Investigating Uncertainties in Single-Molecule Localization Microscopy Using Experimentally Informed Monte Carlo Simulation.

Single-molecule localization microscopy (SMLM) enables the visualization of cellular nanostructures in vitro with sub-20 nm resolution. While substructures can generally be imaged with SMLM, the structural understanding of the images remains elusive. To better understand the link between SMLM images and the underlying structure, we developed a Monte Carlo (MC) simulation based on experimental imaging parameters and geometric information to generate synthetic SMLM images. We chose the nuclear pore complex (NPC), a nanosized channel on the nuclear membrane which gates nucleo-cytoplasmic transport of biomolecules, as a test geometry for testing our MC model. Using the MC model to simulate SMLM images, we first optimized our clustering algorithm to separate >106 molecular localizations of fluorescently labeled NPC proteins into hundreds of individual NPCs in each cell. We then illustrated using our MC model to generate cellular substructures with different angles of labeling to inform our structural understanding through the SMLM images obtained.

GPSmatch: an R package for comparing Genomic-binding Profile Similarity among transcriptional regulators using customizable databases.

SUMMARY: Eukaryotic gene expression requires coordination among hundreds of transcriptional regulators. To characterize a specific transcriptional regulator, identifying how it shares genomic-binding sites with other regulators can generate important insights into its action. As genomic data such as chromatin immunoprecipitation assays with sequencing (ChIP-Seq) are being continously generated from individual labs, there is a demand for timely integration and analysis of these new data. We have developed an R package, GPSmatch (Genomic-binding Profile Similarity match), for calculating the Jaccard index to compare the ChIP-Seq peaks from one experiment to other experiments stored in a user-supplied customizable database. GPSmatch also evaluates the statistical significance of the calculated Jaccard index using a nonparametric Monte Carlo procedure. We show that GPSmatch is suitable for identifying and ranking transcriptional regulators with shared genomic-binding profiles, which may unravel potential mechanistic actions of gene regulation. AVAILABILITY AND IMPLEMENTATION: The software is freely available at https://github.com/Bao-Lab/GPSmatch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Unwinding chromatin at the right places: how BAF is targeted to specific genomic locations during development.

The BAF (SWI/SNF) chromatin remodeling complex plays a crucial role in modulating spatiotemporal gene expression during mammalian development. Although its remodeling activity was characterized in vitro decades ago, the complex actions of BAF in vivo have only recently begun to be unraveled. In living cells, BAF only binds to and remodels a subset of genomic locations. This selectivity of BAF genomic targeting is crucial for cell-type specification and for mediating precise responses to environmental signals. Here, we provide an overview of the distinct molecular mechanisms modulating BAF chromatin binding, including its combinatory assemblies, DNA/histone modification-binding modules and post-translational modifications, as well as its interactions with proteins, RNA and lipids. This Review aims to serve as a primer for future studies to decode the actions of BAF in developmental processes.

Oh, the Mutations You'll Acquire! A Systematic Overview of Cutaneous Squamous Cell Carcinoma.

Nearly two million cases of cutaneous squamous cell carcinoma (cSCC) are diagnosed every year in the United States alone. cSCC is notable for both its prevalence and its propensity for invasion and metastasis. For many patients, surgery is curative. However, patients experiencing immunosuppression or recurrent, advanced, and metastatic disease still face limited therapeutic options and significant mortality. cSCC forms after decades of sun exposure and possesses the highest known mutation rate of all cancers. This mutational burden complicates efforts to identify the primary factors driving cSCC initiation and progression, which in turn hinders the development of targeted therapeutics. In this review, we summarize the mutations and alterations that have been observed in patients' cSCC tumors, affecting signaling pathways, transcriptional regulators, and the microenvironment. We also highlight novel therapeutic opportunities in development and clinical trials.

BIX-01294, a G9a inhibitor, suppresses cell proliferation by inhibiting autophagic flux in nasopharyngeal carcinoma cells.

G9a, a histone methyltransferase, has been found to be upregulated in a range of tumor tissues, and contributes to tumor growth and metastasis. However, the impact of G9a inhibition as a potential therapeutic target in nasopharyngeal carcinoma (NPC) is unclear. In the present study we aimed to investigate the anti-proliferative effect of G9a inhibition in the NPC cell lines CNE1 and CNE2, and to further elucidate the molecular mechanisms underlying these effects. The expression of G9a in NPC tumor tissues was significantly higher than that in normal nasopharyngeal tissues. The pharmacological inhibition of G9a by BIX-01294 (BIX) inhibited proliferation and induced caspase-independent apoptosis in NPC cells in vitro. Treatment with BIX induced autophagosome accumulation, which exacerbated the cytotoxic activity of BIX in NPC cells. Mechanistic studies have found that BIX impairs autophagosomes by initiating autophagy in a Beclin-1-independent way, and impairs autophagic degradation by inhibiting lysosomal cathepsin D activation, leading to lysosomal dysfunction. BIX was able to suppress tumor growth, possibly by inhibiting autophagic flux; it might therefore constitute a promising candidate for NPC therapy.

BIX-01294-enhanced chemosensitivity in nasopharyngeal carcinoma depends on autophagy-induced pyroptosis.

Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and Southeast Asia. Nowadays, radiotherapy is the therapy of choice for NPC patients, and chemotherapy has been found as an alternative treatment for advanced NPC patients. However, finding novel drugs and pharmacologically therapeutic targets for NPC patients is still urgent and beneficial. Our study showed that BIX-01294 (BIX) can induce autophagic vacuoles formation and conversion of LC3B-I to LC3B-II in NPC cells in both dose- and time-dependent manners. Notably, the combination of BIX and chemotherapeutic drugs significantly decreased the cell viability and increased the lactate dehydrogenase release. Meanwhile, BIX plus cis-platinum (Cis) treatment induced pyroptosis in NPC cells as featured by cell swelling and bubble blowing from the plasma membrane, the increased frequency of annexin V and propidium iodide (PI) double-positive cells, as well as the cleavage of gasdermin E (GSDME) and caspase-3. Moreover, the deficiency of GSDME completely shifted pyroptosis to apoptosis. Furthermore, the inhibition of autophagy by chloroquine and the knockout of ATG5 gene significantly blocked the BIX-induced autophagy as well as pyroptosis in both in vitro and in vivo studies. Our data demonstrated that BIX-combined chemotherapeutic drugs could induce the Bax/caspase-3/GSDME-mediated pyroptosis through the activation of autophagy to enhance the chemosensitivity in NPC.

Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila.

In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing.

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark.

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, we demonstrate that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, our findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase.

Domain requirements of the JIL-1 tandem kinase for histone H3 serine 10 phosphorylation and chromatin remodeling in vivo.

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH2-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH2-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.

CASZ1 Is Essential for Skin Epidermal Terminal Differentiation.

The barrier function of skin epidermis is crucial for our bodies to interface with the environment. Because epidermis continuously turns over throughout the lifetime, this barrier must be actively maintained by regeneration. Although several transcription factors have been established as essential activators in epidermal differentiation, it is unclear whether additional factors remain to be identified. In this study, we show that CASZ1, a multi zinc-finger transcription factor previously characterized in nonepithelial cell types, shows highest expression in skin epidermis. CASZ1 expression is upregulated during epidermal terminal differentiation. In addition, CASZ1 expression is impaired in several skin disorders with impaired barrier function, such as atopic dermatitis, psoriasis, and squamous cell carcinoma. Using transcriptome profiling coupled with RNA interference, we identified 674 differentially expressed genes with CASZ1 knockdown. Downregulated genes account for 91.2% of these differentially expressed genes and were enriched for barrier function. In organotypic epidermal regeneration, CASZ1 knockdown promoted proliferation and strongly impaired multiple terminal differentiation markers. Mechanistically, we found that CASZ1 upregulation in differentiation requires the action of both the master transcription factor, p63, and the histone acetyltransferase, p300. Taken together, our findings identify CASZ1 as an essential activator of epidermal differentiation, paving the way for future studies understanding of CASZ1 roles in skin disease.

Clinical and dosimetric predictors of radiation-induced rhinosinusitis following VMAT for nasopharyngeal carcinoma: A retrospective study.

Background: We aimed to investigate the clinical and dosimetric factors associated with radiation-induced rhinosinusitis, and further elucidate the optimal dose-volume constraints for nasopharyngeal cancer patients who underwent volumetric-modulated arc therapy (VMAT). Methods: A retrospective review of 196 nasopharyngeal carcinoma (NPC) patients who underwent definitive VMAT between August 2018 and May 2021 was conducted. Both clinical and dose-volume histogram (DVH) data of NPC patients without rhinosinusitis at baseline were selected for analysis. Results: The cumulative incidence of post-RT rhinosinusitis at the 3-, 6-, 9-, and 12-months, and >1 year were 29.6 %, 41.3 %, 42.9 %, and 45.4 %, and 47.4 %, respectively. Nasal irrigation was negatively associated with post-RT rhinosinusitis (p < 0.001). Higher cumulative incidences of maxillary and ethmoid sinusitis were associated with V70 > 1.16 % and >1.00 %, respectively (p = 0.027 and p = 0.002). Sphenoid sinusitis was more frequent when Dmax(maxillary sinus) exceeded 69.2Gy (p = 0.005). Conclusions: Regular nasal irrigation may reduce the development of rhinosinusitis. Dose-volume constraints of V70 and Dmax to the maxillary sinus are suggested for VMAT planning. Patients exceeding these thresholds should be closely monitored and potentially offered preventative interventions within 3-6 months post-RT.

Acanthopanax senticosus improves cognitive impairment in Alzheimer's disease by promoting the phosphorylation of the MAPK signaling pathway.

Background: Acanthopanax senticosus (AS) can improve sleep, enhance memory, and reduce fatigue and is considered as an effective drug for Alzheimer's disease (AD). The therapeutic effect and mechanism need to be further investigated. Methods: To confirm the AS play efficacy in alleviating memory impairment in mice, 5×FAD transgenic mice were subjected to an open-field experiment and a novelty recognition experiment. Network pharmacology technique was used to analyze the information of key compounds and potential key targets of AS for the treatment of AD, molecular docking technique was applied to predict the binding ability of targets and compounds, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also performed on the targets to derive the possible metabolic processes and pathway mechanisms of AS in treating AD. Quantitative real-time PCR (qRT-PCR) and western blot technique were carried out to validate the candidate genes and pathways. Results: In the open-field experiment, compared with the wild-type (WT) group, the number of times the mice in the AD group crossed the central zone was significantly reduced (P< 0.01). Compared with the AD group, the number of times the mice in the AS group crossed the central zone was significantly increased (P< 0.001). In the new object recognition experiment, compared with the WT group, the percentage of times the AD group explored new objects was significantly reduced (P< 0.05). Compared with the AD group, the AS group had an increase in the percentage of time spent exploring new things and the number of times it was explored (P< 0.05). At the same time, the donepezil group had a significantly higher percentage of times exploring new things (P< 0.01). By using network pharmacology technology, 395 common targets of AS and AD were retrieved. The Cytoscape software was used to construct the protein-protein interaction (PPI) network of common targets. Using the algorithm, nine key targets were retrieved: APP, NTRK1, ESR1, CFTR, CSNK2A1, EGFR, ESR2, GSK3B, and PAK1. The results of molecular docking indicate that 11 pairs of compounds and their corresponding targets have a significant binding ability, as the molecular binding energies were less than -7.0. In comparison to the AD group, the mRNA expression of the key target genes was significantly decreased in the AS treatment group (P< 0.001). The KEGG analysis showed that the MAPK signaling pathway was significantly enriched, and Western blot confirmed that the TRAF6 protein decreased significantly (P< 0.0001). Meanwhile, the levels of MAP3K7 and P38 phosphorylation increased, and there was also an increase in the expression of HSP27 proteins. Conclusion: Our study indicates that the multi-component and multi-target properties of AS play an important role in the alleviation of anxiety and memory impairment caused by AD, and the mechanism is involved in the phosphorylation and activation of the MAPK signaling pathway. The results of this study could provide a novel perspective for the clinical treatment of AD.

Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation.

PBRM1 is frequently mutated in cancers of epithelial origin. How PBRM1 regulates normal epithelial homeostasis, prior to cancer initiation, remains unclear. Here, we show that PBRM1's gene regulatory roles differ drastically between cell states, leveraging human skin epithelium (epidermis) as a research platform. In progenitors, PBRM1 predominantly functions to repress terminal differentiation to sustain progenitors' regenerative potential; in the differentiation state, however, PBRM1 switches toward an activator. Between these two cell states, PBRM1 retains its genomic binding but associates with differential interacting proteins. Our targeted screen identified the E3 SUMO ligase PIAS1 as a key interactor. PIAS1 co-localizes with PBRM1 on chromatin to directly repress differentiation genes in progenitors, and PIAS1's chromatin binding drastically diminishes in differentiation. Furthermore, SUMOylation contributes to PBRM1's repressive function in progenitor maintenance. Thus, our findings highlight PBRM1's cell-state-specific regulatory roles influenced by its protein interactome despite its stable chromatin binding.

The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing in Drosophila melanogaster.

The JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein. By transgenically expressing various truncated versions of JIL-1, with or without kinase activity, and assessing their effect on PEV and heterochromatic spreading, we show that the gross perturbation of polytene chromosome morphology observed in JIL-1 null mutants is unrelated to gene silencing in PEV and is likely to occur as a result of faulty polytene chromosome alignment and/or organization, separate from epigenetic regulation of chromatin structure. Furthermore, the findings provide evidence that the epigenetic H3S10 phosphorylation mark itself is necessary for preventing the observed heterochromatic spreading independently of any structural contributions from the JIL-1 protein.

Radial forearm free flap and a freka-trelumina tube for management of a refractory pharyngocutaneous fistula: A case report.

The management of pharyngocutaneous fistulas (PCFs) is challenging. A multidisciplinary treatment approach according to the clinical needs of a patient is essential for PCF management. Here, we describe the use of a double-layer closure technique involving a radial forearm free flap (RFFF) and a Freka-Trelumina nasojejunal tube in the reconstruction of a refractory PCF.

NUP98 and RAE1 sustain progenitor function through HDAC-dependent chromatin targeting to escape from nucleolar localization.

Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers.

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes' clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors.

Progenitors in epithelial tissues, such as human skin epidermis, continuously make fate decisions between self-renewal and differentiation. Here we show that the Super Elongation Complex (SEC) controls progenitor fate decisions by directly suppressing a group of "rapid response" genes, which feature high enrichment of paused Pol II in the progenitor state and robust Pol II elongation in differentiation. SEC's repressive role is dependent on the AFF1 scaffold, but not AFF4. In the progenitor state, AFF1-SEC associates with the HEXIM1-containing inactive CDK9 to suppress these rapid-response genes. A key rapid-response SEC target is ATF3, which promotes the upregulation of differentiation-activating transcription factors (GRHL3, OVOL1, PRDM1, ZNF750) to advance terminal differentiation. SEC peptidomimetic inhibitors or PKC signaling activates CDK9 and rapidly induces these transcription factors within hours in keratinocytes. Thus, our data suggest that the activity switch of SEC-associated CDK9 underlies the initial processes bifurcating progenitor fates between self-renewal and differentiation.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration.

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.