ARID Domain Research Articles

The missing link: ARID1B non-truncating variants causing Coffin-Siris syndrome due to protein aggregation

ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.

Changes in terrestrial water storage in the Three-North region of China over 2003–2021: Assessing the roles of climate and vegetation restoration

Large-scale vegetation restoration has markedly enhanced ecosystem services in the Three-North (TN) region, concurrently exacerbating the pre-existing water resource crisis stemming from climate change in this arid domain. However, the specific contributions of climate and vegetation changes to the evolution of water resources in the TN region have yet to be elucidated. This study leverages data from the Gravity Recovery and Climate Experiment (GRACE) and its Follow-on mission (GRACE-FO), specifically the terrestrial water storage anomaly (TWSA) from 2003 to 2021. We identify two TWSA rising (TWSA_rⅠ and TWSA_rⅡ) and two declining (TWSA_dⅠ and TWSA_dⅡ) hotspots in the TN region. By exploring the relative importance of climate drivers and vegetation dynamics in influencing the temporal dynamics of TWSA, we found that vegetation restoration assumes a more pivotal role than climate factors in 45.2% of the total area within the identified hotspots, predominantly evident in TWSA declining hotspots. The heightened TWSA in TWSA_rⅠ and TWSA_rⅡ primarily stems from precipitation recharge, with precipitation-dominated areas constituting 19.3% of the area within the identified hotspots. Potential evapotranspiration dominates TWSA changes in only 4.6% of the region within the identified hotspots, primarily scattered in TWSA_dⅠ and TWSA_dⅡ. Furthermore, a comparative assessment of soil moisture and groundwater responses to environmental factors indicates that water storage at shallower layers is more responsive to climate factors, while the impacts of vegetation changes on TWSA are more discernibly reflected in water storage at deeper layers. These findings furnish scientific guidance for future ecological restoration planning and sustainable water resources management.

Abstract 5591: ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer

Abstract The SWI/SNF chromatin remodeler utilizes energy of ATP hydrolysis to slide or evict nucleosomes or histones, respectively, thus enabling nuclear processes by driving an ‘open’ chromatin architecture. The mammalian SWI/SNF, also termed the BrG1/Brm associated factor (BAF) complex, is the major chromatin remodeler in ontogeny and adult life. Three major BAF complexes namely canonical (cBAF), polybromo (pBAF), and non-canonical (ncBAF) have been studied. BAF components, classified as a tumor suppressors, exhibit frequent mutations in cancers. Though, studies have evaluated functional roles of BAF subunits, very few attempted to study BAF mutations identified across cancer types. In our previous studies, we identified tumor specific mutations in the gene encoding ‘A-T rich interaction domain containing 1B’ (ARID1B), within its nuclear localization signal (NLS). NLS-inactivated ARID1B exhibited cytoplasmic localization and promoted tumorigenesis by activating the ERK and Wnt/β-catenin pathways. More recently, we detected inactivating mutations in the gene encoding ARID2, a component of the pBAF complex and an ARID1B paralogue. We now present detailed characterization of tumor-specific ARID2 truncations that were presumed to be inactivating in nature. One such truncation mutation viz. p.Ser989fsArg21*, identified in our previous study, is expected to yield an ARID2 protein truncating at amino acid position 1010 (as against the full length of 1835 amino acids). Scrutiny of The Cancer Genome Atlas cBioPortal and Cancer Cell Line Encyclopedia mutation data bases revealed several ARID2 mutations expected to truncate the protein around the 1010 position. Surprisingly, ectopic expression of truncated ARID2 forms promoted oncogenic function in both ARID2 proficient and deficient backgrounds, thus validating a neomorphic gain of function for the truncated protein. Fluorescence based intracellular localization assays revealed a surprising cytoplasmic localization of truncated ARID2. Further, we mapped the ARID2 NLS by in silico analysis followed by evaluation of several deletion constructs, to be located at 1488-1518 position; thus, explaining the cytoplasmic localization of ARID2 forms truncating around 1010 position. Further, truncated ARID2 was unable to bind other pBAF components and did not sequester other pBAF components to the cytoplasm, in both ARID2 proficient and deficient cells. Tandem Affinity Purification Mass-spectrometry revealed putative interaction partners for truncated ARID2; analysis is currently underway to reveal the mechanism of tumorigenic induction exhibited by truncated ARID2. Immunohistochemistry on colorectal and breast cancer tissue microarrays revealed ARID2 cytoplasmic localization in a significant fraction of samples. We have discovered a novel YIN-YANG function of BAF components expected to yield efficient therapeutic options in cancer. Citation Format: Sanjana Sarkar, Murali Dharan Bashyam. ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5591.

DNA binding affinity of pathogenic missense mutations in the ARID domain of ARID1A

DNA binding affinity of pathogenic missense mutations in the ARID domain of ARID1A

Abstract 3485: Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity

Abstract The mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes collectively represent one of the most frequently mutated cellular entities in cancer, second only to TP53. Mutations across the 29 human genes that encode mSWI/SNF complex subunits occur in over 20% of human cancers, with mutations affecting ARID1A and ARID1B, the largest BAF subunits, being the most frequent. However, the functional contributions of these subunits, particularly their commonly mutated N-terminal intrinsically disordered regions (IDRs) and a highly conserved ARID DNA-binding domain, to BAF function remain poorly understood. Here, we demonstrate that the IDRs of ARID1A/B, coupled with the ARID domain, drive biomolecular condensate formation and BAF chromatin localization in cells. We define ARID1A/B IDRs as two-part systems, facilitating homotypic BAF complex interactions (i.e., valence generated by localized condensation) and heterotypic complex interactions, that together establish a highly specific, sequence-encoded protein interaction network within condensates. Both types of interactions are required for appropriate genome-wide targeting of BAF complexes, DNA accessibility generation, and appropriate gene expression. Replacement of the ARID1A N-terminal IDR with IDRs derived from two unrelated proteins FUS and DDX4, rescues generic condensation of BAF but not chromatin occupancy, DNA accessibility, and heterotypic interactions, highlighting the sequence-specificity embedded in the IDR of ARID1A. Taken together, these data establish a role for the largest and most frequently perturbed IDRs within a major chromatin remodeler and explain how biomolecular condensate formation enables both genomic localization and functional partner recruitment. Furthermore, these findings lay the groundwork for mapping IDR sequence specificity or “grammar”, that dictates the co-condensation network formation, and suggests that targeted disruption of these mechanisms may represent new targeted therapeutic opportunities across multiple cancers. Citation Format: Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, Cigall Kadoch. Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3485.

Biophysical characterization of pathogenic missense mutation in the ARID domain of ARID1A.

The ARID1A, AT- rich interactive domain containing protein 1A, is frequently mutated in cancer (e.g., 6% of every cancer and 45% of all ovarian cancer). ARID1A is a member of the Switch/Sucrose non-Fermentable (SWI/SNF) complex responsible for chromatin remodeling and gene expression. Within ARID1A lies an ancient and highly conserved DNA-binding domain (ARID domain) of ∼100 amino acids with a helix-turn-helix motif. The Cancer Genome Atlas (TCGA) reports on mutations in the ARID domain for which 37% of the mutations are frameshift, another 37% are missense and remaining are stop gain. Here we use experimental and computational approaches to identify how missense mutation impaired the ARID-to-DNA interactions by impacting protein stability, binding affinity, and structural dynamics potentially explaining their pathogenicity. Specifically, we identified the most mutated variants in the TCGA (i.e., R1020S, M1022K, K1047Q, G1063V, Q1098H). Next, we used web server bioinformatic pathogenic predictors tools (SAMPDI, CUPSAT, SAMBA-3D, SAAMBE-SEQ, SAAFEC, SAAFEC-SEQ, nMCS) to compute biophysical properties such as the protein stability and DNA binding energy and their likelihood for pathogenicity. We used denaturation assays to experimentally determine the stability of selected variants and compare it with the wildtype. To determine the binding affinity, we run electrophoresis mobility shift assays, intrinsic fluorescence, fluorescence anisotropy, and microscale thermophoresis approaches. We compare experimental and bioinformatic tools to decipher the subtle impacts of missense mutation in the ARID domain. Our findings will help us understand the impact of missense mutations in the structure-function relationship.

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.

KDM5B protein expressed in viable and fertile ΔARID mice exhibit no demethylase activity.

Post-translational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and small-molecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of Kdm5b knock-out mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔA-T rich interaction domain (ΔARID)-KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARID-KDM5B was found to have no demethylase activity as determined by in vitro demethylase assays and by immunofluorescence in transfected Cos-1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARID-KDM5B structure compared with that in WT-KDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WT-KDM5B. This supports the experimental data that shows the loss of demethylase activity. Since Kdm5b knock-out mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.

Dual Recognition of H3K4me3 and DNA by the ISWI Component ARID5 Regulates the Floral Transition in Arabidopsis.

Chromatin remodeling and histone modifications are important for development and floral transition in plants. However, it is largely unknown whether and how these two epigenetic regulators coordinately regulate the important biological processes. Here, we identified three types of Imitation Switch (ISWI) chromatin-remodeling complexes in Arabidopsis (Arabidopsis thaliana). We found that AT-RICH INTERACTING DOMAIN5 (ARID5), a subunit of a plant-specific ISWI complex, can regulate development and floral transition. The ARID-PHD dual domain cassette of ARID5 recognizes both the H3K4me3 histone mark and AT-rich DNA. We determined the ternary complex structure of the ARID5 ARID-PHD cassette with an H3K4me3 peptide and an AT-containing DNA. The H3K4me3 peptide is combinatorially recognized by the PHD and ARID domains, while the DNA is specifically recognized by the ARID domain. Both PHD and ARID domains are necessary for the association of ARID5 with chromatin. The results suggest that the dual recognition of AT-rich DNA and H3K4me3 by the ARID5 ARID-PHD cassette may facilitate the association of the ISWI complex with specific chromatin regions to regulate development and floral transition.

Resonance assignments for the tandem PWWP-ARID domains of human RBBP1.

Retinoblastoma-binding protein 1 (RBBP1), also known as AT-rich interaction domain 4A (ARID4A), is a tumour suppressor involved in the regulation of the epigenetic programming in leukemia and Prader-Willi/Angelman syndromes. The ARID domain of RBBP1 binds to DNA non-specifically and has gene suppression activity. However, no structural data has been obtained for the human RBBP1 ARID domain so far. Here we report the near-complete 1H, 13C, 15N backbone and side-chain NMR assignment of a 27kDa tandem PWWP-ARID domain construct that spans residues 171-414 with the removal of a short disordered region between the two domains. The predicted secondary structure based on the assigned chemical shifts is consistent with the structures of the isolated PWWP domain of human RBBP1 previously solved and the homologous ARID domains of other proteins.

RETRACTED: Interaction of AtHMGB15, an ARID-HMG family protein, with RING-H2 type E3 ubiquitin ligase AtATL79

Recent studies have shown the importance of Ub/proteasome pathway in regulating transcription for proper synchronization of gene expression. Using yeast two-hybrid screening, we have identified an Arabidopsis RING-H2 type of E3 ubiquitin ligase, AtATL79 that interacts with ARID-HMG protein AtHMGB15 mainly through the ARID domain. Sequence analysis of the RING domain of AtATL79 indicates the presence of conserved six Cys and two His residue that coordinate two Zn⁺² ions. AtATL79 is a membrane-bound protein that colocalizes with AtHMGB15 in the nucleus. AtATL79 is an E3 ubiquitin ligase that self-ubiquitinates at Lys 76 residue in vitro. Moreover, AtHMGB15 was found to be polyubiquitinated by AtATL79 both in vitro and in vivo at lysine residue 420. Interestingly, polyubiquitination of AtHMGB15 was observed in various tissues of Arabidopsis, and its stability significantly increases in the presence of 26S proteasome inhibitor MG132. Our results suggest that the Ub/26S proteasome system regulates cellular AtHMGB15 protein level in different tissues of Arabidopsis to regulate the spatiotemporal activity of the protein.

Structure of the Arabidopsis JMJ14-H3K4me3 Complex Provides Insight into the Substrate Specificity of KDM5 Subfamily Histone Demethylases.

In chromatin, histone methylation affects the epigenetic regulation of multiple processes in animals and plants and is modulated by the activities of histone methyltransferases and histone demethylases. The jumonji domain-containing histone demethylases have diverse functions and can be classified into several subfamilies. In humans, the jumonji domain-containing Lysine (K)-Specific Demethylase 5/Jumonji and ARID Domain Protein (KDM5/JARID) subfamily demethylases are specific for histone 3 lysine 4 trimethylation (H3K4me3) and are important drug targets for cancer treatment. In Arabidopsis thaliana, the KDM5/JARID subfamily H3K4me3 demethylase JUMONJI14 (JMJ14) plays important roles in flowering, gene silencing, and DNA methylation. Here, we report the crystal structures of the JMJ14 catalytic domain in both substrate-free and bound forms. The structures reveal that the jumonji and C5HC2 domains contribute to the specific recognition of the H3R2 and H3Q5 to facilitate H3K4me3 substrate specificity. The critical acidic residues are conserved in plants and animals with the corresponding mutations impairing the enzyme activity of both JMJ14 and human KDM5B, indicating a common substrate recognition mechanism for KDM5 subfamily demethylases shared by plants and animals and further informing efforts to design targeted inhibitors of human KDM5.

ARID1A represses hepatocellular carcinoma cell proliferation and migration through lncRNA MVIH

ARID1A, encoding the BAF250a subunit of SWI/SNF complex, has a high mutation frequency in numerous types of cancer. LncRNAs, a type of non-coding RNAs longer than 200 nucleotides, have been reported to interplay with SWI/SNF complex during cancer progression. However, whether the interaction between ARID1A and lncRNA affects hepatocellular carcinoma (HCC) still needs to be investigated. Here, we reveal that ARID1A interacts with lncRNA MVIH through some region(s) or domain(s) including ARID domain and C-terminal ARID1A protein binding domain. ARID1A upregulates its downstream target CDKN1A and suppresses HCC cell proliferation and migration through inhibiting MVIH. Our data suggests that deficiency or loss of functional mutations of ARID1A in HCC cells might contribute to the increased activity of certain cancer-promoting lncRNAs.

Mediterranean habitat loss under future climate conditions: Assessing impacts on the Natura 2000 protected area network

The Mediterranean basin is a global hotspot of biological diversity and the most rich biodiversity region in Europe. Nevertheless, climate-driven habitat loss is one of the most serious concerns for biodiversity conservation in this region. We assess Mediterranean habitat loss and conversion into arid habitat under scenarios of climate change and evaluate protected areas, including Natura 2000 sites, which will be affected by these changes. We mapped shifts of Mediterranean and arid domains using four bias-corrected simulations from Regional Climate Models for two emission scenarios over this century, disaggregated to a 1 km grid size. Our results indicate that by the end of the century the Euro-Mediterranean domain is projected to shift into other climatic domains by an area equivalent to 53–121% of its current size. However it is projected to lose 11–25% of its current extent, which represents an area close to the size of Greece and Portugal combined. The loss is entirely due to shifts of the arid domain. Additionally, our results indicate that the extent of the arid domain is projected to increase by 228–450% of its current size in the European region. The shrinking of the current Euro-Mediterranean domain is projected to affect 15–23% of the Mediterranean Natura 2000 sites, and the loss in these sites is projected at 13–30% of its current area. Loss is projected to occur in central and southern areas of the Iberian Peninsula, southern Italy and the island of Sicily, south-eastern Greece, Cyprus, Malta and central Turkey. Computed changes in projected climatic parameters indicate that current areas of the Euro-Mediterranean domain will be hotter and drier. Temperature increase and precipitation decrease are projected to be more marked in the summer half of the year. As early as in the 2020s annual temperature is projected to increase by 0.9–1.4 °C with respect to the present reference climate, reaching an increase of 2.2–3.6 °C by the end of the century. By this period, summer precipitation is projected to decrease by 24–46% and annual precipitation by 14–23%. We provide insight into several aspects of adaptation and management of Mediterranean protected areas. A proactive approach taking into consideration landscape connectivity and the concomitant threats triggered by climate change is a priority. Proactive adaptation and management promoting investments in Green Infrastructure and a denser network of interconnected protected areas are necessary instruments for preserving Mediterranean biodiversity from the threats of habitat loss.

Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.

Mutations in the KDM5C ARID Domain and Their Plausible Association with Syndromic Claes-Jensen-Type Disease.

Mutations in KDM5C gene are linked to X-linked mental retardation, the syndromic Claes-Jensen-type disease. This study focuses on non-synonymous mutations in the KDM5C ARID domain and evaluates the effects of two disease-associated missense mutations (A77T and D87G) and three not-yet-classified missense mutations (R108W, N142S, and R179H). We predict the ARID domain’s folding and binding free energy changes due to mutations, and also study the effects of mutations on protein dynamics. Our computational results indicate that A77T and D87G mutants have minimal effect on the KDM5C ARID domain stability and DNA binding. In parallel, the change in the free energy unfolding caused by the mutants A77T and D87G were experimentally measured by urea-induced unfolding experiments and were shown to be similar to the in silico predictions. The evolutionary conservation analysis shows that the disease-associated mutations are located in a highly-conserved part of the ARID structure (N-terminal domain), indicating their importance for the KDM5C function. N-terminal residues’ high conservation suggests that either the ARID domain utilizes the N-terminal to interact with other KDM5C domains or the N-terminal is involved in some yet unknown function. The analysis indicates that, among the non-classified mutations, R108W is possibly a disease-associated mutation, while N142S and R179H are probably harmless.

Abstract 2469: A mutated ARID5B protein found in endometrial cancer has a deleterious function with longer half-life

Abstract The ARID5B gene encodes a member of ARID transcription factors containing a unique DNA binding domain, ARID, which binds to AT-rich regions. Recently, point mutations of ARID5B, including nonsense, missense, and frame-shift mutations, were found in endometrial cancers. This finding strongly suggests that the ARID5B gene is a novel tumor suppressor candidate gene in this disease. However, detailed functions of wild-type/mutated ARID5B have not been examined. To explore the importance of ARID5B in development of endometrial cancer, the property of ARID5B was characterized. Interestingly, half of the point mutations reported previously were nonsense/ frame-shift mutations, generating premature termination codons (PTCs). Generally, genes with PTCs generate unstable mRNA, which are detected and degraded by the nonsense mediated mRNA decay (NMD) unless the PTCs are located in the last exon or within 50 nucleotide from the last exon-intron boundary. Surprisingly, all the PTCs found in endometrial cancer are located in the last exon or within 50 nucleotide upstream of the last exon-intron of the ARID5B gene, suggesting that these mutated mRNA are stable to generate truncated ARID5B proteins. We also found that the wild-type ARID5B protein has PEST sequences, which make proteins unstable through the ubiquitin-proteasome system. In virtue of the PTCs, the truncated ARID5B proteins lack these PEST sequences, but still have the ARID domain. Our study showed that the truncated ARID5B protein had longer half-life than that of wild-type ARID5B. Furthermore, we showed that the C-terminus of ARID5B had a repressive property, which was confirmed by a reporter gene assay with transient transfection. The C-terminus was deleted in the truncated ARID5B. The data suggest that the wild-type ARID5B could suppress the expression of down-stream target genes, but the truncated ARID5B could not. Taken together, the truncated ARID5B may accumulate in cells due to its longer half-life and inhibit repressive function of the wild-type ARID5B in a dominant-negative fashion. We also performed colony formation assays using an endometrial cancer cell line, Ishikawa. Expression of wild-type ARID5B was strongly suppressed in colonies resistant to G418, comparing with expression of GFP transfected into the cells as a control. In summary, we found that the truncated ARID5B is a long half-life protein without its transcriptional property, which may inhibit the transcriptional property of wild-type ARID5B. Our data suggest that ARID5B plays an important role as a transcriptional repressor in development of endometrial cancer and the mutated ARID5B may inhibit the normal function of wild-type ARID5B in the cancer cells. Citation Format: Norihiko Kawamata, Keiichi Itakura. A mutated ARID5B protein found in endometrial cancer has a deleterious function with longer half-life. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2014-2469

Polycomb Recruitment via lncRNA

Molecular Biology There are many protein complexes that modulate gene expression during differentiation and development. Each must be targeted to the correct regulatory sequences in the genome, to orchestrate the appropriate cell- or tissue-specific gene expression programs. Some complexes target DNA by direct binding to a specific DNA sequence. Others must be recruited by auxiliary factors to their place of action. None of the core components of the polycomb repressive complex-2 (PRC2) are able to bind DNA, yet PRC2 is targeted to, and critical for, repressing cell type–specific genes throughout development. The Jumonji family, ARID domain–containing protein JARID2, an accessory subunit of PRC2, is somehow involved in that recruitment process. Kaneko et al. show that JARID2 can bind to long noncoding RNAs (lncRNAs), among them Meg3, and this interaction recruits PRC2 to a subset of its target genes. Meg3 binding to JARID2 also stimulates the interaction of JARID2 with one of the core subunits of PRC2, EZH2, with which Meg3 can also interact. Binding of JARID2 to the Meg3 lncRNA thus serves both to recruit and assemble PRC2 on chromatin. Mol. Cell 53 ,10.1016/j.molcel.2013.11.012 (2014).

Exome sequencing reveals frequent inactivating mutations inARID1A, ARID1B, ARID2andARID4Ain microsatellite unstable colorectal cancer

ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 [16%] samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.

PLU-1/Jarid1B contributes to estrogen-induced cell proliferation in the normal mammary gland and in breast cancer, and is required for embryonic survival

PLU-1/Jarid1B is a nuclear protein that is widely expressed in breast cancer, with higher levels being seen in ER+ cancers. Expression in normal adult issues is largely restricted to testis and the differentiating mammary gland. Through the JmjC domain the protein can demethylate H3K4me3, which correlates with its function as a transcriptional repressor. PLU-1/Jarid1B contains a DNA binding domain, and can be recruited to DNA through binding to transcription factors. We now find that the protein interacts with the ERα receptor and contributes to estrogen-induced survival of MCF-7 cells in in vitro culture and when grown as tumours in nude mice. To investigate the function of Plu-1/Jarid1B in vivo, transgenic mice expressing defective Plu-1/Jarid1B have been developed. The systemic KO is an embryonic lethal with no homozygote embryos being detected at day 7.5. Another strain expressing the protein missing the ARID AT-rich DNA binding domain (which is also required for demethylase function) shows a mammary phenotype. In these ΔARID mice, the development of the mammary tree at puberty and early pregnancy is delayed, but the gland recovers by late pregnancy. The inhibition of development of terminal end buds at puberty, which is crucially dependent on ERα signalling, suggests an involvement of Plu-1/Jarid1B in this signalling that is impaired in the ΔARID mouse. Confirming this, levels of expression of downstream targets of ERα (progesterone receptor and Wnt4) are reduced in the ΔARID mouse. The development of spontaneous mammary tumours in the ΔARID mouse is delayed compared with wild-type mice, suggesting that Plu-1/Jarid1B contributes to tumour growth, and that this action is impaired when the ARID domain is deleted. The data suggest that PLU-1/JARID1B is involved in estrogen-induced growth of normal and malignant mammary epithelial cells.