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The LEAFY Floral Regulator Displays Pioneer Transcription Factor Properties

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The LEAFY Floral Regulator Displays Pioneer Transcription Factor Properties bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425699; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The LEAFY floral regulator displays pioneer transcription factor 2 properties 3 4 Xuelei Lai1,*, Romain Blanc-Mathieu1,*, Loïc GrandVuillemin1,^, Ying Huang2,^, Arnaud 5 Stigliani1,3, Jérémy Lucas1, Emmanuel Thévenon1, Jeanne Loue-Manifel1,4, Hussein Daher1,5, 6 Eugenia Brun-Hernandez1, Gilles Vachon1, David Latrasse2, Moussa Benhamed2,6, Renaud 7 Dumas1, Chloe Zubieta1 and François Parcy1,** 8 9 1Laboratoire Physiologie Cellulaire et Végétale, Univ. Grenoble Alpes, CNRS, CEA, INRAE, 10 IRIG-DBSCI-LPCV, 17 avenue des martyrs, F-38054, Grenoble, France 11 2Université Paris-Saclay, CNRS, INRAE, Univ. Evry, Institute of Plant Sciences Paris-Saclay 12 (IPS2), 91405, Orsay, France. 13 3The Bioinformatics Centre, Department of Biology and Biotech and Research Innovation 14 Centre, University of Copenhagen, Ole Maaløes Vej 5, DK2200 Copenhagen N, Denmark 15 4Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, 16 UCB Lyon 1, CNRS, INRA, F-69342 Lyon, France 17 5Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, Grenoble, France 18 6Université de Paris, Institute of Plant Sciences Paris-Saclay (IPS2), F-75006 Paris, France 19 20 * contributed equally 21 ^ contributed equally to the work 22 ** Correspondence: François Parcy ([email protected]) bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425699; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 Abstract 24 Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to 25 closed chromatin regions in which DNA is wrapped around histones and often highly 26 methylated. Subsequently, they are able to modify the chromatin state to initiate gene 27 expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act 28 as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated 29 and non-methylated DNA using a combination of in vitro genome-wide binding experiments 30 and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin 31 accessibility data suggest that LFY binds a subset of regions occupied by nucleosomes. We 32 confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. 33 Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce 34 chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken 35 together, our study suggests that LFY possesses key pioneer TF features that contribute to 36 launch the floral gene expression program. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425699; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 37 Introduction 38 Proper gene regulation is essential to all living organisms, controlling processes from basic 39 development to environmental response. Gene regulation requires the finely orchestrated 40 activity of transcription factors (TFs) that recognize specific DNA sequences in gene regulatory 41 regions and activate or repress transcription of their target genes. While the binding of most 42 TFs to DNA is restricted to accessible regions of the genome, a specific type of TF, called a 43 “pioneer”, is able to access its cognate binding site even in closed, nucleosome-rich chromatin 44 regions (Magnani et al., 2011; Iwafuchi-Doi and Zaret, 2014; Iwafuchi-Doi and Zaret, 2016; 45 Zaret, 2020). The ability to bind nucleosomal DNA in vivo and in vitro is a defining 46 characteristic of pioneer TFs and has been well-established for diverse mammalian pioneer TFs 47 (Fernandez Garcia et al., 2019). As DNA in closed chromatin regions is often highly 48 methylated, another emerging feature of pioneer TFs is their capability to bind DNA in a 49 methylation insensitive manner (Zhu et al., 2016; Mayran and Drouin, 2018). Some pioneer 50 TFs are even able to directly recruit DNA demethylases at methylated sites, thereby facilitating 51 the remodeling of closed regions (Iwafuchi-Doi, 2018). 52 Pioneer TFs are often master regulators controlling developmental transitions, with the 53 mammalian pluripotency factors OCT4, SOX2, and KLF4 representing some of the most well- 54 studied (Soufi et al., 2015). These factors bind to closed chromatin regions and induce their 55 opening or remodeling, so that genes they contain can be activated by the pioneer TFs 56 themselves or by other TFs called settlers (Sherwood et al., 2014; Slattery et al., 2014). The 57 modification of the chromatin landscape by pioneer TF can be accomplished either directly by 58 triggering DNA detachment from nucleosomes (Dodonova et al., 2020; Michael et al., 2020), 59 or indirectly by the recruitment of ATP-dependent cellular machineries, such as chromatin 60 remodelers that remove or modify adjacent nucleosomes in order to prime downstream 61 regulatory events (Hu et al., 2011; King and Klose, 2017). Such capacity to modify DNA 62 accessibility is another defining feature of pioneer TFs (Iwafuchi-Doi and Zaret, 2014). 63 In plants, the only TF reported as pioneer TF so far is LEAFY COTYLEDON1 (LEC1), a seed 64 specific TF involved in embryonic epigenetic reprogramming (Tao et al., 2017). LEC1 was 65 shown to promote the initial establishment of an active chromatin state of its target gene in 66 silenced chromatin and activate its expression de novo. Pioneer TF activity was also suggested 67 for two types of factors controlling flower development, the MADS homeotic TFs (Pajoro et 68 al., 2014; Denay et al., 2017) and the master floral regulator, LEAFY (LFY). The MADS TFs, bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425699; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 69 including APETALA1 (AP1) and SEPALLATA3, were shown to be able to access closed 70 chromatin regions to specify floral organs, and were thus postulated to act as pioneer TFs 71 (Pajoro et al., 2014). However, mammalian MADS TFs do not seem to act as pioneer factors 72 and thus the identification of AP1 and SEP3 as potential pioneers remains speculative 73 (Sherwood et al., 2014). In contrast to the MADS TFs, one previous study suggest that LFY 74 may have pioneer activity (Sayou et al., 2016). LFY is a master regulator specifying the floral 75 identity of meristems. It directly induces the floral homeotic genes AP1, APETALA3 (AP3) and 76 AGAMOUS (AG) (Parcy et al., 1998; Wagner, 1999; Lohmann et al., 2001; Chae et al., 2008; 77 Yamaguchi et al., 2013; Chahtane et al., 2013). AG and AP3 are known to be under the 78 repression of Polycomb repressive complexes in seedlings (Goodrich et al., 1997; Turck et al., 79 2007; Calonje et al., 2008). This suggests that their activation during flower development 80 requires modifications of their chromatin landscape and that the direct binding of LFY to their 81 regulatory regions might trigger. Consistent with this, LFY was suggested to be able to access 82 closed chromatin regions in vivo (Sayou et al., 2016). Moreover, LFY’s role is not confined to 83 conferring a flower fate to meristems. It can also contribute to meristem emergence (Moyroud 84 et al., 2010; Chahtane et al., 2013; Yamaguchi et al., 2013), and together with its co-regulators 85 such as the homeodomain TF WUSCHEL or the F-Box protein UNUSUAL FLORAL 86 ORGANS, it can even induce meristem formation from root or leaf tissue, respectively (Levin 87 and Meyerowitz, 1995; Gallois et al., 2004; Risseeuw et al., 2013). Taken together, these data 88 indicate that LFY has the full capability of reprogramming cell fate, a property often requiring 89 pioneer activity. However, whether LFY is truly able to directly bind closed chromatin regions 90 and change their status has yet to be demonstrated. 91 Here, we address the pioneer activity of LFY in vitro and in vivo. Firstly, we determined 92 whether LFY binding was sensitive to DNA methylation. For this, we combined in vitro LFY 93 genome-wide binding data using methylated and unmethylated genomic DNA and structural 94 analysis. These experiments demonstrated that LFY binding is only mildly sensitive to DNA 95 methylation. In order to test whether LFY binding was compatible with the presence of 96 nucleosomes, we compared LFY binding data from chromatin immunoprecipitation sequencing 97 (ChIP-seq) and chromatin accessibility data. Based on these comparisons, we found that LFY 98 could access a number of closed chromatin regions and that LFY colocalizes with nucleosomes 99 in some regions in vivo. Using electrophoretic mobility shift assays (EMSA), we further showed 100 that LFY was able to directly bind nucleosomes in vitro. Finally, chromatin accessibility assays 101 demonstrated that LFY constitutive expression was sufficient to increase chromatin bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425699; this version posted January 7, 2021.
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