Histone Demethylase Jumonji AT-rich Interactive Domain 1B (JARID1B) Controls Mammary Gland Development by Regulating Key Developmental and Lineage Specification Genes

The JmjC domain-containing H3K4 histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) (also known as KDM5B and PLU1) is overexpressed in breast cancer and is a potential target for breast cancer treatment. To investigate the in vivo function of JARID1B, we developed Jarid1b^−/− mice and characterized their phenotypes in detail. Unlike previously reported Jarid1b^−/− strains, the majority of these Jarid1b^−/− mice were viable beyond embryonic and neonatal stages. This allowed us to further examine phenotypes associated with the loss of JARID1B in pubertal development and pregnancy. These Jarid1b^−/− mice exhibited decreased body weight, premature mortality, decreased female fertility, and delayed mammary gland development. Related to these phenotypes, JARID1B loss decreased serum estrogen level and reduced mammary epithelial cell proliferation in early puberty. In mammary epithelial cells, JARID1B loss diminished the expression of key regulators for mammary morphogenesis and luminal lineage specification, including FOXA1 and estrogen receptor α. Mechanistically, JARID1B was required for GATA3 recruitment to the Foxa1 promoter to activate Foxa1 expression. These results indicate that JARID1B positively regulates mammary ductal development through both extrinsic and cell-autonomous mechanisms.     

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.     

Inactive yet indispensable: the tale of Jarid2

Methylation of histone tails is believed to be important for the establishment and inheritance of gene expression programs during development. Jarid2/Jumonji is the founding member of a family of chromatin modifiers with histone demethylase activity. Although Jarid2 contains amino acid substitutions that are thought to abolish its catalytic activity, it is essential for the development of multiple organs in mice. Recent studies have shown that Jarid2 is a component of the polycomb repressive complex 2 and is required for embryonic stem (ES) cell differentiation. Here, we discuss current literature on the function of Jarid2 and hypothesize that defects resulting from Jarid2 deficiency arise from a failure to correctly prime genes in ES cells that are required for later stages in development.     

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.     

Overexpression of a histone H3K4 demethylase, JMJ15, accelerates flowering time in Arabidopsis

The methylation of histone 3 lysine 4 (H3K4) is essential for gene activation. Flowering Locus C (FLC), an important flowering repressor, quantitatively regulates flowering time in Arabidopsis and its expression level is coincident with H3K4 trimethylation (H3K4me3) dynamics. The methylation state of FLC chromatin is determined by the balance between methylation and demethylation, which is mediated by histone methyltransferases and demethylases, respectively. However, little is known about the role of histone demethylase(s) in FLC regulation. Here, we characterized the biochemical activity and biological function of a novel JmjC domain-containing H3K4 demethylase, JMJ15, in Arabidopsis. JMJ15, which is a member of the H3K4 demethylase JARID1 family, displayed H3K4me3 demethylase activity both in vitro and in vivo. The mutation of JMJ15 did not produce an obvious phenotype; however, overexpression JMJ15 resulted in an obvious early flowering phenotype, which was associated with the repression of FLC level and reduction in H3K4me3 at the FLC locus, resulting in increased FT expression. Our results suggest that JMJ15 is a novel H3K4 demethylase, involved in the control of flowering time by demethylating H3K4me3 at FLC chromatin when it was overexpressed in Arabidopsis. KEY MESSAGE: Overexpression of a histone H3K4 demethylase, JMJ15, represses FLC expression by decreasing its chromatin H3K4me3 level, thereby controlling flowering time in Arabidopsis.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

JARID1B Expression Plays a Critical Role in Chemoresistance and Stem Cell-Like Phenotype of Neuroblastoma Cells

Neuroblastoma (NB) is a common neural crest-derived extracranial solid cancer in children. Among all childhood cancers, NB causes devastating loss of young lives as it accounts for 15% of childhood cancer mortality. Neuroblastoma, especially high-risk stage 4 NB with MYCN amplification has limited treatment options and associated with poor prognosis. This necessitates the need for novel effective therapeutic strategy. JARID1B, also known as KDM5B, is a histone lysine demethylase, identified as an oncogene in many cancer types. Clinical data obtained from freely-accessible databases show a negative correlation between JARID1B expression and survival rates. Here, we demonstrated for the first time the role of JARID1B in the enhancement of stem cell-like activities and drug resistance in NB cells. We showed that JARID1B may be overexpressed in either MYCN amplification (SK-N-BE(2)) or MYCN-non-amplified (SK-N-SH and SK-N-FI) cell lines. JARID1B expression was found enriched in tumor spheres of SK-N-BE(2) and SK-N-DZ. Moreover, SK-N-BE(2) spheroids were more resistant to chemotherapeutics as compared to parental cells. In addition, we demonstrated that JARID1B-silenced cells acquired a decreased propensity for tumor invasion and tumorsphere formation, but increased sensitivity to cisplatin treatment. Mechanistically, reduced JARID1B expression led to the downregulation of Notch/Jagged signaling. Collectively, we provided evidence that JARID1B via modulation of stemness-related signaling is a putative novel therapeutic target for treating malignant NB.     

猪JARID1C基因的cDNA克隆、生物信息学及组织表达谱分析

JARID1C是高度保守的ARID蛋白家族的成员,该家族的蛋白参与并引起一系列生物学效应,如染色质重塑、细胞增殖与分裂、个体发育以及基因转录调控。JARID1C在人脑中表达丰富,对脑的发育和维持正常功能具有重要作用,突变可引起智力迟钝。本研究采用电子克隆（insilicocloning）的方法并结合5′末端快速扩增技术（RACE）,从猪卵巢中克隆到JARID1C的全长cDNA序列（GenBank登录号：EF139241）。猪JARID1C基因的cDNA全长5,908bp,包括4,551bp的开放阅读框（ORF）、522bp的5′非翻译区（5′UTR）和835bp的3′非翻译区（3′UTR）,polyA加尾信号序列AATAAA位于5,881bp和5,886bp之间。生物信息学分析揭示JARID1C蛋白含有1517个氨基酸残基,定位于细胞核中,该蛋白含有5个保守的结构域：JmjN结构域、ARID结构域、JmjC结构域、C5HC2锌指结构域和PHD锌指结构域。应用ClusterW程序分别对猪、狗、小鼠、大鼠、人和猿的JARID1C核苷酸序列和氨基酸序列进行多重序列比对,发现猪的JARID1C与其他哺乳动物具有很高的相似性。借助Mega3.1软件,采用N-J算法构建JARID1亚家族蛋白的系统进化树,揭示不同物种的进化关系。应用实时荧光定量PCR技术分析该基因在不同组织的表达差异,结果表明该基因在各组织均不同程度地表达,其中在肺和骨骼肌表达水平最低,而在脑和性腺表达水平最高。     

Boundaries between chromosomal domains of X inactivation and escape bind CTCF and lack CpG methylation during early development

Escape from X inactivation results in expression of genes embedded within inactive chromatin, suggesting the existence of boundary elements between domains. We report that the 5' end of Jarid1c, a mouse escape gene adjacent to an inactivated gene, binds CTCF, displays high levels of histone H3 acetylation, and functions as a CTCF-dependent chromatin insulator. CpG island methylation at Jarid1c was very low during development and virtually absent at the CTCF sites, signifying that CTCF may influence DNA methylation and chromatin modifications. CTCF binding sites were also present at the 5' end of two other escape genes, mouse Eif2s3x and human EIF2S3, each adjacent to an inactivated gene, but not at genes embedded within large escape domains. Thus, CTCF was specifically bound to transition regions, suggesting a role in maintaining both X inactivation and escape domains. Furthermore, the evolution of X chromosome domains appears to be associated with repositioning of chromatin boundary elements.     

组蛋白去甲基化酶PHD锌指蛋白8与神经发育

PHD锌指蛋白8(PHF8)是一种Fe2+和α-酮戊二酸依赖的组蛋白赖氨酸去甲基化酶.PHF8属于包含JmjC结构域蛋白家族,在N端还含有一个PHD(planthomeodomain)锌指结构域.人的PHF8基因突变往往破坏组蛋白去甲基化酶活性,从而引发遗传性X-连锁智力迟滞(XLMR)并伴发唇裂的发生.PHF8一方面可催化H3K9me2/1、H4K20me1和H3K27me2的去甲基化,另一方面还通过N端PHD锌指结构域与H3K4me3结合而发挥转录共激活作用.PHF8可调节rRNA和多个涉及神经发育的蛋白质编码基因如JARID1C的表达.这些研究显示,PHF8是一种重要的神经发育调节因子,从而拓宽了对组蛋白甲基化与基因表达关联的理解,同时为XLMR疾病的理解提供了新的线索.