Article

Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex

Emanuele Panatta1,2 , Anna Maria Lena1 , Mara Mancini3, Artem Smirnov1 , Alberto Marini1,2, Riccardo Delli Ponti4,5, Teresa Botta-Orfila4,5 , Gian Gaetano Tartaglia4,5,6 , Alessandro Mauriello1, Xinna Zhang7, George A Calin7, Gerry Melino1,2,* & Eleonora Candi1,3,**

Abstract Introduction

The mechanisms that regulate the switch between epidermal During somatic tissue differentiation, progenitor proliferating cells progenitor state and differentiation are not fully understood. establish a specific programme of activation and silencing that Recent findings indicate that the chromatin remodelling BAF underlies their differentiation into specialized cell types. This complex (Brg1-associated factor complex or SWI/SNF complex) and process is nicely recapitulated during epidermis formation and the transcription factor p63 mutually recruit one another to open epidermis homeostasis. Recent studies demonstrated that epigenetic chromatin during epidermal differentiation. Here, we identify a regulators from multiple classes play a critical role in activating and long non-coding transcript that includes an ultraconserved repressing by affecting the chromatin state. The element, uc.291, which physically interacts with ACTL6A and epigenetic regulators that promote proliferation and inhibit activa- modulates chromatin remodelling to allow differentiation. Loss of tion of terminal differentiation-associated in progenitor cells uc.291 expression, both in primary keratinocytes and in three- include DNA methyltransferase 1 (DNMT1) [1], histone deacety- dimensional skin equivalents, inhibits differentiation as indicated lases 1 and 2 (HDAC1/2) [2], and polycomp components Bmi1, by epidermal differentiation complex genes down-regulation. ChIP Ezh1/2 [3–5] and Cbx4 [6]. Conversely, histone demethylase experiments reveal that upon uc.291 depletion, ACTL6A is bound to Jumonji domain-containing 3 [7], adenosine triphosphate-depen- the differentiation gene promoters and inhibits BAF complex dent (ATP-dependent) chromatin remodeller Brg1/BAF complex targeting to induce terminal differentiation genes. In the presence [6,8,9] and genome organizer Satb1 [10] promote terminal keratino- of uc.291, the ACTL6A inhibitory effect is released, allowing chro- cyte differentiation [11–15]. The BAF pro-differentiation role is matin changes to promote the expression of differentiation genes. prevented by ACTL6A (actin-like 6A, also known as BAF53A), a Thus, uc.291 interacts with ACTL6A to modulate chromatin remod- BAF-associated that is highly expressed in proliferating elling activity, allowing the transcription of late differentiation epithelial precursors [8]. However, the mechanism through which genes. ACTL6A inhibits DNA targeting of the BAF complex is not known, nor is the mechanism to drive ACTL6A down-regulation/inactiva- Keywords ACTL6A/BAF complex; epidermis; keratinocyte; lncRNA tion in epithelial progenitors to allow differentiation. Subject Categories Chromatin, Transcription & Genomics; Development; In the last decade, long non-coding RNAs (lncRNAs) have gener- RNA Biology ated significant interest in controlling progenitor stem cell biology DOI 10.15252/embr.201846734 | Received 13 July 2018 | Revised 23 December and somatic lineage specification and differentiation [16,17]. So far, 2019 | Accepted 8 January 2020 only few lncRNAs have been described to play a role in controlling EMBO Reports (2020)e46734 the proliferation/differentiation switch in keratinocytes: ANCR, anti- differentiation ncRNA, and TINCR, terminal differentiation-induced ncRNA. Although TINCR controls differentiation via a post-tran- scriptional mechanism, the ANCR-mediated mechanism to promote

1 Department of Experimental Medicine, University of Rome “Tor Vergata”, Rome, Italy 2 MRC Toxicology Unit, University of Cambridge, Cambridge, UK 3 IDI-IRCCS, Rome, Italy 4 Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona, Spain 5 Universitat Pompeu Fabra (UPF), Barcelona, Spain 6 Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 7 The Center for RNA Interference and Non-coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX, USA *Corresponding author. Tel: +39 06 7259 6686; E-mail: [email protected] **Corresponding author. Tel: +39 06 7259 6487; E-mail: [email protected]

ª 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license EMBO reports e46734 | 2020 1 of 14 EMBO reports Emanuele Panatta et al

the undifferentiated cell state in the epidermis has not been identi- supra-basal nuclei (Fig 1D). HEKn FISH analysis (Fig 1E) and fied [18,19]. Similarly, LIN00941 has also been recently implicated subcellular fractionation (Fig 1F, fold change 112 29, P < 0.05) in controlling epidermal homeostasis repressing the expression of showed that it is mainly found in the nuclei of keratinocytes, pro-differentiation genes with an not-yet identified mechanisms indicating a nuclear function of this pro-differentiation lncRNA. [20]. Through a bioinformatics analysis comparing mouse, rat and Notably, the uc.291 transcript has the same orientation of its human genomes, a specific sub-class of lncRNAs containing ultra- hosted gene, and it is expressed approximately eightfold higher conserved regions (UCRs) was identified [21–23]. LncRNA- than the antisense transcript (that is the limit of detectability; containing UC regions are transcribed from 481 genomic loci that Figs 1G and H, and EV2A–C); therefore, siRNAs were designed to contain 200–779 bases of highly conserved sequences amongst the specifically target the sense uc.291 transcript. Uc.291 is located three species mentioned before [21], with 100% identity (no inser- on 10 (hg38, chr10:76,523,866–76,524,096) and is tions or deletions). Interestingly, under genomic instability condi- hosted in the sixth intron of the LRMDA gene (previously named tions such as cancer, these UC sequences accumulate mutations C10orf11, Fig 1H). During keratinocyte differentiation, the expres- [24]. A large fraction of these 481 genomic loci are transcribed (T- sion of the LRMDA flanking exons (exon 2–3 and exon 6–7) by UCRs) in normal cells and tissues; some of them are ubiquitously RT–qPCR (Fig 1I) decreases, indicating that uc.291 is transcribed expressed, whereas others follow tissue-specific expression patterns independently from LRMDA mRNA. Originally, only the ultracon- [25–29]. T-UCR functions are largely unknown; the fact that they served sequence (231 bp in Ref. [21]; 424 bp in UCNEbase)of contain a highly conserved region implies that they are important uc.291 was known (Fig 1H); to gain additional information on for mammalian phylogenesis and/or ontogenesis [21]. the transcript, we performed a “cDNA walking” approach using Here, starting from a genome-wide expression profiling, we multiple primer amplifications and cloned a transcript of 3,816 nt demonstrated for the first time a functional link between UC- (nucleotides) that included the original UCR (Appendix Figs S1 containing lncRNAs (T-UCRs) and the switch between the undif- and S2). ferentiated state and the terminal-differentiated state in the epider- mis. We found that uc.291 physically interacts with ACTL6A Silencing of uc.291 alters epidermal proliferation modulating chromatin remodelling to allow differentiation. Chro- and differentiation matin immunoprecipitation (ChIP) experiments show that silencing uc.291 preserves the ACTL6A binding to differentiation gene The biological function of uc.291 was assessed by RNA interfer- promoters and possibly inhibits the BAF complex from targeting ence in primary human keratinocytes growing in differentiating epidermal differentiation complex (EDC) genes; conversely, in the conditions. SiRNA sequence specificity was validated both in presence of uc.291, ACTL6A is released, allowing chromatin HEKn and in FaDu cancer cells (Fig 1J). FISH experiments changes to promote the expression of epithelial differentiation performed after si-uc.291 did not detect signals in contrast to genes. scramble transfected cells, further confirming siRNAs specificity for uc.291 (Fig 1K). Silencing of uc.291 during differentiation strongly impaired this Results process. This was evaluated by RT–qPCR (Fig 2A) and by Western blot (WB) analysis of differentiation genes (Fig 2B). Notably, the Uc.291 expression changes during epidermal differentiation late differentiation marker Loricrin was strongly reduced at both mRNA and protein levels, while the early differentiation marker To investigate the role of UC-containing lncRNAs (T-UCRs) in Keratin 10 (K10) was not affected by uc.291 expression (Fig 2B), progenitor/undifferentiated and differentiated keratinocytes thereby suggesting an important role in the late differentiation steps. (HEKn, human epidermal keratinocytes, neonatal), we performed Interestingly, uc.291 is a pro-differentiation transcript independently a microarray in which we compared T-UCR expression profiles of from the differentiation stimuli used in vitro; beside calcium- undifferentiated proliferating keratinocytes with those of differenti- induced differentiation (Figs 1B and EV3A, CaCl2), uc.291 is up- ated keratinocytes (Fig 1A). We found 79 T-UCRs that are signifi- regulated even with confluence-induced differentiation in both cantly modulated during the differentiation (P < 0.05, 22 up- EpiLife medium and Eagle’s minimum essential medium (Fig EV3A, regulated; 57 down-regulated) (Fig EV1A; Appendix Table S1). Epi and EMEM). Uc.291 knock-down in organotypic human epider- After RT–qPCR validation of 20 T-UCRs (Fig EV1B and C), we mal tissue, a setting that recapitulates the structure and the gene selected uc.291 (fold change 3.8 0.9) for functional studies, expression profile of the human epidermis [30], confirmed the data taking into account that uc.291 was consistently up-regulated shown thus far. Although the uc.291-deficient epidermis was during keratinocyte differentiation also in different human kerati- normally stratified, the expression of key differentiation genes, nocyte donors (Fig 1B); it has also been detected in many human including Loricrin and Filaggrin, was markedly reduced, while K10 epithelial tissues [25]. Uc.291 gradually accumulates during HEKn did not change (Fig 2C). In uc.291-depleted human organotypic calcium-induced differentiation, increasing by 2.2-, 7.5- and 8.5- epidermis, the expression of the proliferation marker Ki67 increased fold over the control after 3, 6 and 9 days of treatment, respec- from 18.9 to 23.1% (P < 0.05) and 24.8% (P < 0.01) in scramble tively (P < 0.05, P < 0.01; Fig 1B). Its expression paralleled the and si-uc.291 sequences 1 and 2, respectively (Fig EV3B). Similarly, expression of early (IVL, KRT10) and late (LOR) differentiation p63-positive keratinocytes increased from 64.2 to 70.6% (P < 0.05) markers (Fig 1C). These in vitro results were confirmed by in situ and 72.2% (P < 0.01) in the same conditions, respectively (Fig 2D hybridization of the human epidermis, showing uc.291 accumula- and E), while we detected a decrease of the cyclin-dependent kinase tion in the epidermis supra-basal layers and a strong staining in inhibitor 1B (p27Kip1) and a higher expression level of DNp63 in

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A Microarray: Validation by Functional studies: 79 T-UCRs modulated in RT-qPCR: • Biological effect HEKn differentiation 10/22 T-UCRs up • Mechanism of action (22 up, 57 down) 10/57 T-UCRs down

undifferentiated Uc.183, Uc.257, Uc.338, Uc.291 vs Uc.63, Uc.291,... differentiated

B C IVL KRT10 LOR D H&E uc.291 NC ** * * * 15 * 120 16 90 100 tnauQ 12 10 80 60

Quant 60 8 epid epid epid Rel Rel 5 40 30 4 ACTB 20 0 0 0 0 0369DD 0369DD 0369DD 0369DD dermis dermis dermis

E Differentiating keratinocytes F uc.291 snRNA U6 ACTB G * ** ** uc.291 (RNA FISH) 150 140 1,2 100 #1 DAPI #2 120 1 80 100 100 0,8 80 60 0,6 60 40 Rel Quant 50 40 0,4 20 20 0,2 0 0 0 % of RNA expression 0 Cyt. Nuc. Cyt. Nuc. Cyt. Nuc. SS AS

H LRMDA I uc.291 Exons 2-3 Exons 6-7

1 2 3 4 5 6 7 10q22

16 ** 1,2 * 1,4 * uc.291 ultra-conserved 14 1,2 LRMDA intron 6 1 region 12 1 10 0,8 Quant 0,8 8 0,6 scale: 231 bp 3816 bp

Rel 0,6 6 Chr10: 0,4 0,4 7.38 4 0,2 Cons. 100 verts 2 0,2 0 0 0 -0.61 0369DD 0369DD 0369DD

uc.291 J uc.291 K SCR si-uc.291 (2) si-uc.291 (1) 1,2 * HEKn FaDu 1,2 1,2 1 * * 1 1 0,8

nauQ 0,8 0,8 0,6 0,6 0,6 Rel Quant 0,4

Rel0,4 t 0,4 0,2 0,2 0,2 0 0 0

uc.291 (RNA FISH) DAPI

Figure 1.

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◀ Figure 1. Uc.291 expression is induced during epidermal differentiation. A Workflow of the study. B Relative uc.291 expression quantification during calcium-induced in vitro differentiation of HEKn (3, 6 and 9 days of differentiation, DD). The quantification is relative to uc.291 expression in proliferating HEKn (0 day). Data shown represent the mean standard deviation (s.d.); n = 4 different human donors; *P < 0.05,**P < 0.01, Student’s t-test. C Involucrin (IVL), Keratin 10 (KRT10) and Loricrin (LOR) expression levels evaluated by RT–qPCR are shown as positive control of differentiation. The quantification is relative to gene expression in proliferating HEKn (0 day). Data shown represent the mean s.d.; n = 3 technical on 1 of the 4 human donors used in panel (B); *P < 0.05, Student’s t-test. D Haematoxylin/eosin (H&E) staining and in situ hybridization of specific (uc.291) or not specific (negative control, NC) probes on human skin section. Scale bars denote 50 and 500 lm. E RNA FISH for uc.291 (red, Quasar 570) on HEKn at 9 days of differentiation. Nuclei were stained with DAPI. Scale bar: 10 lm. F Relative uc.291 quantification after RNA isolation from cytosolic and nuclear cellular compartments. snRNA U6 quantification was used as nuclear fraction positive control, while ACTB as cytosolic fraction positive control. Data shown are mean s.d.; n = 3 technical on 1 of the 4 human donors used in panel (B); *P < 0.05, **P < 0.01, Student’s t-test. G Strand-specific RT–qPCR shows that uc.291 is transcribed in genomic sense orientation in HEKn cells; n = 2 technical (#1 and #2)on1 human donor; SS: sense strand transcript, AS: antisense strand transcript. H Picture showing uc.291 and LRMDA genomic location. I Expression of uc.291 and LRMDA exons 2-3 and exons 6–7 during keratinocyte differentiation. Data shown are mean s.d.; n = 2 different donors (3 technical/donor); *P < 0.05,**P < 0.01, Student’s t-test. J Relative uc.291 quantification after si-uc.291(1) and si-uc.291(2) in HEKn and FaDu cells. Data shown are mean s.d.; HEKn n = 3 technical on 1 human donor; FaDu n = 2 biological replicates (3 technical/biological replicate); *P < 0.05, Student’s t-test. K RNA FISH for uc.291 (red, Quasar 570)onA253 in scramble (SCR) and silenced (si-uc.291(1) and si-uc.291(2)) cells. Nuclei were stained with DAPI. Scale bar: 10 lM. The histogram derives from the RT–qPCR using the leftover cells after removing the slides used for the FISH staining. Data shown are mean s.d; n = 3 biological replicates *P < 0.05, Student’s t-test.

si-uc.291 cells grown in both proliferating and differentiating condi- Uc.291 interacts with ACTL6A tions (Fig 2F). Altogether, these results strongly indicate that uc.291 is required The effect of uc.291 depletion on the expression of EDC genes to allow the expression of terminal differentiation genes and that suggested that uc.291 could interfere with epigenetic modulators alteration of the late differentiation steps also affects the proliferat- that control gene expression during differentiation. To determine ing keratinocyte compartment (Fig 2G). the mechanisms of uc.291 action, we aimed to identify uc.291 binding of relevance to epidermal differentiation. Uc.291 Silencing of uc.291 disrupts the expression of genes involved in sense and antisense (the latter as control) RNA was transcribed epidermal differentiation with Cy5 and independently hybridized to a protein microarray (HuProt v.2 Human Proteome Arrays) containing 50,000 recombi- To gain further information on the role of uc.291 during differentia- nant human proteins (Fig EV4A and B). We performed two repli- tion, transcriptomic profiling of uc.291-depleted differentiating kera- cates, obtaining a correlation of 0.79 for RNA-binding protein tinocytes was performed. We demonstrated that the down- (RBP) interactions and 0.76 for chromatin-related proteins regulation of uc.291 affected the expression of 596 genes (P < 0.05), (Fig EV4C). Using the fluorescence over background (F-B) average 203 of which were up-regulated and 393 were down-regulated calculated for both replicates, we selected uc.291 interactions with (Fig 3A–C; Dataset EV1). Uc.291-regulated genes were enriched for a positive score (15,400; Fig EV4B). The filtering computational skin, epidermis, and epithelia development-, epidermal cell differen- procedure narrowed down the list. To investigate the specific link tiation- and keratinization-related (GO) terms between uc.291 and chromatin modification, we restricted our (Fig 3D and E). The latter is in line with the phenotype of the analysis to chromatin-related proteins (QuickGO and UniProt), uc.291-depleted epidermis (Fig 2). Interestingly, amongst the down- resulting in 40 candidates (Appendix Table S2; Fig EV4D), 20 of regulated genes, 23 belonged to the EDC (Fig 3F and G). The EDC which are epidermis-expressed proteins (Fig 4A and B; comprises several genes that are of crucial importance for the matu- Appendix Table S2). We found that ACTL6A is the 3rd ranked ration/keratinization of the human epidermis and are located in a candidate out of 20 proteins, preceded by BATF2 and SMARCE1. 2 Mb region on human chromosome 1q21 [31,32]. The EDC ACTL6A is a BAF-associated protein and is highly expressed in contains three clustered families of genes that encode a group of proliferating epithelial precursors [8]; it was recently shown to precursor proteins of the cornified envelope (CE), including involu- block the BAF pro-differentiation role that specifically affects EDC crin, Loricrin, SPRRs (small proline-rich proteins) and the “late gene expression [11,15]. Uc.291 and ACTL6A reciprocal binding cornified envelope” (LCE) proteins; calcium-binding proteins was demonstrated by RNA cross-linking immunoprecipitation (S100); and the “fused gene” proteins (SFTPs), including Filaggrin, (CLIP) and by RNA immunoprecipitation (RIP) experiments Filaggrin-2, trichohyalin, trichohyalin-like protein, hornerin, repetin (Fig 4C and D) using a specific anti-ACTL6A antibody. We added and cornulin (Fig 3F and G) [33]. RT–qPCR was used to validate the SMARCC2, another subunit of the complex, and found that it down-regulation of EDC genes (Fig 3H); we also confirmed their interacts with uc.291 by CLIP. As negative control, we tested a down-regulation in the uc.291-depleted epidermis (Fig 3I). These nuclear unrelated transcript FAM83-AS1 [8]. FAM83-AS1 is results confirmed that uc.291 is important for regulating gene expressed in both proliferating and differentiating keratinocytes, expression during differentiation, including the expression of many and RIP experiment showed a negative result confirming that, genes located in the EDC, including LOR. unlike uc.291, FAM83-AS1 does not interact with ACTL6A

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Figure 2.

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◀ Figure 2. Silencing of Uc.291 affects epidermal differentiation. A Relative quantification of the differentiation markers LOR and IVL in HEKn transfected with uc.291 siRNA (1) and (2) or SCR control siRNA and collected after 3 days of differentiation. Data shown represent the mean s.d.; n = 4 different human donors; **P < 0.01, Student’s t-test. B WB showing DNp63a, Klf4,K10 and Loricrin (LOR) protein levels in HEKn transfected cells as described in (A). b-actin was shown as loading control. One representative experiment of three is shown. C IF staining of late differentiation markers Loricrin and Filaggrin in uc.291-depleted, si-uc.291(1) and si-uc.291(2), organotypic human epidermis compared to scramble control one (SCR); haematoxylin/eosin (H&E) staining shows the organotypic epidermis structure (left panels); scale bars: 50 lm. The magnified boxes show expression of Loricrin and Filaggrin in the granular layer; scale bars: 10 lm. Dotted lines represent the insert border. One representative experiment of three is shown. D IF staining and confocal analysis of p63 in the organotypic human epidermis obtained as described in (C). Dotted lines represent the insert border. One representative experiment of three is shown. Scale bars: 50lm. E Box plot showing quantification of p63-positive nuclei on total nuclei. Each central line represents the median; each box represents Q1 (quartile 1, below the median) and Q3 (quartile 3, above the median); the whiskers represent minimum (Q1 1.5*IQR) and maximum (Q3 + 1.5*IQR). IQR = interquartile range; 16, 15 and 19 images were analysed for SCR, si-uc.291 (1) and si-uc.291 (2) respectively; *P < 0.05;**P < 0.01, Student’s t-test. F WB showing cyclin-dependent kinase inhibitor 1B(p27Kip1) and DNp63a protein levels in HEKn cells transfected with uc.291 siRNA (2) or SCR control siRNA and collected in proliferating (0 day) or differentiating (1, 2 and 3 days of differentiation, DD) conditions. b-actin was used as loading control. The numbers below the bands represent the densitometric analysis normalized for the loading control. One representative experiment of three is shown. G Schematic representation of the expression of selected markers in organotypic human epidermis upon uc.291 depletion. Source data are available online for this figure.

(Fig EV5A and B). The predicted uc.291 region interacting with D). Interestingly, the depletion of uc.291 in differentiating condi- ACTL6A (score = 0.59, prediction by Global Score [34]) results in tions resulted in enhanced ACTL6A binding and reduced BRM/ a highly structured region as shown by the CROSS algorithm BRG1 binding (Fig 5B, si-uc.291). Consequently, we detected a modelling [35] (Fig 4E and F; Appendix Fig S2). Our results indi- decrease of histone modification in H3K27ac (Fig 5D) and the cate that uc.291 interacts with ACTL6A to modulate chromatin decreased expression of LOR, FLG and LCE1B at the mRNA levels remodelling. and LOR at the protein level (Fig 5C and E). Controls for an uc.291-unrelated gene (TATA-box-binding protein, TBP) are shown Silencing of uc.291 affects the chromatin status of Loricrin, in Fig EV5C. The role of ACTL6A in repressing differentiation gene Filaggrin and LCE1B promoters expression is confirmed in Fig 5C and D, in which the depletion of ACTL6A resulted in increased LOR, FLG and LCE1B at the mRNA To demonstrate that uc.291 and ACTL6A interaction has a func- levels and LOR at the protein level. To note that uc.291 silencing tional role in controlling the expression of differentiation genes, does not affect ACTL6A expression and that ACTL6A and uc.291 we performed a chromatin immunoprecipitation (ChIP) assay on are partially co-expressed as indicated in in vivo calcium-induced selected differentiation gene promoters (LOR, FLG and LCE1B) that differentiation and in organotypic human epidermis (Appendix Fig were down-regulated upon uc.291 silencing (Figs 2 and 3), to S3A and B). Altogether, these results indicate that uc.291, by inter- examine ACTL6A and BRM/BRG1 binding as a function of uc.291. fering with ACTL6A, participates in the release of ACTL6A inhibi- We confirmed that in proliferating conditions, ACTL6A binds LOR, tory effects on the BAF complex, thus allowing it to bind to and FLG and LCE1B promoters to block the binding of BRM/BRG1, activate differentiated genes. To further confirm our findings, we which are the BAF catalytic subunits [8] (Fig 5A). This is in line compared transcript profiling between ACTL6A- and si-uc.291-defi- with previous results showing that BRM/BRG1 (BAF complex) are cient keratinocytes (Fig 6A). We found that transcript profiling of required to bind and activate differentiation gene promoters. This ACTL6A-deficient keratinocytes [8] showed an overlap with uc.291 binding is repressed by ACTL6A in progenitors and proliferating regulated genes (39 genes). In particular, we found that the genes keratinocytes [8,9]. Instead, differentiated keratinocytes displayed repressed by ACTL6A in proliferating keratinocytes are also down- enhanced binding of BRM/BRG1 at the promoter of the selected regulated by si-uc.291 in differentiating conditions, further support- genes, whereas ACTL6A did not bind them (Fig 5B, SCR). BRM/ ing the observation that the interaction between uc.291 and BRG1 binding paralleled the H3K27ac histone modification and ACTL6A is required to allow the expression of epidermal differenti- LOR, FLG and LCE1B expression at the mRNA levels (Fig 5C and ation genes.

Figure 3. Uc.291 regulates the expression of epidermal differentiation genes, including the epidermal differentiation complex (EDC) genes. ▸ A Heatmap showing gene expression profile of HEKn silenced by either SCR or si-uc.291 (2) siRNAs by gene microarray analysis; n = 4 technical replicates using 1 human donor; P < 0.05, Student’s t-test, abs (FC) > 2. BRT–qPCR confirming the efficiency of silencing. Data shown are mean s.d; the same samples of (A) were used; **P < 0.01, Student’s t-test. C Volcano plot showing fold changes of differently expressed genes from (A): up-regulated genes are shown in red, and down-regulated genes are shown in dark grey. D GO terms analysis for genes down-regulated upon uc.291 depletion from (A). E GSEA comparing a query gene set of modulated genes from (A) with GO biological process signatures associated with epidermis development and keratinocyte differentiation. NES = normalized enrichment score, FDR = false discovery rate. Kolmogorov–Smirnov statistic was used. F Heatmap showing gene expression of the most down-regulated genes located within epidermal differential complex (EDC) upon depletion of uc.291 from (A). G Scheme of the epidermal differentiation complex (EDC) genes. H Validation of array data by RT–qPCR showing mRNA quantification of thirteen EDC genes down-regulated upon si-uc.291. Data shown are mean s.d.; (n = 3 different human donors); **P < 0.01, Student’s t-test). I Validation of array data by RT–qPCR showing mRNA quantification of eight EDC genes down-regulated in uc.291-depleted organotypic human epidermis; n = 2 technical (#1 and #2) from 1 human donor.

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uc.291 ABCHEKn diff (3 days) 7 F epidermal differentiation complex ** SCR si-uc.291 1,2 6 SCR si-uc.291 log2(FC) 1,0 5 HRNR -3.00 0,8 ( P ) IVL -1.20 10

etaluger 4 0,6 LCE1B -3.86 3 LCE1C -2.94 0,4 -log LCE1E -2.83

Rel Quant 2 0,2 LCE2A -2.25 1 -2.93 pu 30 pu 0,0 LCE2B -6 -4 -2 0 2 4 6 LCE2C -2.67 LCE3A -4.21 log2(FC) 2- d LCE3B -3.40 -log10(FDR) LCE3C -3.46 D tissue development 7.6 LCE3D -2.43 cornification 10.4 LCE3E -3.01 epithelium development 10.3 LCE5A -1.79 epithelial cell differentiation 13.5 LCE6A -2.84 epidermis development 16.7 LOR -1.93 peptide cross-linking 18.3 SPRR2A -1.60 -2.81 etaluger epidermal cell differentiation 18.7 SPRR2B skin development 18.6 SPRR2G -2.40 keratinization 18.7 SPRR3 -1.96 keratinocyte differentiation 20.2 FLG -2.67 KPRP -3.74 nwod 39 nwod 0 5 10 15 20 25 PRR9 -3.21 -log10(P) log2 expression level E GO biological process: GO biological process:

3-d epidermis development keratinocyte differentiation -0.84 +6.50 NES -1.804 NES -1.881 G P < 0.001 P < 0.001 epidermal differentiation complex FDR 0.038 FDR 0.030 S100 SPRR LCE S100 family family family family 1q21 log2 expression level 0.3 Mb -5.2 +6.6 up down up down LOR IVL FLG

H ** ** ** ** ** ** ** ** ** ** ** ** ** 1 nauQ

0,5 Rel t

0 LCE1B LCE1C LCE2B LCE2D LCE3A LCE3C LCE6A LOR SPRR2B KPRP HRNR IVL FLG SCR si-uc.291

I 1 nauQ

0,5 Rel t

0 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 LCE1B LCE2D LCE3A LCE6A LOR HRNR IVL FLG SCR si-uc.291 (1) si-uc.291 (2)

Figure 3.

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A B GO of candidate interactors C17orf49 CHCHD2 CPA4 chromatin assembly or disassembly ACTL6A GATA3 BATF2 nucleosome organization SMARCC2 SMARCE1 histone methylation uc.291 histone acetylation VEGFA TRIM16 positive regulation of DNA repair CENPA PRMT3 DEK CRTC2 ATP-dependent

HIST1H1C ATXN7L3 BMI1 regulation of histone acetylation 012345 PRMT6 PRKD2 PHLDB1 -log10 (P) C uc.291 D uc.291 IP 16 16 * * 14 14 IP 12 12 nauQ 10 10 kDa 8 8 * kDa 170 - ACTL6A 6 SMARCC2 Rel Quant 6 Rel4 t 4 50 2 50 - ACTL6A 2 0 0

EF ACTL6A CROSS score 0.2 -0.1 0.0 0.1 0.2 0.3 -0.3 - 0 1000 2000 3000 4000 Uc.291 sequence (nucleotides)

Figure 4. Uc.291 interacts with ACTL6A. A Network of putative interactors, ranked as described in the text, and relative GO analysis. Interactors expressed in the epidermis are shown; see also Appendix Table S1. B GO analysis of the predicted interactors. CRT–qPCR showing the validation of uc.291-ACTL6A interaction by RNA cross-linking immunoprecipitation (CLIP) in HEK293E cells, as fold enrichment over IgG. SMARCC2 was also included. Data shown are mean s.d.; n = 3 (technical); *P < 0.05, Student’s t-test. On the right, control WB for immunoprecipitation of both antibodies. DRT–qPCR showing the validation of uc.291-ACTL6A interaction by RNA immunoprecipitation (RIP) in differentiated HEKn as fold enrichment over IgG. Data shown are mean s.d.; n = 3 technical, on 1 human donor; *P < 0.05, Student’s t-test. On the right, control WB for immunoprecipitation. E Secondary structure of uc.291 predicted binding region (nt 1,700–2,000) using the CROSS Global Score as a soft constraint inside RNA structure [43]. F Uc.291 predicted binding region to interact with ACTL6A (nucleotide 1,763–1,912) by Global Score (score of 0.59). Dotted line defines the CROSS score threshold [43]. Source data are available online for this figure.

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C

D E

Figure 5. Uc.291-ACTL6A interaction modifies chromatin status at LOR, FLG and LCE1B promoters. A Agarose gel electrophoresis of LOR, FLG and LCE1B promoter fragments amplified by end-point PCR after ACTL6A and BRM (left), or BRG1 (right) chromatin IP in proliferating keratinocytes. B Agarose gel electrophoresis of the same PCR products described in (A) after ACTL6A (left), BRM (middle) and BRG1 (right) chromatin IP in uc.291 silenced and differentiated HEKn cells. One representative experiment of three is shown. C Relative quantification by RT–qPCR of LOR, FLG and LCE1B mRNA in uc.291 and ACTL6A silenced and differentiated HEKn cells. Data shown are mean s.d.; n = 3 (technical); *P < 0.05, Student’s t-test. D Quantification by real-time PCR of LOR, FLG and LCE1B promoter fragments after H3K27ac chromatin IP in the same samples described in (B). Data shown are mean s.d.; n = 3 (technical); *P < 0.05, Student’s t-test. E WB analysis of Loricrin expression in uc.291 and ACTL6A silenced and differentiated HEKn cells. b-actin was used as loading control. One representative experiment of three is shown. Source data are available online for this figure.

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A unexplored possibility that uc.291 could also play a role in head and neck squamous cell carcinomas. Many aspects regarding ACTL6A-BAF complex regulation remain unknown, including the mechanistic action that the actin-like proteins employed on the BAF complexes and how ACTL6A inhibits/interferes with DNA targeting of the BAF complex in proliferating/progenitor cells; our data provide some interesting light. Here, we identified a novel mechanism involving a long non-coding RNA that contains an ultraconserved element, named uc.291. Uc.291 participates in the establishment of cell-specific epigenetic changes. Its expression increases during differentiation; by physically interacting with ACTL6A, it releases the ACTL6A-mediated repression of differenti- ation genes (Fig 6B). Following uc.291-ACTL6A interaction, the BRM/BRG1 subunit (BAF complex) is able to interact with dif- ferentiation promoters and releases chromatin to allow the tran- B scription of EDC genes (Fig 6B). Interestingly, in addition to ACTL6A, amongst the putative uc.291 interactors, we identified other chromatin-related subunits belonging to the BAF (SWI/SNF) complex, such as SMARCE1 (chromatin remodelling complex BRG1-associated factor 57), SMARCC2 (BAF170 or mammalian chromatin remodelling complex BRG1-associated factor 170) and SMARCD3 (BAF60C or SWI/SNF-related matrix-associated actin- dependent regulator of chromatin subfamily D member 3). Beside ACTL6A, we validated uc.291-SMARCC2 interaction by CLIP, yet we do not have evidences that this is a direct interaction. These findings further support that amongst the chromatin-associated epigenetic modulators, uc.291 is strongly associated with ATP- dependent nucleosome remodelling complexes. However, other Figure 6. Uc.291 and ACTL6A regulate common genes. chromatin modulators, i.e. BMI1 (component of the polycomb A Venn diagram showing the uc.291 up-regulated genes in si-uc.291 group complex 1) and SIRT1 (NAD-dependent protein deacety- keratinocytes in common with the si-ACTL6A down-regulated genes. The 39 lase), are also putative interactors. The protein microarray also common genes are listed below, and the EDC genes are indicated. The shows that transcription factors and transcription co-activators expression of the genes is inversely correlated. The inverse correlation including BATF2, GATA2, GATA3 and CRTC2 interact with amongst them functionally supports the negative regulation of uc.291 in controlling ACTL6A functions. uc.291 with a high score (Appendix Table S2). Altogether, these B Schematic model of uc.291 role in regulating epidermal differentiation. results suggest that uc.291 may be involved in gene expression regulation with additional mechanisms, yet to be identified. An intriguing issue is the function of the ultraconserved sequence Discussion contained in uc.291 (nt 717–1,140). We modelled the secondary structure of this region using CROSS [35] and found that is highly Many studies have indicated a critical role of chromatin regula- structured. Amongst the 40 interactors identified, none have been tors in controlling somatic stem cell fate and epithelial differentia- predicted to interact with this region, opening new interesting tion [2–6,8]. Amongst them, the BAF (SWI/SNF, switch/sucrose scenarios leading to the investigation of other possible functions. non-fermenting) complex has been shown to have a crucial role Altogether, our results describe a human terminal differentiation- for proper execution of the epidermal differentiation programme, associated lncRNA that interacts with an ATP-dependent nucleo- in which it regulates the transition from the epidermal progenitor some remodelling complex subunit to exert a non-redundant role in state (proliferating basal layer keratinocytes) to the epidermal dif- controlling somatic differentiation and tissue homeostasis. Our ferentiated state (spinous, granular and cornified layers keratino- study expands the understanding of the complexity of the epigenetic cytes) [9,11]. Recently, ACTL6A, which is a part of several regulation that occurs during epithelial differentiation, necessary to epigenetic regulator complexes [36], has been shown to play a balance progenitor compartment and terminally differentiated cells. crucial role in modulating the BAF complex to block keratinocyte differentiation [8]. ACTL6A prevents the BAF complex from bind- ing to promoters of differentiation genes [37]. Indeed, in vivo Materials and Methods ablation of ACTL6A resulted in decreased proliferation, acceler- ated differentiation and skin hypoplasia, whereas over-expression Cell culture and transfection resulted in the expansion of the proliferation compartment. ACTL6A is also amplified and highly expressed together with Human epidermal keratinocytes, neonatal (HEKn) (Cascade, DNp63; it is physically associated with DNp63 in squamous cell Invitrogen) were grown in EpiLife medium supplemented with carcinomas of the head and neck [38–42]. This opens up an HKGS (Invitrogen). Differentiation was induced by 1.2 mM

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CaCl2 addiction to culture medium. HEKn were transfected (G2505B) using one-colour scan setting (scan area 61 × 21.6 mm, using two different si-uc.291 sequences. HEKn were transfected scan resolution 10 lm, dye channel is set to green and green PMT is using two different si-uc.291 sequences HP Custom siRNA (Qia- set to 100%). The scanned images were analysed with Feature gen) or a negative control siRNA (Qiagen) by Lipofectamine Extraction Software 10.3.7.1 (Agilent) using default parameters (pro- RNAiMAX reagent (Invitrogen) according to the manufacturer’s tocol GE1_107_Sep09 and Grid: 039494_D_F_20150612) to obtain protocols. Forty-eight hours after transfection, in vitro differenti- background subtracted and spatially detrended Processed Signal ation was induced. Specific uc.291 siRNA sequences are as intensities. Features flagged in Feature Extraction as feature non- follows: si-uc.291 (i) 50-GUAUUGAUUAAACUUUUAAAUTT-30 uniform outliers were excluded. Microarray data have been depos- and si-uc.291 (ii) 50-ACAGG CUGAGAACAGAUTT-30. HEK293E ited in the NCBI’s Gene Expression Omnibus [GEO: GSE103890]. cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher) supplemented with 10% (vol/vol) foetal cDNA walking bovine serum (Thermo Fisher) and 1% penicillin/streptomycin (Thermo Fisher). HEK293E cells were transfected to express Two lg of total RNA from differentiated HEKn was retrotranscribed full-length TUC-291 lncRNA using Effectene Reagent (Qiagen) as described before to obtain cDNA. PCR was performed using according to the manufacturer protocol. FaDu and A253 cells Phusion High-Fidelity DNA Polymerase (Thermo Scientific) accord- were grown, respectively, in Eagle’s minimum essential medium ing to the manufacturer’s protocol, and the PCR fragments of inter- and McCoy’s 5A medium supplemented with 10% (vol/vol) est were completely sequenced. The primers used are listed in foetal bovine serum and 1% penicillin/streptomycin. FaDu and Appendix Table S3. A253 were transfected using two different si-uc.291 sequences HP Custom siRNA (Qiagen) or a negative control siRNA (Qia- Organotypic epidermis culture gen) by Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocols. After 48-h transfection, 3 × 105 HEKn cells were resuspended in CnT-PR medium (CELLnTEC) and seeded onto Millicell PCF 12 mm Cell fractionation and RNA extraction inserts with 0.4 lm pore size (PIHP01250; Millipore). Twenty-four hours later, medium was replaced inside and outside the insert with Cells were resuspended in 1 ml of RSB (10 mM Tris, pH 7.4, 10 mM CnT-PR-3D medium (CELLnTEC). Twenty-four hours later, air-

NaCl, 3 mM MgCl2), incubated for 3 min on ice and centrifuged at lifting was induced by removing the medium inside. Outside, 4°C. Cell pellet was resuspended in four volumes of lysis buffer medium was changed every 3 days during the following of 20 days

RSBG40 (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 10% glyc- of culture. Inserts were fixed in 10% buffered formalin solution, erol, 0.5% Nonidet P-40, 0.5 mM DTT and 100 U/ml rRNasin processed and paraffin-embedded. Promega, WI). Nuclei were pelleted by centrifugation at 4,600 g for 3 min, and the supernatant was saved as the cytoplasmic fraction. In situ hybridization Nuclear pellets were resuspended in RSBG40, and one-tenth volume of detergent buffer was added (3.3% wt/wt sodium deoxycholate Human tissues were embedded in frozen specimen medium Killik and 6.6% vol/vol Tween 40). Nuclei were incubated 5 min on ice (Bio-Optica) after an overnight incubation in 4% paraformaldehyde and pelleted. Supernatant was saved and added to cytoplasmic frac- followed by an overnight incubation in 0.5 M sucrose. 14-lm-thin tion. Nuclei were collected by centrifugation at 9,400 g for 5 min, sections were cut and mounted on Superfrost glass slides. Slides and the resulting pellet was used for nuclear RNA extraction. RNA were then fixed for 10 min in 4% paraformaldehyde and acetylated was extracted using TRIzol method, according to the manufacturer’s for 10 min in triethanolamine/acetic anhydride. Slides were then instructions (Invitrogen, CA). Cell lysis and nuclear integrity were hybridized in incubation chambers overnight at 46°C using 30 nM monitored during the procedure by light microscopy following detection digoxigenin probes (miRCURY LNA; Exiqon). After trypan blue staining. hybridization, slides were washed (20 min in 5 × SSC, two times for 30 min in Tween 20/SSC at 50°C, and twice for 15 min in Gene array 0.2 × SSC and 15 min in PBS at room temperature (RT)). After 1-h incubation in blocking solution at RT, slides were hybridized for 2 h Total RNA was extracted with mirVana miRNA isolation kit in alkaline phosphatase-conjugated antidigoxigenin Fab fragment (Ambion, AM1561). RNA quality was monitored by Agilent 2100 (1:200 dilution; Roche, 11093274910) at RT. After two washes of Bioanalyzer (Agilent Technologies, Santa Clara, CA). Cyanine-3 20 min, detection was performed by incubating 250 ll nitroblue (Cy3)-labelled cRNA was prepared from 100 ng RNA using the Low tetrazolium/BCIP (1-STEP; Thermo Fisher Scientific) together with Input Quick Amp Labeling Kit one-colour (Agilent) according to the 2 mM levamisole on the slides for 16 h in the dark at RT. manufacturer’s instructions and purified by RNeasy column (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were RNA FISH checked with the NanoDrop ND-1000 Spectrophotometer. 600 ng of Cy3-labelled cRNA (specific activity > 6.0 pmol Cy3/lg cRNA) was Custom Stellaris FISH Probes were designed against Uc. 291 fragmented at 60°C for 30 min following the manufacturer’s instruc- sequence by utilizing the Stellaris RNA FISH Probe Designer tions and then hybridized to Agilent SurePrint G3 Human 8 × 60k (Biosearch Technologies, Inc., Petaluma, CA) available online at Microarrays (G4858A) for 17 h at 65°C. Slides were washed and www.biosearchtech.com/stellarisdesigner. The fixed HEKn cells dried. Slides were scanned by Agilent DNA Microarray Scanner were hybridized with the Uc.291 Stellaris RNA FISH Probe set

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labelled with Quasar 570 dye (Biosearch Technologies, Inc.), follow- Only reactions with a labelling density of 1 Cy5 dye/700–900nt ing the manufacturer’s instructions available online at www.biosea were used in the following experiments. Labelled RNA integrity was rchtech.com/stellarisprotocols. verified with the Agilent 2100 Bioanalyzer. Fifty pmol of labelled RNA was hybridized in the protein arrays HuProt v.2 Human Immunofluorescence Proteome Arrays (Cambridge Arrays, LTD). Two technical replicates were performed. The arrays were dried and immediately scanned at Slides were incubated at 60°C for 1 h and dewaxed by Bio-Clear 635 nm in Microarray Scanner G2505B (Agilent). GenePix Pro 6.1 washing (Bio-Optica) and sections rehydrated by 100, 90, 80, 70 and software (Molecular Devices) was used to determine the signal at 50% ethanol incubations. Antigen retrieval was performed by boil- 635 nm of each spotted protein location and therefore quantify the ing in 0.01 M Sodium Citrate Buffer pH 6.0. IF staining was as RNA-protein interaction. Specifically, the local background intensity follows: permeabilization by Triton X-100 0.2% PBS incubation; 1-h (B635) was subtracted from the intensity (F635) at each of the dupli- blocking in 5% goat serum (Gibco) in PBS at RT; 3-h primary anti- cate spots for a given protein, to quantify. Data were filtered based body incubation at RT; and 1-h secondary antibody and DAPI (40,6- on signal to background ratio for each of the duplicate feature to be diamidino-2-phenylindole) incubation at RT. IF images were greater than 2.5-fold and Z-Score ≥ 3 from the global mean signal acquired by Nikon A1 confocal laser microscope. The following from all the spotted proteins. Finally, the intersection of technical antibodies were used: anti-K14 (Abcam, ab7800); anti-K14 (BioLe- replicates was considered as the final value for quantification. gend, PRB-155P); anti-FLG (Santa Cruz, SC66192); anti-LOR (BioLe- gend, PRB-145P); anti-Ki67 (Cell Signaling, D3B5, 9129S); anti-p63 Chromatin immunoprecipitation (Abcam, ab735); Alexa Fluor goat anti-rabbit 568 (Life Technolo- gies, A11011); goat anti-rabbit 488 (Life Technologies, A11034); ChIP was performed using MAGnify Chromatin Immunoprecipita- goat anti-mouse 568 (Life Technologies, A11019); and goat anti- tion System (Invitrogen) following the manufacturer’s protocol. mouse 488 (Life Technologies, A11017). Chromatin was sonicated at 25% amplitude for 15 min (20″ sonica- tion/30″ pause) using SONOPULS UW3100 (BANDELIN electron- Haematoxylin/eosin staining ics). End-point PCRs were performed using GoTaq G2 Flexi DNA Polymerase (Promega) according to the manufacturer’s protocol. Sections were dewaxed and rehydrated as described before, then Real-time PCR (Fig 5C) was performed using GoTaq qPCR Master incubated 5 min in Mayer’s haematoxylin (Bio-Optica) solution, Mix (Promega); the primers used are listed in Appendix Table S5. extensively washed in distilled water, incubated 5 min in Eosin Y The antibodies used were as follows: anti-ACTL6A (Cell Signaling, alcoholic solution (Bio-Optica), extensively washed in distilled 76682S); anti-SMARCC2 (Cell Signaling, D809V, 12760S); and anti- water and finally dehydrated by 70, 90, 100% ethanol solution incu- H3K27ac (Diagenode, C15410174). bation. Slides were mounted using Bio Mount HM (Bio-Optica). RNA immunoprecipitation Protein microarray RIP was performed using Magna RIP RNA-Binding Protein Immuno- Uc.291 (3,816 nucleotide sequence) sequence was cloned in pBSII- precipitation Kit (Millipore) according to the manufacturer’s proto- SK containing T7 promoter using the following primers: 50-GGCCG col. RNA (80 ng) was retrotranscribed using SuperScript VILO CGGCCGCCCTGGGC ACTTAGCTCTCCAC-30 and 50-GGCCCTCGAGC cDNA Synthesis Kit (Applied Biosystems) following the manufac- TTTACGTTTTAGGGTACACGTG-30. The plasmid was linearized and turer’s protocol. RT–qPCR was performed as described before. The purified with the MinElute PCR Purification Kit (Beckman Coulter) antibodies used were as follows: anti-ACTL6A (Cell Signaling, following the manufacturer’s instructions. In vitro transcription was 76682S) and anti-SMARC2 (Cell Signaling, D809V, 12760S). RNA/ performed with the T7 Megascript T7, High Yield Transcription Kit ChIP was performed using RNA ChIP-IT Kit (Active Motif) according (Thermo Scientific) according to the standard procedure with the to the manufacturer’s protocol. RNA was retrotranscribed using addition of 1% DMSO and 1% RiboLock, overnight at 37°C. The GoScript Reverse Transcription System (Promega) according to the synthetized RNA was treated with TURBO DNAse 2 U/ll (Invitro- manufacturer’s protocols. RT–qPCR was performed as described gen) at 37°C for 15 min, purified using magnetic beads (Agencourt before. The antibodies used were as follows: anti-ACTL6A (Cell RNAClean XP) and eluted in nuclease-free water. The integrity and Signaling, 76682S) and anti-SMARCC2 (Cell Signaling, D809V, specificity of the RNA were checked by RNA denaturing agarose gel 12760S). and Bioanalyzer quality control. Uc.291 was labelled with Cy5 Label IT uArray Labeling Kit (Mirus). Briefly, 5 lg of RNA was mixed with RNA extraction and RT–qPCR analysis 1:5 Label IT Cy5 reagent and incubated at 37°C for 70 min. The labelled RNA was purified by magnetic beads (Agencourt RNAClean Total RNA was isolated from HEKn by RNeasy Mini Kit (QIAGEN) XP). RNA concentration and labelling density were measured using a and retrotranscribed by GoScript Reverse Transcription System NanoDrop 1000 Spectrophotometer (Thermo Scientific) and calcu- (Promega) according to the manufacturer’s protocols. RT–qPCR was lated as follows: performed using GoTaq qPCR Master Mix (Promega). The primers used are listed in Appendix Table S4. The expression of each gene e e Base:dye = (Abase* dye)/(Adye* base) was defined from the threshold cycle (Ct), and the relative expres- DD Abase = A260-(Adye*CF260) sion levels were calculated by using the 2 Ct method after normal- Constants: e dye = 250,000; CF260 = 0.05; e base = 8,250. ization with reference to expression of housekeeping gene.

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Strand-specific retrotranscription was performed using GoScript characterization; EP and AS: si-uc.291 in differentiation, organotypic epidermis, Reverse Transcription System (Promega). Two reverse transcription si-uc.291 array, validation; XZ: in situ hybridization; TBO and RDP: protein reaction mix were made for strand-specific cDNA synthesis: one to array; and EP: ChIP and RNAIP); EC designed the research; EC, GM, GAC, RDP, detect uc.291 sense-oriented transcript (using the following reverse AMau and GGT analysed the data; EC wrote the paper; and all the authors primer, 50-AGTTGTTCCTAGTAAACCATTTTAC-30 and a reverse read the paper and made comments. primer for ACTB,50-AATGTCACGCACGATTTCCCG-30) and one to detect uc.291 antisense-oriented transcript (using the following Conflict of interest forward primer, 50-GGATGA AAAGATGGTGGACAAAC-30 and the Chanel Parfumeur was partially involved in preliminary experiments. However, same reverse primer for ACTB). qPCR was carried on by TaqMan the data included in this article are unrelated to Chanel Parfumeur. probe using the following primers/probe: uc.291FW 50-TTT GTGAGCCCCGCATTC-30; uc.291 REV 50-AATGACACGTCAGCCGT CTTT-30; and uc.291 probe, 50-ACTTTCCTTTGAGGTTGGT-30. References

Western blot 1. Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA (2010) DNMT1 main- tains progenitor function in self-renewing somatic tissue. Nature 463: Total cell extracts were resolved on a SDS–polyacrylamide gel and 563 – 567 blotted on Amersham Hybond P0.45 PVDF membrane (GE Healthcare 2. LeBoeuf M, Terrell A, Trivedi S, Sinha S, Epstein JA, Olson EN, Life Science). Membranes were blocked by non-fat dry milk Blotting- Morrisey EE, Millar SE (2010)Hdac1 and Hdac2 act redundantly to Grade Blocker (Bio-Rad) 5% in PBS 0.1% Tween-20 (Sigma-Aldrich), control p63 and p53 functions in epidermal progenitor cells. Dev incubated with primary antibodies overnight at 4°C, washed and Cell 19: 807 – 818 hybridized for 1 h at RT using the appropriate secondary antibody. 3. Dauber KL, Perdigoto CN, Valdes VJ, Santoriello FJ, Cohen I, Ezhkova E The following antibodies were used: anti-p27 (BD Biosciences, (2016) Dissecting the roles of polycomb repressive complex 2 subunits in 610241); anti-p63 (Abcam, ab735); anti-p63a (Cell Signaling, the control of skin development. J Invest Dermatol 136: 1647 – 1655 CS13109); anti-KLF4 (R&D System, 12173S); anti-K10 (BioLegend, 4. Eckert RL, Adhikary G, Rorke EA, Chew YC, Balasubramanian S (2011) PRB-159P); anti-LOR (BioLegend, PRB-145P); anti-b-actin AC-15 Polycomb group proteins are key regulators of keratinocyte function. J (Sigma, A5441); goat anti-mouse HRP Conjugate (Bio-Rad, 170-5047); Invest Dermatol 131: 295 – 301 goat anti-rabbit HRP Conjugate (Bio-Rad, 170-6517); and bovine anti- 5. Botchkarev VA, Mardaryev AN (2016) Repressing the keratinocyte goat HRP conjugate (Santa Cruz Biotechnology, SC-2350). genome: how the polycomb complex subunits operate in concert to control skin and hair follicle development. J Invest Dermatol 136: Bioinformatics and statistics 1538 – 1540 6. Mardaryev AN, Gdula MR, Yarker JL, Emelianov VU, Poterlowicz K, Sharov The biological process categories were obtained by using Gene AA, Sharova TY, Scarpa JA, Joffe B, Solovei I et al (2014)p63 and Brg1 Ontology Consortium tools (www.geneontology.org/). Experimental control developmentally regulated higher-order chromatin remodelling result significance was evaluated by Student’s t-test as expression of at the epidermal differentiation complex in epidermal progenitor P-value (*P < 0.05; **P < 0.01). cells. Development 141: 101 – 111 7. Sen GL, Webster DE, Barragan DI, Chang HY, Khavari PA (2008) Control of differentiation in a self-renewing mammalian tissue by the histone Data availability demethylase JMJD3. Genes Dev 22: 1865 – 1870 8. Bao X, Tang J, Lopez-Pajares V, Tao S, Qu K, Crabtree GR, Khavari The data reported in this paper have been deposited in GEO: PA (2013)ACTL6a enforces the epidermal progenitor state by GSE103890 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= suppressing SWI/SNF-dependent induction of KLF4. Cell Stem Cell 12: GSE103890). 193 – 203 9. Indra AK, Dupe V, Bornert J-M, Messaddeq N, Yaniv M, Mark M, Cham- Expanded View for this article is available online. bon P, Metzger D (2005) Temporally controlled targeted somatic muta- genesis in embryonic surface ectoderm and fetal epidermal Acknowledgements keratinocytes unveils two distinct developmental functions of BRG1 in The authors thank Dr. Ivano Amelio for gene microarray analysis, Maria Aguilar limb morphogenesis and skin barrier formation. Development 132: from the Genomics Facility at the Centre for Genomic Regulation for the scan- 4533 – 4544 ning arrays, Milena Nicoloso and Simona Rossi for the TUCR array and Simone 10. Fessing MY, Mardaryev AN, Gdula MR, Sharov AA, Sharova TY, Rapisarda Bischetti for 3D-skin histology. This work has been mainly supported by Italian V, Gordon KB, Smorodchenko AD, Poterlowicz K, Ferone G et al (2011) Ministry of Health and IDI-IRCCS (RC to EC) and by AIRC IG grant 22206 to EC. P63 regulates Satb1 to control tissue-specific chromatin remodeling This work has also been partially supported by the European Research Council during development of the epidermis. J Cell Biol 194: 825 – 839 (RIBOMYLOME_309545), the Spanish Ministry of Economy and Competitive- 11. Avgustinova A, Benitah SA (2016) Epigenetic control of adult stem cell ness (BFU2014-55054-P) and “Fundació La Marató de TV3” (PI043296). function. Nat Rev Mol Cell Biol 17: 643 – 658 12. Adam RC, Fuchs E (2016) The Yin and Yang of chromatin dynamics in Author contributions stem cell fate selection. Trends Genet 32: 89 – 100 EP, AML, MM, AS, AMar, XZ and TBO performed the research (AML and MM: 13. Frye M, Benitah SA (2012) Chromatin regulators in mammalian epider- T-UC array, validation, studies on si-uc.291; AMar: uc.291 transcript mis. Semin Cell Dev Biol 23: 897 – 905

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Cold Spring Harb Perspect Biol 2:a004887 26. Jiang J, Azevedo-Pouly ACP, Redis RS, Lee EJ, Gusev Y, Allard D, Sutaria 42. Rivetti di Val Cervo P, Lena AM, Nicoloso M, Rossi S, Mancini M, Zhou DS, Badawi M, Elgamal OA, Lerner MR et al (2016) Globally increased H, Saintigny G, Dellambra E, Odorisio T, Mahe C et al (2012)p63- ultraconserved noncoding RNA expression in pancreatic adenocarci- microRNA feedback in keratinocyte senescence. Proc Natl Acad Sci USA noma. Oncotarget 7: 53165 – 53177 109: 1133 – 1138 27. Ferdin J, Nishida N, Wu X, Nicoloso MS, Shah MY, Devlin C, Ling H, 43. Reuter JS, Mathews DH (2010) RNAstructure: software for RNA Shimizu M, Kumar K, Cortez MA et al (2013) HINCUTs in cancer: hypox- secondary structure prediction and analysis. BMC Bioinformatics 11: 129 ia-induced noncoding ultraconserved transcripts. Cell Death Differ 20: 1675 – 1687 License: This is an open access article under the 28. Marini A, Lena AM, Panatta E, Ivan C, Han L, Liang H, Annicchiarico- terms of the Creative Commons Attribution- Petruzzelli M, Di Daniele N, Calin GA, Candi E et al (2014) Ultracon- NonCommercial-NoDerivs 4.0 License, which permits served long non-coding RNA uc.63 in breast cancer. Oncotarget 8: use and distribution in any medium, provided the 35669 – 35680 original work is properly cited, the use is non- 29. Braconi C, Valeri N, Kogure T, Gasparini P, Huang N, Nuovo GJ, Terrac- commercial and no modifications or adaptations are ciano L, Croce CM, Patel T (2011) Expression and functional role of a made.

14 of 14 EMBO reports e46734 | 2020 ª 2020 The Authors Emanuele Panatta et al EMBO reports

Expanded View Figures

A

BC

Figure EV1. UC RNA is modulated during keratinocyte differentiation. A Heatmap showing microarray data results. Data are expressed as fold change. B Validation by RT–qPCR of down-regulated UCs showed in (A). C Validation by RT–qPCR of up-regulated UCs showed in (A). In (B) and (C), the quantification is relative to the same UC expression in undifferentiated HEKn (undiff., dark grey bars); data shown represent the mean Æ standard error (RT–qPCR has been done in triplicate); n = 1 (technical).

ª 2020 The Authors EMBO reports e46734 | 2020 EV1 EMBO reports Emanuele Panatta et al

A

BC

Figure EV2. Characterization of uc.291 transcript by strand-specific RT–qPCR. A Uc.291 (3,816 bp) genomic location on chromosome 10 in the sixth intron of the C10orf11 gene. B Uc.291 strand-specific RT–qPCR; representative amplification plots (#2 of Fig 1G) of strand-specific RT–qPCR; qPCR curves relative to the sense and the antisense transcripts are shown. C ACTB mRNA strand-specific RT–qPCR was used as control.

EV2 EMBO reports e46734 | 2020 ª 2020 The Authors Emanuele Panatta et al EMBO reports

A Uc.291 * LOR 12 ** * 15000 ** 10 * 10000 8 5000 6 80 * 4 60 2 40 20

Relative Quantification Relative 0 0 0369 0369 0369DD 0369 0369 0369DD KRT10 LCE1B ** ** 400 4000 ** 3000 ** 300 ** 2000 200 1000 100 * 60 25 20 40 15 20 10 5 Relative Quantification Relative 0 0 0369 0369 0369DD 0369 0369 0369DD

CaCl2 Epi EMEM

B 60 SCR si-uc.291 (1) si-uc.291 (2) ** DAPI/Ki67 50 * 40 30 20 Ki67 % of Ki67+ cells Ki67+ % of 10 0

Figure EV3. uc.291 is up-regulated during keratinocyte differentiation, regardless of the differentiation methodology.

ART–qPCR showing uc.291 expression in HEKn grown in calcium-induced differentiation (CaCl2) or confluence-induced differentiation (in Epi and EMEM). CaCl2 = calcium chloride; Epi = EpiLife; EMEM = Eagle’s minimum essential medium. LOR, KRT10 and LCE1B were used as differentiation markers. DD = days of differentiation; the quantification is relative to the same expression in undifferentiated HEKn (0 DD); data shown represent the mean Æ standard deviation; n = 3 (technical); *P < 0.05,**P < 0.01, Student’s t-test. B Box plot showing quantification of Ki67-positive nuclei on total nuclei. In parallel with the increase of p63 expression (Fig 2), depletion of uc.291 affects the proliferation rate of basal keratinocytes. Each central line represents the median; each box represents Q1 (quartile 1, below the median) and Q3 (quartile 3, above the median); the whiskers represent minimum (Q1 À 1.5*IQR) and maximum (Q3 + 1.5*IQR). IQR = interquartile range; 15, 14 and 15 images were analysed for SCR, si- uc.291 (1) and si-uc.291 (2), respectively; *P < 0.05;**P < 0.01, Student’s t-test.

ª 2020 The Authors EMBO reports e46734 | 2020 EV3 EMBO reports Emanuele Panatta et al

AB

CD

Figure EV4. Uc.291 protein microarray. A Schematic depicting high-throughput analysis performed to identify uc.291 protein interactome. B Protein array analysis showing the top 20 chromatin-related candidates; see also Appendix Table S2. C Reproducibility between the two human recombinant protein arrays spotted with 50,000 different proteins. (Pearson’s correlation = 0.76; F-B > 0). D Area under ROC curve (AUC) to assess Global Score [34] ability to separate proteins interacting with uc.291 from those that are non-interacting. The F-B score was employed to select different groups of high-affinity (top ranked) and low-affinity (bottom ranked) interactions reported in the array: (1) RBP, (2) chromatin-related and (3) unfiltered (i.e. without selecting by category). In all cases, Global Score performances increase from poor signal (top/bottom 200) to strong signal (top/bottom 10) interactions, showing AUCs that raise from 0.60 to 0.80.

EV4 EMBO reports e46734 | 2020 ª 2020 The Authors Emanuele Panatta et al EMBO reports

ABRNA-CLIP on HEK293E n.s. 1,2 FAM83H-AS 1 1 0,8 80 0 DD 0,6 0,4 0 0,2 80

Relative Quantification Relative 0 6 DD Differentiation

0 HEKn. Diff. HEKnRIP C 1,4 n.s. 2,5 n.s. 1,2 2 TBP 1 0,8 1,5 Proliferating HEKn 0,6 1 0,4 0,5 0,2 Relative Quantification 0 0 TBP 069DD

SCR si-uc.291 Differentiated HEKn

Figure EV5. Negative controls for RIP, CLIP and ChIP. ART–qPCR amplifying the lncRNA FAM83H-AS1 used as negative control in the RNA-CLIP experiment (Fig 4C). Note that FAM83H-AS1 resulted expressed in HEK293E

(Ct mean in the input = 26.3, data not shown) but was undetectable in all the immunoprecipitated samples (IgG, SMARCC2, ACTL6A). The same samples of Fig 4C were used (n = 3 technical); n.s. = not significant, Student’s t-test; HEK293 = human embryonic kidney 293. B FAM83H-AS1 was used as negative control for the RIP performed in HEKn (Fig 4D). On top, RNA sequencing in HEKn (from Ref. 8) showing FAM83H-AS1 expression is unchanged during keratinocyte differentiation. On bottom, RT–qPCR confirming FAM83H-AS1 is not modulated during the in vitro differentiation process (left histogram; same samples of Fig 1B(n = 4 different human donors) were used) and it does not interact with ACTL6A (right histogram; same samples of Fig 4D(n = 3 replicates on 1 human donor) were used); n.s. = not significant, Student’s t-test; DD = days of differentiation. C On the top, end-point PCR of TATA-box-binding protein (TBP) promoter after BRG1 chromatin IP in proliferating HEKn. On the bottom, end-point PCR of TBP promoter after BRG1 (upper agarose gel) and BRM (lower agarose gel) ChIP in differentiated HEKn silenced for uc.291. The same samples of Fig 5 were used.

ª 2020 The Authors EMBO reports e46734 | 2020 EV5 Appendix for:

The long noncoding RNA uc.291 controls epithelial differentiation by interfering with ACTL6A/BAF complex

Emanuele Panatta, Anna Maria Lena, Mara Mancini, Artem Smirnov, Alberto Marini, Riccardo Delli Ponti, Teresa Botta-Orfila, Gian Gaetano Tartaglia, Alessandro Mauriello, Xinna Zhang, George A Calin, Gerry Melino and Eleonora Candi

Table of contents

Appendix Figure S1. Characterization of uc.291 transcript by complementary DNA (cDNA) “walking”……………………………………………………………...... 2

Appendix Figure S2. Uc.291 transcript sequence……………………………...... 3

Appendix Figure S3. ACTL6A expression during keratinocyte differentiation………4

Appendix Table S1. Modulated T-UCRs during keratinocytes differentiation...... 5-6

Appendix Table S2. List of ranked candidates interacting with uc.291……...... 7

Appendix Table S3. Primers list used in the cDNA walking experiment ...... 8

Appendix Table S4. Primers list used in the RT-qPCR…………………………………...9

Appendix Table S5. Primers list used in the ChIP experiments……………………….10

1 A

Uc.291 transcript EXON EXON 6 Uc.291 7

Chr 10 (5’-3’); C10orf11 locus

B C cDNA walking cDNA walking “up” - 5’

400 500 600 700 800 720 730 750 900 1kb DNA -lad - - - - 1kb DNA lad- - - - 50/+50bp100/+100bp150/+150bp200/+200bp250/+250bp300/+300bp350/+350bp400/+400bp 1kb DNA laduc. sequence------

D cDNA walking “down” - 3’

1kb DNA lad+400 +500 +600 +700 +800 +900 +1000 +1100 1kb DNA lad+1400 +1700 +2000 +2300 1kb DNA lad +3600 +3700 +3800

Appendix Figure S1. Characterization of uc.291 transcript by complementary DNA (cDNA) “walking”. By using a multiple primers amplification approach we defined the length of uc.291 transcript. Primers were designed on 3’ and 5’ side of uc.291 conserved sequence at specific distances. “0 bp” represents the amplification of the genomic reported uc.291 length (424 bp in UCNEbase).(A) Scheme of the walking approach; (B) PCR fragments obtained using the blue primers reported in (A);(C) PCR fragments obtained using the green primers reported in (a);(D) PCR fragments obtained using the red primers reported in (A).

2 5’CTGGGCACTTAGCTCTCCACTTTAATTATTCATCCTTATGAAGAAAAATTTCCCCCGTGTGCTTTATTGTCCTCCAGC CCCGACCTGAGCTCACGCAGACTCTTCCTTCTTTCCCTCCCCAAAGTGGCTTGTGTGTCGAAGGGCTTTCCACTGGGGTG CTTATTCCATTTATGCCTTTAGTATGCATTTTGTGTTCTGCCAAGGGGGTCAGGAGGGCACATGGAGAAGCCAGATGGCT TACCTCTTCCCTCAGGAAACACGCCGCTATTTAGCTGGGCACCTTCCCAACTCAAGGCGGGGTGAGATTCCACTGTTGGA GAGAGAGTTGGTTCAAATGCCACTGTCCCTGAGATCACAGCTCCAAGTTCCTCTAGGCTATGCACTTTTCTACTTCTCTG CCACTTGCTGGAGTTTTCTGCCAGAAACAGCCTTTTAGGAGATATTAGAAAGGGGGGTGGGAAGGGAGGGATGGGAAAAA GCAACTCTTGTTATTTATGGTCCTCGTTCAGGCACAAGTTTCTGTTATTTTAATCCGGCTGAAATAAGAAACCATAATCC ATGGATGAAAAGATGGTGGACAAACTCAGTTAAATCTTGAGCACCCTGAGGATGTGTGTTTTTCCCTATTTCTGGTTTTT GACGTTCCTTCCCTGCTGCCATTTCCCAACTTTAACTTTTGAAAAGCCAAGCAGTTTGGCTGGCCAGGCCCAGGGAACTT ATTTGTATGCAGCACAAATTTCAGAATCTGTTCTCAGCCTGTGCCGAGTGAAAAATGGCCTGCATTTTCTTGATAGCCCA TGTGGTTGTAAAGAATAAATGGCTAATGAATTACAGATGAACATTGACGCAAATTAATCTTCCCGCTGTCCCTGGGTTAT ATGGCAGCCATTTAAAAGTTTAATCAATACACTAAAGTTGAAAACATGCAGGCACTGCAGTTGTTTGGATGTAATAAACA TCAGAGGGAACCGGGAGGTTTGCACCCAGTCCATGTATCATAATGACAGGTATTTATGTTTAATGGACTAAATATTTTTT ATTGGAAGGGAGCAAATGTCAGCTTAACTTTGTGAGCCCCGCATTCAACTTTCCTTTGAGGTTGGTGAAAGACGGCTGAC GTGTCATTGGAAATGAAATGTTAAGGGAGAAAAAAAGCACAACGGAGACAAAGCGAGGATGGGAGCCGTTTGGAGACGTG GTGCAGCGCTTACATTCTGCAGCAATTCATTAACGAAAACGTAAACTCTGATATTTCTAATGTTGCTGTAAAATGGTTTA CTAGGAACAACTTTAATGGTTATTAGTGAAAACAAGTACCCAGATCTGCCAGATATCCTTTTCAGTGCAGGCTTTTGTTT GAAAACTCTTTACCAGGTCATATTTGCTGGAGATTTATAATTAGTCTTTTTCCTTAATGTGAGAAGTGGAGGAGAAACAG CGTGATGGCTCTCAGCTCTTGTATTCCATAGATCTTTCCTTTTGAACGGTGTCCAATGACTTCGAATAACCATGTGTTCT CTTTTGGAATACTTATATCTTAATTACTTTTCAAGTCAATATTTTGTACCTCTGCCGTGAGTCTGGAAGGACCAGGGAGC TAAAAGAGAAAGGAAATTGCCAGTGGCAGTGCCATCTCGCTGGACAAAAACAGGCTGAGACACAGGAGAGCAATGGCTTC ACTTGAACAGAAAAATACAGACTTAGTTCAGCTTTTCATTTTTTTTCCTCCACATATAAAATAAACTAAATATGTCAGTG GAGCATGGGGTCTTAGAAAATAACACACACTTTATAAAAATAATTAGAAACGTTTTAGACAAAGGATATCGTGTTTTTAA TGTCCCTTCTGCAACCAATTGTCTCAGGTCCTCTTCAGTGTCTGAGGGCACATTCAATGGCAGGGCTTGGAAGAAAATTA TTCAGTAGATAGCAAGATGAGGCTGAAGGATGAGAAAAAGGGACAGGGGTTGTGATACTTATCCAGTGTTCAGATCTTGC AAAAGATATGAATTGCACCTTTTTATGTGTTAAATAATTTTGTTAAAATCACAAGAATTTGTGTTTCTAATGGGCCTAAG TTTCATAGGTAATCAGTATTATGTGATACTAAATTAGTAAACCAAACTGATGGGTTCATTGGTCCTTCATCATCCCTTAA GCAAAATGGGGAGAAACTTTCTGTTATGAAAACCGAAGCATGGCTCACTGTTAACCATGATGACATCTCACCCACTACTG CTCCTGAAGTCCTAAACTCTATTTTTCTTCCTACCTCTTTCTACCCTCTTAGAGGATTGAGTCCTTTACCTGTTTGTTCA CTTTGTGTTTCCGCCGTGTGAATATAAGCTCCACAGTAGGGGGTAGGAGGCAAGAACCTGATTTTCTTCTTTGCTTCTAG TGTCCTGGGGCCTGGCCTATAATAGGTTCTTGTAAATGGCAGGTAAGTGAATGGCGGACTACATTTCTTATGGTCTTTAG TAATTTATAACTCCGGCTATTTTTTTTGTTAGTCAGTATGATTTCTAGTCACTGTGTTAGTTGTTTTACATATAATACAA TACTTGATCTTAGTTGTTAACTCTAGAAGATAAGAGTTATTATCTTCATTTTATAGAGGCAAAAACTGATGTCAAGAGAA TTGAAGCTAATTATTTGTGGTTACTGTTAGTATGTAGCAATTCCAGAATTTGAATCCTGACCTGTTTGTCTCTGTATCAG CTATACAGTACTCCCCAAATTCCTGTCACTCATCTTGTCCCTGGGGATTAAAATCTCATTAAGTGACCACAACATTTAAT TCTAGACTATAACCAGCCAGCTCCCCAACTGGCTGATAATGAACAGAAGTATTAGATTCACAATTTGCAACCTAAACCTG GTGTCCTGAGTCCTACCTATAGAAAGCAATCTTTGACTGTACAAAAGTAGCACATTGGCTATTTGATGATGGGTGATATG TCACTCAAATATGTGTGTTCTGTGGTCTAGGGAAATACAAGGAACTCTCTCAAATCATGAGAAAAGTGTTCAGCAACAGA AATCTAACAAATAGTGACTGAAATGCATAGGCATTAATTTCTTCACTCAGAAGTGAGAATGGAGGTAGGCAGCCCAGGAC AGTGATAGTGGCTGAAAGAGCAGATCAGTAGGCACCCAGGTCCTTTCTGTCTTTCTGTCCTACTACCTTTTCAGCATGTG CATTTTAGCCTCATGGTCTCAAAATGGTTGCTACTGCTCCTGGCATCATTTCCTCACCCAAAGCAAGATGAAGAAGGATG TCTTTCCCTTATGAGGCATTCGCTTTTCATTTGTGGATAGACAGCTTCCTAAAGATGTCTGTCTCTTTCTCATTACCCAG ATCATGTCATTGGGAGGCTGGAAAGTCAAATGTTTCAGTTTTCATGCTGTTATAGTAGAGGAAGGCAGGATAAAAGGGAG CCAATGCATAGTGTCTCCTATGTAAGATAAATCGCATTTTTTGTATACCTAATAAAGACTTGAAACTTTGCTAGATGCCA TATGCACATCAACTTATATCATGTTTACAACCACCTTGTTACAGAAGAGTATTTATTCCCATTACACTAAGAAGAGGTGA TCTCATTGTTCAATTCCCACCTATGAGTGAGAATATGCGGTGTTTGAAGGGGAATATCACACTCTGGGGACTGTTGTGGG GTGGGGGGAGGGGGGAGGGATAGATTGGGAGATATACCTAATGCTAGATGACGAGTTAGTGGGTGCAGCGCACCAGCATG GCACATGTATACATATGTAACCTGCACAATGTGCACGTGTACCCTAAAACGTAAAG 3’ Appendix Figure S2. Uc.291 transcript sequence. Uc.291 complete sequence (3816nt) characterized by cDNA walking followed by sequencing of the PCR fragments obtained. Ultraconserved sequence is in red, predicted ACTL6A binding sequence is in light blu.

3 A B K14 ACTL6A DAPI/ACTL6A/K14 -uc.291-uc.291 (1) (2)-uc.291-uc.291 (1) (2) kDa SCRsi si SCRsi si SCR 52 - ACTL6A 48 - β-actin 0 DD 3 DD (1) uc.291 - C si

kDa 0 DD 3 DD 6 DD 9 DD 52 - ACTL6A (2) uc.291 -

si 48 - β-actin

Appendix Figure S3. ACTL6A expression during keratinocyte differentiation. (A) IF staining of K14 (basal layer marker) and ACTL6A in uc.291-depleted, si-uc.291(1) and si-uc.291(2), organotypic human epidermis compared to scramble control (SCR). Bar: 25um. One representative experiment of three is shown. (B) Immunoblot showing ACTL6A protein levels in HEKn cells transfected with scramble sequence (SCR) or si-uc.291(1) and si-uc.291(2) oligos and collected at proliferating (0 day) or differentiating (3 days of differentiation, DD) conditions. β-actin was used as loading control. (C) Immunoblot showing ACTL6A protein levels in HEKn cells collected in proliferating (0 day) or differentiating (3, 6, 9 days of differentiation, DD) conditions. β-actin was used as loading control.

4 Probe for Fold- Parametric FDR Description T-UCR change p-value uc.262 6.9380217 5.00E-07 0.0005516 CAGGCTGAGTACAAATTACTATGCAAGGGAGGCTGAGGGT uc.283 3.9902288 0.003973 0.0439591 CAGGGGCGCGGGATTGGATCAAATCACATAAACTGCAAAA uc.145A 3.4355936 0.0025296 0.0367763 TCCCATCCACGCGCCTCATACATATTGATTTCTGACACCA uc.36 2.935153 0.0042701 0.0455497 ACTTTCCTTGTGTGCTTGCACTATAGGTTTGGGACTGACA uc.88 2.6496588 0.0053214 0.0514358 GGAGGGAAGCAGAAGTCGGGAAGAAAAGAGAAAAGCAGCA uc.231 2.4696586 0.0142377 0.0922464 TGTGCATGTCAGCAGAATTCATAAGAACTTACAAAATCTT uc.291A 2.3985146 0.0144979 0.0932284 TGCCATATAACCCAGGGACAGCGGGAAGATTAATTTGCGT uc.473A 2.2716886 0.0023525 0.035347 CAGACACGGGGAGACACAACAGCACAGAACAGAGAACAAA uc.220 2.1861354 0.0191878 0.1134554 TCAAGATTTGCCAAACCACTAACCGCATGTATCATTTGCA uc.339 2.1658364 0.0007872 0.0230286 ACAGGAATGTAATTTTGCCCGGATGAGGCCCCGAGTTTAA uc.77A 1.9122857 0.0158713 0.0992502 TCTTGGGAGCAGTGTGACAGAAACAGAGTGTGGCGAGAAC uc.117A 1.8411175 0.0018504 0.0321861 AAACTTGTTTATTAAGATGGGCTGGAGGCGCTGGTGTGCA uc.170 1.661361 0.0060615 0.0552691 ACACAGGAACAAAGCAAGAGAACAGAAACTCAGAGGCAGA uc.346 1.6574337 0.0007029 0.0218725 TTCGGAGGCGGCTTTTCTTATTCAAAACAGGCCCACAATG uc.142A 1.6263274 0.0016429 0.0308253 GTCACACCCGAACCGCCAACAAAATTATCTTAAGCTGCCA uc.388A 1.5296091 0.0099989 0.0733348 AGCAACACACCATTTCACAGTCTATCGGGCACAAACACAT uc.20A 1.4471174 0.0436931 0.2063864 CTTGGTGAGGTTTGGGCGGAATAAAGAAAGGTGTTGGTGG uc.110 1.4082587 0.0031046 0.0398286 ACAAGTTGCTGTTAATTTAGTGCAGGGAGGACGGGATGGA uc.398A 1.3920305 0.0497388 0.2267608 GGATCGGGAGGAGGAGAGCCAGCGACCACAAAGAAGACAG uc.478 1.3431458 0.0239285 0.1326632 AAACAAATGGTGGTACGACAAAGGAGAGTGCGGCAGCGGG uc.217A 1.276802 0.021647 0.1232889 AAAGCACCCTTCACAAAGCCACAAACAAGAAGCAGGACAG uc.208A 1.2115439 0.0348729 0.1738692 TCAATGAAGAATGAAGAGAAGTAGAAAGCAAGAGTGAAGT uc.310A 0.65047 0.0210915 0.1214111 GGCCTGTCTCTCCTCTTCTTTTTCAAACCAAATTCTGCTT uc.369 0.6353444 0.0467179 0.2170893 TGATCTCATATTAAATTTTCCCCTGAATCCGCCCCTTGGC uc.198 0.6342153 0.0182638 0.1091729 CTGGTGACAAGTGAAATTGCTGTGCGCCTCATTTAGCTGT uc.262A 0.6117229 0.0442415 0.2085941 ACCCTCAGCCTCCCTTGCATAGTAATTTGTACTCAGCCTG uc.16 0.6081178 0.0409966 0.1964124 CTTGTTCAACACAGAGCTTAGAAACCCTCCTTCCACTCCC uc.478A 0.5942682 0.0280721 0.1492947 CCCGCTGCCGCACTCTCCTTTGTCGTACCACCATTTGTTT uc.352 0.5837183 0.0263843 0.1424184 TTTAAAGGCCAAGTCTAAAATAGATTTGCACCCACGCCCC uc.242 0.5766099 0.0308061 0.1585047 TTTAGTAGTTCCGCTATTCTGCTTAACATGTGGCACCCGT uc.138 0.5757989 0.0400969 0.1932085 CTGACTTCTTTCATCTTCCCCACTTCACACAAATTGCTCA uc.28 0.5717847 0.0282912 0.149858 AACTATATACCAGGCCCTGCTGAAAATACCACCCAACAGT uc.462 0.5621613 0.0064514 0.0572034 GGTGGGTGGAGAGACGAAGTCAGGGCTGTTTGTTGAATAT uc.452 0.5604607 0.0385909 0.1877993 AGCGTGTTTCTAATGCTATTCACCTGCCTTTGATGATTGA uc.346A 0.5596938 0.0026415 0.0374468 CATTGTGGGCCTGTTTTGAATAAGAAAAGCCGCCTCCGAA uc.325 0.5579312 0.0104034 0.0750892 GGGGTTTTGTTGTCAGAGTAGTTCCCGCCTCATGTTTTGT uc.151A 0.5475617 0.0166494 0.1023798 TCTTGCTCACCTGGTCAAACACCCATTCCTCACTGCATTT uc.390 0.5450069 0.0322261 0.1638461 AATTTAGTCAATTACTGTTTCAGCCTGCAAGCCTCCTGGG uc.396 0.5407496 0.0161342 0.1001554 CATCTTGCACAGCTGCGCCCAACTGATTAAATCCTCACAA uc.77 0.5379875 0.0382952 0.1867631 GTTCTCGCCACACTCTGTTTCTGTCACACTGCTCCCAAGA uc.412A 0.5237442 0.031904 0.1631937 TTCAAATCCACACAAGCCTGCCACTTGCACTAGTCATCCC uc.21A 0.5211857 0.0325936 0.1651708 ACCCATAATTTCTCATTTCCCAAACCTCACACATGCTGAA uc.465A 0.5019852 0.0026524 0.0374468 TTCTCTCATCTCCCGCAGCTTAGCACCTGTCTTTGTTTGG uc.400A 0.497497 0.0270436 0.1454441 AATCATCTGACACAAAGATAATTGAGCCAGCCTCTGCCAC uc.457 0.496534 0.0180181 0.1085937 AGACATATTCCAGATCACAACCTTCCCAGAATCCTGCCCT uc.268 0.4928927 0.0027834 0.0383176 ACTATCATCACAACCCTCATTGTTCCATCTCTGCCCTCCC uc.129 0.4880591 0.0251525 0.1374754 CCATTGCCATCATGTGCTCGCTGCCTGCTAATTAAGACTC uc.223A 0.4698592 0.0008607 0.0242558 GGCTGATTTATGTTGTCATTCCCTCTCACCAGTCCTGCAT uc.373A 0.46421 0.0193354 0.1138975 TTGAAAGGAGACAACATTGTCCCGATTTCCCACTGCGACA uc.473 0.4634708 0.0022747 0.03482 TTTGTTCTCTGTTCTGTGCTGTTGTGTCTCCCCGTGTCTG uc.133A 0.4487399 0.0259639 0.1407152 ACTTTCCGATTTAATTTCCTCCATGCCCTTGACATCCACA uc.476A 0.4466684 0.0276283 0.1476632 GCTGATTGATGGCTCTCTCCCTCACTGCTGAAGGCTGTAT uc.422 0.4430617 0.0099478 0.0730988 GAGATTCTGCATTACTACACCTCAGTGCCTGCCTGGTTCC uc.250 0.4364047 0.0070389 0.0603978 GCAAATATGATTATACACTATCAAACTGGCACCACCAGGC uc.10A 0.4228508 0.0180147 0.1085937 TCTGCATCTCCTAATGGCCTCTGCTCCTCTACACTCAATT

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5 uc.335A 0.4227937 0.0146008 0.0936515 CAGCAGAAGCAGGTCTCAGGTCCCAGTGGTTAATGTAGTG uc.281A 0.4197648 0.0082006 0.0648906 GCCTTTATTGGAATTCTGAAACCTCTCTGCCTCCCTGGCT uc.391A 0.4037248 0.0289005 0.1520426 AAATTATTGCATTGCTGGTGGCAATTTGTGGGCATGTGTT uc.305A 0.3992737 0.0028265 0.0385672 CGGCAGGCTCTTCCCATCTTCCCATCCATTCATCATCTCG uc.161A 0.3980419 0.0007734 0.0228909 CCATCATAATTTGTTCATTTGGTGCCCATCAGCCCAGCCT uc.177A 0.3960284 0.048816 0.224542 CACAATTTCAAAACTGCCTGCTAATTTCCACCACCTGTCA uc.159A 0.3946312 0.0031571 0.0401024 GGTCCTCCACAGTCCCAAATTCACCCGTTTAAGCCATAAT uc.416A 0.3747073 0.0023867 0.0355154 GCTGCCATGCACACCGGCTCTTACGGCTACAATTACAATG uc.134A 0.3737976 0.0088282 0.0679086 GCTCATAAATTACTGCTCTTTTGTTGCTGGCTGGCCTTGT uc.1 0.3736053 0.0064382 0.0571786 CACTTCCCCACCGCAGGCTTTGCTGAGCAGTTTTAGTATC uc.181A 0.3669021 0.0016712 0.0308772 GAATGAGCAGGCCCCAAGGTGTTTCAACTGGAGTGTTTTC uc.349 0.3633513 0.003553 0.0415567 ATGTTTTCCACAGCCCAAGACCTTTGGTGCATGCTTTTGA uc.266A 0.3471284 0.0207761 0.1199206 AGACCACTTCAATTTCAGCTTCTGCCAGCCTCGTTTGAGA uc.136 0.3449797 0.0143047 0.0925777 CAATAACGCTTTGATAGATCCCTGTCTGCCCGGTGCTTGA uc.411 0.3341136 0.0003292 0.0146432 ACCCATTTGTCACCCAGCATTTCCCTTTGTGTATAGAACA uc.164A 0.3221855 0.0045111 0.0470165 TTACCACATGCTGTCACAGTTAGCTCAGCCAGGTCTTGTT uc.329A 0.2860004 0.0025932 0.0370876 TGTAGTCATCAAGTGAGAAAGACATCCTCCTCCTGGCTGC uc.232 0.2818089 0.0006993 0.0218725 AAGTGTTGTGTCAATAACGCAGCCAAGATCTCGCCAATCG uc.63A 0.2714599 0.0012258 0.0280916 CAGTGTTTGCCTGTTTGCTTGCCTGGTAAATTTTGCTTTT uc.257 0.2624234 0.0002364 0.0124403 CTGATGGATTCCCAGGCCCATTGCTGTCACTGATGAACTA uc.338A 0.2565722 0.0010626 0.026549 CTGTCGCTGCCTGTGCTGTACCTGTCCTGTGGATAAATAG uc.183A 0.250974 6.32e-05 0.0068978 CCCAGTGTCAGATGTCTCCAGGTGCCTCTCTTCTTTCTTG uc.252A 0.2239623 0.0002429 0.01259 TCTCAGGAAGCCCAGCCAATTAAGGACTTGACCATATTGA uc.204A 0.1862553 2.7e-06 0.001604 GCTCTAATTTACAAGCCGCCTGCCTAATGAATTGTCTGGG

Appendix Table S1. Modulated T-UCRs during keratinocytes differentiation Description: Summary of Results Number of classes: 2 Sorted by p-value of the univariate test. Type of univariate test used: Two-sample T-test Class 1: differentiated; Class 2: undifferentiated. Nominal significance level of each univariate test: 0.05 The genes are significant at the nominal 0.05 level of the univariate test

6 Gene Symbol Expressed in epidermis* BATF2 yes SMARCE1 yes ACTL6A yes TRIM16 yes TADA2A CENPO DEK yes CRTC2 yes UBE2B BRCC3 CHCHD3 SETMAR CHD2 SMARCC2 yes VEGFA yes PRMT3 yes C17orf49 yes GATA2 MRGBP BMI1 yes CPA4 yes PRMT6 yes CHCHD2 yes DCAF17 SMARCD3 SETDB1 EYA1 UBE2U ZCRB1 GATA3 yes EPC1 PRKD2 yes ATXN7L3 yes HJURP CENPA yes SIRT1 PRKCA HIST1H1C yes ZNF451 PHLDB1 yes Appendix Table S2. List of ranked candidates interacting with uc.291. Table showing the top 40 uc.291 interactors derived from the protoarray. See also Figure 4 and Figure EV4. * The expression in human epidermis was extrapolated from The Human Protein Atlas (http://www.proteinatlas.org).

7 cDNA Walking Name Sequence 5'-3' F-50 TTCCCAACTTTAACTTTTGAAAAGC R+475 AAACGGCTCCCATCCTCGCTTTG F-100 ATGTGTGTTTTTCCCTATTTCTGG R+525 CGTTTTCGTTAATGAATTGCTGCAG F-150 GGATGAAAAGATGGTGGACAAAC R+575 AGTTGTTCCTAGTAAACCATTTTAC F-200 CACAAGTTTCTGTTATTTTAATCCG R+625 AAAGGATATCTGGCAGATCTGGG F-250 AGGGAGGGATGGGAAAAAGCAAC R+675 TCCAGCAAATATGACCTGGTAAAG F-300 GTTTTCTGCCAGAAACAGCCTTTTAG R+725 GCTGTTTCTCCTCCACTTCTCAC F-350 CCAAGTTCCTCTAGGCTATGCAC R+775 CACCGTTCAAAAGGAAAGATCTATG F-400 CTGTTGGAGAGAGAGTTGGTTC R+825 AAGATATAAGTATTCCAAAAGAGAAC cDNA Walking UP Name Sequence 5'-3' R5'-UP GGCTGTTTCTGGCAGAAAACTCC F-456 CGCCGCTATTTAGCTGGGCACC F-505 GGAGGGCACATGGAGAAGCCAG F-620 CAGACTCTTCCTTCTTTCCCTC F-647 GTCCTCCAGCCCCGACCTGAGC F-685 CATCCTTATGAAGAAAAATTTCCC F-715 CTGGGCACTTAGCTCTCCAC F-732 AACTACAGTGAGGCCTGGGCAC F-861 GAGTTGCTGCCTTGTACAGCTG cDNA Walking DOWN Name Sequence 5'-3' F3'-DOWN CTTTACCAGGTCATATTTGCTGG R+875 TCCTTCCAGACTCACGGCAGAGG R+923 GCGAGATGGCACTGCCACTGGC R+1060 CTAAGACCCCATGCTCCACTGAC R+1177 AATGTGCCCTCAGACACTGAAG R+1288 CTTTTGCAAGATCTGAACACTGG R+1440 GGGATGATGAAGGACCAATGAAC R+1578 CTTCTAAGAGGGTAGAAAGAGG F3'-1500-DOWN CCCACTACTGCTCCTGAAGTCC R+1826 CAACTAACAGAGTGACTAGAAATC R+2118 CAGCCAGTTGGGGAGCTGGCTGG R+2400 CCTGGGCTGCCTACCTCCATTC R+2664 CCAGCCTCCCAATGACATGATC F3-2700-DOWN GGATGTCTTTCCCTTATGAGGC R+3040 CTGGTGCGCTGCACCCACTAAC R+3100 CTTTACGTTTTAGGGTACACGTG R+3200 GTCAGACCTGATAATTTAGGGC Appendix Table S3. Primers list used in the cDNA walking experiment (see also Appendix Figure S1).

8 Name Sequence 5'-3' T-UC291 FW GCGTCAATGTTCATCTGTAATTC T-UC291 REV CTGTTCTCAGCCTGTGCCGAG T-UC183 FW TGCTTCTTCTCTTCCTTCCTTTTTG T-UC183 REV TGCTCTCTACCAGGTTGGCG T-UC257 FW GCTGATGGATTCCCAGGCCCATTG T-UC257 REV GTTCTGATCTTGAGGTAATTAGCC T-UC338 FW AGTGAGCCTTGGAGACTGAACATCC T-UC338 REV ACAGCCCTGGAGACTGAAATCCTC T-UC63 FW CAGTGTTTGCCTGTTTGCTTGC T-UC63 REV CCTGTTGCTTTCTTTCTGTTCCTC T-UC36 FW AAATGTGTGTGAGTGCAAGCAG T-UC36 REV GGGTCATTACCGCATGAAGGCC T-UC88 FW TGTCAAAACTGCCAGGAGCAAG T-UC88 REV GCCAATCTGTCACCGTTCAGC IVL FW CAGGTCCAAGACATTCAACC IVL REV CAAGTTCACAGATGAGACGG KRT10 FW AGGAGGAGTGTCATCCCTAAG KRT10 REV AAGCTGCCTCCATAACTCCC LOR FW CTCTGTCTGCGGCTACTCTG LOR REV CACGAGGTCTGAGTGACCTG U6 FW GTGCTCGCTTCGGCAGCACATATAC U6 REV AAAAATATGGAACGCTTCACGAATTT ACTIN FW GTTGCTATCCAGGCTGTGC ACTIN REV AATGTCACGCACGATTTCCCG LCE1B FW GCTACAACTACACTAAGCAGTTC LCE1B REV CTATTCTTGCCCTTTCAGCATCA LCE1C FW AATCCAGGACCGCAAACTG LCE1C REV TGGACCTGTGAGCCTCTCAG LCE2B FW GGTTGACTAAACTCTGCCAGG LCE2B REV GGCACTGGGGCAGGCATTTA LCE2D FW CTGCAGAAGAGCTCTGGTACTG LCE2D REV CTCCATCAAGCACAAAGTTCTG LCE3A FW CTGAGTCACCACAGATGCC LCE3A REV CTTGCTGACCACTTCCCCTG LCE3C FW CCAGTTGTCCCTCACCCAAG LCE3C REV CCGGAGCTTAGAGCACAGTC LCE6A FW CAGGGAGCTGAAAAGCCAGA LCE6A REV GAGGGGAGCATTTGGGAACA SPRR2B FW AGGAACCTTGGCTTTGTCAGT SPRR2B REV AGCTCATGCCCAGGTGAAAG KPRP FW CCTGAAGGACTGCTTGCCTG KPRP REV CTGGGCAAAGGGGGATTGAG HRNR FW GAAGTGCTCAGGGCAAGAT HRNR REV GCACCTCTGCTCTTGGACAT FLG FW CAATCAGGCACTCATCACAC FLG REV ACTGTTAGTGACCTGACTACC TBP FW TCAAACCCAGAATTGTTCTCCTTAT TBP REV CCTGAATCCCTTTAGAATAGGGTAGA Appendix Table S4. Primers list used in the RT-qPCR

9 Name Sequence 5'-3' LOR FW TCTCCTTTCCTGCATTCGCT LOR REV TTCGTTTGCCAGCCATTCCT FLG FW CCAAGGCAAAGGGCAAGATTC FLG REV GCTACGCTGTCTAGCTCTCTG LCE1B FW TGAAGGAGGGAGCCTCAAGA LCE1B REV TACAGGTGAACATGGCAGGC TBP FW CTGACAGGTAAGGAGGACGC TBP REV AGTTACCTGACCTCTCCCCC

Appendix Table S5. Primers list used in the ChIP experiments (see also Figure 5).

10