Molecular Neurobiology https://doi.org/10.1007/s12035-020-01971-w

Molecular Mechanisms for Krüppel-Like Factor 13 Actions in Hippocampal Neurons

José Ávila-Mendoza1 & Arasakumar Subramani1 & Christopher J. Sifuentes 1,2 & Robert J. Denver1

Received: 5 March 2020 /Accepted: 1 June 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract Krüppel-like factors (KLFs) play key roles in nervous system development and function. Several KLFs are known to promote, and then maintain neural cell differentiation. Our previous work focused on the actions of KLF9 in mouse hippocampal neurons. Here we investigated genomic targets and functions of KLF9’s paralog KLF13, with the goal of understanding how these two closely related transcription factors influence hippocampal cell function, proliferation, survival, and regeneration. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf13 expression with doxycycline. We also generated HT22 Klf13 knock out cells, and we analyzed primary hippocampal cells from wild type and Klf13−/− mice. RNA sequencing showed that KLF13, like KLF9, acts predominantly as a transcriptional repressor in hippocampal neurons and can regulate other Klf . Pathway analysis revealed that genes regulated by KLF13 are involved in cell cycle, cell survival, cytoarchitecture regulation, among others. Chromatin-streptavidin sequencing conducted on chromatin isolated from HT22 cells expressing biotinylated KLF13 identified 9506 genomic targets; 79% were located within 1-kb upstream of transcription start sites. Transfection-reporter assays confirmed that KLF13 can directly regulate transcriptional activity of its target genes. Comparison of the target genes of KLF9 and KLF13 found that they share some functions that were likely present in their common ancestor, but they have also acquired distinct functions during evolution. Flow cytometry showed that KLF13 promotes cell cycle progression, and it protects cells from glutamate-induced excitotoxic damage. Taken together, our findings establish novel roles and molecular mechanisms for KLF13 actions in mammalian hippocampal neurons.

Keywords Krüppel-like factors . Transcription . Chromatin . Hippocampus . Cell cycle . Cell survival

Introduction eighteen transcription factors characterized by three C- terminal C2H2 motifs that recognize GC/GT rich Krüppel-like factors (KLFs) are zinc finger transcription fac- sequences in DNA [2, 3]. They are grouped into three sub- tors that have recently emerged as important regulators of families based on the primary structure of their N-terminal neural development and function. During development, they domains, which are highly variable, and comprise sites for play essential roles in neural cell differentiation, and in interaction with coregulators that modulate the activities of adults, they contribute to several activities to maintain nor- KLFs. The KLFs of subfamily 3 (KLF9, KLF10, KLF11, mal brain function [1]. The KLFs constitute a family of KLF13, KLF14, and KLF16) can act as transcriptional acti- vators or repressors depending on the cellular context [2]. Members of this subfamily share a common domain that Electronic supplementary material The online version of this article binds the co-repressor Swi-independent 3a (Sin3a) (https://doi.org/10.1007/s12035-020-01971-w) contains supplementary [4]. They are expressed in multiple tissues, including the material, which is available to authorized users. central nervous system (CNS) [5], where they are implicated in the regulation of cell proliferation, differentiation, surviv- * Robert J. Denver al, and regeneration [6–8]. [email protected] Krüppel-like factor 9 (KLF9; formerly basic transcription – 1 Department of Molecular, Cellular and Developmental Biology, The element binding protein 1 BTEB1), one of the most studied University of Michigan, Ann Arbor, MI 48109, USA KLFs with regard to CNS development and function, shows a 2 Present address: Takara Bio USA Inc., Mountain View, dramatic increase in expression in rat and mouse brain through CA 94043, USA the first 3–4 weeks of postnatal life, reaching a peak around Mol Neurobiol postnatal day (PND) 30 [9, 10]. The increase in Klf9 mRNA and survival. We investigated the consequences of Klf13 deletion parallels the postnatal rise in circulating thyroid hormone on these biological processes in HT22 cells, in which Klf13 was (T3), and we showed previously that Klf9 is a direct T3 recep- inactivated by CRISPR/Cas9 genome editing, and in primary tor target [10, 11]. The postnatal increase in Klf9 expres- hippocampal cultures from Klf13−/− mice. Our findings support sion is in accordance with its ability to promote and maintain that KLF13 promotes cell cycle progression and has neuronal differentiation [12–15]. The Klf9 gene is induced in cytoprotective actions in mammalian neurons. mature neurons by extracellular stimuli, including several hor- mones [11, 16], electrical activity [12], cellular damage [8], and stress [15, 17], reflecting the important roles that KLF9 Materials and Methods plays in both developing and mature neurons [18]. To under- stand the molecular mechanisms for KLF9 actions in neurons, Animals we recently identified KLF9 target genes in mouse hippocampus-derived neuronal cells, and discovered that We purchased wild type mice (C57/Bl6J strain) from Jackson KLF9 functions predominantly as a transcriptional repressor Laboratories. Mice null for Klf13 were kindly provided by Dr. [18]. One of the most strongly regulated genes by KLF9 is the Raul A. Urrutia and described previously [30]. We re-derived paralogous gene Klf13, also a member of subfamily 3; the two the Klf13−/− mice into the C57/Bl6J strain (using the genes arose through a gene or genome duplication event in the University of Michigan Transgenic Mouse Core) and vertebrate lineage [19]. backcrossed them for at least five generations prior to Krüppel-like factor 13 (formerly basic transcription conducting experiments. Mice received food and water ad element-binding protein 3-BTEB3) was initially described as libitum, and were euthanized by rapid decapitation. All pro- an SP1-like capable of activating tran- cedures involving animals were conducted under an approved scription of SV40 [20], but repressing Cyp1a1 in the human animal use protocol (PRO00008816) in accordance with the liver cancer cell line Hep G2 through binding to the basic guidelines of the Institutional Animal Care and Use transcription element (BTE) [21], a GC-box sequence origi- Committee at the University of Michigan. nally identified as a binding site for KLF9 [22]. The Klf13 gene is expressed in multiple tissues, with highest levels in Generation of Clonal HT22 Cell Lines with the spleen [23] and thymus where it is involved in the control Doxycycline-Inducible V5Klf13 of erythropoiesis and the activation of T lymphocytes [24]. In addition, KLF13 plays a role in the early stages of We engineered the mouse hippocampus-derived cell line cardiogenesis [25] and mediates the protective actions of glu- HT22 to control Klf13 expression by addition of doxycycline cocorticoids on cardiomyocytes [26]. It has also been shown (dox) following the strategy that we reported previously for to have diverse roles in different cancers; it inhibits cell pro- Klf9 [18]. We generated a plasmid for transfection to confer liferation in prostate and glioma tumors [27, 28], but promotes tetracycline-inducible expression of a Klf13 transgene con- the proliferation of oral cancer cells [29]. taining a V5 tag fused to the N-terminus of KLF13. We used Krüppel-like factor 13 is expressed in neurons and, similar pCMV-Entry-Klf13 (MR224297; Origene) as template to to KLF9, is capable of inhibiting neurite outgrowth of mature PCR-amplify the Klf13 open reading frame, and we sub- cortical neurons [7]. However, virtually nothing is known cloned this DNA fragment into pENTR4-V5 (12426; about the cellular actions of KLF13 in neurons, what its target Addgene) at the BclI and XbaI sites. We then used this plas- genes are (in any cell type), or how it functions in chromatin. mid to PCR-amplify the full-length V5-Klf13 DNA fragment, Moreover, it is unknown if the actions of KLF13 overlap with, and sub-cloned it into the pCDNA4:TO vector (Invitrogen) at or are different from those of its paralog KLF9. the KpnI and ApaI sites to create pTO-V5Klf13. To create In the present study, we identified KLF13 target genes in the stably transfected cell lines, we cultured the HT22 parent line adult mouse hippocampus-derived cell line HT22, and confirmed in high-glucose DMEM (Invitrogen) supplemented with 10% and extended these findings in mouse hippocampus in vivo and fetal bovine serum (Corning) and penicillin G (100 U/ ml) + in primary hippocampal neurons. We engineered HT22 cells to streptomycin (100 μg/ml; Gibco) at 37 °C under an atmo- control Klf13 expression, then conducted RNA sequencing; or sphere of 5% CO2. We first created a cell line to express the we expressed a biotinylated form of KLF13 and conducted chro- tet repressor (TetR; HT22-TR) by culturing cells in 10-cm matin streptavidin precipitation (ChSP) to identify KLF13 target dishes to 60% confluence, then transfecting them with 5 μg genes. We found that KLF13 functions predominantly as a tran- of the pCDNA6:TR vector (Invitrogen; this vector has a scriptional repressor through association in chromatin at proxi- blasticidin resistance cassette) using Fugene6 Transfection mal promoters of target genes. We compared the pathways reg- Reagent following the manufacturer’s instructions ulated by KLF9 and KLF13 in hippocampal neurons. We also (Promega). Twenty-four hours after transfection, we replaced investigated roles for KLF13 in neuronal cell cycle progression the medium with selective medium containing 5 μg/ml Mol Neurobiol blasticidin (Research Products International) and continued We cultured the HT22 parent cell line in 10-cm dishes, the cell culture for 10 days. We harvested the stably transfected cells with 5 μg of pCas-Klf13gRNA-EF1a-GFP transfected cells and randomly sorted cells into a 96-well plate (Origene), then harvested and sorted EGFP-positive cells by (1 cell/well) at the University of Michigan Flow Cytometry fluorescence-activated cell sorting (FACAS) to 1 cell/well in a Core. We expanded, then tested 6 clonal lines by transient 96-well plate. We expanded eight clonal cell lines and transfection with the pTO-Egfp vector (we subcloned a screened them for mutations at the Klf13-gRNA target region cDNA encoding EGFP into the pCDNA4:TO vector at the by direct DNA sequencing at the University of Michigan KpnI and ApaI sites), then selected a clone (HT22-TR-3) that DNA Sequencing Core. We purified genomic DNA from cells had nondetectable baseline EGFP that could be induced by cultured in 6-well plates using the DNeasy Blood & Tissue Kit addition of dox (1 μg/ml for 8 h; Sigma Chemical Co., St. (Qiagen) following the manufacturer’s instructions, and we Louis, MO). To generate V5Klf13 dox-inducible cell lines, we used this DNA as template to amplify the region flanking transfected HT22-TR-3 with 5 μg of the pTO-V5Klf13 plas- the gRNA target region (see Supplementary Table 1 for mid, selected with 100 μg/ml Zeocin (InvivoGen), and sorted oligonucleotide primer sequences). We sub-cloned the PCR cells by flow cytometry as described above. We tested 6 clonal products into the pGEMT easy vector (Promega), then cell lines for baseline and dox-inducible V5Klf13 mRNA by screened at least 10 bacterial colonies per clonal line for mu- culturing them in 6-well plates and treating them with dox tations by isolating plasmid DNA and conducting direct DNA (1 μg/ml) for 8 h. We quantified Klf13 mRNA by reverse sequencing. We identified a clonal line (clone number 5) that transcriptase quantitative polymerase chain reaction contains 4 types of inactivating mutations: 2 insertions and 2 (RTqPCR; see below), and we selected a clonal line (HT22- deletions (Supplementary Fig. 1) which create nonsense or TR/TO-V5Klf13-1) for further study that had a baseline prematurely truncated . We used this clonal line, des- mRNA level similar to that of HT22-TR-3, and that could ignated HT22-Klf13-KO, for further experiments. be induced fivefold after 8 h of exposure to dox. We first conducted a time course experiment with HT22- Generation of Primary Cell Cultures from Neonatal TR/TO-V5Klf13-1 cells to investigate the kinetics of dox- Mouse Hippocampus induction of the V5Klf13 transgene using RTqPCR and Western blotting. We cultured cells in 6-well plates to 70– We isolated primary cells from neonatal mouse hippocampus 80% confluence, then induced them by addition of dox following the protocol of Cazares and colleagues [31], which (1 μg/ml) for different times before harvesting cells for RNA generates cultures that are enriched for neurons. Briefly, for each (described below) or protein extraction. For Western blot anal- biological replicate (n = 3), we dissected the region of the hip- ysis, we prepared nuclear extracts from cells using the NE-PER pocampus from four postnatal day (PND) 1 mice and kept the Nuclear and Cytoplasmic extraction reagents (Thermo- tissues in ice-cold NDM buffer (10 mM HEPES Hank’s Scientific) and fractionated 50 μg of protein per well by SDS- Balanced Salt Solution; Gibco) until preparation of primary PAGE, transferred to nitrocellulose and detected the expressed cells. We rinsed tissues with NDM buffer and digested at V5KLF13 using a V5 tag polyclonal antibody (1:1000 dilution; 37 °C for 10 min in 10 ml of papain solution (NDM buffer; Millipore). Primary immune complexes were revealed by incu- 200 μl of 2× crystallized papain activated with 3.4 mg L-cyste- bation with a goat-anti rabbit IgG-HRP secondary antibody ine; Sigma). Digestion was stopped by rinsing the tissue with (1:10,000; Jackson ImmunoResearch) and detection by NDM buffer followed by tissue dissociation using gentle tritu- chemiluminescenc (Denville Scientific). We hypothesized that ration. After removing large pieces of undissociated tissue, we Klf9 isregulatedbyKLF13,sowealsoanalyzedKlf9 mRNA pelleted the cells at 400×g for 3 min at 4 °C, then resuspended in by RTqPCR at each of the time points. 2 ml of Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco) and 1X Glutamax (Gibco). We seeded cells in Generation of HT22-Klf13-Knockout Cell Lines Using 12-well tissue culture plates coated with 150–300 kDa poly-L- CRISPR/Cas9 Genome Editing lysine (Sigma) at 1.5–2×105 cells/well, and cultured them at 37 °C under an atmosphere of 5% CO2. We replaced half of the To introduce inactivating mutations into the Klf13 gene in culture medium every 3–4days. HT22 cells, we designed a guide RNA (gRNA) using two web tools: https://zlab.bio/guide-design-resoucers and RNA Extraction, Reverse Transcription, and https://chopchop.cbu.uib.no. We selected the sequence 5′ Quantitative PCR gcg cgg tcg tgc acg agc cg 3′, which was predicted by both algorithms and targets a sequence in the 5′ region of the Klf13 We extracted total RNA from HT22 cells or primary hippocam- gene. We sub-cloned this oligonucleotide into the pCas- pal cells using the TRIzol Reagent (Invitrogen) following the Guide-EF1a-GFP (Origene) plasmid; this vector also ex- manufacturer’s instructions. For each sample, we treated 1 μg presses Cas9 and Egfp. total RNA with 20 IU of DNase I (Promega) for 30 min at 37 °C, Mol Neurobiol then synthesized cDNA using the High Capacity Reverse members of the KLF subfamily 3. We amplified the DNA Transcription Kit with ribonuclease inhibitor (Applied fragment from pCMV-Entry-Klf13 and subcloned it into the Biosystems). We conducted quantitative real-time PCR using a pMCSG7 vector [32] using ligation independent cloning. The StepOne Systems machine (Applied Biosystems) with resulting plasmid (pMSCG7-Klf13) had the DNA fragment qPCRBIO SyGreen Blue Mix Lo-Rox (PCRBiosystems). We inserted in-frame with a 6x-His tag at the N-terminus. We designed oligonucleotide primers to span exon-exon boundaries produced the recombinant KLF13 antigen in the E. coli where possible (Supplementary Table 1) using the BLAST prim- Rosseta2 DE3 strain (Millipore) by inducing with 0.4 mM er algorithm (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). IPTG for 16 h at 37 °C. We purified the recombinant protein We generated standard curves by pooling cDNA samples and from cellular extracts using a HiTrap SP cation exchange preparing serial dilutions for relative quantification. We chromatography column (GE Healthcare Life Sciences). normalized all genes to the geometric mean of the mRNA Proteins from each step of the purification were resolved by levels of the reference genes Gapdh and Ppia, whose mRNAs SDS-PAGE and stained with Coomassie blue (Supplementary were unaffected by the treatments (data not shown). Fig. 3b). We used the purified antigen to produce two polyclonal RNA Sequencing (RNA-Seq) antiserums in rabbits (27293 and 27294; Lampire Biologicals Laboratories.) We monitored the antiserum titers We extracted total RNA as described above from HT22-TR/ using enzyme-linked immunosorbent assay (ELISA). Briefly, TO-V5Klf13-1 cells treated with vehicle or dox (n =3/treat- we coated Immulon 1B plates (Thermo-Scientific) with 1 μg/ ment) for 8 h. We analyzed RNA quality using a Bioanalyzer well of antigen, blocked with 0.5% BSA in PBST, incubated (Aligent) which gave RNA integrity numbers of approximate- with 1:10 serial dilutions of antiserum, followed by detection ly 9. The libraries were prepared at the University of Michigan with alkaline phosphatase goat anti rabbit IgG (1:5000). We DNA Sequencing Core using the TruSeq Stranded mRNA Kit developed the signal using p-nitrophenylphosphate (pNPP) (Illumina), and sequenced in one lane (6 samples) using an substrate (Sigma) and selected the antiserum with the highest Illumina 4000 Hi-Seq machine. titer (27293) for further study. We purified IgG from both pre- We checked the quality of the raw reads data for each sample immune serum and antiserum 27293 using Protein A-Agarose using FastQC (version v0.11.3) (Supplementary Fig. 2)toiden- Fast Flow (Millipore). We further purified the anti-KLF13 tify features of the data that may indicate quality problems (e.g., IgG by affinity column purification using Affi Gel-10 low quality scores, over-represented sequences, inappropriate (Biorad) following the manufacturer’s instructions. We ana- GC content). Briefly, we aligned reads to the reference genome lyzed the titer of the affinity-purified anti-KLF13 IgG using (GRCm38) using Hisat2 (version 2.1.0). We used default pa- ELISA with the IgG from the pre-immune serum serving as rameter settings for alignment, with the exception of: “–dta”, control, and based on this analysis, we chose 0.0002 μg/ml for which in addition to the way that the genome index was built, Western blotting, and 1 μg/ml for ChIP assays allows for reads to be mapped across splice-junctions. Stringtie (Supplementary Fig. 4a). (version 1.3.3) was used for gene and isoform-level quantifica- To test if the anti-KLF13 IgG recognizes endogenous tion, using the “-e -G” option which quantifies transcripts based KLF13, we conducted Western blots on protein isolated from on an input GTF file, rather than taking a de novo approach. spleen and hippocampus from wild type and Klf13−/− mice. This also allowed us to use count-based differential expression We also used the cell line HT22-TR/TRO-V5Klf13-1 after callers such as DESeq2. We pre-filtered the data to remove dox induction (24 h) as a positive control. We prepared nucle- genes with 0 counts in all samples. We conducted normalization ar extracts using the Nuclear and Cytoplasmic Extraction and differential expression analysis with DESeq2, using a neg- Reagents (Thermo-Scientific) and determined protein concen- ative binomial generalized linear model. Plots were generated tration using the BCA assay (Pierce). We resolved 50 μgof using variations or alternative representations of native DESeq2 nuclear protein by 10% SDS-PAGE, transferred proteins to plotting functions, ggplot2, plotly, and other packages within the nitrocellulose membrane and blocked with 5% BSA. We in- R environment. Some of the quality control analyses are shown cubated the membrane with affinity purified anti-KLF13 IgG in Supplementary Fig. 2. (0.0002 μg/ml), followed by incubation with anti-rabbit HRP- conjugated secondary antibody (1:10000; Jackson Production and Validation of Rabbit Polyclonal ImmunoResearch). We detected immune complexes by Antiserums to KLF13 chemiluminescence using the ECL reagent (Denville Scientific), which revealed a band for the expressed To produce custom anti-KLF13 antiserums, we selected an V5KLF13 at the expected molecular weight of 43 kDa in epitope corresponding to the 74-169 aa position HT22-TR/TO-V5Klf13-1 cells (Supplementary Fig. 4b). We (Supplementary Fig. 3a) of KLF13 (NP_067341.2) based on also identified a protein of approximately 39 kDa (the estimat- the uniqueness of this sequence in comparison with other ed molecular weight of native KLF13) in the spleen of wild Mol Neurobiol type mice, which was absent in Klf13−/− animals chromatin with 10 U of Proteinase K (New England (Supplementary Fig. 4b). To further test the specificity of BioLabs)at40°Cfor2hfollowedby2μgofRNase our antiserum, we preabsorbed the anti-KLF13 IgG before A (Applied Biosystems) at 37 °C for 1 h. The DNA was Western blotting by incubating 5 μg of the IgG with a 100 extracted with phenol:choloroform:isoamyl alcohol X molar excess of the recombinant antigen overnight at 4 °C, (Invitrogen) and precipitated with 0.3 M sodium acetate which extinguished the signal (Supplementary Fig. 4b). We and 100% ethanol. We resuspended the DNA in 100 μl were unable to reliably detect KLF13 in hippocampus by nuclease-free water and analyzed it by targeted qPCR. We Western blotting. generated standard curves by preparing serial dilutions of To further validate the anti-KLF13 serum, we conducted genomic DNA isolated from HT22 cells using the DNeasy ChIP assay on chromatin isolated from the hippocampus of Blood & tissue Kit (Qiagen). Precipitated samples were PND30 wild type and Klf13−/− mice. This showed specific quantified as a percentage of the corresponding input KLF13 association in chromatin at both the Klf13 and Klf16 sample. promoters, which was absent at the region of the Klf16 distal For chromatin immunoprecipitation (ChIP), we used (control). The KLF13 ChIP signal at these two pro- the HT22-TR/TO-V5Klf13-1 cell line treated with dox moters was absent in chromatin isolated from the hippocam- for 16–20 h. We prepared chromatin as described above pus of Klf13−/− mice (Supplementary Fig. 4c). and conducted immunoprecipitation by incubating 50– 100 μg of chromatin with 1 μg of our custom affinity- Chromatin Extraction and Precipitation purified anti-KLF13 IgG, or with 1 μg of IgG purified from pre-immune serum as control. After 16 h of incuba- To identify regions of the genome where KLF13 associates in tion, we added 70 μl of Protein A Agarose (Millipore) chromatin, we expressed in HT22-BirA cells a KLF13 fusion slurry (2 ml of protein A-agarose diluted in 10 ml of protein containing a C-terminal biotin ligase recognition pep- 10 mM Tris-HCl, pH 8.0; 0.2 mg/ml Sheared Salmon tide (BIO) that can be biotinylated in vivo (we previously DNA; 0.16 mg/ml BSA), and rocked the samples at 4 °C engineered HT22 to constitutively express the biotin-protein for 4 h. Precipitated immune complexes were washed and ligase BirA) [18]. We constructed a plasmid to express the DNA purified as described above. We also conducted KLF13 fusion protein by amplifying by PCR a DNA fragment ChIP assay on hippocampal tissue isolated from PND30 containing the entire Klf13 ORF (using the pCMV-Entry- mice following a method described by Denver and Klf13 plasmid as template, MR224297; Origene), then we Williamson [10]. fused the BIO sequence (amplified from the vector pEF1a- FLBIO) [33]tothe3′ end of the Klf13 ORF by ligation. We Chromatin Streptavidin Precipitation Sequencing then directionally cloned this DNA fragment (Klf13-Bio)into (ChSP-Seq) the pCDNA4:TO vector at the KpnI and XbaI sites to generate pTO-Klf13Bio. We transfected HT22-BirA cells with 6 μgof For ChSP-seq, we precipitated DNA from HT22-BirA cells pTO-Klf13Bio or pTO-Egfp plasmids in 10 cm dishes, then transfected with the pTO-Klf13Bio plasmid (cells harvest- conducted chromatin streptavidin precipitation (ChSP) as de- ed 24 h after transfection) as described above. We quanti- scribed [18]. Briefly, cells were washed, crosslinked with 1% fied the precipitated DNA using the Qubit dsDNA HS paraformaldehyde and chromatin extracted, and sonicated Assay Kit (Invitrogen), and combined DNA from several using an M220 Focused-Ultrasonicator (Covaris) for 20 min independent precipitations to produce at least 5 ng per bi- with a 5% duty factor. The chromatin shearing (200–600 bp) ological replicate for sequencing. Libraries were prepared was confirmed by agarose gel electrophoresis. by the University of Michigan Sequencing Core from 4 We conducted ChSP following the method that we de- input samples and 4 streptavidin-precipitated samples scribed previously [18] with minor modifications. Briefly, using the SMARTer ThruPLEX DNA-Seq Kit (Takara), we precipitated 50–100 μg of cross-linked chromatin (5% and sequenced (50 bp single read) in 1 lane using an of chromatin was reserved as input) with 50 μlofMyOne Illumina 4000 Hi-Seq machine. We analyzed the quality T1 streptavidin-conjugated Dynabeads (Invitrogen) in of the raw reads data using FastQC, then we quality- 1 ml of dilution buffer (16.7 Tris-HCl, pH 8.1; 150 mM trimmed using bbduk, and mapped reads to the GRCm38 NaCl; 0.01% SDS; 1.1% triton X-100; 1.2 mM EDTA), reference genome using BWA mem. Additional QC checks and rocked overnight at 4 °C. Chromatin complexes were for enrichment, correlation analyses, and binding profiles washed with low salt, high salt, LiCl, and TE buffers as were performed with deepTools, phantomtools, and described by Gade and Kalvakolanu [34], and we re- ngsplot. Some informative quality analyses are given in moved the crosslinks by incubation at 65 °C overnight Supplementary Fig. 5. We identified and annotated peaks (after this step the input samples were processed simulta- using PePr and ChIPseeker, respectively. We analyzed mo- neously with the ChSP samples). We treated the tif enrichment using the program HOMER. Mol Neurobiol

Transfection-Reporter Assay Cell Proliferation Assay Using 5-Ethynyl-2′- Deoxyuridine (EdU) Incorporation To investigate direct transcriptional regulation by KLF13 of genomic targets that we identified by ChSP-seq, we We forced expression of Klf13 by transfection in primary cells generated luciferase reporter constructs and conducted isolated from PND1 mouse hippocampus, and used EdU in- transfection assays using the HT22-TR-3 (control) and corporation to investigate possible effects of KLF13 on cell HT22-TR/TO-V5Klf13-1 cell lines. We isolated genomic proliferation. We prepared primary cells as described above, DNA from mouse tails using the DNeasy Blood & tissue andafterpelletingthembycentrifugationat400×g,weresus- Kit (Qiagen) and used this as template to amplify genomic pended 1–3×106 cells in 100 μl of nucleofection buffer (P3 regions that corresponded to KLF13 ChSP peaks for Klf16 Primary Cell 4D-Nucleofector Kit; Lonza) and added 2 μgof (2200 bp),Casp3(500 bp) and (500 bp). We pTO-Egfp or pTO-V5Klf13 vector. We transfected cells using directionally cloned the PCR products into the pGL4.23 a Nucleofector (Lonza) with the EM110 program following vector (Promega) at the NheI and HindIII sites and con- the manufacturer’s instructions. We then plated transfected ducted direct DNA sequencing to verify the constructs. cells in 24-well plates (pre-coated with 150–300 kDa Poy-L- We plated cells in 24-well tissue culture plates at a Lysine; Sigma) at a density of 5 × 105 cells per well, and 4 density of 5 × 10 cells/well in growth medium (DMEM cultured cells at 37 °C under an atmosphere of 5% CO2. with 10% FBS). The following day, we replaced the me- Twenty four hours later, we added EdU to a final concentra- dium with DMEM plus 1% FBS containing vehicle or dox tion of 20 μM for 1.5 h, then washed cells with PBS, and fixed (1 μg/ml), then we co-transfected cells with 200 ng of them with 4% paraformaldehyde for 30 min. We perme- pGL4.23 plasmids (firefly) plus 10 ng of the promoter- abilized cells in 0.1% Triton for 10 min, washed 2 times with less pRenilla vector using Fugene6. Twenty-four hours PBS, then incubated with developer cocktail (100 mM TBS; later, we harvested cells and conducted Dual Luciferase 1mMCuSO4 5H2O; 100 mM Ascorbate; 25 μM Sulfo-Cy3 Reporter Assay (Promega) following the manufacturer’s azide; Invitrogen) for 30 min. Cells were counter-stained with instructions. All transfection reporter assays were repeated DAPI (5 μg/ml; Invitrogen). We counted all Cy3-positive at least two times with 4 replicates/treatment. cells in each well and normalized to the total cell number (cells stained with DAPI).

Analysis of Cell Cycle by Flow Cytometry Glutamate-Induced Excitotoxicity Assay

We analyzed cell cycle progression after forced expression We used the glutamate-induced excitotoxicity assay to ana- of Klf13 (dox treatment of HT22-TR/TO-V5Klf13–1 cells) lyze survival of HT22 cells and primary mouse hippocampal and deletion of Klf13 (using Klf13-KO HT22 cells). We cells. We used two independent assays to assess cell viability. plated 5 × 105 HT22-TR/TO-V5Klf13-1 cells in 10 cm For MTT assay, we plated 5 × 104 HT22 cells per well in 24- dishes in growth medium, and 16 h later, we treated with well plates and cultured them for 24 h before treatment. For vehicle or dox (1 μg/ml) for 24 h before harvest for cell lactate dehydrogenase (LDH) release, we plated 2 × 104 cells cycle analysis. We synchronized the Klf13-KO cells by per well in 96-well plates and cultured them for 24 h before replacing the growth medium (DMEM with 10% FBS) treatment. We plated 5 × 104 primary hippocampal cells per with DMEM containing 1% FBS for 24 h, then changed well in 96-well plates for both assays, and cultured them for to growth medium for 24 h before harvest for cell cycle 15–20 days before treatment. analysis. We induced excitotoxicity in HT22 by exposing cells to L- To prepare cells for flow cytometry, we pelleted them glutamic acid monosodium salt (MP Biomedicals) in 1% at 1000×g, rinsed the pellet with DPBS plus 1% BSA, DMEM for 24 h. We treated HT22-TR/TO-V5Klf13-1 cells then fixed cells in 4% formaldehyde for 20 min at RT. with vehicle or dox (1 μg/ml) for 4 h before addition of glu- We collected the fixed cells by centrifugation, washed tamate to a final concentration of 10 mM. We treated primary them, and resuspended in 100 μl of 1× saponin-based hippocampal cells with 1 mM glutamate for 24 h. Permeabilization Buffer (eBioscience) containing 1% For MTT assay, we rinsed cells with DPBS and then incu- BSA. We then stained cells in darkness for 1 h using bated them for 2 h with 1.2 mM MTT (ThermoFisher) in FxCycle Violet (Thermo Fisher) following the manufac- DMEM with 1% FBS (HT22) or complete Neurobasal medi- turer’s instructions. Cells were then analyzed using an um (primary cells). We then dissolved the formazan crystals in Attune Cytometer (Applied Biosystems) at 405 nm wave- DMSO for 10 min, and quantified absorbance using a micro- length counting at least 8 × 105 events. The relative pro- plate reader at 540 nm. We expressed the percentage viability portion of cells in G1/G0 and G2/M phases was deter- relative to vehicle-treated cells. We analyzed LDH release in mined using ModFit LT (Verity Software House). 50 μl of culture medium using the LDH Cytotoxicity Assay Mol Neurobiol

Kit (Pierce) following the manufacturer’s instructions. The RNA-seq results using RTqPCR for 4 induced and 4 re- data are presented as percentage cytotoxicity, determined as pressed genes (Fig. 1d). LDH activity of (glutamate–vehicle)/(maximum LDH activi- Our RNA-seq analysis showed that like KLF9 [18], ty − vehicle × 100). KLF13 regulates the expression of several members of the KLF superfamily in HT22 cells. Forced expression of Statistical Analysis V5Klf13 in HT22 repressed Klf3, Klf9, Klf10, Klf11, Klf13, and Klf16; the control gene Klf6 (not found to be regulated All data are expressed as the mean ± standard error of the in the RNA-seq dataset) was unaffected (Fig. 2a). mean (SEM). We analyzed data by unpaired Student’s t test Conversely, inactivation of Klf13 by CRISPR/Cas9 ge- or by one-way analysis of variance (ANOVA) followed by nome editing in HT22 cells led to increased expression of Tukey’s multiple comparison test using Prism8 (GraphPad). each of the Klf genes that were repressed by V5KLF13 Derived values were tested for homogeneity of variance using (Fig. 2b). These findings in HT22 were confirmed using the Brown-Forsythe test, and when appropriate data were primary hippocampal cells isolated from wildtype and −/− Log10 transformed before analysis. A p value ≤ 0.05 was con- Klf13 mice, where the mRNA levels for all 6 Klf genes sidered significant. were elevated in the knockout cells (Fig. 2c; although this was not statistically significant for Klf16).

Results KLF13 Impacts Diverse Biological Processes and Cellular Signaling Pathways Validation of the V5Klf13 Dox-Inducible HT22 Cell Line We analyzed the 3336 differentially expressed genes using iPathway Guide (Advaita Bioinformatics) in the We analyzed Klf13 mRNA using RTqPCR in HT22-TR/TO- context of pathways obtained from the Kyoto V5Klf13-1 cells and found that treatment with dox induced Encyclopedia of Genes and Genomes (KEGG) database time-dependent expression of the V5Klf13 transgene (Fig. 1a; [36, 37]. After Bonferroni’s correction, we found 45 path- dox had no effect in the control TR cells). The V5Klf13 ways to be significantly impacted (Table 2). This analysis mRNA level increased by 3.9-fold after 2 h and remained showed that KLF13 has diverse actions in neuronal cells elevated through 24 h. We also analyzed V5KLF13 protein related to cell cycle, cell survival, cytoarchitecture regula- synthesis by Western blotting, which showed that the protein tion, among others. was elevated by 2 h after dox, increased through 16 h, and remained elevated through 24 h (Fig. 1a). We assessed the KLF13 Associates in Chromatin at Proximal Promoter bioactivity of the V5KLF13 fusion protein by quantifying Regions in HT22 Cells Klf9 mRNA, which may be regulated by KLF13 [35]. This showed a time-dependent, inverse relationship between the To identify sites across the genome where KLF13 associates in Klf9 mRNA and the transgene mRNA and protein levels; chromatin, we conducted ChSP-seq on chromatin isolated from the Klf9 mRNA was reduced by 8 h after dox treatment in HT22 cells (n = 4) expressing a biotinylated KLF13-Bio fusion HT22-TR/TO-V5Klf13-1 cells (dox had no effect in the TR protein (validation details given in Supplementary Fig. 4). We cells), and continued to decrease through 16 h and remained obtained an average of 30 and 50 million 50 bp reads per lowat24h(Fig.1b; F(5,12) =8.934, p < 0.001; one way sample for input and precipitated samples, respectively. Using ANOVA, n = 3/time point). the program PePr, we identified 9506 peaks with an average length of 698 bp. The coordinates, nearest gene, and distance to KLF13 Acts Predominantly as a Transcriptional transcription start site (TSS) are given in Supplementary Repressor in HT-22 Cells Table 3. The majority of peaks (79.94%) are located 1 kb up- stream of TSSs; 2.6% are within 1–2 kb, and 1.6% are within We used RNA-seq to analyze the global 2–3 kb upstream of TSSs; and 7.34% are found in intergenic pattern in HT22-TR/TO-V5Klf13-1 cells treated with ve- regions(morethan3kbfromagene;Fig.3a). The remainder of hicle or dox (1 μg/ml)for8h(n = 3/treatment). We found the peaks are found within gene bodies. that expression of V5KLF13 caused statistically signifi- Next, we analyzed the intersection of genes identified as cant changes in the mRNA levels of 3336 genes (log fold differentially expressed by RNA-seq with the genes associat- change > 0.58; adjusted p < 0.05; FDR ≤ 0.01); 2354 were ed with KLF13 peaks. We found that of the 2354 genes that downregulated and 982 were upregulated (Fig. 1c and were repressed by V5KLF13, 1521 (64%) had a KLF13 peak. Supplementary Table 2). The top 10 most induced and By contrast, of the 982 genes that were induced by V5KLF13, repressed genes are given in Table 1. We validated the only 188 (19%) had a KLF13 peak (Fig. 3b). The genes Mol Neurobiol

Fig. 1 Identification of genes regulated by forced expression of V5Klf13 HT22-TR-3 cells. c. Volcano plot: All 3336 significantly differentially in HT22 cells. a Treatment of HT22-TR/TO-V5Klf13-1 cells with expressed (DE) genes are represented in terms of their measured expres- doxycycline (dox; 1 μg/ml) caused a time-dependent induction of the sion change (x-axis) and the significance of the change (y-axis). The V5Klf13 transgene measured by RTqPCR, which peaked at 2 h and significance is represented in terms of the negative log (base 10) of the remained elevated through 24 h (F(5,12) = 9.964, p = 0.0006; one way p value. The dotted lines represent the thresholds used to select the DE ANOVA followed by Turkey’s post-hoc test). Points represent the genes: 0.585 for expression change and 0.05 for significance. The upreg- mean +SEM(n = 3/time point). The endogenous Klf13 mRNA in the ulated genes (positive log fold change) are shown in black, while the control cell line HT22-TR-3 was unaffected by dox treatment. Time- downregulated genes are white. d Validation of a subset of KLF13- dependent synthesis of V5KLF13 protein was confirmed by Western regulated genes identified by RNA-seq using RTqPCR. HT22-TR-3 blotting using a V5 antibody (shown above the graph). b Doxycycline and HT22-TR/TO-V5Klf13-1 cells were treated with vehicle or dox for induction of V5Klf13 in HT22-TR/TO-V5Klf13-1 cells caused a time- 8 h before harvest for RNA isolation. Gene expression was unaffected by dependent suppression of Klf9 mRNA, thereby confirming V5KLF13 dox in HT22-TR-3 cells. Bars represent the mean +SEM(n = 4/treat- bioactivity. The Klf9 mRNA showed a statistically significant reduction ment; Phf13: p < 0.0001; Fshc2: p = 0.0053; Cand2: p < 0.0001; Nlgn2: at 8 h after dox treatment, and remained low through 24 h (Fig. 1b; p < 0.0001; B4gal: p =0.01;Tctn2: p =0.025;Grk4: p = 0.0035; Dgat2: F(5,12) = 8.934, p < 0.001; one-way ANOVA followed by Turkey’s p = 0.033; Student’sunpairedt test). Asterisks indicate statistically sig- post-hoc test; n = 3/time point); dox had no effect on Klf9 mRNA in the nificant differences with p <0.05.

associated with the remaining 7797 KLF13 peaks (the major- present in 51.42% (p =1e−27) of all peaks. In addition, we ity of these peaks were found within or in close proximity to identified two other motifs that were significantly enriched genes) were unaffected by forced expression of V5Klf13 (Fig. in 27.37% (CGCAGCGACA) and 26.99% 3b). De novo motif analysis using the program Homer found (GKCGGGGCWG) of KLF13 peaks (Fig. 3c). All discovered that the most highly enriched motif (CCGCAACT) was motifs are listed in Supplementary Tables 4 and 5. Mol Neurobiol

Table 1 The top ten most down- and upregulated genes after Gene symbol Gene name Log fold change Adjusted p value forced expression of V5Klf13 in HT-22 cells Prl2c1 Prolactin family 2, subfamily c, member 1 − 4.96 0.000001 Fchsd1 FCH and double SH3 domains 1 − 4.08 0.000001 Gm5946 Predicted gene 5946 − 3.95 0.000001 Phf13 PHD finger protein 13 − 3.65 0.000001 Cand2 Cullin-associated and neddylation- − 3.63 0.000001 dissociated 2 (putative) Mapk11 Mitogen-activated protein kinase 11 − 3.60 0.000001 Sox12 SRY (sex determining region Y)-box 12 − 3.54 0.000001 Nlgn2 Neuroligin 2 − 3.52 0.000001 Xkr8 X-linked Kx blood group related 8 − 3.34 0.000001 Prmt2 Protein arginine N-methyltransferase 2 − 3.31 0.000001 Gm8289 Predicted gene 8289 5.23 0.000001 Kif9 Kinesin family member 9 2.71 0.000001 Actn3 Actinin alpha 3 1.87 0.000001 Tctn2 Tectonic family member 2 1.85 0.000001 B4galnt2 Beta-1,4-N-acetyl-galactosaminyl transferase 2 1.85 0.000001 Lrguk Leucine-rich repeats and guanylate 1.78 9.4E-06 kinase domain containing Grk4 G protein-coupled kinase 4 1.76 0.000001 1700003E16Rik RIKEN cDNA 1700003E16 gene 1.74 0.000001 4833417C18Rik RIKEN cDNA 4833417C18 gene 1.71 0.000001 Coq8a Coenzyme Q8A 1.70 0.000001

Validation of KLF13 ChSP-Seq Peaks in HT22 Cells Sin3a, Casp3, Casp9, Homer,andSnapin); these genes code for proteins involved with the regulation of cell cycle, cell To validate the ChSP-seq dataset, we used targeted ChSP- survival, and synaptogenesis. The mapped positions of qPCR at 8 genomic loci with KLF13 peaks that were associ- KLF13 occupancy at each gene (KLF13 ChSP-seq peaks) ated with genes that showed differential expression by RNA- are shown in Fig. 4a. We found significantly greater seq (induced genes: Pde4a, E2f2; repressed genes: Klf16, ChSP-qPCR signal at the 8 loci tested in HT22 cells

Fig. 2 KLF13 regulates the expression of several members of the KLF Klf10 = 2.7-fold, p = 0.0035; Klf11 = 5-fold, p = 0.0056; and superfamily. a Treatment of HT22-TR/TO-V5Klf13-1 cells with Klf16 = 2.2-fold, p = 0.0023; Student’sunpairedt test). Asterisks indicate doxycycline (dox; 1 μg/ml) for 8 h decreased mRNA levels of Klf3, 10, statistically significant differences with p < 0.05. The mRNA of the con- 11, 13,and16 compared with vehicle-treated cells (see Fig. 1 for Klf9 trol gene Klf6 was unaffected by loss of Klf13. c The mRNA levels of mRNA). We analyzed gene expression by RTqPCR; the endogenous Klf3, 9, 10,and11 were significantly elevated in primary hippocampal Klf13 mRNA (#) was analyzed by targeting the Klf13 5’UTR. Bars rep- neurons from Klf13-null mice compared with cells from wild-type mice. resent the mean +SEM(n = 4/treatment; % reduction: Klf3 = 71%, p = Cells were isolated from PND1 mice and cultured for 15 days before 0.0012; Klf13 = 90.8%, p < 0.0001; Klf10 = 46.8%, p =0.009; harvest. Bars represent the mean +SEM(n = 4/genotype; fold Klf11 = 64.1%, p = 0.0005; and Klf16 = 97%, p < 0.0001; Student’sun- increase: Klf3 = 2.2.-fold, p =0.001; Klf9 = 1.7-fold, p =0.03; paired t test). The Klf6 mRNA, which was not found to be regulated in the Klf10 = 1.5-fold, p =0.034;andKlf11 = 2.2-fold, p = 0.0010; Student’s RNA-seq dataset, was unaffected by dox. b The mRNA levels of Klf3, 9, t test). Asterisks indicate statistically significant differences p < 0.05. The 10, 11,and16 were increased in HT22-Klf13-KO cells compared with the level of Klf6 mRNA was unaffected in Klf13-deficient cells, and although parent cell line. Bars represent the mean +SEM(n = 4/genotype; fold the mean Klf16 mRNA level was greater, this difference was not statisti- increase: Klf3 = 1.3-fold, p = 0.0061; Klf9 = 1.6-fold, p = 0.0029; cally significant Mol Neurobiol

Table 2 Cellular signaling pathways affected by forced Pathway name p value* Pathway name p value* expression of V5Klf13 in HT22 cells Pathways in cancer 0.0000013 AGE-RAGE signaling pathway in diabetic 0.0054554 complications Ras signaling pathway 0.0000072 Phosphatidylinositol signaling system 0.0057907 Signaling pathways regulating 0.0000500 Choline metabolism in cancer 0.0057963 pluripotency of stem cells Proteoglycans in cancer 0.0002072 Endocrine resistance 0.0083161 Chagas disease (American 0.0003528 cGMP-PKG signaling pathway 0.0128971 trypanosomiasis) Fc gamma R-mediated phagocytosis 0.0004713 Longevity regulating pathway 0.0135293 Phospholipase D signaling pathway 0.0005452 Circadian entrainment 0.0140493 Sphingolipid signaling pathway 0.0005700 HTLV-I infection 0.0142487 Estrogen signaling pathway 0.0006312 signaling pathway 0.0182016 Adherens junction 0.0007809 Hepatitis C 0.0187759 Oxytocin signaling pathway 0.0008702 PI3K-Akt signaling pathway 0.0193298 Platelet activation 0.0011659 EGFR tyrosine kinase inhibitor resistance 0.0240422 cAMP signaling pathway 0.0013778 Adrenergic signaling in cardiomyocytes 0.0262535 Cholinergic synapse 0.0015566 Endocytosis 0.0283002 Rap1 signaling pathway 0.0016173 Inositol phosphate metabolism 0.0294264 Regulation of lipolysis in adipocytes 0.0017748 VEGF signaling pathway 0.0325453 Hippo signaling pathway 0.0025141 Measles 0.0366411 Axon guidance 0.0030830 Glutamatergic synapse 0.0425898 Prostate cancer 0.0035223 Breast cancer 0.0435059 Regulation of actin cytoskeleton 0.0040904 Melanogenesis 0.0442750 MAPK signaling pathway 0.0043617 Central carbon metabolism in cancer 0.0473981 Gap junction 0.0048939 AMPK signaling pathway 0.0489105 Toxoplasmosis 0.0053272

Differentially expressed genes were analyzed in the context of pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) *Bonferroni expressing biotinylated KLF13 compared with control cells treated with dox for 24 h. This showed that pTO-Egfp cells (Fig. 4b). We also conducted targeted V5KLF13 associated in chromatin at each of the ChIP-qPCR assays using our custom KLF13 antiserum KLF13 target genes, but not at the control region tested in HT22-TR-3 (control) and HT22-TR/TO-V5Klf13-1 (the Klf16 intron; Fig. 4c).

Fig. 3 The KLF13 cistrome in HT22 cells. a We conducted ChSP-seq for circle includes all ChSP peaks; genes that were induced or repressed by KLF13 in HT22 cells as described in the “Materials and Methods.” RNA-seq after forced expression of V5Klf13 are shown in the two smaller Shown is a pie chart depicting the percentage of KLF13 peaks within circles. c The three most commonly occurring DNA motifs found within each genomic feature. b Venn diagram showing the intersection of the KLF13 ChSP peaks using the program Homer, represented as posi- KLF13-regulated genes discovered by RNA-seq with genes associated tion weight matrices with KLF13 peaks within 2 kb of their TSS found by ChSP-seq. The large Mol Neurobiol

Fig. 4 Confirmation of KLF13 occupancy in chromatin at KLF13- peaks by ChIP-qPCR using a custom affinity-purified antiserum to regulated genes. a Genome traces from screen shots of the Integrative KLF13. We treated HT22-TR-3 and HT22-TR/TO-V5Klf13-1 cells Genome Viewer (IGV) showing the locations of KLF13 peaks deter- with doxycycline (dox; 1 μg/ml) for 24 h, harvested the cells then isolated mined by ChSP-seq at 8 KLF13-regulated genes. The gene structures chromatin for ChIP assay as described in the “Materials and Methods”. are shown below the genome traces; lines and black filled bars represent Controls included precipitation with IgG purified from pre-immune rabbit and exons, respectively. b Validation of KLF13 ChSP-seq peaks serum and analysis of a genomic region that did not have a KLF13 peak using targeted ChSP-qPCR in HT22 cells. We isolated chromatin from (the Klf16 intron). Enrichment was analyzed comparing normal IgG to HT22-BirA cells transfected with pTO-Egfp (control) or pTO- anti-KLF13 within cell lines. Bars represent the mean + SEM (n =4/ Klf13FLBio to express biotinylated KLF13, precipitated DNA with treatment; HT22-TR-3: Klf16: p =0.045;Pde4a: p =0.033;Casp9: p = streptavidin, and analyzed enrichment of target sequences under KLF13 0.004. HT22-TR/TO-V5Klf13-1: Klf16: p = 0.0028; Sin3a: p = 0.0024; peaks using qPCR. Bars represent the mean + SEM (n = 4/treatment; E2f2: p =0.014;Pde4a: p =0.047;Casp3: p =0.023;Casp9: p =0.039; Klf16: p = 0.0008; Sin3A: p =0.004; E2f2: p =0.027; Pde4a: p = Homer3: p = 0.0026; Snapin: p = 0.0013; Student’s unpaired t test). 0.0004; Casp3: p = 0.049; Casp9: p = 0.007; Homer3: p = 0.003; and Asterisks indicate statistically significant differences (p <0.05) Snapin: p = 0.034; Student’sunpairedt test). Asterisks indicate statisti- cally significant differences (p <0.05).c Validation of KLF13 ChSP-seq

Transfection-Reporter Assays Showed that KLF13 Can compared with vehicle treatment. By contrast, dox treatment Directly Regulate Gene Transcription increased RLA by 45% in HT22-TR/TO-V5Klf13-1 cells transfected with pGL-E2f2 (Fig. 5). Treatment with dox had We conducted transfection-reporter assays in HT22-TR/TO- no effect on the RLA in the control HT22-TR-3 cells V5Klf13-1 cells to investigate if KLF13 can directly regulate transfected with either of the three reporter-promoter transcription of genes with KLF13 ChSP peaks. We selected constructs. three genes for analysis whose mRNAs were changed by ex- pression of V5KLF13 and had associated KLF13 ChSP-seq KLF9 and KLF13 Share Some Common Target Genes in peaks, two that were repressed (Klf16 and Casp3) and one that Neuronal Cells was induced (E2f2). Cells were transfected, then treated with dox for 24 h before harvest for dual luciferase assay. Our RNA-seq analyses after forced expression of KLF9 or Treatment with dox reduced relative luciferase activity KLF13 in HT22 cells suggest that KLF13 (this study) has a (RLA) in HT22-TR/TO-V5Klf13-1 cells transfected with broader regulatory landscape than KLF9 [18]; we re-analyzed pGL-Klf16 or pGL-Casp3 by 93% and 76% respectively, the KLF9 dataset generated by Knoedler and colleagues [18] Mol Neurobiol

Fig. 5 KLF13 represses transcriptional activity of the Klf16 and Casp3 or dox (1 μg/ml) for 24 h, then harvested cells and conducted dual lucif- gene promoters, but activates the E2f2 promoter. We transfected HT22- erase assay. RLA, relative luciferase activity. Bars represent the TR or HT22-TR/TO-V5Klf13-1 cells with luciferase reporter vectors mean + SEM (n = 4/treatment). We used Student’s unpaired t test to containing promoter fragments from the Klf16, Casp3,andE2f2 genes compare vehicle vs. dox within a cell line (Klf16: p < 0.0001; Casp3: corresponding to genomic regions with KLF13 peaks discovered by p < 0.0001; E2f2: p < 0.0001) ChSP-seq. Twenty hours after transfection, we treated cells with vehicle using DeSeq2, which increased the total number of regulated KLF13 Promotes Cell Cycle Progression genes from 238 to 944. In this cell type and under these con- ditions, KLF13 influenced the expression of 3336 genes, Our analysis of differential gene expression showed that while KLF9 affected 944 genes (33% of KLF13-regulated KLF13 impacts several pathways involved with cell prolifer- genes). We compared the differentially regulated genes for ation (Table 2). To test if KLF13 can influence cell cycle, we the two KLFs and found overlap of 461 genes, which repre- treated the HT22-TR/TO-V5Klf13-1 cell line with vehicle or sents 48% of KLF9-regulated genes, and 13% of KLF13- dox, then analyzed cell cycle progression by flow cytometry. regulated genes (Fig. 6). We also looked at peaks discovered Forced expression of V5Klf13 increased the percentage of by ChSP-seq and found that 1657 overlapped between KLF9 cells in G2/M phase, and reduced the percentage in G0/G1 and KLF13, which represents 57% of the KLF9 and 21% of compared with vehicle-treated HT22-TR/TO-V5Klf13-1 the KLF13 peaks (Fig. 6). Supplementary Tables 6 and 7 cells, or HT22-TR-3 cells treated with vehicle or dox (Fig. 7a). show the top ten pathways that we found by analyzing the We also analyzed cell cycle in HT22-Klf13-KO cells and intersections of the KLF9 and KLF13 RNA-seq or ChSP- found that they had a lower percentage of cells in G2/M phase, seq datasets. This analysis showed that four pathways were and a higher percentage in G0/G1 phase compared with the overrepresented for both KLFs in both the RNA-seq and the parent cell line (Fig. 7b). We also looked at cell proliferation ChSP-seq datasets: regulation of actin cytoskeleton, pathways in primary hippocampal neurons isolated from wild type mice in cancer, ubiquitin-mediated proteolysis, and the and transfected with the expression plasmids pTO-V5Klf13 or neurotrophin signaling pathway. pTO-Egfp (control). Forced expression of V5KLF13 in

Fig. 6 KLF13 shares some target genes with KLF9. Venn diagrams showing the overlap among differentially expressed genes discovered by RNA-seq follow- ing forced expression of Klf9 or V5Klf13 in HT22 cells (left) and genes with KLF9 or KLF13 as- sociated peaks discovered by ChSP-seq (right) Mol Neurobiol

Fig. 7 KLF13 promotes cell cycle progression in HT22 cells and primary staining, and conducting flow cytometry. Bars represent the mean (n =4/ hippocampal neurons. a The percentage of HT22 cells in G1 and G2/M treatment; G1: p = 0.008; G2/M: p = 0.009; Student’s unpaired t test). phases after forced expression of V5Klf13. We treated HT22-TR-3 Asterisks indicate statistically significant differences in G1 between cell (control) and HT22-TR/TO-V5Klf13-1 cells with vehicle or doxycycline genotypes; hashtag indicates statistically significant differences in G2/M (dox; 2 μg/ml) for 24 h, harvested cells, fixed and stained them with the between cell genotypes. c Incorporation of EdU in primary hippocampal nuclear stain FxCycle Violet (Thermo Fisher), then conducted flow cy- cellsisolatedfromwildtypePND1miceandtransfectedwithpTO-Egfp tometry. Bars represent the mean (n = 4/treatment). The baseline of cy- (control) or pTO-V5Klf13. We transfected primary cells before plating tometry value in G1 and G2/M was different between cell lines treated using a Lonza nucleofector, and cultured them for 24 h before addition of with vehicle (G1: p =0.038;G2/M:p =0.048;Student’s unpaired t test). 20 μM EdU. After 1.5 h, we harvested cells and analyzed EdU incorpo- Treatment with dox affected the cytometry value of G1 and G2/M in ration using the sulfo-Cy3 fluorophore paired with epifluorescence mi- HT22-TR/TO-V5Klf13-1 (p< 0.0001; Student’s unpaired t test) but croscopy. Bars represent the mean + SEM (n = 3/treatment; p = 0.0022; not in HT22-TR-3 cells. Asterisks indicate statistically significant differ- Student’sunpairedt test). d Analysis of cell cycle-related gene expression ences in G1 or G2/M between HT22-TR-3 and HT22 TR/TO-V5Klf13-1 by RTqPCR in HT22 after forced expression of V5Klf13. We treated treated with vehicle; hashtag indicate statistically significant differences HT22-TR/TO-V5Klf13-1 cells with vehicle or dox (1 μg/ml) for 8 h in G1 or G2/M in HT22-TR/TO-V5Klf13-1 vehicle vs doxycycline treat- before harvest and RNA isolation. The expression of was used as ments. b Comparison of the percentage of the HT22 parent and HT22- a negative control. Gene expression was normalized to the geometric Klf13-KO cell lines in G1 and G2/M phases. Cell cycle of the two cell mean of the mRNA levels of the reference genes Gadph and Ppia.Bars lines was first synchronized with DMEM containing 1% FBS for 24 h. represent the mean +SEM(n = 4/treatment; E2f2: p =0.021;Orc1: p = We then incubated cells in growth medium for 24 h before fixing and 0.001; Orc2: p =0.028;Cdk7: p = 0.0003; Student’sunpairedt test)

primary hippocampal cells led to an approximate doubling of levels of E2f2, Orc1, Orc2,andCdk7 (Fig. 7d). The EdU incorporation compared with cells transfected with the mRNA level of E2f1, which was unchanged in the RNA- control vector (Fig. 7c), indicating greater DNA synthesis in- seq dataset, was not affected. duced by V5KLF13. To investigate mechanisms by which KLF13 promotes KLF13 Has Cytoprotective Actions in Neurons cell proliferation, we quantified the expression of several KLF13 target genes whose protein products are effectors of Previous work showed that KLF13 protects cardiomyocytes cell cycle progression (selected for differential expression from DNA damage [26]. Our RNA-seq analysis showed that from the RNA-seq dataset). Treatment of HT22-TR/TO- KLF13 represses some genes in the apoptotic pathway in V5Klf13-1 cells with dox for 8 h increased the mRNA HT22 cells (Supplementary Table 2), and some (Casp3 and Mol Neurobiol

Casp9) are direct targets of KLF13 (see Fig. 4). We also found To differentiate the effect of KLF13 on cell survival that the glutamatergic synapsis pathway is impacted by KLF13 from its effect on cell cycle, we quantified LDH release (Table 2). Therefore, we investigated a possible role for KLF13 (an indicator of cytotoxicity) after treatment with gluta- in neuronal survival using the glutamate-induced excitotoxicity mate. This showed that glutamate exposure increased assay. Forced expression of V5Klf13 for 4 h before exposure to LDH release as expected, which was reduced in the glutamate improved cell viability, as evidenced by increased HT22-TR/TO-V5Klf13-1 cells after treatment with dox MTT signal compared with the HT22-TR-3 control cell line (Fig. 8d). By contrast, LDH release was higher in (Fig. 8a). By contrast, HT22- Klf13-KO cells and primary hip- HT22-Klf13-KO cells compared with the parent cell line, pocampal cells derived from Klf13−/− mice had lower MTT sig- and also in primary hippocampal cells isolated from nal after glutamate exposure than the parent HT22 cell line or Klf13−/− mice compared with cells from wild type mice wild type primary cells, respectively (Fig. 8b;HT22:p = 0.0069; (Fig. 8e; HT22: p = 0.0019; Fig. 8f; primary cells: Fig. 8c; primary cells: p = 0.0242; Student’s t test; n =4). p < 0.0001; Student’s t test; n =4).

Fig. 8 KLF13 has cytoprotective actions in HT22 and primary from wild type or Klf13−/− PND1 mice (* indicates p =0.023).d–f We hippocampal cells. a–c We analyzed cell viability in HT22 and primary analyzed LDH release in HT22 and primary hippocampal cells after ex- hippocampal cells after exposure to glutamate (10 mM for 24 h) using the posure to glutamate (10 mM for 24 h). Bars represent the mean +SEM MTT assay. Bars represent the mean + SEM (n = 4/treatment). a We (n = 4/treatment). d We treated HT22-TR-3 and HT22-TR/TO-V5Klf13- treated HT22-TR-3 and HT22-TR/TO-V5Klf13-1 cells with doxycycline 1 cells with dox (1 μg/ml) for 4 h before exposure to (dox; 1 μg/ml) for 4 h before exposure to glutamate. Glutamate caused a glutamate. Glutamate caused a significant increase in LDH release in both significant decrease in cell viability in both cell lines (HT22-TR-3: cell lines (TR-3: p < 0.0004; TR/TO-V5Klf13-1: p < 0.0001) but this ef- p < 0.0001; HT22-TR/TO-V5Klf13-1: p = 0.002; Student's unpaired t- fect was lesser in HT22-TR/TO-V5Klf13-1 cells (*indicates p < 0.05 test) but this effect was greater in HT22-TR-3 cells (* indicates p between the two cell genotypes within the glutamate treatment). e We < 0.05 between the two cell genotypes within the glutamate treatment). compared the parent HT22 cell line with HT22-Klf13-KO cells (* b We compared the parent HT22 cell line with HT22-Klf13-KO cells (* indicates p =0.002).f We compared primary hippocampal cells isolated indicates p = 0.0069). c.We compared primary hippocampal cells isolated from wild type or Klf13−/− PND1 mice (* indicates p < 0.0001) Mol Neurobiol

Discussion 1 kb of TSSs. Using de novo motif analysis, we found three motifs that were enriched at the genomic regions where KLF13 Here we report the first genome-wide analysis of KLF13 geno- associates, and these resemble the characteristic GC boxes to mic targets and its impact on cellular signaling pathways in which other members of the KLF/SP family bind [19]. When mammalian hippocampal neurons. To our knowledge, this is we looked for known motifs, we discovered that 82% of the the first analysis of its kind in any cell type. Using the adult KLF13 peaks had GGCGGCGG, which contains two repetitions mouse hippocampus-derived cell line HT22, we found that of the GGCG sequence, a GC box found in the Basic KLF13 regulates a diverse set of genes and cellular pathways, Transcriptional Element (BTE) first reported by Imataka and and it functions predominantly as a transcriptional repressor, colleagues [22] as a binding site for KLF9 (at the time referred associating in chromatin at proximal promoters of target genes. to as BTEB) in the rat Cyp1A1 gene. However, the KLF13 We also found that, like its paralog KLF9, KLF13 directly consensus motifs that we found here are different from those that represses transcription of other Klf genes, and thus participates we found previously for KLF9 [18], thus providing one mecha- in a transcriptional regulatory network that modulates KLF ac- nism for differential gene regulation by these two KLFs. Other tivities in cells. Our signaling pathway analyses revealed that previously described consensus motifs that we found to be KLF13 is involved with a diversity of cellular functions, includ- enriched within genomic regions covered by the KLF13 peaks ing the control of cell cycle progression and cell survival, include those for KLF3, , KLF5, KLF6, KLF9, KLF10, among others. In addition, we demonstrate that KLF9 and and KLF14, supporting that multiple other KLFs may regulate KLF13 share some common target genes, supporting that these the KLF13 target genes that we found. These findings support paralogous transcription factors can function cooperatively, but the conclusion that multiple KLFs function within a transcrip- also antagonistically. Taken together, our findings represent an tional network to regulate target genes, either cooperatively or important advance in understanding the molecular mechanisms antagonistically [2]. In this regard, and based in our RNA-seq and physiological roles that these two closely related KLFs dataset, we found that the HT22 cells express twelve members of have in mammalian neurons. the Klf gene family, which includes 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, It is well known that KLF13 has important functions in the 15, and 16. Six of these Klf genes (3, 9, 10, 11, 13, and 16) were immune system; hence, it was previously known as RANTES repressed after forced expression of V5Klf13, while their mRNAs Factor of Late Activated T Lymphocytes-1 (RFLAT-1) [38]. (for 3, 9, 10, and 11) were increased in the HT22-Klf13-KO cell However, its expression is not restricted to the immune sys- line and in primary neurons isolated from Klf13-null mice. The tem, and it is expressed in several tissues, including the central presence of KLF13 peaks at the promoters of these genes sup- nervous system (CNS) [18]. Moore and colleagues [7]studied ports that KLF13 directly regulates their transcription. This pro- the effects of forced expression of multiple KLFs in cortical vides further evidence for the participation of KLF13 in a KLF neurons and found that KLF13, like KLF9 and several other transcriptional network in neurons. KLFs, can inhibit neurite-outgrowth. However, virtually noth- The cross-regulation between members of the KLF superfam- ing is known about other cellular functions, or the molecular ily has been described in several contexts. Dang and colleagues mechanisms for KLF13 actions in mammalian neurons. Using [41] found that KLF4 and KLF5 have opposing effects on Klf4 the mouse HT22 cell line, which has characteristics of adult promoter activity by competing for binding with the same DNA hippocampal neurons [39, 40], we found that KLF13 func- sequence, which could result in coordinated actions to regulate tions predominantly as a transcriptional repressor; of the the proliferation of intestinal epithelial cells. In another example, 3336 differentially regulated genes discovered by RNA-seq Eaton and colleagues [42] demonstrated that KLF1 activates after forced expression of V5Klf13, 2354 were repressed transcription of Klf3 and Klf8, while KLF3 competes for binding (70.5%), while 982 were induced. Using ChSP-seq, we found with KLF1 to repress transcription of Klf8. In addition, KLF13 that 64% of the repressed genes have a KLF13 peak, com- has been implicated in a cross-regulatory network with KLF9. pared with only 19% of induced genes. These findings are For example, Pablona and colleagues [35] showed that KLF9 consistent with our previous work on KLF9 in the same cell and KLF13 are expressed sequentially to mediate the actions of line, where we found that KLF9 functions predominantly as a progesterone on uterine cell decidualization, and subsequent transcriptional repressor; a majority of the KLF9-repressed blastocyst implantation and maintenance of pregnancy through genes had a KLF9 ChSP-seq peak, while very few of the their regulation of bone morphogenic protein 2. It was also induced genes had peaks [18]. This suggests that although shown that KLF9 can compensate for the loss of KLF13, via KLF9 and KLF13 may directly repress transcription of most upregulation of KLF9 protein levels in the uterus of Klf13-null of their target genes, and directly induce transcription of some mice, which resulted in mice with normal fertility [43]. genes, the majority of the induced genes may be due to sec- Moreover, in a recent study, we showed that KLF9 and KLF13 ondary responses to these KLFs. both regulate transcription of the cellular circadian output Using ChSP-seq, we discovered that KLF13, like KLF9, as- gene Dbp [44]. The circadian rhythm in Dbp mRNA was not sociates in chromatin primarily at genomic regions located within affected by single gene knock out of Klf9 or Klf13 in HT22 cells. Mol Neurobiol

However, double gene knockout caused complete loss of Dbp contrast, knockout of Klf13 in HT22 led to a greater percentage mRNA rhythmicity, suggesting cooperative and compensatory of cells in G1 phase compared with the parent cell line, actions of these paralogous transcription factors. These findings supporting impaired cell cycle progression when the Klf13 gene support that evolution has produced redundancy in the pathways is inactivated. In complement, we found that forced expression regulated by KLFs, which provides robustness to the regulation of KLF13 increased incorporation of EdU in primary hippo- of intracellular signaling pathways. campal cells, demonstrating that KLF13 promotes the transition To understand the mechanisms underlying the cooperative, from G1 to M phase (DNA synthesis). Although the action of compensatory, or antagonist actions of KLF9 and KLF13, we KLF13 on the genes found in the pathways described above is compared genes differentially regulated by both KLFs and primarily inhibitory, KLF13 induced the expression of some discovered that KLF13 influenced the expression of three genes, such as E2f2, Orc1, Orc2,andCdk7, whose protein times more genes than KLF9, and that 48% of the KLF9- products are effectors of cell cycle progression [50–52]. regulated genes are also regulated by KLF13. This is consis- Members of the family of transcription factors accumulate tent with the number of peaks that we found by ChSP-seq: during G1 phase and play critical roles in inducing genes 9506 for KLF13 and 2452 for KLF9 [18]. By analyzing the expressed during S phase [53]. Indeed, members of this family overlap between KLF9 and KLF13 RNAseq or ChSP can drive S-phase entry in the absence of growth factor stimu- datasets, we found that of the top-ten most highly represented lation [54]. This suggests that one mechanism by which KLF13 pathways, four (regulation of actin cytoskeleton, pathways in can promote progression to S-phase is by inducing expression cancer, ubiquitin-mediated proteolysis, and neurotrophin sig- of E2f2. By contrast, we previously found that KLF9 sup- naling pathways) were enriched for both KLFs. It is well presses cell cycle progression in HT22 cells [18]. Intriguingly, known that genes found in the “Regulation of actin cytoskel- by analyzing our KLF9 RNA-seq and ChSP-seq dataset, we eton” [45] and the “Neurotophin signaling” pathways are in- found that KLF9 does not associate with the promoters and volved in neurite outgrowth and axon regeneration [46]. does not affect the expression of the effector cell cycle genes Results from the present study reveal possible molecular that we found were regulated by KLF13. These findings raise mechanisms to explain findings reported by Moore and col- the interesting possibility that these two closely related KLFs leagues [7] who showed that both KLF9 and KLF13 (and may regulate cell cycle antagonistically, in part by regulating seven other KLFs) inhibited neurite outgrowth of mouse cor- each other. This implies that when Klf9 is induced, one expects tical neurons. Complementary experiments that will be pub- to see a decrease in cell proliferation, in part through its sup- lished in a separate manuscript confirm that KLF9 and KLF13 pression of Klf13, and, consequently, suppression of genes such both block neurite-outgrowth in primary hippocampal neu- as Orc1, Orc2, E2f2,andCdk7. On the other hand, when Klf13 rons, in part by inhibiting the cAMP pathway (J. Avila is induced, one expects to see an increase in cell proliferation, in Mendoza, A. Subramani and R.J. Denver, unpublished data). part through direct activation of cell cycle genes, but also by the We analyzed the genes identified by RNA-seq in the con- inhibition of Klf9. text of pathways impacted by forced expression of V5Klf13 to We found that KLF13 associates with promoters of the understand how KLF13 influences neuron cell physiology apoptotic effector genes Casp3 and Casp9, and it is able to and structure. We found that forty-three pathways were sig- inhibit the promoter activity of Casp3. The repression of these nificantly impacted, with the most significant being genes, and possibly other genes involved in apoptosis, may “Pathways in cancer”. The role of KLFs in cancer biology underlie the cytoprotective actions of KLF13 that we discov- has been studied extensively, and it is well known that mem- ered in hippocampal neurons. These findings are consistent bers of this family of transcription factors function in cancer with a previous report that identified KLF13 as a mediator pathogenesis in multiple ways, having roles as oncogene or of signaling in the protection of tumor suppressor genes depending on the cell type [47]. In cardiomyocytes from toxins [26]. Cruz-Topete and colleagues this regard, KLF13 has been shown to suppress the growth of [26] reported that one potential mechanism for KLF13 prostate carcinoma cells through inhibition of AKT activation cytoprotection is its regulation of both anti- and pro-cell death [27]. Intriguingly, we found that “Prostate cancer” and the genes. Our results strongly suggest that the cytoprotective “PI3K-AKT” pathways were also enriched in our dataset, actions of KLF13 could be through the repression of pro- which confirms and expands the mechanisms by which apoptotic genes. Taken together, the results support the im- KLF13 functions as an inhibitor of prostate cancer. portance of KLF13 have as a cytoprotective factor. Moreover, other cellular pathways related to the control of In summary, our findings show that KLF13 works predom- cell cycle were influenced by KLF13. These include the “Ras, inantly as a transcriptional repressor in mouse hippocampal cAMP, Rap1a, MAPK and AMPK signaling Pathways” [48, neurons, associating in chromatin at proximal promoters of 49]. These findings caused us to test if KLF13 might affect cell target genes like its paralog KLF9. Similar to KLF9, KLF13 cycle of hippocampal neurons. We found that forced expression functions within a transcriptional regulatory network that in- of V5Klf13 promoted cell cycle progression of HT22 cells. By volves other members of the KLF superfamily. KLF13 Mol Neurobiol promotes cell cycle and survival of neuronal cells, actions that 7. Moore DL, Blackmore MG, Hu Y et al (2009) KLF family mem- may be opposite to those of KLF9. These differences in func- bers regulate intrinsic axon regeneration ability. Science (80- ) 326: 298–301. https://doi.org/10.1126/science.1175737 tion indicate that neofunctionalization occured since the orig- 8. Tang X, Liu K, Hamblin MH, Xu Y, Yin KJ (2018) Genetic dele- inal gene (or genome) duplication event that produced the two tion of Krüppel-like factor 11 aggravates ischemic brain injury. Mol paralogous genes; whereas, the overlap between KLF13 and Neurobiol 55:2911–2921. https://doi.org/10.1007/s12035-017- KLF9 actions suggest that their shared functions are ancient, 0556-9 9. Denver RJ, Ouellet L, Furling D, Kobayashi A, Fujii-Kuriyama Y, andhavebeenmaintainedbystabilizingselection.Ourfind- Puymirat J (1999) Basic transcription element-binding protein ings advance our understanding of the molecular mechanisms (BTEB) is a thyroid hormone- regulated gene in the developing that underlie the cooperative, compensatory, or antagonist ac- central nervous system: evidence for a role in neurite outgrowth. J tions of members of the KLF superfamily in neurons. Biol Chem 274:23128–23134. https://doi.org/10.1074/jbc.274.33. 23128 10. Denver RJ, Williamson KE (2009) Identification of a thyroid hor- Authors’ Contributions JAM conceived the project, designed and conduct- mone response element in the mouse Kruppel-like factor 9 gene to ed experiments, analyzed data, and wrote the manuscript. AS conducted explain its postnatal expression in the brain. Endocrinology 150: experiments, analyzed data, and assisted in writing the manuscript. CJS 3935–3943. https://doi.org/10.1210/en.2009-0050 conducted the bioinformatics analyses and assisted in writing the manu- 11. Bagamasbad PD, Bonett RM, Sachs L, Buisine N, Raj S, Knoedler script. RJD conceived the project, designed experiments, analyzed data, and JR, Kyono Y, Ruan Y et al (2015) Deciphering the regulatory logic wrote the manuscript. All authors read and approved the final manuscript. of an ancient, ultraconserved enhancer module. Mol Endocrinol 29:856–872. https://doi.org/10.1210/me.2014- Funding Information This work was supported by a grant from the National 1349 Science Foundation (IOS 1456115) and funding from the College of 12. Scobie KN, Hall BJ, Wilke SA, Klemenhagen KC, Fujii-Kuriyama Literature, Science, and the Arts of the University of Michigan to R.J.D. Y, Ghosh A, Hen R, Sahay A (2009) Krüppel-like factor 9 is nec- J.A.M. was supported by a postdoctoral fellowship from Consejo Nacional essary for late-phase neuronal maturation in the developing dentate de Ciencia y Tecnología (CONACYT), CVU-267642. gyrus and during adult hippocampal neurogenesis. J Neurosci 29: 9875–9887. https://doi.org/10.1523/JNEUROSCI.2260-09.2009 Availability of Data and Material The RNA-seq and ChSP-seq data re- 13. 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