RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

PHF20 is a Novel Prognostic Biomarker and Correlated with Immune Status in Breast Cancer.

Increasing evidence has demonstrated that enhanced PHF20 expression plays a crucial role in cancer development and progression. However, little is known about the prognostic value of PHF20 expression in breast cancer (BRCA). In this study, we attempted to explore the expression pattern of PHF20 and its associations with prognosis and immune status in BRCA. The gene expression data and clinical information of 1109 BRCA samples were downloaded from TCGA database. The expression level of PHF20 protein was determined using UALCAN and HPA database. Kaplan-Meier method and CIBERSORT algorithm were used to analyze the associations of PHF20 expression with overall survival (OS) and immune microenvironment, respectively. Besides, GSEA analysis was conducted to explore potential biological functions and molecular mechanisms of PHF20 in BRCA. Moreover, starBase database was applied to construct a ceRNA nework. PHF20 was highly expressed in BRCA samples both at the transcriptional and protein level, and was strongly correlated with the OS and immune status. Univariable and multivariate Cox regression analyses identified PHF20 as an independent prognostic factor. Additionally, GSEA analysis showed that high PHF20 expression was closely associated with TGF-ß signaling pathway, Wnt signaling pathway, and adherens junction. Furthermore, three ceRNA networks (AC037198.1/hsa-miR-223-3p/PHF20, CBR3-AS1/hsa-miR-223-3p/PHF20, and ZNF561-AS1/hsa-miR-223-3p/PHF20) were identified by starBase analysis. Functional experiments validated that PHF20 knockdown inhibited the cell viability and progression in BRCA cells. PHF20 overexpression was significantly associated with poor prognosis and immune status in BRCA, and could act as a potential novel prognostic biomarker for BRCA.

PHF20L1 mediates PAX2 expression to promote angiogenesis and liver metastasis in colorectal cancer through regulating HIC1.

Colorectal cancer (CRC) is a common cancer with poor prognosis. The research was designed to explore the role of PHF20L1 in angiogenesis and liver metastasis in CRC and discuss its molecular mechanism. Expression levels of PHF20L1, HIC1 and PAX2 in CRC tissues collected from CRC patients were detected using qRT-PCR, WB and immunohistochemical staining. CRC cells were transfected with PHF20L1, HIC1 and PAX2 overexpression or knockdown vectors and the proliferation, apoptosis, EMT and angiogenesis of the cells were determined. WB was utilized to assess protein levels of PHF20L1, HIC1, PAX2 and angiogenesis factor (ANGPT2, FGF1, PDGFA and VEGFA). The role of PHF20L1 regulating tumor formation and liver metastasis in vivo was detected as well. PHF20L1 was observed to express at a high level of CRC tissues. PHF20L1 promoted CRC cell growth, EMT and angiogenesis, and inhibited cell apoptosis. Knockdown of PHF20L1 had opposite effects on CRC cells. PHF20L1 negatively regulated HIC1 expression to promote PAX2 expression, thus promoting CRC cell progression. The in vivo results showed that PHF20L1 contributed to tumor formation and liver metastasis. PHF20L1 increases PAX2 expression to promote angiogenesis in CRC by inhibiting HIC1, therefore facilitating CRC cell EMT and liver metastasis. Our finding may provide a novel insight for CRC pathogenesis.

PHD finger protein 20-like protein 1 (PHF20L1) in ovarian cancer: from its overexpression in tissue to its upregulation by the ascites microenvironment.

BACKGROUND: Ovarian cancer is the most aggressive gynecological malignancy. Transcriptional regulators impact the tumor phenotype and, consequently, clinical progression and response to therapy. PHD finger protein 20-like protein 1 (PHF20L1) is a transcriptional regulator with several isoforms, and studies on its role in ovarian cancer are limited. We previously reported that PHF20L1 is expressed as a fucosylated protein in SKOV-3 cells stimulated with ascites from patients with ovarian cancer. METHODS: We decided to analyze the expression of PHF20L1 in ovarian cancer tissues, determine whether a correlation exists between PHF20L1 expression and patient clinical data, and analyze whether ascites can modulate the different isoforms of this protein. Ovarian cancer biopsies from 29 different patients were analyzed by immunohistochemistry, and the expression of the isoforms in ovarian cancer cells with or without exposure to the tumor microenvironment, i.e., the ascitic fluid, was determined by western blotting assays. RESULTS: Immunohistochemical results suggest that PHF20L1 exhibits increased expression in sections of tumor tissues from patients with ovarian cancer and that higher PHF20L1 expression correlates with shorter progression-free survival and shorter overall survival. Furthermore, western blotting assays determined that protein isoforms are differentially regulated in SKOV-3 cells in response to stimulation with ascites from patients with epithelial ovarian cancer. CONCLUSION: The results suggest that PHF20L1 could play a relevant role in ovarian cancer given that higher PHF20L1 protein expression is associated with lower overall patient survival.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

Identification of genes and pathways potentially related to PHF20 by gene expression profile analysis of glioblastoma U87 cell line.

BACKGROUND: Glioblastoma is the most common and aggressive brain tumor associated with a poor prognosis. Plant homeodomain finger protein 20 (PHF20) is highly expressed in primary human gliomas and its expression is associated with tumor grade. However, the molecular mechanism by which PHF20 regulates glioblastoma remains poorly understood. METHODS: Genome wide gene expression analysis was performed to identify differentially expressed genes (DEGs) in U87 cells with PHF20 gene knockdown. Gene ontology (GO) and pathway enrichment analyses were performed to investigate the functions and pathways of DEGs. Pathway-net and signal-net analyses were conducted to identify the key genes and pathways related to PHF20. RESULTS: Expression of 540 genes, including FEN1 and CCL3, were significantly altered upon PHF20 gene silencing. GO analysis results showed that DEGs were significantly enriched in small molecule metabolic and apoptotic processes. Pathway analysis indicated that DEGs were mainly involved in cancer and metabolic pathways. The MAPK, apoptosis and p53 signaling pathways were identified as the hub pathways in the pathway network, while PLCB1, NRAS and PIK3 s were hub genes in the signaling network. CONCLUSIONS: Our findings indicated that PHF20 is a pivotal upstream regulator. It affects the occurrence and development of glioma by regulating a series of tumor-related genes, such as FEN1, CCL3, PLCB1, NRAS and PIK3s, and activation of apoptosis signaling pathways. Therefore, PHF20 might be a novel biomarker for early diagnosis, and a potential target for glioblastoma therapies.

Training intensity modulates changes in PGC-1α and p53 protein content and mitochondrial respiration, but not markers of mitochondrial content in human skeletal muscle.

Exercise training has been associated with increased mitochondrial content and respiration. However, no study to date has compared in parallel how training at different intensities affects mitochondrial respiration and markers of mitochondrial biogenesis. Twenty-nine healthy men performed 4 wk (12 cycling sessions) of either sprint interval training [SIT; 4-10 × 30-s all-out bouts at ∼200% of peak power output (WPeak)], high-intensity interval training (HIIT; 4-7 × 4-min intervals at ∼90% WPeak), or sublactate threshold continuous training (STCT; 20-36 min at ∼65% WPeak). The STCT and HIIT groups were matched for total work. Resting biopsy samples (vastus lateralis) were obtained before and after training. The maximal mitochondrial respiration in permeabilized muscle fibers increased significantly only after SIT (25%). Similarly, the protein content of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, p53, and plant homeodomain finger-containing protein 20 (PHF20) increased only after SIT (60-90%). Conversely, citrate synthase activity, and the protein content of TFAM and subunits of the electron transport system complexes remained unchanged throughout. Our findings suggest that training intensity is an important factor that regulates training-induced changes in mitochondrial respiration and that there is an apparent dissociation between training-induced changes in mitochondrial respiration and mitochondrial content. Moreover, changes in the protein content of PGC-1α, p53, and PHF20 are more strongly associated with training-induced changes in mitochondrial respiration than mitochondrial content.

Mitochondrial adaptations to high-volume exercise training are rapidly reversed after a reduction in training volume in human skeletal muscle.

Increased mitochondrial content and respiration have both been reported after exercise training. However, no study has directly compared how different training volumes influence mitochondrial respiration and markers of mitochondrial biogenesis. Ten healthy men performed high-intensity interval cycling during 3 consecutive training phases; 4 wk of normal-volume training (NVT; 3/wk), followed by 20 d of high-volume training (HVT; 2/d) and 2 wk of reduced-volume training (RVT; 5 sessions). Resting biopsy samples (vastus lateralis) were obtained at baseline and after each phase. No mitochondrial parameter changed after NVT. After HVT, mitochondrial respiration and citrate synthase activity (∼40-50%), as well as the protein content of electron transport system (ETS) subunits (∼10-40%), and that of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), NRF1, mitochondrial transcription factor A (TFAM), PHF20, and p53 (∼65-170%) all increased compared to baseline; mitochondrial specific respiration remained unchanged. After RVT, all the mitochondrial parameters measured except citrate synthase activity (∼36% above initial) were not significantly different compared to baseline (all P > 0.05). Our findings demonstrate that training volume is an important determinant of training-induced mitochondrial adaptations and highlight the rapid reversibility of human skeletal muscle to a reduction in training volume.-Granata, C., Oliveira, R. S. F., Little, J. P., Renner, K., Bishop, D. J. Mitochondrial adaptations to high-volume exercise training are rapidly reversed after a reduction in training volume in human skeletal muscle.

Methyllysine reader plant homeodomain (PHD) finger protein 20-like 1 (PHF20L1) antagonizes DNA (cytosine-5) methyltransferase 1 (DNMT1) proteasomal degradation.

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Long non-coding RNA PCAT5 regulates the progression of Esophageal Squamous Cell Carcinoma via miR-4295/PHF20.

Long non-coding RNAs (lncRNAs) have been discovered through many studies to play a crucial role in tumor progression. LncRNA PCAT5 has been identified as a human cancer-related gene in diverse cancers. However, the specific role of PCAT5 in esophageal squamous cell carcinoma (ESCC) still needs further study. The study aimed to test the PCAT5 expression and find its biological function in ESCC. Functional experiments, including EdU, transwell and TUNEL, were done in the chosen ESCC cell lines under silenced PCAT5. Luciferase reporter and Western blot experiments were implemented to ensure the possible regulatory mechanism involved in ESCC. PCAT5 presented higher expression in ESCC cells in comparison to normal cells. The silence of PCAT5 restrained ESCC cell abilities of proliferation, migration and invasion. On the contrary, it accelerated ESCC cell apoptosis. The results of rescue experiments showed that PCAT5 regulated ESCC cell proliferative, migrated, invasive and apoptotic abilities via sponging miR-4295 to up-regulate PHF20.

Corrigendum: PHF20 promotes glioblastoma cell malignancies through a WISP1/BGN-dependent pathway.

[This corrects the article DOI: 10.3389/fonc.2020.573318.].

N6-methyladenosine demethylase ALKBH5 suppresses colorectal cancer progression potentially by decreasing PHF20 mRNA methylation.

BACKGROUND: As the most widespread mRNAs modification, N6-methyladenosine (m6 A) is dynamically and reversibly modulated by methyltransferases and demethylases. ALKBH5 is a major demethylase, and plays vital roles in the progression of cancers. However, the role and mechanisms of ALKBH5 in colorectal cancer (CRC) is unclear. RESULTS: Herein, we discovered that in CRC, downregulated ALKBH5 was closely related to poor prognosis of CRC patients. Functionally, our results demonstrated that knockdown of ALKBH5 enhanced the proliferation, migration and invasion of LOVO and RKO in vitro, while overexpression of ALKBH5 inhibited the functions of these cells. The results also demonstrated that knockdown of ALKBH5 promoted subcutaneous tumorigenesis of LOVO in vivo, while overexpression of ALKBH5 suppressed this ability. Mechanistically, results from joint analyses of MeRIP-seq and RNA-seq indicated that PHF20 mRNA was a key molecule that was regulated by ALKBH5-mediated m6 A modification. Further experiments indicated that ALKBH5 may inhibit stability of PHF20 mRNA by removing the m6 A modification of PHF20 mRNA 3'UTR. CONCLUSIONS: ALKBH5 suppresses CRC progression by decreasing PHF20 mRNA methylation. ALKBH5-mediated m6 A modification of PHF20 mRNA can serve as a hopeful strategy for the intervention and treatment of CRC.

Assessing kinetics and recruitment of DNA repair factors using high content screens.

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

An insight into the promoter methylation of PHF20L1 and the gene association with metastasis in breast cancer.

BACKGROUND: Plant homeodomain finger protein 20-like 1 (PHF20L1) is a protein reader involved in epigenetic regulation that binds monomethyl-lysine. An oncogenic function has been attributed to PHF20L1 but its role in breast cancer (BC) is not clear. OBJECTIVES: To explore PHF20L1 promoter methylation and comprehensive bioinformatics analysis to improve understanding of the role of PHF20L1 in BC. MATERIAL AND METHODS: Seventy-four BC samples and 16 control samples were converted using sodium bisulfite treatment and analyzed with methylation-specific polymerase chain reaction (PCR). Bioinformatic analysis was performed in the BC dataset using The Cancer Genome Atlas (TCGA) trough data visualized and interpreted in the MEXPRESS website. Methylation, gene expression and survival evaluation were performed with R v. 4.0.2 software. Using multiple bioinformatic tools, we conducted a search for genes co-expressed with PHF20L1, analyzed its ontology and predicted associated miRNAs and miRNA-PHF20L1 networks. The expression and prognostic value of PHF20L1 and co-expressed genes were analyzed. RESULTS: We found demethylation in PHF20L1 promoter in both BC samples and healthy tissues. Data mining with 241 patients demonstrated changes in methylation of promoter regions in basal-like and luminal A subtypes. Expression of the PHF20L1 gene had a negative correlation with methylation. Twelve genes were co-expressed. PHF20L1 is a target of miR96-5p, miR9-5p and miR182-5p, which are involved in proliferation and metastasis. PHF20L1 gene expression was not associated with overall survival (OS), or relapse-free survival (RFS), but was associated with distant metastasis-free survival (DMFS). CONCLUSIONS: Our findings showed differences in methylation of PHF20L1 promoter region near TSS and upstream in BC subtypes; its overexpression impacted DMFS. We found that PHF20L1 is targeted by miR96-5p, miR9-5p and miR182-5p, which are involved in proliferation and metastasis, and regulates genes engaged in processes such as alternative splicing.

PHF20 Promotes Glioblastoma Cell Malignancies Through a WISP1/BGN-Dependent Pathway.

Glioblastoma (GBM) stem cells are resistant to cancer therapy, and therefore responsible for tumor progression and recurrence after conventional therapy. However, the molecular mechanisms driving the maintenance of stemness and dedifferentiation are poorly understood. In this study, we identified plant homeodomain finger-containing protein 20 (PHF20) as a crucial epigenetic regulator for sustaining the stem cell-like phenotype of GBM. It is highly expressed in GBM and tightly associated with high levels of aggressiveness of tumors and potential poor prognosis in GBM patients. Knockout of PHF20 inhibits GBM cell proliferation, as well as its invasiveness and stem cell-like traits. Mechanistically, PHF20 interacts with WDR5 and binds to the promoter regions of WISP1 for its expression. Subsequently, WISP1 and BGN act in concert to regulate the degradation of ß-Catenin. Our findings have identified PHF20 as a key driver of GBM malignant behaviors, and provided a potential target for developing prognosis and therapy.

Ascites from Ovarian Cancer Induces Novel Fucosylated Proteins.

Ovarian cancer is considered to be the most lethal type of gynecological cancer. During the advanced stages of ovarian cancer, an accumulation of ascites is observed. Fucosylation has been classified as an abnormal post-translational modification that is present in many diseases, including ovarian cancer. Ovarian cancer cells that are cultured with ascites stimulation change their morphology; concomitantly, the fucosylation process is altered. However, it is not known which fucosylated proteins are modified. The goal of this work was to identify the differentially fucosylated proteins that are expressed by ovarian cancer cell lines that are cultured with ovarian cancer patients' ascites. Aleuria aurantia lectin was used to detect fucosylation, and some changes were observed, especially in the cell membrane. Affinity chromatography and mass spectrometry (MALDI-TOF) were used to identify 6 fucosylated proteins. Four proteins (Intermediate filament family orphan 1 [IFFO1], PHD finger protein 20-like protein 1 [PHF20L1], immunoglobulin gamma 1 heavy chain variable region partial [IGHV1-2], and Zinc finger protein 224 [ZNF224]) were obtained from cell cultures stimulated with ascites, and the other two proteins (Peregrin [BRPF1] and Dystrobrevin alpha [DTNA]) were obtained under normal culture conditions. The fucosylated state of some of these proteins was further analyzed. The experimental results show that the ascites of ovarian cancer patients modulated the fucosylation process. The PHD finger protein 20-like protein 1, Zinc finger protein 224 and Peregrin proteins colocalize with fucosylation at different levels.

Structure-based analysis of curcumin and conventionaldrugs targeting tumor-inducing protein PHF20.

Recently, the PHF20 has been reported as tumor inducer protein by suppressing the activity of tumor suppressor protein p53. Conventional drugs (albendazole, doxazosin, and propranolol) are used for treatment of cancer causing side effect. The secondary metabolite curcumin is employed in various diseases treatment including cancer. The present study is to explore curcumin in comparison to selected conventional drugs by using molecular docking. The online database "Molinspiration online server" detected the physicochemical pharmacokinetics and drug likeness score of curcumin and conventional drugs. Results from Molinspiration online server showed that curcumin did not violate the "Lipinski five rule" for drug. The lead compound for molecular docking exhibited the potential interaction to the active site of PHF20. The resulted binding energy of albendazole and doxazosin were -21.97 and -26.64 respectively. The binding energy (-18.12 kcal/mol) of curcumin was higher than propranolol (17.62 kcal/mol). Thus, curumin has greater potential to interact for further consideration as an anti-cancerous regimen.

JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

Epigenetic regulator JMJD3 plays an important role in both tumor progression and somatic cell reprogramming. Here, we explored the effect of JMJD3 on the stem cell-like characteristics of breast cancer and its underlying mechanism involving stemness-related transcription factor Oct4. Our data revealed that, in breast cancer cells lines and an orthotopic xenograph mouse model of breast cancer, ectopic overexpression of JMJD3 suppressed stem cell-like characteristics of breast cancer cells, whereas knockdown of JMJD3 promoted these characteristics. Oct4 mediated the suppressive effects of JMJD3 on the stemness of breast cancer cells. The inhibitory effect of JMJD3 on Oct4 was independent of demethylase activity, but mediated via degradation of PHF20. Furthermore, we applied an agonist of the vitamin D receptor, paricalcitol, and found that it induced JMJD3 in breast cancer cells. Our data showed that administration of paricalcitol suppressed stem cell-like characteristics and Oct4 expression. Taken together, JMJD3 inhibits the stem cell-like characteristics in breast cancer by suppression of stemness factor Oct4 in a PHF20-dependent manner. Administration of paricalcitol leads to upregulation of JMJD3 that suppresses Oct4 expression and the stem cell-like characteristics in breast cancer.

PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation.

PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD) finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT), p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin.

An integrated genomic analysis of Tudor domain-containing proteins identifies PHD finger protein 20-like 1 (PHF20L1) as a candidate oncogene in breast cancer.

Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer.