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Regulation of integrin and extracellular matrix genes by HNRNPL is necessary for epidermal renewal

['Jingting Li', 'Institute Of Precision Medicine', 'The First Affiliated Hospital', 'Sun Yat-Sen University', 'Guangzhou', 'Yifang Chen', 'Department Of Dermatology', 'Department Of Cellular', 'Molecular Medicine', 'Ucsd Stem Cell Program']

Date: 2021-09

Abstract Stratified epithelia such as the epidermis require coordinated regulation of stem and progenitor cell proliferation, survival, and differentiation to maintain homeostasis. Integrin-mediated anchorage of the basal layer stem cells of the epidermis to the underlying dermis through extracellular matrix (ECM) proteins is crucial for this process. It is currently unknown how the expression of these integrins and ECM genes are regulated. Here, we show that the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to these genes on chromatin to promote their expression. HNRNPL recruits RNA polymerase II (Pol II) to integrin/ECM genes and is required for stabilizing Pol II transcription through those genes. In the absence of HNRNPL, the basal layer of the epidermis where the stem cells reside prematurely differentiates and detaches from the underlying dermis due to diminished integrin/ECM expression. Our results demonstrate a critical role for RBPs on chromatin to maintain stem and progenitor cell fate by dictating the expression of specific classes of genes.

Citation: Li J, Chen Y, Tiwari M, Bansal V, Sen GL (2021) Regulation of integrin and extracellular matrix genes by HNRNPL is necessary for epidermal renewal. PLoS Biol 19(9): e3001378. https://doi.org/10.1371/journal.pbio.3001378 Academic Editor: Bon-Kyoung Koo, IMBA, AUSTRIA Received: January 26, 2021; Accepted: July 30, 2021; Published: September 20, 2021 Copyright: © 2021 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files (S1 Data). The RNA-Seq and ChIP-Seq has been deposited in GEO (GSE162546) at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162546. Funding: This work was supported by grants from the National Institutes of Health (NIH R01AR072590, R01AR066530, and R01CA225463) to GLS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: ChIP-Seq, chromatin immunoprecipitation sequencing; DMEM, Dulbecco’s Modified Eagle Medium; ECM, extracellular matrix; FLG, filaggrin; FN1, fibronectin1; GO, gene ontology; HNRNP, heterogeneous nuclear ribonucleoprotein; HNRNPK, heterogeneous nuclear ribonucleoprotein K; HNRNPL, heterogeneous nuclear ribonucleoprotein L; IP, immunoprecipitation; K10, keratin 10; lncRNA, long noncoding RNA; LOR, loricrin; MAPK, mitogen-activated protein kinase; Pol II, polymerase II; qPCR, quantitative PCR; RBP, RNA-binding protein; RNA-seq, RNA sequencing; rpm, reads per million; RT-qPCR, reverse transcription quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; TES, transcription end site; TNF, tumor necrosis factor; TSS, transcription start site

Introduction Integrins are large, transmembrane spanning proteins that are essential for tissue organization, survival, homeostasis, differentiation, and signal transduction [1]. Integrins form heterodimers between 18 α and 8 β subunits that can form up to 24 different receptors [2,3]. Integrins are important for epithelial tissues such as the epidermis to communicate as well as attach to the extracellular matrix (ECM) [4]. The human epidermis acts as an essential barrier between the body and the outside environment. It is a stratified epithelium that requires a proper balance of cell proliferation and differentiation to maintain its homeostasis and integrity [5]. Stem and progenitor cells in the basal layer of the epidermis maintain a population of proliferative cells while also giving rise to differentiated progeny. Those cells entering the differentiation program undergo various stages of differentiation as they migrate outwards through the epidermis, where they will eventually form the protective outer layer known as the stratum corneum. In order for the basal layer stem and progenitor cells to stay undifferentiated, they must attach to the underlying dermis through hemidesmosomes. Hemidesmosomes are made of cell surface integrins (i.e., α6β4 and α3β1) that connect to laminins and collagens in the basement membrane [6]. Human genetic mutations in these proteins (integrins, laminins, and collagens) lead to debilitating and life-threatening skin blistering diseases known as epidermolysis bullosa, which is characterized by the detachment of the epidermis from the dermis [7–11]. β1 integrin activation blocks epidermal differentiation and promotes stem cell expansion [12]. In addition, integrins such as α5β1 are necessary for epithelial movement [13]. Despite the importance of integrins and ECM proteins such as laminins to epithelial integrity, stem cell fate decisions, migration, and tumorigenesis, it is currently unclear how these genes are regulated. Potential regulators of integrin expression include RNA-binding proteins (RBPs). The human genome encodes approximately 1,500 RBPs that are involved in all aspects of RNA metabolism such as splicing, export, stability and translation [14]. While most studies have focused on RBPs function in regulating RNA, there is increasing evidence that they may have direct roles in transcription. This includes SRSF2, which has been shown to be part of the 7SK complex and is critical for transcription pause release [15]. A recent large-scale analysis of 45 RBPs in K562 cells and 58 RBPs in HepG2 cells showed that approximately 60% of these factors associated with chromatin [16]. Binding to chromatin occurred through 2 ways. First, RBPs can directly bind DNA or be part of complexes assembled at gene promoters. Second, RBPs can associate with chromatin indirectly through nascent RNA to control gene expression. Despite major efforts to understand RBPs and their potential function on chromatin, >95% of RBPs mechanism of action as well as tissue function remains uncharacterized [16]. We have recently shown that in human epidermis, the RBP heterogeneous nuclear ribonucleoprotein K (HNRNPK) is necessary for epidermal proliferation and prevention of premature differentiation through its impacts on both the RNA and DNA level [17]. HNRNPK degrades the mRNAs of genes that code for differentiation promoting transcription factors such as GRHL3 and KLF4 in progenitor cells to prevent premature differentiation [17,18]. HNRNPK also binds to chromatin and is necessary to recruit RNA polymerase II (Pol II) to proliferation genes to promote epidermal growth. Because of HNRNPK’s importance to epidermal growth and differentiation, we hypothesized that other related RBPs might also be critical to epidermal function. Through targeting candidate RBPs, we found that heterogeneous nuclear ribonucleoprotein L (HNRNPL) is necessary to maintain epidermal stem and progenitor cell identity. HNRNPL does this by binding to hemidesmosome and ECM genes and promoting their transcription by stabilizing Pol II binding to those genes. HNRNPL-mediated expression of those genes allows attachment of basal layer stem and progenitor cells to the underlying dermis, which prevents premature differentiation and anoikis. Our results have identified an RBP acting on chromatin to regulate integrin and ECM expression, which, in turn, maintains the stem and progenitor cell compartment of the epidermis.

Discussion HNRNPL has been shown to be involved in most aspects of RNA regulation including RNA stability, splicing, as well as association with long noncoding RNAs (lncRNAs) to control gene expression [37–40]. It has been mostly studied in the context of cancer where it is required for tumor growth and metastasis [41]. In this context, HNRNPL interacts with various lncRNAs (specific lncRNAs dependent on tumor type) to promote tumorigenesis [42–46]. For example, the complex of lncRNA, DSCAM-AS1, and HNRNPL are necessary for breast cancer progression and tamoxifen resistance [42]. In hepatocellular carcinoma, the lncRNA SPRY4-IT1 associates with HNRNPL to promote growth and metastasis through the tumor necrosis factor (TNF) pathway [43]. Intriguingly, very little is known about the role of HNRNPL during normal tissue homeostasis as well as how it impacts gene expression by binding chromatin. In a large-scale study, HNRNPL was found to bind chromatin, but not much characterization was done on how it impacted gene expression [16]. Our results suggest HNRNPL is necessary to sustain epidermal stem and progenitor cells in the basal layer of the epidermis. In its absence, primary human keratinocytes undergo apoptosis, stop proliferating, and prematurely differentiate. In regenerated human skin, HNRNPL loss leads to terminal differentiation proteins being expressed in the basal layer of the epidermis, suggesting that the stem cell compartment has been lost. Apoptosis could be observed throughout the epidermis. In addition, the epidermis could no longer attach to the underlying dermis due to loss of expression of hemidesmosome/ECM genes. This phenotype resembled what is seen in epidermolysis bullosa patients who have mutations in integrin, laminin, or collagen. Integrin and ECM genes has been extensively studied; however, there is limited understanding on how these crucial genes are regulated on the transcriptional level [47,48]. Surprisingly, our HNRNPL ChIP-Seq data showed that HNRNPL bound genes were significantly enriched for hemidesmosome and cell-substrate junction assembly GO terms. This includes genes such as ITGB4 and LAMC2 that are crucial for the attachment of the basal layer to the dermis. It should be noted that HNRNPL binding to chromatin is weaker than other factors that have been traditionally characterized such as transcription or epigenetic factors. This weaker association was also observed by Xiao and colleagues when they mapped the DNA binding of a large number of RBPs [16]. Interestingly, HNRNPL binding to chromatin resembled that of Pol II and the 2 proteins also associate. This suggests that HNRNPL may be crucial for Pol II–mediated transcription on HNRNPL bound genes. Supporting this, knockdown of HNRNPL substantially decreased Pol II binding across HNRNPL bound genes that were enriched for hemidesmosome and ECM genes. Importantly, Pol II was only minimally impacted on genes not bound by HNRNPL. Our data suggest that HNRNPL indirectly regulates differentiation and apoptosis since HNRNPL did not bind to these gene classes. Genes up-regulated (or gained Pol II peaks) upon HNRNPL depletion and were bound by HNRNPL were not enriched for differentiation or apoptosis genes. Instead, these genes were involved in the regulation of transcription from RNA Pol II promoter. Thus, HNRNPL indirectly prevents premature differentiation and anoikis by promoting the expression of hemidesmosome/ECM genes. This, in turn, allows attachment of the basal layer to the underlying dermis [6,49,50]. Previous studies have shown that keratinocytes grown in suspension without anchorage to the ECM spontaneously differentiate as it is mimicking the process of delamination from the basal layer [6,29,30]. A prior study showed that HNRNPL binds to muscle differentiation genes and activates their expression [51]. This is in contrast to our study where HNRNPL does not bind to differentiation genes in keratinocytes. HNRNPL is also necessary for maintaining the undifferentiated state in keratinocytes rather than promoting differentiation as shown in the muscle study. It is currently unclear whether the differences are due to cell type–specific differences (keratinocytes versus myoblasts) or the primary cells that were used in this study as compared to the transformed muscle cell line used in the previous study. Since HNRNPL can bind to RNA, it is also possible that HNRNPL binds to RNA to control epidermal growth and differentiation. For example, HNRNPL could potentially control the mRNA stability or translation of skin master transcription factors such as P63 to promote expression of hemidesmosome/ECM genes. However, the major phenotypes of premature differentiation, apoptosis, and detachment of the epidermis from the dermis can be explained through transcriptional control of hemidesmosome and ECM genes through HNRNPL. Our model suggests that lineage-specific transcription factors open the chromatin to hemidesmosome/ECM genes, which then allows HNRNPL to maintain Pol II transcription at those genes. This is supported by our findings that HNRNPL bound genes were enriched with SOX9 and RUNX2 binding motifs. With HNRNPL being expressed throughout the epidermis, basal layer–specific transcription factors such as SOX9 [34,35] could potentially recruit HNRNPL to target genes to achieve layer-specific gene expression. In summary, we have elucidated how HNRNPL maintains epidermal renewal by promoting the expression of adhesion genes through RNA Pol II, which then allows attachment of the epidermis to the dermis.

Methods Cell culture Primary human epidermal keratinocytes (derived from neonatal foreskin) were purchased from Life Technologies (Carlsbad, California, USA) (C0015C) and cultured in EpiLife medium (Life Technologies: MEPI500CA). This media was supplemented with penicillin/streptomycin (HyClone SV30010) and human keratinocyte growth supplement (Life Technologies: S0015). Cells were induced to differentiate in full confluence and 1.2 mM calcium for 3 and 5 days. Phoenix cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal calf serum. Gene knockdown siRNAs were placed into EpiLife media with the transfection reagent Lipofectamine RNAiMAX (25 ul for 10-cm plate transfection and 50 ul for 15-cm plate transfection) (Thermo Fisher Scientific (Waltham, MA): 13778) and incubated for 5 minutes at room temperature. This siRNA media was then diluted 1:10 and added to subconfluent keratinocytes, with the siRNA at a final concentration of 10 nM. The keratinocytes were then incubated in this media for a minimum of 18 hours to carry out siRNA knockdown. The siRNAs used in this study are as follows: Control siRNA (Ambion (Carlsbad, CA) Silencer Select Negative Control 4390844), HNRNPL siRNA (Ambion Silencer Select s6742), HNRNPF (Dharmacon (Lafayette, CO) M-013449-02-0005), HNRNPH1 siRNA (Dharmacon M-012107-00-0005), and HNRNPH2 siRNA (Dharmacon M-013245-01-0005). The retroviral constructs (3 ug) expressing shRNAs were transfected using Fugene 6 (Roche, Basel, Switzerland) into amphotrophic phoenix cells to knockdown genes. Viral supernatants were collected 48 hours posttransfection and used to infect primary human keratinocytes. Cells were incubated in the viral supernatants and centrifuged at 1,000 reads per million (rpm) for 1 hour with the addition of polybrene (5 ug/ml). Cells were transduced on 2 consecutive days with the shRNA retroviral constructs and then selected using puromycin (2 ug/ml). Retroviral constructs Retroviral constructs to knockdown HNRNPL were generated by cloning oligos into the pSuper retroviral vector as previously described [25,52]. The following sequences targeting HNRNPL were used: HNRNPL A shRNA: GTTACAAAGACTTCAGTGA; HNRNPL B shRNA: GAACCATTACCAGATGAAA. Regenerated human epidermis Human dermis obtained from the New York Firefighters Skin Bank was devitalized, cut, and placed upon an organotypic cassette. The bottom of the dermis was coated in matrigel, and the cassette was placed in keratinocyte growth medium, with the bottom of the dermis contacting the media and the top at the air interface. A total of 1 million keratinocytes were then seeded on the top of the dermis and allowed to regenerate and stratify for 4 days. The regenerated human skin were then harvested and embedded in OCT (Optimal Cutting Temperature) for sectioning and staining. Additional details for regenerating human skin have been previously described [25,36,52–54]. Immunofluorescent staining Sectioned tissue–derived regenerated human skin or normal adult human skin was fixed with 10% formalin solution (Sigma (St. Louis, MO) HT5012) for 12 minutes. Sections were blocked (PBS, 2% bovine serum albumin, 2.5% normal goat serum, and 0.3% Triton X-100) for 30 minutes. Primary antibodies were then added to blocking buffer and put onto sections for 1 hour. The following antibodies were used at the following concentrations: FLG at 1:200 (Abcam (Waltham, MA): ab3137), LOR at 1:400 (Abcam: ab198994), KI67 (Abcam: Ab16667) at 1:300, cleaved caspase-3 at 1:300 (Cell Signaling Technology (Danvers, MA): 9661S), HNRNPL (Bethyl (Montgomery, TX): A303-895A) at 1:400, K10 (Abcam: ab9025) at 1:300, and ITGB4 (Cell Signaling Technology: 14803S) at 1:150. Secondary antibodies were used at 1:500 for 30 minutes and included Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific: A21424) and Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific: A21206). Hoechst 33342 (Thermo Fisher Scientific: H3570) was used at 1:1,000 to stain nuclei. Hematoxylin–eosin staining Sectioned tissue derived from regenerated human skin was fixed with 10% formalin solution (Sigma HT5012) for 12 minutes. Sections were then dipped in 0.25% Triton X-100 in PBS for 5 minutes. Hemotoxylin (Vector H-3401) staining was performed for 8 minutes, rinsed in water, and then dipped in acid alcohol (1% HCL in 70% ethanol). After subsequent rinsing, the sections were dipped in 0.2% ammonia water for 1 minute, rinsed again, and then dipped in 95% ethanol. Eosin (Richard-Allan Scientific (Kalamazoo, MI) 71304) staining was performed for 30 seconds followed by 95% ethanol rinsing for 1 minute. Sections were then put into 100% ethanol for 4 minutes followed by 2 minutes in Xylene. RNA extraction and analysis by RT-qPCR The GeneJET RNA purification kit (Thermo Fisher Scientific: K0732) was used to extract RNA from cultured keratinocytes. RNA concentration for each sample was measured by nanodrop, and 1 μg of RNA was used for generating cDNA. This cDNA was generated using the Maxima cDNA Synthesis Kit (Thermo Fisher Scientific: K1642). qPCR was performed using this cDNA on the Bio-Rad (Hercules, CA) CFX96 real-time system. The housekeeping gene L32 was used for normalization of signal. Primer sequences for all genes tested are listed in S1 Data. Apoptosis assay Four days after transfection, control and HNRNPL knockdown cells were stained with Annexin V conjugated to Alexa Fluor 488 (Life Technologies: A13201) and analyzed using the Guava flow cytometer (Millipore, Burlington, MA) according to manufacturer’s instructions and as previously described [17]. Western blotting IP samples or 20 to 80 ug of cell lysates were loaded into 4% to 12% Bis-Tris (Thermo Fisher Scientific: NW04122BOX) or 3% to 8% Tris-acetate (Thermo Fisher Scientific: EA03752BOX) and transferred to PVDF membranes. Membranes were blocked in 5% BSA in TBS. Membranes were exposed to primary antibodies in blocking buffer overnight at 4 degrees. The following primary antibodies were used: ITGB4 (Cell Signaling Technology: 14803S) at 1:200, ITGB1 (Cell Signaling Technology: 9699S) at 1:250, ITGA5 (Cell Signaling Technology: 4705S) at 1:200, ITGAV (Cell Signaling Technology: 4711S) at 1:250, HNRNPL (Bethyl: A303-895A) at 1:1,000, and RNA Pol II (Active Motif (Carlsbad, CA): 91151) at 1:1,000. The loading control Beta-Actin (Santa Cruz Biotechnology (Dallas, TX) sc-47778) was used at 1:5,000. The secondary antibodies used were donkey anti-rabbit IRDye 680RD (Li-Cor 926–68073) and donkey anti-mouse IRDye 800CW (Li-Cor 926–32212) at 1:5,000. RNA sequencing and bioinformatic analysis Control or HNRNPLi cells were harvested 4 days after siRNA transfection. Independent biological duplicates were obtained for both CTLi and HNRNPLi, and total RNA was isolated using the GeneJET RNA purification kit (Thermo Fisher Scientific: K0732) and quantified by Nanodrop. RNA-seq was performed using the Illumina (San Diego, CA) NovaSeq 6000. RNA-seq libraries were prepared with NEBNext Ultra II Library Prep Kit (Illumina (San Diego, CA): E7760) then multiplexed, and approximately 40 million reads per sample were obtained. Pair-end reads were aligned to the GENCODE v19 transcriptome hg19 using TopHat2 with default settings [55]. Differential expression among samples was calculated using ANOVA from the Partek Genomic Suite (Partek, St. Louis, MO). Analysis of the read count distribution indicated that a threshold of 10 reads per gene generally separated expressed from unexpressed genes, so all genes with fewer than 10 reads were excluded from ANOVA analysis. Gene lists for significantly up-regulated or down-regulated genes were created using p-value < 0.05 and ≥2-fold change. Enriched GO terms for RNA-seq differentially expressed gene sets were identified using Enrichr [56,57]. Heatmaps for the RNA-seq data were generated using Partek Genomic Suite (http://www.partek.com/partek-genomics-suite/). ChIP sequencing and bioinformatic analysis A total of 10 million cells and 5 μg of antibody were used for each antibody pulldown experiment for ChIP [17,27,54]. ChIP was performed using the following antibodies: HNRNPL (Bethyl: A303-895A), RNA Pol II (Active Motif: 91151), mouse IgG (Abcam: AB18413), and rabbit IgG (Millipore: 12–370). Cells for the RNA Pol II ChIP-qPCR or ChIP-Seq were fixed at a final concentration of 1% formaldehyde. Cells for the HNRNPL ChIP-qPCR or ChIP-Seq were fixed in both formaldehyde (1% final concentration, Thermo Fisher Scientific: 28908) and disuccinimidyl glutarate (2 mM final concentration, Thermo Fisher Scientific: 20593). qPCR results are represented as a percentage of input DNA. For ChIP-Seq, the ChIP DNA library was prepared using the TrueSeq DNA sample prep kit (Illumina). Sequencing was done on the HiSeq 4000 System (Illumina) using single 1 × 75 reads at the Institute for Genomic Medicine Core, UCSD. The ChIP-seq reads were processed by the ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2) on our local workstation. The reads were first trimmed based on quality score before alignment to reference hg19; upon alignment and deduplication, the peak calling was then carried out by MACS2.2.4 with a cutoff q-value of 0.05 [58,59]. The heatmaps and metagene plots for the ChIP-Seq data were generated using ngs.plot [60]. Gene tracks were visualized using UCSC genome browser along with annotation tracks. The differential peaks between CTLi and HNRNPLi Pol II ChIP-Seq were determined using diffReps [61]. Co-immunoprecipitation Primary human keratinocytes were harvested in IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) and sheared with a syringe. Moreover, 5 ug of HNRNPL (Bethyl: A303-895A) or control rabbit IgG (Millipore: 12–370) was conjugated to 50 ul of Protein G dynabeads (Life Technologies: 10004D) for 30 minutes at room temperature. Lysis buffer was diluted to 1 ml per sample and was added to the antibody conjugated beads and incubated overnight at 4 degrees. The following day, the beads were washed with IP lysis buffer and boiled in RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with NuPAGE LDS Sample Buffer (Life Technologies: NP0008) to elute. Samples were then loaded for western blotting. Statistics Statistical analyses were performed using GraphPad Prism 9. Histogram data are presented as the mean ± SD, and significant differences between 2 samples were determined by Student t tests. For groups of 3 or more, 1-way ANOVA with Tukey was used. Significant changes were defined as p ≤ 0.05.

Supporting information S1 Fig. Knockdown of HNRNP genes shows that only HNRNPL is necessary for epidermal growth and blockade of premature differentiation. (A) Primary human keratinocytes were knocked down with control (CTLi), HNRNPFi, HNRNPH1i, or HNRNPH2i siRNAs, and the remaining mRNA levels of each gene was measured using RT-qPCR. qPCR results were normalized to L32 levels. (B) CTLi, HNRNPFi, HNRNPH1i, or HNRNPH2i cells were seeded at 150,000 cells and counted 4 days later. Cell number was reported as a percentage of CTLi levels. (C, D) RT-qPCR for IVL (C) and FLG (D) mRNA expression in CTLi, HNRNPFi, HNRNPH1i, or HNRNPH2i cells after 4 days of knockdown. qPCR results were normalized to L32 levels. (E) Primary human keratinocytes were knocked down with control (CTL shRNA) or HNRNPL (HNRNPL shRNA-A or shRNA-B) shRNAs delivered by retroviruses. HNRNPL shRNAs A and B are distinct shRNAs that target 2 different regions of the gene. Knockdown was measured using RT-qPCR. qPCR results were normalized to L32 levels. (F) CTL shRNA, HNRNPL shRNA-A, and HNRNPL shRNA-B cells were seeded at 150,000 cells and counted 6 days later. Cell number was reported as a percentage of CTLi levels. (G) RT-qPCR for epidermal differentiation gene expression in CTL shRNA, HNRNPL shRNA-A, and HNRNPL shRNA-B cells after 6 days of knockdown. qPCR results were normalized to L32 levels. N = 3 independent experiments for the entire figure. Mean values are shown with error bars = SD. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 (t test for A–D and 1-way ANOVA with Tukey for E–G). Primary data for this figure can be found in S1 Data. HNRNPL, heterogeneous nuclear ribonucleoprotein L; RT-qPCR, reverse transcription quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA. https://doi.org/10.1371/journal.pbio.3001378.s001 (EPS) S2 Fig. HNRNPL is expressed throughout the epidermis and is necessary to prevent apoptosis of the tissue. (A) Regenerated human skin using three-dimensional organotypic cultures made from CTLi or HNRNPLi cells were harvested after 4 days of culture. Immunostaining of apoptotic marker cleaved caspase 3 (green) and FLG (red) are shown. Nuclei are shown in blue (Hoechst staining). White arrows denote large areas of apoptotic cells. The dashed white lines denote the basement membrane zone. Scale bar = 40 μm. (B) Immunostaining of HNRNPL (green) and early differentiation marker K10 (red) on adult human skin. Nuclei are shown in blue (Hoechst staining). The dashed white lines denote the basement membrane zone. Scale bar = 40μm. (C) Western blot for HNRNPL in undifferentiated (day 0) and differentiated (days 3 and 5) primary human keratinocytes. Actin was used as a loading control. Representative images are shown. Primary data for this figure can be found in S1 Data. FLG, filaggrin; HNRNPL, heterogeneous nuclear ribonucleoprotein L; K10, keratin 10. https://doi.org/10.1371/journal.pbio.3001378.s002 (EPS) S3 Fig. HNRNPL binds to regions enriched for skin transcription factors such as RUNX2 and SOX9. (A) De novo motif analysis of the 2,843 HNRNPL bound peaks using HOMER. (B) Gene track of ITGB4 and its adjacent genes (SAP30BP and UNK). HNRNPL ChIP-Seq is shown in red. Y-axis shows RPM and red bar over gene tracks represent significant peaks. X-axis shows position along gene. (C) HNRNPL ChIP-qPCR on CTLi and HNRNPLi cells. IGG pulldowns in CTLi and HNRNPLi cells were used as a negative control. qPCR was used to determine the amount of binding to genes listed on the X-axis. Primers were targeted toward the TSS of each gene. Results are plotted as a percent of input. N = 3 independent experiments. Mean values are shown with error bars = SD. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 (t test was used and each sample compared to CTLi). Primary data for this figure can be found in S1 Data. ChIP-Seq, chromatin immunoprecipitation sequencing; HNRNPL, heterogeneous nuclear ribonucleoprotein L; IGG, immunoglobulin; RPM, reads per million; TSS, transcription start site. https://doi.org/10.1371/journal.pbio.3001378.s003 (EPS) S4 Fig. HNRNPL binds and promotes the transcription of integrin/ECM genes through RNA Pol II. (A) Heatmap of Pol II ChIP-Seq in CTLi and HNRNPLi cells. Pol II ChIP-Seq signal is ranked by decreasing Pol II occupancy. X-axis shows −2 kb to +2 kb from the TSS. (B) Overlap of the HNRNPL bound genes that lost Pol II peaks with the down-regulated genes upon HNRNPL knockdown. (C) GO terms of the 111 overlapped genes from (B). (D–F) Gene tracks of LAMA3 (D), ITGA3 (E), and FN1 (F). Pol II ChIP-Seq is shown in in CTLi (red) and HNRNPLi (green) cells. Y-axis shows RPM. Red or green bar over gene tracks represent significant peaks. X-axis shows position along gene. (G) RNA Pol II ChIP on CTLi and HNRNPLi cells. IGG pulldowns in CTLi and HNRNPLi cells were used as a negative control. qPCR was used to determine the amount of binding to genes listed on the X-axis. Primers were targeted toward the TSS of each gene. Results are plotted as a percent of input. N = 3. Mean values are shown with error bars = SD. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 (t test was used and each sample compared to CTLi). (H) Overlap of the HNRNPL bound genes (HNRNPL ChIP-Seq) with the genes that gain Pol II peaks upon HNRNPL depletion. (I) GO terms of the 430 overlapped genes from (H). Primary data for this figure can be found in S1 Data. ChIP-Seq, chromatin immunoprecipitation sequencing; ECM, extracellular matrix; GO, gene ontology; HNRNPL, heterogeneous nuclear ribonucleoprotein L; IGG, immunoglobulin; Pol II, polymerase II; RPM, reads per million; TSS, transcription start site. https://doi.org/10.1371/journal.pbio.3001378.s004 (EPS) S1 Table. RNA-seq of HNRNPL knockdown primary human keratinocytes. Primary human keratinocytes were grown in proliferation conditions and knocked down for control (CTLi) or HNRNPL (HNRNPLi). Gene lists for significantly up-regulated or down-regulated genes were created using p-value < 0.05 and ≥2-fold change. HNRNPL, heterogeneous nuclear ribonucleoprotein L; RNA-seq, RNA sequencing. https://doi.org/10.1371/journal.pbio.3001378.s005 (XLSX) S2 Table. ECM and hemidesmosome genes down-regulated upon HNRNPL depletion. List of the 69 ECM and hemidesmosome genes that are decreased in expression upon HNRNPL knockdown. ECM, extracellular matrix; HNRNPL, heterogeneous nuclear ribonucleoprotein L. https://doi.org/10.1371/journal.pbio.3001378.s006 (XLSX) S3 Table. ChIP-Seq of HNRNPL in proliferating keratinocytes. List of the genes bound by HNRNPL including the coordinates where the peaks were found. ChIP-Seq, chromatin immunoprecipitation sequencing; HNRNPL, heterogeneous nuclear ribonucleoprotein L. https://doi.org/10.1371/journal.pbio.3001378.s007 (XLSX) S4 Table. HNRNPL-bound genes that lost RNA Pol II peaks upon HNRNPL knockdown. List of the 495 genes bound by HNRNPL that lost RNA Pol II peaks upon HNRNPL depletion. HNRNPL, heterogeneous nuclear ribonucleoprotein L; Pol II, polymerase II. https://doi.org/10.1371/journal.pbio.3001378.s008 (XLSX) S1 Data. Primary data for Figs 1–4 and S1–S4 Figs. https://doi.org/10.1371/journal.pbio.3001378.s009 (XLSX)

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