CXXC Zinc Finger Research Articles

Structural insights into an atypical histone binding mechanism by a PHD finger

Complex associating with SET1 (COMPASS) is a histone H3K4 tri-methyltransferase controlled by several regulatory subunits including CXXC zinc finger protein 1 (Cfp1). Prior studies established the structural underpinnings controlling H3K4me3 recognition by the PHD domain of Cfp1's yeast homolog (Spp1). However, metazoans Cfp1PHD lacks structural elements important for H3K4me3 stabilization in Spp1, suggesting that in metazoans, Cfp1PHD domain binds H3K4me3 differently. The structure of Cfp1PHD in complex with H3K4me3 shows unique features such as non-canonical coordination of the first zinc atom and a disulfide bond forcing the reorientation of Cfp1PHD N-terminus, thereby leading to an atypical H3K4me3 binding pocket. This configuration minimizes Cfp1PHD reliance on canonical residues important for histone binding functions of other PHD domains. Cancer-related mutations in Cfp1PHD impair H3K4me3 binding, implying a potential impact on epigenetic signaling. Our work highlights a potential diversification of PHD histone binding modes and the impact of cancer mutations on Cfp1 functions.

Cfp1 Controls Cardiomyocyte Maturation by Modifying Histone H3K4me3 of Structural, Metabolic, and Contractile Related Genes.

Cardiomyocyte maturation is the final stage of heart development, and abnormal cardiomyocyte maturation will lead to serious heart diseases. CXXC zinc finger protein 1 (Cfp1), a key epigenetic factor in multi-lineage cell development, remains underexplored in its influence on cardiomyocyte maturation. This study investigates the role and mechanisms of Cfp1 in this context. Cardiomyocyte-specific Cfp1 knockout (Cfp1-cKO) mice died within 4 weeks of birth. Cardiomyocytes derived from Cfp1-cKO mice showed an inhibited maturation phenotype, characterized by structural, metabolic, contractile, and cell cycle abnormalities. In contrast, cardiomyocyte-specific Cfp1 transgenic (Cfp1-TG) mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing Cfp1 displayed a more mature phenotype. Mechanistically, deficiency of Cfp1 led to a reduction in trimethylation on lysine 4 of histone H3 (H3K4me3) modification, accompanied by the formation of ectopic H3K4me3. Furthermore, Cfp1 deletion decreased the level of H3K4me3 modification in adult genes and increased the level of H3K4me3 modification in fetal genes. Collectively, Cfp1 modulates the expression of genes crucial to cardiomyocyte maturation by regulating histone H3K4me3 modification, thereby intricately influencing the maturation process. This study implicates Cfp1 as an important molecule regulating cardiomyocyte maturation, with its dysfunction strongly linked to cardiac disease.

Lysine Demethylase KDM2A Promotes Proteasomal Degradation of TCF/LEF Transcription Factors in a Neddylation-Dependent Manner.

Canonical Wnt signaling is essential for a plethora of biological processes ranging from early embryogenesis to aging. Malfunctions of this crucial signaling pathway are associated with various developmental defects and diseases, including cancer. Although TCF/LEF transcription factors (TCF/LEFs) are known to be essential for this pathway, the regulation of their intracellular levels is not completely understood. Here, we show that the lysine demethylase KDM2A promotes the proteasomal destabilization of TCF/LEFs independently of its demethylase domain. We found that the KDM2A-mediated destabilization of TCF/LEFs is dependent on the KDM2A zinc finger CXXC domain. Furthermore, we identified the C-terminal region of TCF7L2 and the CXXC domain of KDM2A as the domains responsible for the interaction between the two proteins. Our study is also the first to show that endogenous TCF/LEF proteins undergo KDM2A-mediated proteasomal degradation in a neddylation-dependent manner. Here, we reveal a completely new mechanism that affects canonical Wnt signaling by regulating the levels of TCF/LEF transcription factors through their KDM2A-promoted proteasomal degradation.

A novel view to varicose veins pathogenesis: Proteomic profiling suggests a pivotal role of extracellular matrix degradation.

Although morphological and anatomical studies indicate that venous wall weakening and subendothelial fibrosis characterize varicose veins (VV), the pathogenesis of VV remains poorly understood. The aim of this study is to obtain protein expression profiles in patients with VV and thereby get a step closer to understanding the pathogenesis of VV. Specimens were obtained from total of 10 patients, that is, from 5 patients undergoing VV surgical stripping and from 5 non-VV patients undergoing bypass surgery. Specimens were collected from the same layers of venous wall. Proteins were extracted from each specimen and analyzed by ion mobility spectrometry (IMS-MS). In total, 1387 were identified and 486 proteins were identified in all samples. From these, 15 proteins were differentially expressed between VV and non-VV samples (p < .05) and 12 of these showed a fold change >1.5. Interestingly, among the differentially expressed proteins, only two proteins were significantly increased in the VV tissue, that is, GAPDH (p = .028, fold change 2.74), where several proteins involved in maintaining the homeostasis in the extracellular matrix, that is, the CXXC zinc finger protein 5 (CXXC5) and nucleoporin (SEH1) were prominently downregulated (p = .049, fold change 37.8, and p = .040, fold change 3.46). The downregulation in protein expression of CXXC5 and SEH1 as well as upregulation of GAPDH were validated by Western blotting. The identified differentially expressed proteins suggest an altered profile of the connective tissue proteins as well as an increased proteolytic enzyme activity which both may be central in the pathophysiology of varicose veins.

β-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced β-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model.

Pancreatic β-cells are susceptible to oxidative stress, leading to β-cell death and dysfunction due to enhanced ROS levels and type 2 diabetes. To inhibit the β-cells damages induced by the oxidative stress, the present study investigates the beneficial effect of various peptides (WL15, RF13, RW20, IW13 and MF18) of immune related proteins (cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively). Also, the molecular mechanism of WL15 from cysteine and glycine-rich protein 2 on β-cell regeneration was identified through PEPCK and insulin pathway. In this study, a total of five peptides including WL15, RF13, RW20, IW13, and MF18 were derived from immune-related proteins such as cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively. These protein sequences were obtained from an earlier constructed transcriptome database of a teleost Channa striatus. The identified peptides were evaluated for their antioxidant as well as antidiabetic activity. Based on the in silico analysis and in-vitro screening experiments, WL15 was predicted to have better antioxidant and antidiabetic activity among the five different peptides. Therefore, WL15 alone was further analyzed for apoptosis, antioxidant capacity, glucose metabolism, and gene expression performance, which was investigated on the alloxan (500µM) induced zebrafish in vivo larval model. The results showed alloxan exposure to zebrafish larvae for a day, the ROS was generated in the β-cells. Interestingly, WL15 treatment showed a protective effect by reducing the toxicity of alloxan exposed zebrafish larvae by increasing their survival and heart rate. Moreover, WL15 reduced the intracellular ROS level and apoptosis in alloxan-induced larvae. The superoxide anion and lipid peroxidation levels are also reduced by improving the glutathione content after the WL15 treatment. Besides, WL15 treatment increased the proliferation rate of β-cells and decreased the glucose level. Further, the gene expression studies revealed that WL15 treatment normalized the PEPCK expression while upregulating the insulin expression in alloxan exposed larvae. Overall, the findings indicate that WL15 of cysteine and glycine-rich protein 2 can act as a potential antioxidant for type 2 diabetes patients in respect of improving β-cell regeneration.

CpG-binding protein CFP1 promotes ovarian cancer cell proliferation by regulating BST2 transcription

Epigenetic alterations have been functionally linked to ovarian cancer development and occurrence. The CXXC zinc finger protein 1 (CFP1) is an epigenetic regulator involved in DNA methylation and histone modification in mammalian cells. However, its role in ovarian cancer cells is unknown. Here, we show that CFP1 protein is highly expressed in human ovarian cancer tissues. Loss of CFP1 inhibited the growth of human ovarian cancer cells, promoted apoptosis, and increased senescence. CFP1 knockdown resulted in reduced levels of SETD1 (a CFP1 partner) and histone H3 trimethylation at the fourth lysine residue (H3K4me3). RNA-sequencing revealed that deletion of CFP1 resulted in mRNA reduction of bone marrow stromal cell antigen 2 (BST2). Bioinformatics analysis and chromatin immunoprecipitation showed that CFP1 binds to the promoter of BST2 and regulates its transcription directly. Overexpression of BST2 rescued the growth inhibitory effect of CFP1 loss. Furthermore, depletion of cullin-RING ubiquitin ligases 4 (CRL4) components ROC1 or CUL4A had significantly inhibited the expression of CFP1 and BST2 similar to MLN4924 treatment that blocked cullin neddylation and inactivated CRL4s. In conclusion, CFP1 promotes ovarian cancer cell proliferation and apoptosis by regulating the transcription of BST2, and the expression of CFP1 was affected by CRL4 ubiquitin ligase complex.

A prelude to the proximity interaction mapping of CXXC5

CXXC5 is a member of the zinc-finger CXXC family proteins that interact with unmodified CpG dinucleotides through a conserved ZF-CXXC domain. CXXC5 is involved in the modulation of gene expressions that lead to alterations in diverse cellular events. However, the underlying mechanism of CXXC5-modulated gene expressions remains unclear. Proteins perform their functions in a network of proteins whose identities and amounts change spatiotemporally in response to various stimuli in a lineage-specific manner. Since CXXC5 lacks an intrinsic transcription regulatory function or enzymatic activity but is a DNA binder, CXXC5 by interacting with proteins could act as a scaffold to establish a chromatin state restrictive or permissive for transcription. To initially address this, we utilized the proximity-dependent biotinylation approach. Proximity interaction partners of CXXC5 include DNA and chromatin modifiers, transcription factors/co-regulators, and RNA processors. Of these, CXXC5 through its CXXC domain interacted with EMD, MAZ, and MeCP2. Furthermore, an interplay between CXXC5 and MeCP2 was critical for a subset of CXXC5 target gene expressions. It appears that CXXC5 may act as a nucleation factor in modulating gene expressions. Providing a prelude for CXXC5 actions, our results could also contribute to a better understanding of CXXC5-mediated cellular processes in physiology and pathophysiology.

DNA Methyltransferases Contribute to Cold Tolerance in Ticks Dermacentor silvarum and Haemaphysalis longicornis (Acari: Ixodidae).

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum, and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days (P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly (P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts (P < 0.01), and mortality increased significantly (P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis. The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks.

A CpG island promoter drives the CXXC5 gene expression

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

Analyzing the impact of CFP1 mutational landscape on epigenetic signaling.

CXXC Zinc finger protein 1 (CFP1) is a multitasking protein playing essential roles during various developmental processes. Its ability to interact with several proteins contribute to several epigenetic events. Here, we review CFP1's functions and its impact on DNA methylation and the post-translational modification of histone proteins such as lysine acetylation and methylation. We will also discuss the potential role of CFP1 in carcinogenesis and the impact of the mutations identified in patients suffering from various cancers.

Downregulation Of The Wnt Inhibitor CXXC5 Predicts a Better Prognosis In Acute Myeloid Leukemia

▪Different CXXC zinc finger proteins like MLL, DNMT1 and TET1 have been identified to play a role in leukemia development. The CXXC family member CXXC5 is located on 5q31, a region recurrently deleted in acute myeloid leukemia (AML), suggesting that inactivation of CXXC5 might be involved in leukemogenesis. CXXC5 has previously been shown to exert tumor suppressor functions in solid tumor cells and to negatively regulate Wnt signaling, a known pro-survival pathway in leukemia. Here, we investigated prognostic and biological implications of CXXC5expression in AML.CXXC5 mRNA expression was determined by real time quantitative PCR in pretreatment bone marrow samples of 186 AML patients enrolled on the Medical Research Council (MRC) AML15 and UK National Cancer Research Institute (NCRI) AML17 trials. We found downregulation of CXXC5 in AML compared to healthy controls, particularly in cases with del(5q)/-5, MLL rearrangements and t(8;21). To investigate CXXC5-associated gene expression, microarray data from 351 normal karyotype AML derived from the Microarray Innovations in LEukemia study and 529 unselected AML patients treated in Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group (HOVON/SAKK) trials were analyzed. CXXC5 was associated with expression of several genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1), indicating that alteration of CXXC5 expression is a downstream effect of different leukemic pathways. Data from the HOVON/SAKK cohort (median age 46 years (range 15-77) was further used to perform outcome analyses according to microarray-based CXXC5 expression. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P=0.007) and a better overall survival (OS: 46% vs 28%; P<0.001) and event-free survival (EFS: 36% vs 21%; P<0.001) at 5 years. This was of independent significance in multivariate analyses after adjustment for age, white blood cell count, cytogenetic risk group, FLT3-ITD status, biallelic CEBPA mutations, as well as mutations of NPM1, DNMT3A and ASXL1.DNA sequencing of CXXC5 exons 1-3 on 84 AML cases revealed no mutations, suggesting that CXXC5 is predominantly inactivated by gene regulatory mechanisms in AML. Pyrosequencing analysis showed that lower expression of CXXC5 in AML samples was correlated with hypermethylation within the promoter region. Treatment of NB4 and U937 cell lines with 5-Aza-2´-deoxycytidine led to demethylation of this region and induction of CXXC5 at the mRNA and protein level.Functional analyses confirmed Wnt inhibitor function of CXXC5 in leukemic cells. K562 cells overexpressing pEGFP-C2-CXXC5 showed impaired upregulation of active β-catenin protein, decreased upregulation of Wnt target genes Axin2 and Survivin, as well as decreased Wnt reporter activity after Wnt3a stimulation. In addition, we found evidence for involvement of CXXC5 in the DNA damage-induced p53 response in leukemia. Knockdown of CXXC5in OCI-AML3 cells harboring wildtype p53 led to attenuated p53 upregulation and cell cycle arrest after γ-irradiation.In summary, we have identified CXXC5 as a novel independent prognostic factor in AML. Our data suggest a tumor suppressor function of CXXC5 in AML and association of the gene with different leukemic pathways. These functional and prognostic implications build the framework for further investigating the role of CXXC5 in leukemia. AML patients with downregulation of CXXC5 might be candidates for evaluation of Wnt inhibition and cases with epigenetically silenced CXXC5 expression may particularly benefit from demethylating agents. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Molecular Evolution of DNA Topoisomerase III Beta (TOP3B) in Metazoa.

DNA topoisomerase III beta (TOP3B) is unique by operating on both DNA and RNA substrates to regulate gene expression and genomic stability. Mutations in human TOP3B are linked to neurodevelopmental and cognitive disorders, highlighting its relevance for human health. Despite the emerging importance of TOP3B, its precise cellular functions and evolutionary history remain poorly understood. Here, we show that TOP3B is conserved across main metazoan groups and evolved under strong purifying selection. Subdomain IV was identified as the most conserved TOP3B region, in agreement with its role in providing the structural foundation of the protein. On the contrary, subdomain II is the less conserved, possibly because it is the most structurally flexible region of all TOP3B regions. Interestingly, TOP3B residue at position 472, previously associated with schizophrenia, is highly variable across animals, suggesting a more specific role in humans and related species. Finally, we show that all TOP3B CXXC zinc finger motifs previously identified at the protein C-terminal region are retained across metazoans. We also found that the two major methylation sites known to regulate TOP3B activity are located in the most conserved region of the C-terminal arginine-glycine-glycine (RGG) box, suggesting that a similar regulatory mechanism may operate throughout animals. Overall, our results provide a better understanding of the evolution and functional roles of TOP3B.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

CxxC Zinc Finger Protein Derived Peptide, MF18 Functions Against Biofilm Formation.

The major threat in modern medicine was biofilm forming bacterial related infections and they were highly tolerant to conventional antibiotics and a boundless demand for new drugs. In this regard, antimicrobial peptide (AMP) have been considered as potential alternative agents to conventional antibiotics. In this study, we have reported a CxxC zinc finger protein derived peptide, MF18 and its various biological role including activity against biofilm forming bacteria. Zinc finger protein are important in regulation of several cellular processes and wide range of molecular functions. The CxxC zinc finger protein identified from the cDNA library of a teleost fish; further it was characterised using various online bioinformatics programs. During the in-silico analysis, an AMP named MF18 was identified from the CxxC zinc finger protein, then it was synthesised for further biological activity studies. The antimicrobial activity of MF18 was confirmed against the biofilm clinical isolates such as Staphylococcus aureus and Escherichia coli. The MIC of the antimicrobial peptide at the concentration of 320µM was observed against these two biofilm bacteria. The mechanism of the peptides was determined using bacteria on its membrane permeabilization ability by scanning electron microscopy. It is exhibited that the MF18 potentially influenced in damaging the morphology of the bacteria. The toxicity of MF18 against the continuous cell line (RAW 264.7) was demonstrated by MTT assay and also using peripheral red blood cells by haemolytic assay; both assays showed that the peptide have no toxicity on the cells at lower concentration. Overall, the study showed the potential therapeutic application of the peptide in pharma industry.

A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS.

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS’s subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis.

KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

Role of KDM2B and Non-Canonical PRC1.1 As a Tumor Suppressor in T-ALL

Introduction: Polycomb repressive complexes (PRCs) play an important role for the transcriptional repression of their target genes through histone modification. KDM2B is a component of non-canonical PRC1.1 and has a role for the recruitment of the complex to the target gene loci. It has a zinc finger-CxxC (ZF-CxxC) domain which specifically binds to unmethylated sequences in CpG islands (CGIs) and deletion of the CxxC domain induces complete loss of KDM2B occupancy and removal of other PRC1.1 components from CGIs. Recent studies revealed that loss of function mutations of several PRC component genes such as EZH2, EED, SUZ12 and BCOR were frequently detected in human T-cell acute lymphoblastic leukemia (T-ALL), which suggested PRCs have a tumor suppressive role in T cell development. Our group have reported conditional knock out of Bcor, encoding a component protein of non-canonical PRC1.1, induced T-ALL in mice. However, it is still unknown how KDM2B and non-canonical PRC1.1 regulate T cell leukemogenesis. Therefore, we performed detailed analysis of mice deficient for Kdm2b ZF-CxxC domain (Kdm2bΔCxxC/ΔCxxC) specifically in hematopoietic cells.Methods: We used the conditional Kdm2b allele (Kdm2bfl) mice, which contains LoxP sites flanking Kdm2b exon 13 encoding the ZF-CxxC domain. To generate hematopoietic cell specific Kdm2b KO mice, we transplanted Kdm2bfl/fl;Cre-ERT total BM cells into lethally irradiated CD45.1+ recipient mice and deleted Kdm2b 4 weeks after transplantation by intraperitoneally injecting of tamoxifen.Results: During the observation period of 300 days, almost all of the Kdm2b KO mice developed lethal T-ALL. They showed thymomegaly and splenomegaly and presented infiltration of donor-derived leukemic cells into the bone marrow, spleen, thymus and peripheral blood. Flow cytometric analysis revealed that T-ALL cells were mainly CD4 and CD8-double positive (DP). Notch1 active mutations in exons 26, 27, 28 or 34 were found in over a half of the T-ALL cases, indicating the Notch1 activation could be a driver for leukemic transformation in this mouse model. RNA sequence analysis of the DP cells revealed activation of Myc, which plays a key role in the development of T-ALL, and their downstream target genes in Kdm2b-deficient T-ALL. ChIP sequence analysis of DP thymocytes expressing 3xFLAG-KDM2B confirmed the binding peaks of KDM2B at the promotor of Myc. Peak calling analysis of the ChIP sequence data revealed that KDM2B was mainly located at transcript start sites (TSS), where KDM2B was co-localized with H2AK119ub1 and H3K27me3 histone marks. In addition, ChIP sequencing of H3K4me3 revealed that the KDM2B target genes include more bivalent genes than non-target genes. We next compared histone modification status around TSS in WT and Kdm2b KO DP cells and Kdm2b-deficient T-ALL cells. Global levels of H2AK119ub1 were significantly decreased in T-ALL cells and the reduction was mainly observed at the promoters of KDM2B target genes. Direct target genes of NOTCH1 including Myc also lost H2AK119ub1 at their promotors in T-ALL cells. Peak calling analysis of KDM2B, BCOR and NOTCH1 ChIP sequence data revealed that their target genes were closely overlapped at the promotor region. Moreover, EZH2 binding peaks were also overlapped with those of KDM2B and NOTCH1, suggesting that non-canonical PRC1.1 and PRC2 cooperatively antagonize NOTCH1-mediated gene activation. KEGG pathway analysis of the genes with overlapping binding peaks among KDM2B, EZH2 and NOTCH1 showed significant enrichment of T cell receptor signaling, Notch1 signaling and cell cycle pathway, all of which play an important role for the development of T-ALL. Of interest, H3K27me3 levels of the common target genes of EZH2, KDM2B, and NOTCH1 were much lower than the EZH2-specific target genes, indicating that H2AK119ub1 plays a key role in the repression of NOTCH1 targets.Conclusions: Our findings suggest that KDM2B recruits non-canonical PRC1.1 at the promotor regions of NOTCH1 targets to restraint thymocytes from transformation in concert with PRC2. DisclosuresNo relevant conflicts of interest to declare.

Description of the molecular and phenotypic spectrum of Wiedemann-Steiner syndrome in Chinese patients

BackgroundWiedemann–Steiner syndrome (WDSTS) is a rare genetic disorder characterized by facial gestalt, neurodevelopmental delay, skeletal anomalies and growth retardation, which is caused by variation of KMT2A gene. To date, only 2 Chinese WDSTS patients have been reported. Here, we report the phenotypes and KMT2A gene variations in 14 unrelated Chinese WDSTS patients and investigate the phenotypic differences between the Chinese and French cohorts. MethodsNext generation sequencing was performed for each patient, and the variants in the KMT2A gene were validated by Sanger sequencing. The phenotypes of 16 Chinese WDSTS patients were summarized and compared to 33 French patients.ResultsGenetic sequencing identified 13 deleterious de novo KMT2A variants in 14 patients, including 10 truncating, 2 missenses and 1 splicing variants. Of the 13 variants, 11 are novel and two have been reported previously. One of the patients is mosaic in the KMT2A gene. The variation spectra and phenotypic profiles of the Chinese WDSTS patients showed no difference with patients of other ethnicities; however, differ in the frequencies of several clinical features. We demonstrated that variations in the KMT2A gene can lead to both advanced and delayed bone age. We identified 6 novel phenotypes, which include microcephaly, deep palmar crease, external ear deformity, carpal epiphyseal growth retardation, dyslipidemia, and glossoptosis. In addition, patients harbored missense variants in the CXXC zinc finger domain of KMT2A showed more severe neurophenotypes.ConclusionOur study consists of the largest cohort of Chinese WDSTS patients that continues to expand the WDSTS phenotypic and variation spectrum. Our results support the notion that the CXXC zinc finger domain of KMT2A gene is a hotspot for missense variants associated with more severe neurophenotypes.

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

BackgroundThe mechanism by which protein complexes interact to regulate the deposition of post-translational modifications of histones remains poorly understood. This is particularly important at regulatory regions, such as CpG islands (CGIs), which are known to recruit Trithorax (TrxG) and Polycomb group proteins. The CxxC zinc finger protein 1 (CFP1, also known as CGBP) is a subunit of the TrxG SET1 protein complex, a major catalyst of trimethylation of H3K4 (H3K4me3).ResultsHere, we used ChIP followed by high-throughput sequencing (ChIP-seq) to analyse genomic occupancy of CFP1 in two human haematopoietic cell types. We demonstrate that CFP1 occupies CGIs associated with active transcription start sites (TSSs), and is mutually exclusive with H3K27 trimethylation (H3K27me3), a marker of polycomb repressive complex 2. Strikingly, rather than being restricted to active CGI TSSs, CFP1 also occupies a substantial fraction of active non-CGI TSSs and enhancers of transcribed genes. However, relative to other TrxG subunits, CFP1 was specialised to TSSs. Finally, we found enrichment of CpG-containing DNA motifs in CFP1 peaks at CGI promoters.ConclusionsWe found that CFP1 is not solely recruited to CpG islands as it was originally defined, but also other regions including non-CpG island promoters and enhancers.