A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

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.

Driver and novel genes correlated with metastasis of non-small cell lung cancer: A comprehensive analysis.

Although mutations of genes are crucial events in tumorigenesis and development, the association between gene mutations and lung cancer metastasis is still largely unknown. The goal of this study is to identify driver and novel genes associated with non-small cell lung cancer (NSCLC) metastasis. Candidate genes were identified using a novel comprehensive analysis, which was based on bioinformatics technology and meta-analysis. Firstly, EGFR, KRAS, ALK, TP53, BRAF and PIK3CA were identified as candidate driver genes. Further meta-analysis identified that EGFR (Pooled OR 1.33, 95% CI 1.19, 1.50; P < .001) and ALK (Pooled OR 1.52, 95% CI 1.22, 1.89; P < .001) mutations were associated with distant metastasis of NSCLC. Besides, ALK (Pooled OR 2.40, 95% CI 1.71, 3.38; P < .001) mutation was associated with lymph node metastasis of NSCLC. In addition, thirteen novel gene mutations were identified to be correlated with NSCLC metastasis, including SMARCA1, GGCX, KIF24, LRRK1, LILRA4, OR2T10, EDNRB, NR1H4, ARID4A, PRKCI, PABPC5, ACAN and TLN1. Furthermore, elevated mRNA expression level of SMARCA1 and EDNRB was associated with poor overall survival in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), respectively. Additionally, pathway and protein-protein interactions network analyses found the two genes were correlated with epithelial-mesenchymal transition process. In conclusion, mutations of EGFR and ALK were significantly correlated with NSCLC metastasis. In addition, thirteen novel genes were identified to be associated with NSCLC metastasis, especially SMARCA1 in LUAD and EDNRB in LUSC.

Structural basis for the DNA-binding activity of human ARID4B Tudor domain.

Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity. Structure determination and DNA titration analysis indicated that the ARID4B Tudor domain is also an interdigitated double Tudor domain with a DNA-binding surface similar to ARID4A. We identified a residue close to the DNA-binding site of the Tudor domain that differs between ARID4A and ARID4B. The Leu50 in ARID4A is Glu50 in ARID4B, and the latter forms salt bridges with two lysine residues at the DNA-binding surface. This causes a decrease in the strength of positive charge, thus reducing DNA-binding affinity while significantly increasing protein stability. We also found that a C-terminal extension region enhances the DNA-binding affinity of the ARID4B Tudor domain. This C-terminal extension is disordered and contains a positively charged RGR motif, providing an additional DNA-binding site. Finally, sequence and phylogenetic analyses indicated that the residue differences and the presence of the RGR extension region are conserved. These results provide new insight into the functional differences between ARID4A and ARID4B proteins, as well as elucidating the function of the disordered regions in these proteins.

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 27 kDa 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.

HPV-associated neuroendocrine carcinomas of the head and neck in FNA biopsies: Clinicopathologic features of a rare entity.

BACKGROUND: The majority of human papillomavirus (HPV)-associated oropharyngeal carcinomas are squamous cell carcinomas; however, there are rare reports of HPV-associated neuroendocrine carcinomas (HPV-NECs) in the upper aerodigestive tract. The aim of this study was to characterize the diagnostic features of fine-needle aspiration (FNA) cases of head and neck HPV-NEC. METHODS: Cytology cases of HPV-NEC were identified over a 3-year period from 2 institutions. Clinical, cytomorphologic, and ancillary test results were evaluated. RESULTS: Five FNA cases of HPV-NEC were identified from 4 patients with cervical lymph node metastases with primaries in the oropharynx (n = 2), nasopharynx (n = 1), and larynx (n = 1). Three cases showed mixed small cell and large cell neuroendocrine morphologies; 1 case was a small cell carcinoma, and the last case appeared as a large cell neuroendocrine carcinoma. All tumors were strongly positive for synaptophysin and p16 and negative for p63/p40. Two cases tested for INSM1 showed diffuse nuclear staining. HPV was confirmed by in situ hybridization in 4 cases, and HPV-18 was detected by polymerase chain reaction in the fifth case. Retinoblastoma (Rb) staining was moderate to weak (5/5), and p53 was weakly positive (5/5). CONCLUSIONS: Head and neck HPV-NEC is a rare, aggressive entity that can show mixed small and large cell features and p16 upregulation; p53 and Rb are variable with limited diagnostic utility. Because p16 positivity can be nonspecific, confirmatory HPV testing is required and may be helpful in determining the primary site for neuroendocrine carcinoma of an unknown primary. The accurate diagnosis of HPV-NEC is also important because of its worse prognosis in comparison with HPV-associated squamous cell carcinoma.

Functional haplotypes of ARID4A affect promoter activity and semen quality of bulls.

The AT-rich interaction domain 4 A (ARID4A) has an important role in regulating Sertoli cell function and male fertility. Its molecular mechanisms, however, remain largely unknown. In this study, two single nucleotide polymorphisms (SNPs) (g.53 G > T, ss 1966531596, and g.826 G > A, rs 210809648) were identified in the promoter region of ARID4A in 215 Chinese Holstein bulls using polymerase chain reaction (PCR)-restriction fragment length polymorphism and created restriction site-PCR. Results revealed that bulls with g.53 G > T-GG and g.826 G > A-G G genotype exhibited higher sperm deformity rate than those with g.53 G > T-TT and g.826 G > A-AA genotype (P < 0.01). Furthermore, three haplotypes (H1 (GG), H3 (TG), H4 (TA)) and six haplotype combinations (H1H1, H1H3, H1H4, H3H3, H3H4, H4H4) were obtained. The bulls with H4H4 exhibited lower sperm deformity rate than those with H1H1 and H1H3 (P < 0.05). In addition, results of bioinformatics analysis revealed that ARID4A has two promoters and that two SNPs of ARID4A are located in transcription factor binding sites. Compared with g.53 G > T-G and g.826 G > A-G allele, there was a greater fluorescence intensity in g.53 G > T-T and g.826 G > A-A allele by transient transfection in MLTC-1 cells and the luciferase report assay. qRT-PCR indicated the ARID4A expression was greater in bull spermatozoa with H4H4 haplotype combination than those with H1H1 haplotype combination (P < 0.05). Results of the present study indicate that g.53 G > T and g.826 G > A are functional mutations that are involved in regulation of ARID4A gene expression by affecting promoter activity and thus semen quality of Chinese Holstein bulls.

Downregulation of ARID4A and ARID4B promote tumor progression and directly regulated by microRNA-30d in patient with prostate cancer.

AT-rich interaction domain 4A (ARID4A) and AT-rich interaction domain 4B (ARID4B), which are both the AT-rich interaction domain (ARID) family, have been reported to be oncogene or tumor suppressor gene in various human malignances, but there is no involvement about their functions in prostate cancer (PCa). Our previous study has reported that microRNA-30d (miR-30d) expression can predicted poor clinical prognosis in PCa, however, the underlying mechanisms of miR-30d have not been fully described. The aim of our study is to investigate the expression relevance between miR-30d and ARID4A or ARID4B, and examine the clinical significance and biological function of ARID4A and AIRD4B in PCa. In this study, both ARID4A and ARID4B were identified as the target genes of miR-30d. In addition, the mRNA expression of miR-30d in PCa tissues were significantly negative correlated with ARID4A (Pearson correlation coefficient = -0.313, P = 0.001) and ARID4B (Pearson correlation coefficient = -0.349, P < 0.001), while there was a positive correlation between ARID4A and ARID4B (Pearson correlation coefficient = 0.865, P < 0.001). Moreover, both ARID4A and ARID4B were significantly downregulated in PCa tissues with high Gleason scores (P = 0.005, P = 0.033), PSA failure (P = 0.012, P = 0.05) and short biochemical recurrent-free survival (P = 0.033, P = 0.031). Furthermore, the knockout expression of ARID4A and ARID4B promoted PCa cell proliferation, migration and invasion in vitro. In conclusion, our results indicated that ARID4A and ARID4B may serve as tumor suppressor in PCa progression, suggesting that they might be the potential therapeutic targets in prostate cancer.

Crystal structure of chromo barrel domain of RBBP1.

RBBP1 is a retinoblastoma protein (pRb) binding protein acting as a repressor of gene transcription. RBBP1 is a multidomain protein including a chromo barrel domain, and its chromo barrel domain has been reported to recognize histone H4K20me3 weakly, and this binding is enhanced by the simultaneous binding of DNA. However, the molecular basis of this DNA-mediated histone binding by the chromo barrel domain of RBBP1 is unclear. Here we attempted to co-crystallize the chromo barrel domain of RBBP1 with either a histone H4K20me3 peptide alone or with both a histone H4K20me3 peptide and DNA, but only solved the peptide/DNA unbound crystal structure. Our structural analysis indicates that RBBP1 could interact with histone H4K20me3 similar to other histone binding chromo barrel domains, and the surface charge representation analysis of the chromo barrel domain of RBBP1 suggests that the chromo barrel domain of RBBP1 does not have a typical DNA binding surface, indicating that it might not bind to DNA. Consistently, our ITC assays also showed that DNA does not significantly enhance the histone binding ability of the chromo barrel domain of RBBP1.

miR-376c promotes carcinogenesis and serves as a plasma marker for gastric carcinoma.

Gastric carcinoma is highly prevalent throughout the world. Understanding the pathogenesis of this disease will benefit diagnosis and resolution. Studies show that miRNAs are involved in the tumorigenesis of gastric carcinoma. An initial screening followed by subsequent validation identified that miR-376c is up-regulated in gastric carcinoma tissue and the plasma of patients with the disease. In addition, the urinary level of miR-376c is also significantly increased in gastric carcinoma patients. The plasma miR-376c level was validated as a biomarker for gastric carcinoma, including early stage tumors. The induction of miR-376c was found to enrich the proliferation, migration and anchorage-independent growth of carcinoma cells and, furthermore, the repression of the expression of endogenous miR-376c was able to reduce such oncogenic phenotypes. ARID4A gene is a direct target of miR-376c. Knockdown of endogenous ARID4A increased the oncogenicity of carcinoma cells, while ARID4A was found to be drastically down-regulated in tumor tissue. Thus, expression levels of miR-376c and ARID4A mRNA tended to be opposing in tumor tissue. Our results demonstrate that miR-376c functions by suppressing ARID4A expression, which in turn enhances the oncogenicity of gastric carcinoma cells. It seems likely that the level of miR-376c in plasma and urine could act as invaluable markers for the detection of gastric carcinoma.

miR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18-25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated ß-galactosidase positive cells, the expression of p16(INK4a) and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1.

Upregulation of microRNA-132 in gastric cancer promotes cell proliferation via retinoblastoma 1 targeting.

Gastric cancer is one of the most frequent malignancies and a leading cause of cancer-related mortality worldwide. MicroRNAs (miRs), a class of small non­coding RNAs, have been shown to be critical in tumorigenesis. In the present study, the expression levels of miR­132 were analyzed in gastric cancer samples using quantitative reverse transcription­polymerase chain reaction. In addition, the cell viability, proliferation and invasion abilities were determined in two gastric cancer cell lines, NCI­N87 and MGC80­3, that were transfected with miR­132 mimics or antisense oligos. It was found that miR­132 expression was significantly upregulated in gastric cancer tissues when compared with adjacent non­cancerous tissues. At the molecular level, the data demonstrated that miR­132 inhibits the protein levels of retinoblastoma 1 (RB1) by targeting the 3'­untranslated region. Furthermore, reintroduction of RB1 markedly attenuated the proliferative roles of miR­132 overexpression. Therefore, the present results indicate that the miR­132/RB1 regulatory axis may be a potential novel diagnostic and therapeutic target for the treatment of gastric cancer.

Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 µM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

Exome sequencing reveals frequent inactivating mutations in ARID1A, ARID1B, ARID2 and ARID4A in 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.

ARID4A and ARID4B regulate male fertility, a functional link to the AR and RB pathways.

ARID4A and ARID4B are homologous members of the ARID (AT-rich interaction domain) gene family. ARID4A and ARID4B physically interact with each other. ARID4A is a retinoblastoma (RB)-binding protein. Biological function of these interactions is still unknown. Here, we report that mice with complete deficiency of Arid4a, combined with haploinsufficiency of Arid4b (Arid4a(-/-)Arid4b(+/-)), showed progressive loss of male fertility, accompanied by hypogonadism and seminal vesicle agenesis/hypodysplasia. Arid4a and Arid4b are expressed mainly in Sertoli cells of testes, which implies that their roles in Sertoli cell function are to support spermatogenesis and create the impermeable blood-testis barrier. In fact, evaluation of germ cell development in the Arid4a(-/-)Arid4b(+/-) mice showed spermatogenic arrest at the stages of meiotic spermatocytes and postmeiotic haploid spermatids. Analysis of the integrity of the blood-testis barrier showed increased permeability of seminiferous tubules in the Arid4a(-/-)Arid4b(+/-) testes. Interestingly, phenotypic Sertoli cell dysfunction in the Arid4a(-/-)Arid4b(+/-) mice, including spermatogenic failures and the impaired blood-testis barrier, recapitulated the defects found in the Sertoli cell-specific androgen receptor (AR) knockout mice and the Sertoli cell-specific RB knockout mice. Investigation of the molecular mechanism identified several AR- and RB-responsive genes as downstream targets of ARID4A and ARID4B. Our results thus indicate that ARID4A and ARID4B function as transcriptional coactivators for AR and RB and play an integral part in the AR and RB regulatory pathways involved in the regulation of Sertoli cell function and male fertility.

Reactive oxygen species mediate microRNA-302 regulation of AT-rich interacting domain 4a and C-C motif ligand 5 expression during transitions between quiescence and proliferation.

Normal cell growth consists of two distinct phases, quiescence and proliferation. Quiescence, or G(0), is a reversible growth arrest in which cells retain the ability to reenter the proliferative cycle (G(1), S, G(2), and M). Although not actively dividing, quiescent cells are metabolically active and quiescence is actively maintained. Our results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular proliferation. Results from a Q-RT-PCR and dual-luciferase-3'-UTR reporter assays identified ARID4a (AT-rich interacting domain 4a, also known as RBP1) and CCL5 (C-C motif ligand 5) as targets for miR-302. Ionizing radiation decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide as well as SOD2-overexpressing cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as an ROS-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.

Structural insight into recognition of methylated histone tails by retinoblastoma-binding protein 1.

Retinoblastoma-binding protein 1 (RBBP1), also named AT-rich interaction domain containing 4A (ARID4A), is a tumor and leukemia suppressor involved in epigenetic regulation in leukemia and Prader-Willi/Angelman syndromes. Although the involvement in epigenetic regulation is proposed to involve its chromobarrel and/or Tudor domains because of their potential binding to methylated histone tails, the structures of these domains and their interactions with methylated histone tails are still uncharacterized. In this work, we first found that RBBP1 contains five domains by bioinformatics analysis. Three of the five domains, i.e. chromobarrel, Tudor, and PWWP domains, are Royal Family domains, which potentially bind to methylated histone tails. We further purified these domains and characterized their interaction with methylated histone tails by NMR titration experiments. Among the three Royal Family domains, only the chromobarrel domain could recognize trimethylated H4K20 (with an affinity of ∼3 mm), as well as recognizing trimethylated H3K9, H3K27, and H3K36 (with lower affinities). The affinity could be further enhanced up to 15-fold by the presence of DNA. The structure of the chromobarrel domain of RBBP1 determined by NMR spectroscopy has an aromatic cage. Mutagenesis analysis identified four aromatic residues of the cage as the key residues for methylated lysine recognition. Our studies indicate that the chromobarrel domain of RBBP1 is responsible for recognizing methylated histone tails in chromatin remodeling and epigenetic regulation, which presents a significant advance in our understanding of the mechanism and relationship between RBBP1-related gene suppression and epigenetic regulation.

Identification of chromatin remodeling genes Arid4a and Arid4b as leukemia suppressor genes.

BACKGROUND: Leukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized. METHODS: The leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a(-/-) mice or Arid4a(-/-)Arid4b(+/-) mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription-polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided. RESULTS: Young (2-5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a(-/-) mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a(-/-) mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a(-/-) mice and 10 of 12 Arid4a(-/-)Arid4b(+/-) mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4a(-/-)Arid4b(+/-) bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice. CONCLUSIONS: Arid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)-like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.

Alterations of BRMS1-ARID4A interaction modify gene expression but still suppress metastasis in human breast cancer cells.

The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3.histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1(mut)) and mapped ARID4A interactions. BRMS1(L174D) disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1(DeltaCC1)) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1(mut). Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1(L174D) and BRMS1(DeltaCC1), both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1(mut), which modifies the composition of a SIN3.histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/cellular functions may prove important for future strategies targeting metastasis.

Deficiency of Rbbp1/Arid4a and Rbbp1l1/Arid4b alters epigenetic modifications and suppresses an imprinting defect in the PWS/AS domain.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by deficiency of imprinted gene expression from paternal or maternal chromosome 15q11-q13, respectively. Genomic imprinting of the PWS/AS domain is regulated through a bipartite cis-acting imprinting center (PWS-IC/AS-IC) within and upstream of the SNRPN promoter. Here, we show that two Rb-binding protein-related genes, Rbbp1/Arid4a and Rbbp1l1/Arid4b, are involved in the regulation of imprinting of the IC. We recovered these two genes from gene trap mutagenesis selecting for altered expression of an Snrpn-EGFP fusion gene strategy. RBBP1/ARID4A is an Rb-binding protein. RBBP1/ARID4A interacts with RBBP1L1/ARID4B and with the Snrpn promoter, implying that both are part of a protein complex. To further elucidate their roles on regulation of imprinting, we deleted the Rbbp1/Arid4a and Rbbp1l1/Arid4b genes in mice. Combined homozygous deficiency for Rbbp1/Arid4a and heterozygous deficiency for Rbbp1l1/Arid4b altered epigenetic modifications at the PWS-IC with reduced trimethylation of histone H4K20 and H3K9 and reduced DNA methylation, changing the maternal allele toward a more paternal epigenotype. Importantly, mutations of Rbbp1/Arid4a, Rbbp1l1/Arid4b, or Rb suppressed an AS imprinting defect caused by a mutation at the AS-IC. These data identify Rbbp1/Arid4a and Rbbp1l1/Arid4b as new members of epigenetic complexes regulating genomic imprinting at the PWS/AS domain.