Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Backbone and sidechain 1H, 13C and 15N resonance assignments of the Bright/ARID domain from the human JARID1C (SMCX) protein

We have assigned ¹H, ¹⁵N and ¹³C resonances of the Bright/ARID DNA-binding domain from the human JARID1C protein, a newly discovered histone demethylase belonging to the JmjC domain-containing protein family.     

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.     

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.     

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.     

1H, 13C, 15N backbone and side-chain resonance assignments of the Bright/ARID domain from the human histone demethylase JARID1B

We report backbone and side-chain resonance assignments of the Bright/ARID domain from the human JARID1B protein. These assignments provide a basis for the detailed structural investigation of the interaction between DNA and ARID domains.     

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.     

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.     

Solution structure of SWI1 AT-rich interaction domain from Saccharomyces cerevisiae and its nonspecific binding to DNA

SWI1 is a subunit of the SWI/SNF complex involved in chromatin remodeling. It contains an AT-rich interaction domain (ARID) which has the potential DNA binding activity. In this study, we determined the solution structure of the SWI1 ARID domain from Saccharomyces cerevisiae by nuclear magnetic resonance spectroscopy. Yeast SWI1 ARID domain is composed of seven alpha helices, six of which are conserved among the ARID family. In addition, the DNA-binding activity of the SWI1 ARID domain was confirmed by chemical shift perturbation assay. Similar to its human homolog, the yeast SWI1 ARID domain binds DNA nonspecifically.     

Chromatin Sensing by the Auxiliary Domains of KDM5C Regulates Its Demethylase Activity and Is Disrupted by X-linked Intellectual Disability Mutations

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.     

The DNA-binding properties of the ARID-containing subunits of yeast and mammalian SWI/SNF complexes

SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that are highly conserved from yeast to human. From yeast to human the complexes contain a subunit with an ARID (A-T-rich interaction domain) DNA-binding domain. In yeast this subunit is SWI1 and in human there are two closely related alternative subunits, p270 and ARID1B. We describe here a comparison of the DNA-binding properties of the yeast and human SWI/SNF ARID-containing subunits. We have determined that SWI1 is an unusual member of the ARID family in both its ARID sequence and in the fact that its DNA-binding affinity is weaker than that of other ARID family members, including its human counterparts, p270 and ARID1B. Sequence analysis and substitution mutagenesis reveals that the weak DNA-binding affinity of the SWI1 ARID is an intrinsic feature of its sequence, arising from specific variations in the major groove interaction site. In addition, this work confirms the finding that p270 binds DNA without regard to sequence specificity, excluding the possibility that the intrinsic role of the ARID is to recruit SWI/SNF complexes to specific promoter sequences. These results emphasize that care must be taken when comparing yeast and higher eukaryotic SWI/SNF complexes in terms of DNA-binding mechanisms.