Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

Two patients with KDM3B variants and new presentations of Diets-Jongmans syndrome.

KDM3B is located on chromosome 5q31 and encodes KDM3B, which is involved in histone demethylation and epigenetic regulation. Pathogenic KDM3B variants cause a dominantly inherited disorder presenting with intellectual disability (ID), short stature, and facial dysmorphism, named Diets-Jongmans syndrome. We describe two patients with KDM3B variants presenting with Diets-Jongmans syndrome. Genetic testing was performed because of the clinical data and a lack of a clear diagnosis in both patients. Candidate variants were verified by Sanger sequencing. After KDM3B variants were detected, in silico tools were used to predict the pathogenicity of the missense variants. A minigene assay was performed to evaluate the splicing effects of the c.5070 + 1G > A variant on KDM3B. Patient 1 mainly presented with repetitive upper respiratory tract infection and patient 2 presented with palpitation, shortness of breath, and pitting edema; both had ID. Whole exome sequencing identified variants of KDM3B. Patient 1 had the de novo KDM3B c.5070 + 1G > A variant, whereas patient 2 had the c.2828G > A (p.R943Q) variant. Transcriptional experiments of the splicing variant c.5070 + 1G > A revealed aberrant transcripts leading to truncated protein products. We found two pathogenic variants in KDM3B, one of which is novel. Both patients had additional clinical presentations, and patient 1 had transient neutropenia. KDM3B c.5070 + 1G > A is the first KDM3B splice-site variant and was identified as a germline variant. Neutropenia and cardiomyopathy are newly found presentations of Diets-Jongmans syndrome. Our report enriches our knowledge of the genotypic spectrum of the KDM3B variants and phenotypic diversity of Diets-Jongmans syndrome.

Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity.

Epigenetic modifications operate in concert to maintain cell identity, yet how these interconnected networks suppress alternative cell fates remains unknown. Here, we uncover a link between the removal of repressive histone H3K9 methylation and DNA methylation during the reprogramming of somatic cells to pluripotency. The H3K9me2 demethylase, Kdm3b, transcriptionally controls DNA hydroxymethylase Tet1 expression. Unexpectedly, in the absence of Kdm3b, loci that must be DNA demethylated are trapped in an intermediate hydroxymethylated (5hmC) state and do not resolve to unmethylated cytosine. Ectopic 5hmC trapping precludes the chromatin association of master pluripotency factor, POU5F1, and pluripotent gene activation. Increased Tet1 expression is important for the later intermediates of the reprogramming process. Taken together, coordinated removal of distinct chromatin modifications appears to be an important mechanism for altering cell identity.

Histone Demethylase KDM3 (JMJD1) in Transcriptional Regulation and Cancer Progression.

Methylation of histone H3 lysine 9 (H3K9) is a repressive histone mark and associated with inhibition of gene expression. KDM3 is a subfamily of the JmjC histone demethylases. It specifically removes the mono- or di-methyl marks from H3K9 and thus contributes to activation of gene expression. KDM3 subfamily includes three members: KDM3A, KDM3B and KDM3C. As KDM3A (also known as JMJD1A or JHDM2A) is the best studied, this chapter will mainly focus on the role of KDM3A-mediated gene regulation in the biology of normal and cancer cells. Knockout mouse studies have revealed that KDM3A plays a role in the physiological processes such as spermatogenesis, metabolism and sex determination. KDM3A is upregulated in several types of cancers and has been shown to promote cancer development, progression and metastasis. KDM3A can enhance the expression or activity of transcription factors through its histone demethylase activity, thereby altering the transcriptional program and promoting cancer cell proliferation and survival. We conclude that KDM3A may serve as a promising target for anti-cancer therapies.

De Novo and Inherited Pathogenic Variants in KDM3B Cause Intellectual Disability, Short Stature, and Facial Dysmorphism.

By using exome sequencing and a gene matching approach, we identified de novo and inherited pathogenic variants in KDM3B in 14 unrelated individuals and three affected parents with varying degrees of intellectual disability (ID) or developmental delay (DD) and short stature. The individuals share additional phenotypic features that include feeding difficulties in infancy, joint hypermobility, and characteristic facial features such as a wide mouth, a pointed chin, long ears, and a low columella. Notably, two individuals developed cancer, acute myeloid leukemia and Hodgkin lymphoma, in childhood. KDM3B encodes for a histone demethylase and is involved in H3K9 demethylation, a crucial part of chromatin modification required for transcriptional regulation. We identified missense and truncating variants, suggesting that KDM3B haploinsufficiency is the underlying mechanism for this syndrome. By using a hybrid facial-recognition model, we show that individuals with a pathogenic variant in KDM3B have a facial gestalt, and that they show significant facial similarity compared to control individuals with ID. In conclusion, pathogenic variants in KDM3B cause a syndrome characterized by ID, short stature, and facial dysmorphism.

A 7-nt nucleotide sequence variant within the sheep KDM3B gene affects female reproduction traits.

Lysine demethylase 3B (KDM3B) gene is a histone demethylase, demonstrating specific demethylation of the histone H3 lysine 9. It was detected as a sheep reproductive candidate gene by genome-wide scans, and related studies also showed its significance in female reproductive process. However, rare study researched its polymorphism. Herein, we hypothesized that the polymorphisms of KDM3B gene were associated with sheep reproduction traits. A 7-nt nucleotide sequence variant (rs1088697156) within KDM3B gene was identified in a total of 888 individuals, including the Australian White (AUW) sheep and Lanzhou Fat-tailed (LFT) sheep. II (insertion/insertion) and ID (insertion/deletion) genotypes of 7-nt variant were detected, which were at Hardy-Weinberg equilibrium (HWE) in detected breeds. Association analysis illustrated the 7-nt variant was significantly associated with the litter size, duration of pregnancy, live lamb number, live lamb rate, stillbirth number, stillbirth rate of average and different parity (P < 0.05) in AUW sheep. Moreover, 'ID' was the dominant genotype with excellent consistency in reproductive traits. It is instrumental to select individuals with ID genotype for improving the sheep reproduction traits. These findings suggest that the 7-nt variant within KDM3B gene can be used as a candidate marker of reproduction traits for sheep breeding improvement by marker-assisted selection.

Inhibition of ALKBH5-mediated m6 A modification of PPARG mRNA alleviates H/R-induced oxidative stress and apoptosis in placenta trophoblast.

The alpha-ketoglutarate-dependent (ALKB) homolog 5 (ALKBH5), an m6 A demethylase, has been reported to be involved in the pathogenesis of preeclampsia (PE), but the exact mechanism requires further investigation. RT-qPCR or Western blotting were used to determine ALKBH5 and peroxisome proliferator-activated receptor gamma (PPARG) expression in placentas from PE patients and normal volunteers, as well as in HTR-8/SVneo cells treated with hypoxia/reoxygenation (H/R). Our results showed that the expression of ALKBH5 was significantly upregulated and PPARG was downregulated in preeclamptic placentas and H/R-treated cells. ALKBH5 interference reduced m6 A levels of PPARG mRNA, and increased PPARG mRNA stability and promoted PPARG translation level. In addition, ALKBH5 silencing increased the cell proliferation, migration, and vimentin protein level, and inhibited cell apoptosis, oxidative stress, and protein levels of endoglin (ENG) and E-cadherin in H/R-treated cells, whereas PPARG interference reversed these effects. Furthermore, PPARG repressed the H3K9me2 levels at activated leukocyte cell adhesion molecule (ALCAM) promoter region by increasing the expression and activity of lysine demethylase 3B (KDM3B). ALCAM inhibition reversed the effects of PPARG overexpression on H/R-treated cell functions. PKF115-584 suppressed the effects of ALKBH5 interference on the behaviors of H/R-treated cells. Finally, inhibition of ALKBH5 alleviates PE-like features in pregnant mice. Inhibition of ALKBH5 promotes KDM3B-mediated ALCAM demethylation by facilitating PPARG mRNA m6 A modification, and further activates the Wnt/ß-catenin pathway, and in turn alleviates PE progression.

Small molecular modulators of JMJD1C preferentially inhibit growth of leukemia cells.

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.

KDM3B suppresses APL progression by restricting chromatin accessibility and facilitating the ATRA-mediated degradation of PML/RARα.

BACKGROUND: A hallmark of acute promyelocytic leukemia (APL) is the expression of PML/RARα fusion protein. Treatment with all-trans retinoic acid (ATRA) results in the terminal differentiation of neutrophil granulocytes. However, the underlying mechanisms remain largely unknown. Here, we identify and elucidate a novel differentiation-suppressive model of APL involving the histone demethylase KDM3B, which has been identified as a suppressor of the tumor genes involved in hematopoietic malignancies. METHODS: First, we established a KDM3B knockdown NB4 cell model to determine the functional characteristics of KDM3B by cell proliferation assay and flow cytometry. Then, we performed ChIP-seq and ATAC-seq to search for potential relationships among KDM3B, histone modification (H3K9me1/me2) and the chromatin state. Finally, molecular biological techniques and a multi-omics analysis were used to explore the role of KDM3B in differentiation of the leukemia cells after ATRA treatment. RESULTS: We found that knocking down KDM3B contributed to the growth of NB4 APL cells via the promotion of cell-cycle progression and blocked granulocytic differentiation. Through global and molecular approaches, we provided futher evidence that knocking down KDM3B altered the global distribution of H3K9me1/me2 and increased the chromatin accessibility. Moreover, knocking down KDM3B inhibited the ATRA-induced degradation of the PML/RARα oncoprotein. CONCLUSION: Our study suggested that KDM3B was able to inhibit APL progression by maintaining chromatin in a compact state and facilitating the ATRA-mediated degradation of PML/RARα. Taken together, the results show that KDM3B may be an alternative target for the treatment regimens and the targeted therapy for APL by sustaining the function of PML/RARα fusion protein.

A rare presentation of childhood interstitial lung disease attributed to KDM3B gene mutation: a case report.

Childhood Interstitial Lung Disease (chILD) encompasses various respiratory conditions affecting children's lung airspaces and tissues, with diverse causes. One rare cause involves structural vascular changes. We describe a case of a 10-year-old boy diagnosed with chILD who exhibited specific dysmorphic features, developmental delay, and intellectual disability. He was diagnosed with severe pulmonary arterial hypertension (PAH) due to venous thromboembolic disease, an unusual underlying condition for chILD. A Whole Exome Sequence showed mutations in KDM3B and SIN3A genes, respectively responsible for Diets-Jongmans syndrome (DIJOS) and Witteveen-Kolk syndrome (WITKOS). Both syndromes can explain our patient´s phenotype and KDM3B mutation has been previously described to be associated with PAH. Our case suggests a potential association between KDM3B mutation and PAH leading to chILD. It also enriches the knowledge of genotypic diversity in KDM3B and SIN3A genes as well as the spectrum of clinical associations with DIJOS and WITKOS syndromes.

Rapid detection of InDel within the KDM3B gene in five sheep breeds using the mathematical expectation (ME) method.

Lysine demethylase 3B (KDM3B), a candidate gene associated with bone formation and growth, and differentiation of osteoblast, might affect the animal growth traits. Herein, the insertion/deletion (InDel) of the KDM3B gene was quickly detected in 882 sheep from five breeds using the mathematical expectation (ME) method. The results showed that there were two genotypes of 7-bp variation in KDM3B, including II (insertion/insertion) and ID (insertion/deletion), and the frequency of two genotypes varied among the five sheep breeds. Association analysis results demonstrated that the 7-bp indel was significantly associated with chest depth of LFT sheep (P = 0.012), and body weight (P = 0.006), body height (P = 0.030), chest depth (P = 0.043), chest circumference (P = 0.016), abdominal width (P = 0.035) and height at hip cross (P = 0.022) in LXBH sheep. Moreover, II genotype was the predominant genotype with excellent consistency in sheep growth traits (P < 0.05). Collectively, the above results suggest that this locus can be used as an effective molecular marker to improve the sheep growth traits and provide a scientific basis for the development of sheep breeding.

A novel de novo pathogenic variant in KDM3B gene at the first Albanian case of Diets-Jongmans syndrome: DIJOS.

Diets-Jongmans syndrome, DIJOS, is a very recently described autosomal dominant condition, which is caused by heterozygous pathogenic variants in KDM3B gene and characterized by impaired intellectual development, short stature, as well as facial dysmorphism. We describe a new DIJOS patient harboring a heterozygous, novel, de novo and likely pathogenic variant in KDM3B gene, which is the first case reported after Diets et al.`s publication, to the best of our knowledge.

Kdm3b haploinsufficiency impairs the consolidation of cerebellum-dependent motor memory in mice.

Histone modifications are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifications in cerebellum-dependent motor learning and memory are not well understood. To test whether changes in histone methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+/-) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di-methylation is significantly increased selectively in the granule cell layer of the cerebellum of Kdm3b+/- mice. In the cerebellum-dependent optokinetic response (OKR) learning, Kdm3b+/- mice show deficits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA-seq analyses revealed that the expression levels of several plasticity-related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory.

Histone demethylase KDM3B protects against ferroptosis by upregulating SLC7A11.

Ferroptosis is a type of adaptive cell death driven by cellular metabolism and iron-dependent lipid peroxidation. Though multiple genes (including SLC7A11 and GPX4) have been demonstrated to play key roles in ferroptosis, little is known about the epigenetic regulation of this process. Here, we report that KDM3B, a histone H3 lysine 9 demethylase, can protect against ferroptosis induced by Erastin, an inhibitor of SLC7A11. KDM3B overexpression in HT-1080 cells results in decreased histone H3 lysine 9 methylation. Furthermore, KDM3B upregulates the expression of SLC7A11 through cooperation with the transcription factor ATF4. In summary, we identify here KDM3B as a potential epigenetic regulator of ferroptosis.

JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells.

The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B-/- mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis.

Knockout of the Histone Demethylase Kdm3b Decreases Spermatogenesis and Impairs Male Sexual Behaviors.

Kdm3b is a JmjC domain-containing histone H3 (H3) demethylase and its physiological functions are largely unknown. In this study, we found that Kdm3b protein is highly expressed in multiple cell types in the mouse testes, including Leydig cells, Sertoli cells, spermatogonia and spermatocytes at different differentiation stages. We also observed Kdm3b protein in the epithelial cells of the caput epididymis, prostate and seminal vesicle. Breeding tests revealed that the number of pups produced by the breeding pairs with Kdm3b knockout (Kdm3bKO) males and wild type (WT) females was reduced 68% because of the decreased number of litters when compared with the breeding pairs with WT males and females. Further analysis demonstrated that Kdm3bKO male mice produced 44% fewer number of mature sperm in their cauda epididymides, displaying significantly reduced sperm motility. No significant differences in the circulating concentration of testosterone and the expression levels of androgen receptor and its representative target genes in the testis were observed. However, the circulating levels of 17ß-estradiol, a modulator of sperm maturation and male sexual behaviors, was markedly reduced in Kdm3bKO male mice. Strikingly, abrogation of Kdm3b in male mice significantly increased the latencies to mount, intromit and ejaculate and decreased the number of mounts and intromissions, largely due to their loss of interest in female odors. These findings indicate that Kdm3b is required for normal spermatogenesis and sexual behaviors in male mice.

Differential expression of histone demethylase KDM3B and JMJD1C in acute myeloid leukemia / 中国生化药物杂志

Objective To investigate the synergistic regulation of KDM3B and JMJD1C in leukemia. Methods The expression level of JMJD1C and KDM3B were analyzed in multiple acute myeloid leukemia (AML) cell lines. AML cell lines NB4 and HL-60 were treated with Daminozide, followed by determination of H3K9 mono-methylation and di-methylation. AML cell lines NB4 and HL-60 were treated with Daminozide, ATRA (retinoid acid All-trans), C Vitamin and the expression of KDM3B and JMJD1C were detected by real-time quantitative PCR. Results The expression level of KDM3B and JMJD1C in the AML cell lines was negatively correlated. In NB4 and HL-60 cells treated by daminozide, H3K9 mono-methylation and di- methylation level showed a rising trend in these two cell groups. After treatment of NB4 cells with the 3 reagents, the level of mRNA of KDM3B was down-regulated while the level of mRNA of JMJD1C was up-regulated. In HL-60 cells treated by daminozide, the mRNA level of KDM3B was up-regulated and the mRNA level of JMJD1C was down-regulated. Conclusion The expression of KDM3B and JMJD1C is negatively correlated in patients with AML.