Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy.

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.

A transcriptionally repressed quiescence program is associated with paused RNA polymerase II and is poised for cell cycle re-entry.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition. This article has an associated First Person interview with the first authors of the paper.

AFF4 globally affects the release of paused RNA polymerase II in HEL cells.

P-TEFb, a heterodimer of the kinase CDK9 and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb complex, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. We and others recently found that AFF1, ENL and AF9 are capable of regulating transcriptional initiation, but it is unknown yet whether AFF4 is also capable of doing so. With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex, one recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. However, it is unclear whether those mechanisms are general. In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation, we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II. Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag. Notably, we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

CircRNA AFF4 induced by KDM1A promotes osteogenic differentiation through FNDC5/Irisin pathway.

BACKGROUND: Circular RNA (circ) AFF4 was documented to regulate osteogenesis but the underlying mechanism remains to be elucidated. The preliminary study showed that circ_AFF4 may promote osteogenesis via FNDC5/Irisin. Furthermore, the online prediction tool indicated the interaction of circ_AFF4, insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3), FNDC5 and lysine (K)-specific demethylase 1 A (KDM1A). Therefore, this study aims to elucidate the relationships of KDM1A, circ_AFF4, IGF2BP3 and FNDC5/Irisin during osteogenesis. METHODS: The alkaline phosphatase (ALP) activities and osteogenic-related factors were determined using ALP and alizarin red S (ARS) staining, real-time quantitative PCR(RT-qPCR) and western blot. Immunoprecipitation (RIP), pull-down assay and fluorescence in situ hybridization (FISH) were used to examine the interactions among circ_AFF4/IGF2BP3/FNDC5. A mouse in vivo model was utilized to further confirm the regulatory effect on bone formation. RESULTS: Circ_AFF4 and KDM1A expression levels were increased during osteoinduction of BM-MSCs. Knockdown of circ_AFF4 and KDM1A significantly suppressed BM-MSC osteogenesis. We also proved that KDM1A directly bound to circ_AFF4 and FNDC5 promoter and induced circ_AFF4 and FNDC5 expression. Furthermore, circ_AFF4 enhanced the stability of FNDC5 by generating a circ_AFF4, IGF2BP3 and FNDC5 RNA-protein complex, and thereby induced Irisin and osteogenesis. The in vitro data was confirmed with in vivo model. CONCLUSION: These findings elucidate that KDM1A induces circ_AFF4, which promotes promote osteogenesis via IGF2BP3. This study indicates that circ_AFF4 may potentially represent a critical therapeutic target for the diseases.

AFF4 facilitates melanoma cell progression by regulating c-Jun activity.

Melanoma is characterized by high mortality and poor prognosis due to metastasis. AFF4 (AF4/FMR2 family member 4), as a scaffold protein, is a component of the super elongation complex (SEC), and is involved in the progression of tumors, e.g., leukemia, head and neck squamous cell carcinoma (HNSCC). However, few studies on AFF4 have focused on melanoma. Here, AFF4 expression levels and clinicopathological features were evaluated in melanoma tissue samples. Then, we performed cell proliferation, migration and invasion assays in A375 and A2058 cells lines in vitro to evaluate the role of AFF4 in melanoma. The effects of AFF4 knockdown in vivo were characterized via a xenograft mouse model. Finally, the correlation between c-Jun and AFF4 protein levels in melanoma was analyzed by rescue assay and immunohistochemistry (IHC). We found that AFF4 expression was upregulated in melanoma tumor tissues and that AFF4 protein expression was also closely related to the prognosis of patients with cutaneous melanoma. Moreover, AFF4 could promote the invasion and migration of melanoma cells by mediating epithelial to mesenchymal transition (EMT). AFF4 might regulate c-Jun activity to promote the invasion and migration of melanoma cells. Importantly, c-Jun was regulated by the AFF4 promoted melanoma tumorigenesis in vivo. Taken together, AFF4 may be a novel oncogene that promotes melanoma progression through regulation of c-Jun activity.

Structure of the super-elongation complex subunit AFF4 C-terminal homology domain reveals requirements for AFF homo- and heterodimerization.

AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription. AFF4 consists of an intrinsically disordered N-terminal region that interacts with other super-elongation complex subunits and a C-terminal homology domain (CHD) that is conserved among AF4/FMR2 family proteins, including AFF1, AFF2, AFF3, and AFF4. Here, we solved the X-ray crystal structure of the CHD in human AFF4 (AFF4-CHD) to 2.2 Å resolution and characterized its biochemical properties. The structure disclosed that AFF4-CHD folds into a novel domain that consists of eight helices and is distantly related to tetratrico peptide repeat motifs. Our analyses further revealed that AFF4-CHD mediates the formation of an AFF4 homodimer or an AFF1-AFF4 heterodimer. Results from fluorescence anisotropy experiments suggested that AFF4-CHD interacts with both RNA and DNA in vitro Furthermore, we identified a surface loop region in AFF4-CHD as a substrate for the P-TEFb kinase cyclin-dependent kinase 9, which triggers release of polymerase II from promoter-proximal pausing sites. In conclusion, the AFF-CHD structure and biochemical analyses reported here reveal the molecular basis for the homo- and heterodimerization of AFF proteins and implicate the AFF4-CHD in nucleic acid interactions. The high conservation of the CHD among several other proteins suggests that our results are also relevant for understanding other CHD-containing proteins and their dimerization behavior.

Long noncoding RNA ZFPM2-AS1 is involved in lung adenocarcinoma via miR-511-3p/AFF4 pathway.

Lung cancer is the dominating cause of cancer-induced death and can be classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Lung adenocarcinoma (LUAD) is the most common histological subtype of NSCLC and its pathology remains unclear. Mounting reports have revealed that lncRNAs could regulate cellular activities in cancers. Yet the role of ZFPM2 antisense RNA 1 (ZFPM2-AS1) in LUAD has not been elucidated. Using GEPIA online dataset, we identified the amplification of ZFPM2-AS1 in LUAD tissues. Through quantitative real-time reverse transcription-polymerase chain reaction analysis, we observed an upregulation of ZFPM2-AS1 in LUAD cell lines. Conducting loss-of-function assays, we found that ZFPM2-AS1 depletion impaired cell viability, suppressed cell migration, and reversed epithelial-mesenchymal transition progress in LUAD cells. Mechanism investigation manifested that ZFPM2-AS1 was distributed in the cytoplasm of LUAD cells. Moreover, ZFPM2-AS1 functioned as a molecular sponge of miR-511-3p, which was a suppressor in LUAD. Moreover, ZFPM2-AS1 sponged miR-511-3p and thereby deregulated AF4/FMR2 family member 4 (AFF4), a target of miR-511-3p. At length, rescue assays indicated that AFF4 overexpression revived the inhibiting effects of ZFPM2-AS1 knockdown on the biological processes in LUAD. All in all, this study uncovered the function and the mechanism of ZFPM2-AS1 in LUAD.

AFF4 enhances odontogenic differentiation of human dental pulp cells.

AFF4 is a component of super elongation complex (SECs) and functions as a scaffold protein to bridge the transcription elongation factors. It is associated with leukemia, HIV transcription, and head neck cancer. However, its role in odontogenic differentiation of dental pulp cells (DPCs) is unclear. Here, we show the expression of AFF4 is increased during odontogenesis. Depletion of AFF4 in human DPCs leads to a decrease of alkaline phosphatase (ALP) activity, calcium mineralization and odontogenic-related genes expression. On the contrary, Lentivirus-mediated overexpression of AFF4 induces the odontogenic potential of DPCs. Mechanistically, we found AFF4 regulates the transcription of NFIC, a key factor for tooth root formation. Overexpression of NFIC successfully rescues the restricted differentiation of AFF4-depleted cells. Our data demonstrate that AFF4 serves as a previously unknown regulator of odontogenesis.

Clinical and molecular spectrum of CHOPS syndrome.

CHOPS syndrome is a multisystem disorder caused by missense mutations in AFF4. Previously, we reported three individuals whose primary phenotype included cognitive impairment and coarse facies, heart defects, obesity, pulmonary involvement, and short stature. This syndrome overlaps phenotypically with Cornelia de Lange syndrome, but presents distinct differences including facial features, pulmonary involvement, and obesity. Here, we provide clinical descriptions of an additional eight individuals with CHOPS syndrome, as well as neurocognitive analysis of three individuals. All 11 individuals presented with features reminiscent of Cornelia de Lange syndrome such as synophrys, upturned nasal tip, arched eyebrows, and long eyelashes. All 11 individuals had short stature and obesity. Congenital heart disease and pulmonary involvement were common, and those were seen in about 70% of individuals with CHOPS syndrome. Skeletal abnormalities are also common, and those include abnormal shape of vertebral bodies, hypoplastic long bones, and low bone mineral density. Our observation indicates that obesity, pulmonary involvement, skeletal findings are the most notable features distinguishing CHOPS syndrome from Cornelia de Lange syndrome. In fact, two out of eight of our newly identified patients were found to have AFF4 mutations by targeted AFF4 mutational analysis rather than exome sequencing. These phenotypic findings establish CHOPS syndrome as a distinct, clinically recognizable disorder. Additionally, we report three novel missense mutations causative for CHOPS syndrome that lie within the highly conserved, 14 amino acid sequence of the ALF homology domain of the AFF4 gene, emphasizing the critical functional role of this region in human development.

Drosophila melanogaster positive transcriptional elongation factors regulate metabolic and sex-biased expression in adults.

BACKGROUND: Transcriptional elongation is a generic function, but is also regulated to allow rapid transcription responses. Following relatively long initiation and promoter clearance, RNA polymerase II can pause and then rapidly elongate following recruitment of positive elongation factors. Multiple elongation complexes exist, but the role of specific components in adult Drosophila is underexplored. RESULTS: We conducted RNA-seq experiments to analyze the effect of RNAi knockdown of Suppressor of Triplolethal and lilliputian. We similarly analyzed the effect of expressing a dominant negative Cyclin-dependent kinase 9 allele. We observed that almost half of the genes expressed in adults showed reduced expression, supporting a broad role for the three tested genes in steady-state transcript abundance. Expression profiles following lilliputian and Suppressor of Triplolethal RNAi were nearly identical raising the possibility that they are obligatory co-factors. Genes showing reduced expression due to these RNAi treatments were short and enriched for genes encoding metabolic or enzymatic functions. The dominant-negative Cyclin-dependent kinase 9 profiles showed both overlapping and specific differential expression, suggesting involvement in multiple complexes. We also observed hundreds of genes with sex-biased differential expression following treatment. CONCLUSION: Transcriptional profiles suggest that Lilliputian and Suppressor of Triplolethal are obligatory cofactors in the adult and that they can also function with Cyclin-dependent kinase 9 at a subset of loci. Our results suggest that transcriptional elongation control is especially important for rapidly expressed genes to support digestion and metabolism, many of which have sex-biased function.

The Drosophila melanogaster model for Cornelia de Lange syndrome: Implications for etiology and therapeutics.

Discovery of genetic alterations that cause human birth defects provide key opportunities to improve the diagnosis, treatment, and family counseling. Frequently, however, these opportunities are limited by the lack of knowledge about the normal functions of the affected genes. In many cases, there is more information about the gene's orthologs in model organisms, including Drosophila melanogaster. Despite almost a billion years of evolutionary divergence, over three-quarters of genes linked to human diseases have Drosophila homologs. With a short generation time, a twenty-fold smaller genome, and unique genetic tools, the conserved functions of genes are often more easily elucidated in Drosophila than in other organisms. Here we present how this applies to Cornelia de Lange syndrome, as a model for how Drosophila can be used to increase understanding of genetic syndromes caused by mutations with broad effects on gene transcription and exploited to develop novel therapies. © 2016 Wiley Periodicals, Inc.

The Biological Significance Of AFF4: Promoting Transcription Elongation, Osteogenic Differentiation and Tumor Progression.

As a member of the AF4/FMR2 (AFF) family, AFF4 is a scaffold protein in the superelongation complex (SEC). In this mini-view, we discuss the role of AFF4 as a transcription elongation factor that mediates HIV activation and replication and stem cell osteogenic differentiation. AFF4 also promotes the progression of head and neck squamous cell carcinoma, leukemia, breast cancer, bladder cancer and other malignant tumors. The biological function of AFF4 is largely achieved through SEC assembly, regulates SRY-box transcription factor 2 (SOX2), MYC, estrogen receptor alpha (ESR1), inhibitor of differentiation 1 (ID1), c-Jun and noncanonical nuclear factor-κB (NF-κB) transcription and combines with fusion in sarcoma (FUS), unique regulatory cyclins (CycT1), or mixed lineage leukemia (MLL). We explore the prospects of using AFF4 as a therapeutic in Acquired immunodeficiency syndrome (AIDS) and malignant tumors and its potential as a stemness regulator.

Finding meaning in chaos: a selection signature for functional interactions and its use in molecular biology.

A primary goal of biomedical research is to elucidate molecular mechanisms, particularly those responsible for human traits, either normal or pathological. Yet achieving this goal is difficult if not impossible when the traits of interest lack tractable models and so cannot be dissected through time-honoured approaches like forward genetics or reconstitution. Arguably, no biological problem has hindered scientific progress more than this: the inability to dissect a trait's mechanism without a tractable likeness of the trait. At root, forward genetics and reconstitution are powerful approaches because they assay for specific molecular functions. Here, we discuss an alternative way to uncover important mechanistic interactions, namely, to assay for positive natural selection. If an interaction has been selected for, then it must perform an important function, a function that significantly promotes reproductive success. Accordingly, selection is a consequence and indicator of function, and uncovering multimolecular selection will reveal important functional interactions. We propose a selection signature for interactions and review recent selection-based approaches through which to dissect traits that are not inherently tractable. The review includes proof-of-principle studies in which important interactions were uncovered by screening for selection. In sum, screens for selection appear feasible when screens for specific functions are not. Selection screens thus constitute a novel tool through which to reveal the mechanisms that shape the fates of organisms.

PI3K/ c-Myc/AFF4 axis promotes pancreatic tumorigenesis through fueling nucleotide metabolism.

MLL-AFF4 fusion gene has been discovered in acute leukemia, whether AFF4 alone plays a role in tumor, especially pancreatic tumorigenesis, is still elusive. Increasing evidence suggests that cancer cells altered nucleotide metabolism during tumorigenesis. In present study, we observed AFF4 overexpression promoted cell proliferation, colony formation and cell cycle progression while loss of AFF4 impairs above phenotypes of pancreatic ductal carcinoma (PDAC) cells. Using RNA-profiling, we revealed that HPRT1 and IMPDH2, two enzymes in the nucleotide metabolism pathway, were upregulated following AFF4 overexpression. Simultaneous expression of HPRT1 and IMPDH2 would mainly rescue the phenotypes of cells lacking AFF4. Additionally, xenograft study proved HPRT1 and IMPDH2 genetically function in the downstream of AFF4, which was recruited by PAX2 when CDK9 mediated AFF4 phosphorylation at S388 and drove HPRT1 and IMPDH2 expression. We further discovered PI3K/c-Myc axis is required for AFF4 expression in PDAC cells. Finally, we obtained the positive correlation between c-Myc and AFF4 or AFF4 and HPRT1/IMPDH2 in clinical PDAC samples. Otherwise, we conducted data-mining and found that the expression levels of AFF4 and HPRT1/IMPDH2 are correlated with patients' prognosis, establishing AFF4 as a potential biomarker and therapeutic target for PDAC.

Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation.

The super elongation complex (SEC) containing P-TEFb plays a critical role in regulating transcription elongation. AFF1 and AFF4, members of the AF4/FMR2 family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. We here reveal differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSSs, while AFF4 is enriched downstream of the TSSs. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4 bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate Pol II transcription.

Unexpected appearance of KMT2A::MLLT10 fusion transcript in acute myeloid leukemia with t(5;11)(q31;q23.3).

As an uncommon but nonrandom translocation in acute myeloid leukemia (AML) t(5;11)(q31;q23) results in fusion between KMT2A at 11q23 and ARHGAP26 at 5q31. The 5q31 region has another KMT2A partner, AFF4, which was identified in acute lymphoblastic leukemia harboring ins(5;11)(q31;q13q23). We report here a 65-year-old woman with AML M5b. G-banding and spectral karyotyping demonstrated 46,XX,t(5;11)(q31;q23.3). Fluorescence in situ hybridization revealed not only separated 5' and 3' KMT2A signals but a faint 5' KMT2A signal. Reverse transcription polymerase chain reaction (RT-PCR), using a KMT2A sense primer and ARHGAP26 antisense primer, detected no band whereas RT-PCR with a AFF4 antisense primer revealed an amplified band. However, sequence analysis unexpectedly disclosed that KMT2A exon 6 was connected with MLLT10 exons 15 to 18. This may be due to cross-hybridization between MLLT10 exon 18 and AFF4 antisense primer derived from AFF4 exon 10 since both exons had eight identical bases (AAGCAGCT). The MLLT10 gene is located at 10p12.31; a faint 5' KMT2A signal was probably present at this locus. These findings indicate that in AML the 5' KMT2A fragment containing exons 1 to 6 may be cryptically inserted into MLLT10 intron 14 when a reciprocal translocation t(5;11)(q31;q23.3) involving KMT2A occurred.

Circular RNA hsa_circ_0057452 facilitates keloid progression by targeting the microRNA-1225-3p/AF4/FMR2 family member 4 axis.

The circular RNA, hsa_circ_0057452, is highly expressed in keloids, but its specific mechanism of action remains unknown. The levels of hsa_circ_0057452, microRNA (miR)-1225-3p, and AF4/FMR2 family member 4 (AFF4) in keloid tissues and keloid fibroblasts (KFs) were determined using quantitative reverse transcription-polymerase chain reaction. Changes in KFs viability, proliferation, apoptosis, and migration were investigated using the cell counting kit-8, bromodeoxyuridine, flow cytometry, and Transwell assays. Luciferase, RNA immunoprecipitation, and RNA pull-down assays were performed to identify the binding relationship among hsa_circ_0057452, miR-1225-3p, and AFF4. We found that hsa_circ_0057452 and AFF4 expression levels were upregulated, whereas miR-1225-3p expression levels were downregulated in keloids. Knockdown of hsa_circ_0057452 or AFF4 suppressed the viability, proliferation, and migration of KFs and induced apoptosis, whereas hsa_circ_0057452 overexpression and miR-1225-3p knockdown showed the opposite trend. Furthermore, hsa_circ_0057452 affected the biological behavior of KFs by releasing AFF4 via sponging of miR-1225-3p. Therefore, our results show that hsa_circ_0057452 promotes keloid progression by targeting miR-1225-3p and regulating AFF4 levels.

AFF4 Predicts the Prognosis of Colorectal Cancer Patients and Suppresses Colorectal Cancer Metastasis via Promoting CDH1 Expression.

INTRODUCTION: AF4/FMR2 family member 4 (AFF4) is a core component of super elongation complex (SEC) and regulates the transcription elongation of many genes. AFF4 depletion or amplification is associated with multiple cancers, but its role in colorectal cancer (CRC) has not been investigated so far. METHODS: qRT-PCR and Western blot analyzed AFF4 expression in the paired clinical CRC tissues. The patients' overall survival curve was determined using the Kaplan-Meier plotter. In vitro experiments, such as cell proliferation, migration, and invasion, were used to preliminarily ascertain the role of AFF4 in CRC. A CRC cell liver metastasis animal model was well established. Livers were harvested and examined histologically by a series of indicators, such as tumor nodules, liver weight, ALT/AST activity, and tumor cell identification by hematoxylin-eosin (HE) staining. RESULTS: We firstly examined the expression of AFF4 in colorectal cancer and normal tissues by collecting paired CRC tissues and adjacent normal tissues, revealing that AFF4 was significantly downregulated in CRC patients and lower expression of AFF4 was correlated with poor prognosis. Next, we observed that presence or absence of AFF4 in CRC cells had no effect on cancer cell proliferation, while AFF4 depletion significantly promoted the migration or invasion of CRC cells in vitro. Furthermore, we confirmed that AFF4 deficiency enhanced the metastatic capacity of CRC cells in vivo. Mechanistically, we found that AFF4 upregulated the transcription of CDH1 gene, which encodes E-cadherin and suppresses the epithelial-mesenchymal transition (EMT). Knockdown of AFF4 interfered with CDH1 transcription, resulting in downregulation of E-cadherin expression and the progression of CRC. Moreover, restored CDH1 expression could rescue the phenotype of CRC cells without AFF4. CONCLUSIONS: Collectively, our data demonstrated that AFF4 served as a significant novel regulator of CRC via CDH1 transcriptional regulation and a potential effective therapy target for patients with CRC.

A case of CHOPS syndrome accompanied with moyamoya disease and systemic vasculopathy.

BACKGROUND: CHOPS syndrome, caused by a mutation in the AFF4 gene, is a recently established and extremely rare genetic disorder, which has moderate phenotypic overlap with Cornelia de Lange syndrome. The main phenotypes include characteristic facial features, short stature, obesity, skeletal and pulmonary involvement, and neurodevelopmental impairment. CASE REPORT: We report on a Korean girl with CHOPS syndrome presenting with an atypical manifestation. The patient was referred to the out-patient clinic to evaluate the underlying etiology of short stature, obesity, developmental delay, and Moyamoya disease. The patient showed characteristic facial features including a round face, thick eyebrows, and synophrys. Her developmental milestones had been delayed since infancy and a moderate degree of intellectual disability persisted. She was also diagnosed with Moyamoya disease at 6 years of age and had undergone synangiosis surgery thrice. Her renal arteries and infrarenal aorta were diffusely narrowed. A novel de novo missense variant, c.758C > T (p.Pro253Leu) in AFF4 was identified by whole exome sequencing. No additional candidate variants for her vascular manifestation were found except a susceptibility variant, c.14429G > A (p.Arg4810Lys) in RNF213, inherited from asymptomatic mother. CONCLUSION: This is the first case of CHOPS syndrome accompanied by systemic vasculopathy. More clinical observations and functional studies are required to clarify this association.

MiR-425-5p accelerated the proliferation, migration, and invasion of ovarian cancer cells via targeting AFF4.

BACKGROUND: Accumulating data have established that microRNAs (miRNAs) play significant regulatory roles in the carcinogenesis and progression of ovarian cancer (OC). MiR-425-5p was reported to function in various tumors. However, the roles and underlying mechanism of miR-425-5p involvement in OC development and progression are unclear. METHODS: A comprehensive strategy of data mining, computational biology, and real-time polymerase chain reaction was employed to identify the involvement of miR-425-5p in OC progression. The effect of miR-425-5p on the proliferation, migration, and invasion of OC cells was determined using Cell Counting Kit-8, wound-healing, and Matrigel invasion assays, respectively. Luciferase assay was performed to evaluate the interactions between miR-425-5p and MAGI2-AS3 or AFF4. RESULTS: miR-425-5p was significantly up-regulated in OC tissues and cells. The luciferase reporter assay revealed that miR-425-5p was negatively regulated by MAGI2-AS3. Silencing miR-425-5p inhibited the proliferation, migration, and invasion of OC cells in vitro. Bioinformatics analysis and luciferase reporter assay revealed that AFF4 was the target gene of miR-425-5p. Moreover, AFF4 expression was significantly decreased in OC and was closely related to the good prognosis of patients with OC. AFF4 overexpression inhibited the proliferation, migration, and invasion of OC cells in vitro. By contrast, silencing AFF4 promoted the proliferation, migration, and invasion of OC cells in vitro. Finally, AFF4 suppression rescued the inhibitory effect of silencing miR-425-5p on the proliferation, migration, and invasion of OC cells. CONCLUSION: To the best our knowledge, this is the first study to demonstrate that miR-425-5p overexpression in OC is negatively regulated by MAGI2-AS3. Moreover, miR-425-5p promotes the proliferation, migration, and invasion of OC cells by targeting AFF4, suggesting that miR-425-5p/AFF4 signaling pathway represented a novel therapeutic target for patients with OC.