP-tag tracking, immunoelectron microscopy, and immunoprecipitation (IP). To examine the role of MTA1 in rRNA synthesis, we performed quantitative polymerase chain reaction analysis. We revealed that both endogenous and exogenous MTA1 showed apparent granule-like nucleolar subcellular localization. MTA1 interacts with two major resident nucleolar proteins, nucleolin and nucleophosmin. Immunofluorescent colocalization analyses showed that MTA1 localizes to the fibrillarin-deficient regions of the nucleolus, and Co-IP experiments indicated that there was no interaction between MTA1 and fibrillarin; further, fibrillarin was not identified in the MTA1 interactome. Loss- and gain-of-function studies indicated that MTA1 promotes pre-rRNA transcription in cancer cells. Collectively, our data identify MTA1 as a novel nucleolar protein, and activation of pre-rRNA transcription in cancer cells may be an alternative mechanism by which MTA1 promotes malignancies.

MTA3-SOX2 Module Regulates Cancer Stemness and Contributes to Clinical Outcomes of Tongue Carcinoma.

Cancer cell plasticity plays critical roles in both tumorigenesis and tumor progression. Metastasis-associated protein 3 (MTA3), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex and multi-effect coregulator, can serve as a tumor suppressor in many cancer types. However, the role of MTA3 in tongue squamous cell cancer (TSCC) remains unclear although it is the most prevalent head and neck cancer and often with poor prognosis. By analyzing both published datasets and clinical specimens, we found that the level of MTA3 was lower in TSCC compared to normal tongue tissues. Data from gene set enrichment analysis (GSEA) also indicated that MTA3 was inversely correlated with cancer stemness. In addition, the levels of MTA3 in both samples from TSCC patients and TSCC cell lines were negatively correlated with SOX2, a key regulator of the plasticity of cancer stem cells (CSCs). We also found that SOX2 played an indispensable role in MTA3-mediated CSC repression. Using the mouse model mimicking human TSCC we demonstrated that the levels of MTA3 and SOX2 decreased and increased, respectively, during the process of tumorigenesis and progression. Finally, we showed that the patients in the MTA3low/SOX2high group had the worst prognosis suggesting that MTA3low/SOX2high can serve as an independent prognostic factor for TSCC patients. Altogether, our data suggest that MTA3 is capable of repressing TSCC CSC properties and tumor growth through downregulating SOX2 and MTA3low/SOX2high might be a potential prognostic factor for TSCC patients.

Abstract 5229: The mutually exclusive and diverse NuRD chromatin remodeling complex in cancer progression

Abstract 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.

Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation.

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2β, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2β arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2β also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2β loss. Mi-2β stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2β shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2β promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.

YAP and TAZ Negatively Regulate Prox1 During Developmental and Pathologic Lymphangiogenesis.

The Hippo pathway governs cellular differentiation, morphogenesis, and homeostasis, but how it regulates these processes in lymphatic vessels is unknown. We aimed to reveal the role of the final effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), in lymphatic endothelial cell (LEC) differentiation, morphogenesis, and homeostasis. During mouse embryonic development, LEC-specific depletion of Yap/Taz disturbed both plexus patterning and valve initiation with upregulated Prox1 (prospero homeobox 1). Conversely, LEC-specific YAP/TAZ hyperactivation impaired lymphatic specification and restricted lymphatic sprouting with profoundly downregulated Prox1. Notably, lymphatic YAP/TAZ depletion or hyperactivation aggravated or attenuated pathological lymphangiogenesis in mouse cornea. Mechanistically, VEGF (vascular endothelial growth factor)-C activated canonical Hippo signaling pathway in LECs. Indeed, repression of PROX1 transcription by YAP/TAZ hyperactivation was mediated by recruitment of NuRD (nucleosome remodeling and histone deacetylase) complex and endogenous binding activity of TEAD (TEA domain family members) to the PROX1 promoter. Furthermore, YAP/TAZ hyperactivation enhanced MYC signaling and inhibited CDKN1C, leading to cell cycle dysregulation and aberrant proliferation. We find that YAP and TAZ play promoting roles in remodeling lymphatic plexus patterning and postnatal lymphatic valve maintenance by negatively regulating Prox1 expression. We further show that YAP and TAZ act as plastic regulators of lymphatic identity and define the Hippo signaling-mediated PROX1 transcriptional programing as a novel dynamic checkpoint underlying LEC plasticity and pathophysiology.

Structural and functional characterization of the RBBP4-ZNF827 interaction and its role in NuRD recruitment to telomeres.

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD-ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.

A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans.

Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development.

Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

ZMYND8 Co-localizes with NuRD on Target Genes and Regulates Poly(ADP-Ribose)-Dependent Recruitment of GATAD2A/NuRD to Sites of DNA Damage

NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles intranscription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide bindingsites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, theMYND domain in ZMYND8 facilitates the rapid,poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes.

Identification of CASZ1 NES reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma.

As a transcription factor, localization to the nucleus and the recruitment of co-factors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc finger transcription factor CASZ1 to function as neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation we define a critical region at the CASZ1 N-terminus (AA23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identify a nuclear export signal (NES) at the N-terminus (AA176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1 (CRM1)-dependent manner. An analysis of CASZ1 protein expression in a primary neuroblastoma tissue microarray shows that high nuclear CASZ1 staining is detected in tumors from NB patients with good prognoses. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumors from patients with poor prognoses. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as neuroblastoma tumorigenesis.

Ikaros-Dependent Downregulation of MYC with IMiDs in Myeloma (MM) Cells Is Mediated through the Depletion of the Acetylated Chromatin Reader BRD4 at Super-Enhancer Loci

Abstract Background: IMiDs are now recognized to promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC and IRF4. MYC locus rearrangement is a recurrent somatic event in MM and results in MYC repositioning near superenhancers (such as IGH@, IGL@ and others). The mechanisms of IMiDs-mediated downregulation of MYC in MM cells is not well understood, in particular it is unclear how IMiDs alter the activity of the super-enhancers driving MYC transcription. Methods and results: Transcriptome analysis (RNAseq) of MM cells (OPM2 and MM1S) treated with lenalidomide or upon the silencing of IKZF3 (lentiviral delivery of dox-inducible IKZF3 shRNA) confirmed the downregulation of MYC, IRF4 and among others the upregulation of other genes of interest such as CD38, DKK1, PDL1, FOXO3 and a host of genes involved in interferon response. Gene set enrichment analysis (GSEA) confirmed the enrichment of genes signatures associated with MYC as well as the interferon response. Notably, in t(4;14) MM cell lines we observed a significant downregulation of FGFR3 while the expression of WHSC1 was not affected. This finding was confirmed in a library of t(4;14) MM cell lines by qRT-PCR analysis. As FGFR3 is driven by the 3' IGH enhancer while WHSC1 is under the control of the intronic Em enhancer in t(4;14) MM cells, and in view of the known role of IKZF1/3 in class switch recombination, we postulated that IKZF1/3 regulate the 3' IGH enhancer activity and MYC expression in cells harbouring a MYC-IGH rearrangement. To validate this hypothesis we established the genome-wide distribution of IKZF1 in OPM2 and MM1S cells (both cell lines harbour an IGH-MYC rearrangement) by ChIP-Seq. Ikaros bound to 17660 loci of which 43% were associated with gene targets. Ikaros-binding motifs and other motifs such as STAT1, E2A, RUNX1 were also identified (MEME Tomtom and Jasper motif analysis) in the vicinity of Ikaros enrichment peaks. Importantly IKZF1 peaks also mapped to the IGH 3' enhancer loci and these results were confirmed by ChIP-PCR analysis. Of interest while no Ikaros peaks mapped to MYC (within 5 kb of TSS) by ChIP-seq, modest enrichment of Ikaros at MYC promoter was identified by ChIP-PCR in MM1S and KMS11 cells, and this enrichment was significantly attenuated by lenalidomide treatment. Since inhibition of MYC occurs as a consequence of depletion of the acetylated chromatin reader BRD4 at enhancers that drive MYC expression (Delmore et al., 2011), we examined the interaction between BRD4, IKZF1 and IMiDs treatment. Chip-Seq analysis revealed that BRD4 and IKZF1 are associated with most active enhancers and promoters in MM1S tumor cells. In particular, BRD4 and IKZF1 were enriched at the IGH 3'α2 enhancer locus. Importantly, treatment with lenalidomide (10μM, 4 hours), dramatically depleted IKZF1 and BRD4 enrichment at the 3' IGH enhancers (ChIP-PCR analysis). In contrast, treatment with the bromodomain inhibitor JQ1 depleted BRD4, but not IKZF1 from the IGH 3' enhancer locus. These findings suggest that IKZF1 regulates BRD4 binding to the 3' IGH enhancers and explain the selective effect of IMiDs on MYC transcription in transformed cells harboring a MYC rearrangement. In addition, these results suggest that IMiDs will affect MYC expression only in cells were MYC is driven by an IKZF1/IKZF3 responsive enhancer. Lastly we sought to explain how IKZF1 may regulate BRD4 enrichment at H3K27Ac marks in super-enhancer loci. Since Ikaros is a known integral component of the NuRD (nucleosome remodelling and histone deacetylase) complex, we examined whether lenalidomide treatment and the ensuing Ikaros depletion lead to local gain of NuRD at MYC TSS and depletion of H3K27 acetylation. CHD4 (helicase subunit of the NURD complex) and H3K27Ac ChIP-PCR analysis revealed significant accumulation of the NuRD complex at MYC TSS as well as reduced H3K27Ac at 3'IGH enhancers after IMiDs exposure in MM1S and OPM2 cells. Importantly, CHD4 silencing abrogated lenalidomide induced MYC transcriptional repression and cell death. Conclusion: In summary, our studies suggest that Ikaros drives MYC expression in MM cells harbouring an IGH-MYC rearrangement by regulating the accumulation of the acetylated chromatin reader BRD4 at the IGH 3' enhancer and preventing the NURD complex mediated depletion of H3K27Ac marks at these enhancers. Our findings also explain the IKZF1/3 dependent mechanism of IMiDs-mediated MYC transcriptional repression. Disclosures Neri: Celgene: Research Funding. Duggan:Jansen: Honoraria; Celgene: Honoraria. Keats:Translational Genomic Research Institute: Employment. Bahlis:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Johnson & Johnson: Consultancy; Johnson & Johnson: Speakers Bureau; Johnson & Johnson: Research Funding; Amgen: Consultancy.

Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.

Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs.

Abstract 476: Loss of chd5-mediated tumor suppression accelerates MYCN-driven neuroblastoma tumorigenesis in zebrafish

Abstract Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system (PSNS) that originates from cells of the primitive neural crest. For the 40% of patients with high-risk disease, current therapies are often ineffective and long-term survival remains obstinately low. A major risk factor in neuroblastoma is hemizygous loss of the 1p36 chromosomal region, which has long been suspected to harbor one or more powerful tumor suppressor genes. Our studies indicate that loss of Chromodomain helicase DNA-binding protein 5 (chd5), which is in this deleted region, cooperates with MYCN overexpression to accelerate in vivo neuroblastoma tumorigenesis. Zebrafish chd5-null alleles were created utilizing both zinc-finger nuclease and TALEN technology. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). In order to examine the effect of chd5 haploinsuficiency on in vivo neuroblastoma tumorigenesis, chd5 mutant fish were crossed with the dbh:MYCN transgenic model resulting in neuroblastoma tumors. Consistent with a tumor suppressor function, chd5 haploinsufficient fish exhibit an accelerated neuroblastoma phenotype with tumors present beginning as early as 6 weeks compared to 15 weeks observed in wildtype fish. The chd5 protein can serve as one of two enzymatic components of the nucleosome remodeling and histone deacetylase (NuRD) complex, which is a repressor of gene expression and is reported to have diverse roles in regulating chromatin organization, developmental signaling and gene stability. Future studies will examine the mechanism and function of chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established. Citation Format: Mark W. Zimmerman, Shuning He, Jimann Shin, Shizhen Zhu, Marc Mansour, Keith Joung, Jinhua Quan, Timur Yusufzai, A. Thomas Look. Loss of chd5-mediated tumor suppression accelerates MYCN-driven neuroblastoma tumorigenesis in zebrafish. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 476. doi:10.1158/1538-7445.AM2015-476

MTA family of proteins in DNA damage response: mechanistic insights and potential applications

The DNA damage, most notably DNA double-strand breaks, poses a serious threat to the stability of mammalian genome. Maintenance of genomic integrity is largely dependent on an efficient, accurate, and timely DNA damage response in the context of chromatin. Consequently, dysregulation of the DNA damage response machinery is fundamentally linked to the genomic instability and a likely predisposition to cancer. In turn, aberrant activation of DNA damage response pathways in human cancers enables tumor cells to survive DNA damages, thus, leading to the development of resistance of tumor cells to DNA damaging radio- and chemotherapies. A substantial body of experimental evidence has established that ATP-dependent chromatin remodeling and histone modifications play a central role in the DNA damage response. As a component of the nucleosome remodeling and histone deacetylase (NuRD) complex that couples both ATP-dependent chromatin remodeling and histone deacetylase activities, the metastasis-associated protein (MTA) family proteins have been recently shown to participate in the DNA damage response beyond its well-established roles in gene transcription. In this thematic review, we will focus on our current understandings of the role of the MTA family proteins in the DNA damage response and their potential implications in DNA damaging anticancer therapy.