Neuronal and Muscle Differentiation of Mammalian Cells Is Accompanied by a Change in PHF10 Isoform Expression.

The PBAF chromatin remodeling complex of the SWI/SNF family plays a critical role in the regulation of gene expression during tissue differentiation and organism development. The subunits of the PBAF complex have domains responsible for binding to N-terminal histone sequences. It determines the specificity of binding of the complex to chromatin. PHF10, a specific subunit of the PBAF complex, contains a DPF domain, which is a unique chromatin interaction domain. A PHF10 isoform that lacks the DPF domain is also present in vertebrate cells. This work shows that during neuronal and muscle differentiation of human and mouse cells, the expression of PHF10 isoforms changes: the form that does not have DPF replaces the form in which it is present. Replacement of PHF10 isoforms in the PBAF complex may affect its selectivity in the regulation of genes in differentiating cells.

New Approach for Studying of Isoforms and High-Homology Proteins in Mammalian Cells.

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.

Contact Inhibition of Proliferation Is Accompanied by Expression of the PHF10D Subunit of the Chromatin Remodeling Complex PBAF in Mouse and Human Cell Lines.

PHF10 is a subunit of the PBAF complex, which regulates the expression of many genes in developing and maturing organisms. PHF10 has four isoforms that differ in domain structure. The PHF10A isoform, containing a DPF domain at the C-terminus and 46 amino acids at the N-terminus, is necessary for the expression of proliferation genes; the functions of the other isoforms are less studied. In this work, we have established that, upon contact inhibition of mouse and human cell proliferation caused by the establishment of a tight junction and adherence junction between cells, the expression of the PHF10A isoform stops and instead the PHF10D isoform is expressed, which does not contain DPF-domain and N-terminal sequence. The function of the PHF10D isoform may be associated with the establishment of intercellular contacts.

Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1-3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1-3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.

[PHF10, a Subunit of the PBAF Chromatin Remodeling Complex, Changes Its Localization and Interacts with c-FOS during the Initiation of Long-Term Potentiation in Neuronal Culture].

The PBAF chromatin remodeling complex interacts with many transcriptional activators and is recruited to target chromatin regions. PBAF plays an important role in maintaining and modifying the chromatin structure in mammalian cells. A subunit of the PBAF complex, the PHF10 transcription factor, is required for proliferation of neuronal precursors in the early stages of mouse brain development and gene expression in differentiated neurons. We showed that PHF10 interacts with the protein product of the early response gene c-FOS, the c-FOS transcriptional activator, which is expressed in response to the induction of long-term potentiation (LTP). LTP induction triggers the transcription of genes and the synthesis of proteins that provide changes that lead to the establishment of long-term contacts between neurons. We showed that in cells in differentiated neuronal culture, after the induction of LTP, expression of c-FOS, which is initially localized in the cytoplasm and then moves to the nucleus, begins. PHF10 is expressed in neuronal cells prior to LTP induction and has nuclear localization. However, 1 h after LTP induction, PHF10 is detected in the cytoplasm together with c-FOS, and then moves into the nucleus with it. Importantly, this behavior of PHF10 in response to KC1 stimulation is specific for neuronal cultures. It is assumed that during LTP, PHF10 together with c-FOS participates in the activation of secondary response genes that regulate the maintenance of plastic modifications and homeostasis of neuronal synapses. The PHF10 export from the nucleus and its rapid return together with c-FOS to the nucleus is possibly necessary for the rapid modulation of expression of target secondary response genes during LTP.

[PHF10 isoforms are phosphorylated in the PBAF mammalian chromatin remodeling complex].

Chromatin remodeling complex PBAF(SWI/SNF) alters the structure of chromatin and controls gene expression. PHF10 is a specific subunit of PBAF complex and is expressed as four isoforms in mammalian cells. We demonstrated that all isoforms are expressed in various human cell types of different histological origins. All four isoforms are extensively phosphorylated and their phosphorylation level is depended on the cell type. Phosphorylation of PHF10 isoforms occurs while they are incorporated as a subunit of the PBAF complex, and therefore phosphorylation of PHF10 isoforms may play an essential role in regulation of PBAF complex's function and mechanism of action.

PHD finger protein 10 promotes cell proliferation by regulating CD44 transcription in gastric cancer.

PHD finger protein 10 (PHF10) plays an important role in the tumorigenesis of gastric cancer (GC). However, clinical significance and underlying molecular mechanisms about PHF10 is unclear. In the article, it suggested that PHF10 involved in tumor progression and metastasis based on the analysis of datasets and 190 cases of tumor tissues in GC. And PHF10 provided the diagnostic value with areas under the receiver operating characteristics curve of 0.71 ± 0.069. Then we established GC cell lines MKN28 with PHF10 overexpression and SGC7901 with PHF10 knockdown. CCK8 assay and tumor xenograft experiment showed that upregulation of PHF10 could promote MKN28 cell proliferation, while PHF10 knockdown would inhibit the proliferation of SGC7901 in vitro and vivo. Nevertheless, PHF10 could upregulate CD44 mRNA expression by acting on its promoter at the level of transcription. This effect could be associated with BRG, BAF155 and SNF5, which were conserved subunits of switch/sucrose non-fermentable (SWI/SNF) complex. In conclusion, PHF10 targeting CD44 plays an essential part during the modulation of proliferation of GC cell and may offer a new therapeutic direction for GC.

CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts.

The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.

A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

ZC3H13-mediated N6-methyladenosine modification of PHF10 is impaired by fisetin which inhibits the DNA damage response in pancreatic cancer.

DNA damage repair is a major barrier for chemotherapy efficacy of pancreatic ductal adenocarcinoma (PDAC), including the efficacy of platinum-based and gemcitabine/nab-paclitaxel treatments. N6-methyladenosine modifications (m6A) have recently been reported to play a role in homologous recombination (HR) repair of DNA double strand breaks (DSBs); however, the mechanism of action remains unknown. Our previous work indicated that fisetin may be a promising anti-tumour agent that induces DNA damage. In this study, we reported that fisetin induced DSBs and suppressed HR repair through m6A modification in PDAC cells. The m6A writer ZC3H13 and PHF10, which is a subunit of the PBAF chromatin remodelling complex, were identified as the main molecules affected by fisetin treatment. To our knowledge, it's the first time that PHF10 was found and involved in the DNA damage response. PHF10 loss-of-function resulted in elevated recruitment of γH2AX, RAD51, and 53BP1 to DSB sites and decreased HR repair efficiency. Moreover, ZC3H13 knockdown downregulated the m6A methylation of PHF10 and decreased PHF10 translation in a YTHDF1-dependent manner. In conclusion, our study demonstrates that fisetin enhanced DSBs via ZC3Hl3-mediated m6A modification of PHF10, which may provide insight into novel therapeutic approaches for PDAC.

Enhancer RNA LINC00242-Induced Expression of PHF10 Drives a Better Prognosis in Pancreatic Adenocarcinoma.

Enhancer RNA is a kind of non-coding RNA, which is transcribed from the enhancer region of gene and plays an important role in gene transcription regulation. However, the role of eRNA in pancreatic adenocarcinoma (PAAD) is still unclear. In this study, we identified the key eRNA and its target gene in PAAD. The transcriptome data and clinical information of pancreatic cancer were downloaded from the UCSC Xena platform. A total of 2,695 eRNAs and its target gene predicted by the PreSTIGE method were selected as candidate eRNA-target pairs. After survival analysis, we found that LINC00242 was the eRNA most related to patients' survival, and correlation analysis further indicated that LINC00242 and its target gene PHF10 had a significant co-expression relationship. Downregulation of LINC00242 was significantly associated with unfavorable clinicopathological features. Based on pan-cancer analysis, we found that LINC00242 was associated with the survival of multiple cancers, and LINC00242 was co-expressed with its target genes in multiple cancer types. External experiments further demonstrated that PHF10 was the downstream target gene of LINC00242. After ssGSEA analysis, PAAD patients were classified as high, medium, and low immune cell infiltration clusters. Compared with the low and medium immune infiltration clusters, the expression level of PHF10 was significantly upregulated in the high immune infiltration clusters. After performing the CIBERSORT algorithm, we found that there was a significant difference in the abundance of immune infiltrating cells between the PHF10 high- and low-expression groups. Additionally, the web tool TIMER was used to detect the distribution and expression of PHF10 in pan-cancer.

Circ_0001023 Promotes Proliferation and Metastasis of Gastric Cancer Cells Through miR-409-3p/PHF10 Axis.

BACKGROUND: Circular RNAs (circRNAs) have been well documented to regulate the gene expression via sponging microRNA (miRNA) in diverse neoplasms including gastric cancer (GC). METHODS: In the present study, the expressions of circ_0001023, miR-409-3p, and plant homeodomain finger 10 (PHF10) in GC tissues were detected by qRT-PCR. Chi-square test was performed to analyze the associations between circ_0001023 and pathological parameters. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, and transwell assay were adopted to detect the role of circ_0001023/miR-409-3p axis in the proliferation, apoptosis, and migration of GC cells, respectively. The targeting relationship between circ_0001023 and miR-409-3p was investigated by dual-luciferase gene reporter gene assay. Additionally, subcutaneous xenotransplanted tumor model in nude mice was established to detect the function of circ_0001023 on GC growth in vivo. RESULTS: Compared with adjacent tissues, the expression of circ_0001023 was significantly upregulated and correlated with lymph node invasion and higher T stage of GC patients. It has also been proved that circ_0001023 could target miR-409-3p. Silencing circ_0001023 can impede the proliferation of GC cells and promote apoptosis, while miR-409-3p inhibitors can partially reverse the biological behavior of GC cells mentioned above. Moreover, the expression of circ_0001023 was reversely associated with miR-409-3p expression but positively correlated with PHF10, a downstream oncogene of miR-409-3p. CONCLUSION: Collectively, it is concluded that circ_0001023 promotes the progression of GC via regulating miR-409-3p/PHF10 axis.

The sequential phosphorylation of PHF10 subunit of the PBAF chromatin-remodeling complex determines different properties of the PHF10 isoforms.

The mammalian PBAF subfamily of SWI/SNF chromatin remodeling complexes plays a wide role in the regulation of gene expression. PHF10 is a subunit of the signature module of PBAF, responsible for its interaction with chromatin. PHF10 is represented by four different isoforms, which are alternatively incorporated in the complex. Two of PHF10 isoforms lacking C-terminal PHD domains contain a cluster of phosphorylated serine residues, designated as X-cluster. In the present study, we explore the phosphorylation of the X-cluster in detail. We identified additional phosphorylated serine residues and designated them as either frequently or rarely phosphorylated. The X-cluster consists of two independently phosphorylated subclusters. Phosphorylation of the second subcluster depends on phosphorylation of a primary serine 327. These two subclusters surround a sequence, which is predicted to be a nuclear localization sequence (NLS3). The NLS3 does not affect localization of PHF10 isoforms. However, it is essential for X-cluster phosphorylation and increased stability of isoforms that lack PHD. Conversely, the presence of NLS3 signal in isoforms that contain C-terminal PHD domains reduces their stability. Thus, phosphorylation of PHF10 isoforms regulates their cell level, determining the rate of incorporation in PBAF. This may alter the pattern of PBAF regulated genes.

The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

PBAF lacking PHD domains maintains transcription in human neutrophils.

The myeloid precursor cell differentiation requires an extensive chromatin remodeling. We show that the level of the PBAF chromatin remodeling complex decreases following the start of differentiation of myeloid precursors, becoming very low in the terminally differentiated peripheral blood (PB) neutrophils where it co-localizes with Pol II on the transcriptionally active chromatin. Previously, we have shown that the PHF10 subunit of the PBAF signature module has four isoforms, two of them (PHF10-P) contain a tandem of C-terminal PHD domains. We found that out of four PHF10 isoforms present in the myeloid precursor cells, only the PHF10-Ss isoform lacking PHD domains, is actively expressed in the PB neutrophils. In particular, the longest of the PHF10 isoforms (PHF10-Pl), which is essential for proliferation, completely disappears in PB neutrophils. In addition, in the myeloid precursors, promoters of neutrophil-specific genes are associated with the PHD-containing isoforms, together with PBAF and Pol II, when these genes are inactive and only during their activation stage. However, at the later stages of differentiation, when neutrophil-specific genes are actively transcribed, PHF10-P isoforms on their promoters are replaced by the PHF10-S isoforms. Evidently, PHD domains of PHF10 are essential for active chromatin remodeling during transcription activation, but are dispensable for the constantly transcribed genes.

Mammalian cells contain two functionally distinct PBAF complexes incorporating different isoforms of PHF10 signature subunit.

The PBAF subtype of the mammalian chromatin remodeling SWI/SNF complex has wide and diverse functions in transcription regulation and development, being both transcription activator and repressor. However, a mechanism accounting for such functional diversity remains unclear. Human PHF10/BAF45a subunit of the PBAF complex plays an important role in brain development but has not been studied sufficiently. We have shown that the PHF10 gene encodes 2 types of evolutionarily conserved, ubiquitously expressed isoforms that are incorporated into the PBAF complex in a mutually exclusive manner. One isoform contains C-terminal tandem PHD fingers, which in the other isoform are replaced by the consensus sequence for phosphorylation-dependent SUMO 1 conjugation (PDSM). PBAF complexes containing different PHF10 isoforms can bind to the promoters of the same genes but produce different effects on the recruitment of Pol II to the promoter and on the level of gene transcription. In addition, it is only the PBAF with PHD-containing isoform that activates proliferation. Our study demonstrates the existence of functionally different PBAF complexes in mammalian cell. It also provides an insight into the molecular structure and role of human PHF10/BAF45a and characterizes it as an essential PBAF subunit.