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KMT2D Regulates P63 Target Enhancers to Coordinate Epithelial Homeostasis

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KMT2D Regulates P63 Target Enhancers to Coordinate Epithelial Homeostasis Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis Enrique Lin-Shiao,1,2,7 Yemin Lan,1 Mariel Coradin,1 Amy Anderson,3 Greg Donahue,1 Cory L. Simpson,3 Payel Sen,1 Rizwan Saffie,4 Luca Busino,4 Benjamin A. Garcia,1 Shelley L. Berger,1,5,6 and Brian C. Capell1,3,7 1Penn Epigenetics Institute, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 2Department of Biochemistry and Molecular Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 3Department of Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 4Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 5Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; 6Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA Epithelial tissues rely on a highly coordinated balance between self-renewal, proliferation, and differentiation, dis- ruption of which may drive carcinogenesis. The epigenetic regulator KMT2D (MLL4) is one of the most frequently mutated genes in all cancers, particularly epithelial cancers, yet its normal function in these tissues is unknown. Here, we identify a novel role for KMT2D in coordinating this fine balance, as depletion of KMT2D from undiffer- entiated epidermal keratinocytes results in reduced proliferation, premature spurious activation of terminal dif- ferentiation genes, and disorganized epidermal stratification. Genome-wide, KMT2D interacts with p63 and is enriched at its target enhancers. Depletion of KMT2D results in a broad loss of enhancer histone modifications H3 Lys 4 (H3K4) monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) as well as reduced expression of p63 target genes, including key genes involved in epithelial development and adhesion. Together, these results reveal a critical role for KMT2D in the control of epithelial enhancers and p63 target gene expression, including the re- quirement of KMT2D for the maintenance of epithelial progenitor gene expression and the coordination of proper terminal differentiation. [Keywords: chromatin; enhancers; epigenetics; epithelial homeostasis; KMT2D; p63] Supplemental material is available for this article. Received August 23, 2017; revised version accepted January 12, 2018. Proper regulation of epigenetic information through the cancer worldwide (http://cancer.sanger.ac.uk/cosmic; precise control of gene enhancer and transcriptional net- Cerami et al. 2012; Gao et al. 2013). works is essential for establishing and maintaining cell Functionally, KMT2D and the related protein KMT2C fate and identity (Sur and Taipale 2016). Disruption of (MLL3) are the major histone methyltransferases that these pathways critical for differentiation can lead to a deposit histone H3 Lys 4 (H3K4) monomethylation loss of proliferative control, ultimately driving carcino- (H3K4me1), which poises enhancers for activation and genesis (Gonda and Ramsay 2015). In line with this, the transcription factor binding (Herz et al. 2012; Hu et al. gene encoding the chromatin modifier and master 2013; Lee et al. 2013). Furthermore, in addition to its enhancer regulator KMT2D (MLL4) is among the most fre- catalytic role in H3K4me1 deposition, KMT2D is also re- quently mutated genes in human cancer (Kandoth et al. quired for the binding of the histone acetyltransferase 2013; Ford and Dingwall 2015; Rao and Dou 2015; Sze complex CBP/p300, which acetylates H3K27 (H3K27ac) and Shilatifard 2016). There is an exceptionally high prev- at enhancers, resulting in enhancer activation during alence of KMT2D mutations in self-renewing epithelial cell identity gene induction and changes in cellular fate tissues ranging from the lung and esophagus to the skin (Wang et al. 2016, 2017; Lai et al. 2017). In mice, complete and bladder, with the highest frequencies seen in kerati- deletion of Kmt2d is embryonic-lethal (Lee et al. 2013). nocyte cancers (cutaneous squamous cell carcinoma and Tissue-specific deletion studies have demonstrated that basal cell carcinoma), the two most common forms of KMT2D is essential for the normal development and © 2018 Lin-Shiao et al. This article is distributed exclusively by Cold 7These authors contributed equally to this work. Spring Harbor Laboratory Press for the first six months after the full-issue Corresponding authors: [email protected], bergers@ publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). pennmedicine.upenn.edu After six months, it is available under a Creative Commons License Article published online ahead of print. Article and publication date are (Attribution-NonCommercial 4.0 International), as described at http:// online at http://www.genesdev.org/cgi/doi/10.1101/gad.306241.117. creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 32:181–193 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/18; www.genesdev.org 181 Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Lin-Shiao et al. Figure 1. KMT2D loss leads to reduced keratinocyte proliferation and a broad loss of epithelial development and adhesion genes. (A) KMT2D mRNA expression is significantly reduced in both NHEKs (P = 0.0036) and HaCaTs (P = 0.0312) treated with shKMT2D as deter- mined by RNA-seq. (B) shKMT2D keratinocytes display reduced levels of KMT2D but normal-appearing nuclear morphology. (C) shKMT2D keratinocytes display re- duced proliferation in comparison with shSC keratino- cytes. (D) Representative genes from shKMTD NHEKs that are significantly reduced in expression along with corresponding adjusted P-values. (E) Gene ontology (GO) analysis of the 134 common genes lost with shKMT2D treatment between both shKMT2D NHEKs and HaCaTs demonstrates that genes involved in key ep- ithelial signaling pathways (RARG and VDR), epithelial cell growth and morphogenesis, polarity, and adhesion are enriched among those genes with reduced expression. (F) shKMT2D keratinocytes display reduced expression of RARγ and VDR by IF (40×). function of enhancers during adipogenesis and myogene- cytes (NHEKs) and spontaneously immortalized human sis (Lee et al. 2013), cardiac development (Ang et al. epidermal keratinocytes (HaCaTs) using shRNAs directed 2016), and B-cell lymphocyte development (Ortega-Molina against KMT2D (shKMT2D) compared with scrambled et al. 2015; Zhang et al. 2015). control shRNA (shSC). RNA sequencing (RNA-seq) car- Despite the high incidence of KMT2D mutations in the ried out in both the shRNA-treated NHEKs and HaCaTs epidermis and established roles in enhancer function and demonstrated a significant reduction in KMT2D expres- developmental programs in other tissues, little is under- sion (Fig. 1A). This was confirmed by using three distinct stood about KMT2D function in normal epithelial ho- shKMT2D hairpins that displayed a consistent twofold to meostasis. Given the poor understanding yet potential threefold reduction in KMT2D expression using RT- importance of KMT2D in development and cancer, here qPCR, Western blotting, and immunofluorescence (IF) we explore the role of KMT2D in self-renewing epithelia (Fig. 1B; Supplemental Fig. S1A,B). In addition, prolifera- and provide the first description of KMT2D’s role at epi- tive growth of shKMT2D keratinocytes was reduced by thelial enhancers, including its genome-wide interactions KMT2D loss compared with shSC keratinocytes (Fig. with the transcription factor p63, a master regulator of 1C), consistent with previous reports in other cell types epithelial transcription, in coordinating key gene expres- (Issaeva et al. 2007; Guo et al. 2013). To confirm that the sion programs required for both epithelial progenitors reduced proliferative capacity did not reflect a simple and the coordination of proper epithelial differentiation global stress response, we examined levels of the senes- and stratification. cence-associated tumor suppressor p16/CDKN2A and the stress and senescence-associated transcription factor phospho-NF-κB p65 (Ser536) and did not observe any dif- Results ferences between either shSC or shKMT2D keratinocytes (Supplemental Fig. S1C). To study KMT2D in the epithelium, we reduced KMT2D Given KMT2D’s importance in transcriptional enhancer levels in primary neonatal human epidermal keratino- function, we examined the transcriptional consequences 182 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press KMT2D coordinates epithelial homeostasis of KMT2D depletion in keratinocytes. We performed while introns (and likely intronic enhancers) made up three biological replicates of RNA-seq from NHEKs treat- the majority of the remaining binding sites (40%) (Fig. ed with shKMT2D. Strikingly, we found a broad reduction 2A). Analysis of the gene nearest to each peak showed in the expression of genes critical for epithelial develop- that 76% of peaks were associated with protein-coding ment and differentiation as well as adherence to the genes, while the majority of the others occurred near non- epidermal basement membrane (Fig. 1D). These encode coding RNAs (ncRNAs; 22%) (Fig. 2B). proteins involved in epithelial
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