Mouse CHD4-NURD is Required for Neonatal Spermatogonia Survival and Normal Gonad Development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of CHD4 and CHD3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of Chd4 early in gametogenesis, but not Chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that CHD4 controls expression of Dmrt1 and its downstream target Plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of CHD4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

Background Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. Results Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. Conclusions These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Discovery of small molecule antagonists of human Retinoblastoma Binding Protein 4 (RBBP4)

RBBP4 is a nuclear WD40 motif-containing protein widely implicated in various cancers and a putative drug target. It interacts with multiple proteins within diverse complexes such as nucleosome remodeling and deacetylase (NuRD) complex and polycomb repressive complex 2 (PRC2), as well as histone H3 and H4 through two distinct binding sites. B-cell lymphoma/leukemia 11A (BCL11A), friend of GATA-1 (FOG-1), plant homeodomain finger protein 6 (PHF6) and histone H3 bind to the top of the donut-shaped seven-bladed β-propeller fold of RBBP4, while suppressor of zeste 12 (SUZ12), metastasis associated protein 1 (MTA1) and histone H4 bind to a pocket on the side of the WD40 repeats of this protein. Here, we report the discovery of the first small molecule antagonists of the RBBP4 top pocket, competing with interacting peptides from proteins such as BCL11A and histone H3. We also determined the first crystal structure of RBBP4 in complex with a small molecule (OICR17251), paving the path for structure-guided design and optimization towards more potent antagonists.

Identification of Germline Monoallelic Mutations in IKZF2 in Patients with Immune Dysregulation

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3, encoding Ikaros and Aiolos, have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK-cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of pro-inflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.

Low NCOR2 levels in multiple myeloma patients drive multidrug resistance via MYC upregulation

MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.

GFI1 tethers the NuRD complex to open and transcriptionally active chromatin in myeloid progenitors

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information is available on the complex and a detailed understanding of its mechanism is lacking. We investigated the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1N-HDAC1-MBD3GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2 (NuDe) using Bayesian integrative structure determination with IMP (Integrative Modeling Platform), drawing on information from SEC-MALLS, DIA-MS, XLMS, negative stain EM, X-ray crystallography, NMR spectroscopy, secondary structure and homology predictions. The structures were corroborated by independent cryo-EM maps, biochemical assays, and known cancer-associated mutations. Our integrative structure of the 2:2:2 MHM complex shows asymmetric binding of MBD3, whereas our structure of the NuDe complex shows MBD3 localized precisely to a single position distant from the MTA1 dimerization interface. Our models suggest a possible mechanism by which asymmetry is introduced in NuRD, and indicate three previously unrecognized subunit interfaces in NuDe: HDAC1C-MTA1BAH, MTA1BAH-MBD3, and HDAC160-100-MBD3. We observed that a significant number of cancer-associated mutations mapped to protein-protein interfaces in NuDe. Our approach also allows us to localize regions of unknown structure, such as HDAC1C and MBD3IDR, thereby resulting in the most complete structural characterization of these NuRD sub-complexes so far.

Beta-Catenin Forms Repressive Complexes with Ikzf1 and Ikzf3 to Orchestrate Tumor-Suppression in B-Cell Malignancies

Introduction: Oncogenic WNT/β-catenin signaling and activating mutations that increase stability and transcriptional activity of β-catenin are among the most frequent lesions throughout all main types of cancer (Morin et al. 1997; Rubinfeld et al. 1997). Results: Strikingly, pan-cancer analysis of activating mutations of β-catenin in patient samples and global proteomic analysis of human cancer cell lines revealed that B-cell malignancies are exempt from oncogenic activation of β-catenin. While nuclear accumulation of β-catenin was observed in >80% of cancer samples studied, nuclear β-catenin was consistently absent in normal and malignant B-lymphoid cells. To model the effects of inducible β-catenin activation in B-ALL, pre-B cells from Ctnnb1 ex3fl/+ mice were transformed with BCR-ABL1 or NRAS G12D oncogenes. Cre-mediated excision of exon 3 removes GSK3β and CK1 phosphorylation sites (S 33, S 37, T 41 and T 45) required for β-catenin degradation, therefore resulting in accumulation of β-catenin. Stabilization of β-catenin rapidly impaired competitive fitness of B-ALL cells, abolished colony forming ability and induced cell cycle arrest. Likewise, expression of constitutively active form of β-catenin in patient derived B-ALL cells (n=6) or B-cell lymphoma (n=4) compromised proliferation, clonal fitness, and induced cell death. Interestingly, activation of β-catenin signaling in myeloid leukemia (n=4) accelerated proliferation. Corroborating the lineage specific effects of β-catenin, CEBPα-mediated reprogramming of B-ALL cells into myeloid lineage leukemia cells, reversed the deleterious effect of inducible β-catenin-activation. In epithelial cell types, β-catenin/TCF complexes drive transcriptional activation of MYC (He et. al.1998). In striking contrast to epithelial cells, however, our transcriptomic analysis showed that inducible activation of β-catenin in B-cell malignancies resulted in transcriptional repression of Myc and its target genes. Surprisingly, proximity-based labeling and interactome studies in B-ALL cells revealed Ikzf1 and Ikzf3 as top-ranking interaction partners for β-catenin. Furthermore, several components of the Nucleosome Remodeling and Deacetylase (NuRD) complex including Mta1/2, Gatad2a/b, Chd4, Hdac1/2 were among the most significantly enriched proteins within the β-catenin interactome. Interaction of Ikzf1 and Ikzf3 with NuRD complex induced loss of histone H3 lysine 27 acetylation (H3K27Ac) and suppressed Myc expression. To test whether repressive complexes of β-catenin with Ikzf1, Ikzf3:NuRD subvert proliferation and survival by transcriptional repression of Myc, we introduced Cas9-RNP for genetic deletion of Ikzf1 and Ikzf3 in B-ALL cells. Interestingly, deletion of both Ikzf1 and Ikzf3 was required to restore clonal fitness, proliferation and Myc-driven transcriptional programs. Conversely, genetic deletion of β-catenin was sufficient to abrogate Ikzf1/Ikzf3-mediated tumor suppression and transcriptional repression of Myc. Together, these results suggest that engagement of b-catenin set the threshold for Ikzf1/Ikzf3-mediated tumor suppression in B-ALL and mature B-cell malignancies. Together with B-lineage-specific expression of Ikzf1 and Ikzf3, these findings provide a mechanistic explanation as to why activating lesions of β-catenin are not detected in B-cell malignancies. Therapeutic implication: To leverage the unique sensitivity of B-cell malignancies to β-catenin-activation, we tested pharmacological activation of β-catenin based on small molecule-inhibition of GSK3β, a central negative regulator of β-catenin. Testing six different GSK3β inhibitors, we identified LY2090314 as the most potent inhibitor in killing B-ALL cells and suppressing Myc (IC50=4.5 nM). Treatment of NSG mice bearing patient derived B-ALL xenografts with LY2090314 substantially reduced leukemia burden and significantly extended overall survival compared to vehicle treated mice (n=9, P= 6.5E-05). These findings suggest that β-catenin activation is a unique vulnerability and small molecule GSK3β inhibition represents a novel opportunity to overcome drug-resistance in refractory B-ALL. Disclosures No relevant conflicts of interest to declare.

Hemogen/BRG1 Cooperativity Is Required for Erythrocyte Maturation and Hemoglobin Production during Mammalian Erythropoiesis

Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unclear. 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 of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to occupy chromatin at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization. Disclosures No relevant conflicts of interest to declare.

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes

This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.