The human genome contains more than 4.5 million inserts derived from transposable elements (TEs), the result of recurrent waves of invasion and internal propagation throughout evolution. For new TE copies to be inherited, they must become integrated in the genome of the germline or pre-implantation embryo, which requires that their source TE be expressed at these stages. Accordingly, many TEs harbor DNA binding sites for the pluripotency factors OCT4, NANOG, SOX2, and KLFs and are transiently expressed during embryonic genome activation. Here, we describe how many primate-restricted TEs have additional binding sites for lineage-specific transcription factors driving their expression during human gastrulation and later steps of fetal development. These TE integrants serve as lineage-specific enhancers fostering the transcription, amongst other targets, of KRAB-zinc finger proteins (KZFPs) of comparable evolutionary age, which in turn corral the activity of TE-embedded regulatory sequences in a similarly lineage-restricted fashion. Thus, TEs and their KZFP controllers play broad roles in shaping transcriptional networks during early human development. The human genome harbors more than 4.5 million transposable element (TE)-derived insertions, the result of recurrent waves of invasion and internal propagation. Here they show that TEs belonging to evolutionarily recent subfamilies go on to regulate later stages of human embryonic development, notably conditioning the expression of genes involved in gastrulation and early organogenesis. Most are retroelements, whether ERVs (endogenous retroviruses), LINEs (long interspersed nuclear elements), SINEs  or SVAs , which replicate through a copy-and-paste mechanism with reverse transcription of an RNA intermediate followed by insertion of its DNA copy. TEs are increasingly recognized as major drivers of genome evolution owing to their recombinogenic and regulatory potential, even though most are unable to spread further due to inactivating mutations6. The same subset of TEs is enriched in acetylated histone, a hallmark of enhancers, in human embryonic stem cells (hESCs) derived from the pre-implantation embryo9, where many are bound and controlled by pluripotency transcription factors (TFs)13. This broad induction of transcriptionally active TE loci likely contributes to the efficiency of EGA, and their cis-regulatory influences shape the gene regulatory landscape of the pre-implantation embryo. In a remarkable regulatory feedback loop, the pluripotency factor-mediated activation of primate-restricted TE-embedded enhancers leads to the expression of evolutionary contemporaneous Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs), which act as sequence-specific repressors of these EGA-induced TEs14.TEs are tightly controlled by epigenetic silencing mechanisms, yet many are expressed when these mechanisms are put on hold in the context of genome reprograming during gametogenesis or in pre-implantation embryos, when transposition results in inheritable new integrants. Correspondingly, thousands of TEs, albeit mostly primate-restricted ERVs , SVAs, Alus, and young LINE-1s, display marks of open chromatin and are transcribed during embryonic genome activation (EGA)18. However, it has become established that TE-embedded regulatory sequences (TEeRS) influence multiple aspects of human or mouse biology, and it is increasingly recognized that KZFPs exert profound influences on their actions24. Here, we report that many TEeRS induced during EGA are re-expressed during gastrulation and exhibit an open chromatin in fetal tissues, with a high degree of lineage specificity reflecting their activation no longer by pluripotency factors but by cell-type-restricted TFs. We also determine that many of these TE-derived enhancers are enriched near KZFP genes, the secondary stimulation of which is responsible for lineage-specific heterochromatin formation during germ layer formation. We thus conclude that evolutionary recent TEs and their KZFP controllers strongly influence and thereby confer a high degree of species specificity to the conduct of human gastrulation and fetal development.KZFP-induced heterochromatin formation and DNA methylation are often viewed as responsible for maintaining TE-embedded regulatory sequences in a repressed state at later stages of development and in adult tissues25. Gene expression patterns allowed the grouping of cells in clusters corresponding to epiblast, primitive streak, primordial germ cells (PGCs), ectoderm, to nascent, emergent, advanced, yolk sac and axial mesoderm, to endoderm and to early hematopoietic compartment 9, observing that many TEs expressed in specific cells of the gastrula exhibited some level of chromatin accessibility in the pre-implantation embryo and in hESC  repressed upon LTR5 CRISPRi-mediated silencing during endodermal differentiation reside in one such cluster, which we found to be enriched in LTR5B inserts . Correspondingly, we determined that GATA6 bound directly to numerous LTR5B integrants during endodermal differentiation and induced expression of LTR5B-HERVK and nearby KZFP genes when overexpressed in hESC  with harvest after 3 days of endodermal differentiation followed by 3 days of foregut differentiation. Gastruloid differentiation was performed as in ref. 2 in mTSER replaced 48–76 h post-splitting by Nutristem media with 3 μM CHIR-99021. After 24 h cells were split in single-cell passaging with TryplE, embryoid bodies were formed with 800 cells per well of low adherence 96-well plate Cell Star in E6 media supplemented with 3 μM CHIR-99021 and 10 μM Rock inhibitor for 18 h, then replaced with E6 twice to be harvested at 72 h.H9 and H1 human ESC line were maintained in mTSER plus on Matrigel and were passaged using TryplE in single cells. Endodermal differentiation was performed as in ref. 55. hESCs in H9 media were transduced with dCAS9-KRAB lentiviral vector, selected, and maintained in puromycin (0.25 μg/mL) for 5 to 10 days before differentiation experiments.sgRNA design was performed by taking the Dfam consensus of LTR5B common sequence (TTGCAGTTGAGATAAGAGGAAGG). Furthermore, sgRNA design for LTR6B  perform to uniquely match chr1:33109510-33110065 (hg19). Specificity was predicted with the CRISPOR software v5.01ChIP were performed as in ref. 56. MACS 2.2.457 was used for peak calling. Peaks were merged using bedtools v2.27.158. FeatureCounts59 was used to count uniquely mapped reads (MAPQ > 10) on the peaks. Samtools tools v1.1 was used to convert in bam files. Library size correction was performed using the TMM method as implemented in the limma package of R, using the total number of aligned reads as size factor. All ChIP-seq binding locations from the literature were extracted from ChIP-Atlas database32 containing data for more than a thousand of chromatin associated proteins including more than 15'000 different datasets. Differential analysis on the uniquely mapped counts between conditions was performed with voom60. Heatmaps and profile averages were calculated using deeptools v3.5.161 over 5 kb windows around the peak/repeat center from bigwigs. Enrichment analysis over TE subfamilies was performed with HOMER software v4.10.462 and visualized in IGV v2.8.4 and ggplots v3.l.1 in R v4.l.2.Sequenced reads were aligned to the reference human genome hg19 with bowtie263. Counts on genes and TEs were generated using featureCounts v1.6.258 and only uniquely mapped reads with MAPQ > 10 were kept. To avoid read assignation ambiguity between genes and TEs, a gtf file containing both, genes and TEs was provided to featureCounts. For repetitive sequences, an in-house curated version of the Repbase database was used . Only uniquely mapped reads were used for counting on genes and repetitive sequences integrants. TEs overlapping exons or that did not have at least one sample with 3 reads were discarded from the analysis. Normalization for sequencing depth was done for both genes and TEs using the TMM method as implemented in the limma package of Bioconductor64, with the counts on genes as library size. Finally, for each transgene, differential gene expression analysis was performed using Voom60 as it has been implemented in the limma package of Bioconductor v3.13 and assessing only genes (or TEs) that had 3 reads in at least one sample. A gene (or TE) was considered differentially expressed when the fold change between groups was bigger than 2 and the p-value was smaller than 0.05. A moderated t-test (as implemented in the limma package of R) was used to test significance. P-values were corrected for multiple testing using the Benjamini–Hochberg’s method. For counting on TE subfamilies, we added up reads on repetitive sequences without filtering out for multi-mapped reads and added them up per subfamily.Total RNA from cell lines was isolated with NucleoSpin™ RNA Plus kit (Machery-Nagel). cDNA was prepared with Maxima Reverse Transcriptase (Thermo Scientific). Sequencing libraries were performed with Illumina Truseq Stranded mRNA LT kit. Reads were mapped to the human (hg19) genome using Hisat2 v2.l.033. We excluded TEs overlapping exons and TE subfamilies that colocalized less than 150 times near the promoters of protein coding genes. The coefficients of this linear regression problem can be interpreted as the deviations of logged gene expression values explained by the presence of one TE for each TE subfamily. We define these coefficients as the cis-regulator activity of TE subfamilies. Similar models were proposed to infer the activity of DNA motifs from gene expression data65. To find TE subfamilies with significant cis-regulatory activities, we performed null significance hypothesis testing on the linear regression coefficients and accounted for multiple testing using the Benjamini–Hochberg procedure.To identify TE subfamilies exerting putative cis-regulatory activities directly from RNA-seq data, we modeled treatment vs control deviations in gene expression of protein coding genes as a linear combination of occurrences of nearby (within 50 kb of the TSS of protein coding genes) TE subfamily integrantsWe used a lentiviral vector containing a minimal promoter followed of GFP cDNA  containing a full LTR from LTR5 subfamily amplified from a LTR5B . Then a single mutation was generated using Agilent Technologies QuikChange II XL (Cat#200522-5). H9 hESC were transduced by either of these enhancer-containing vectors followed by 3 days of endodermal differentiation and then analyze by FACS (FlowJo LCC v8.8.7).q < 10e-5, and using the --bampe option when PE reads.Reads were aligned with bowtie2. Mitochondrial reads were removed before peak calling. Peak calling was done with MACS2 with 66 was used to obtain counts on genes and peaks using default parameters. The hg38 reference genome provided by cellranger-arc was used. We identified five main clusters in this experimental system: TBXT-expressing axial mesoderm, SOX2-expressing neuro-mesenchymal progenitors (NMP), and three different stages of paraxial mesoderm differentiation preferentially expressing TBX6 (somitic), or PAX3/TWIST1/SIX1  with a different degree of somitic HOX gene markers 29. Significance for TE family chromatin accessibility enrichment was assessed by random permutations: First, for each TE subfamily, the total number of detected peaks for a given cell type on the selected TE subfamily was computed using R GenomicRanges library v1.48.0. Then, TE subfamilies were randomly shuffled 10 times using bedtools with options –chrom and –noOverlapping and the total number of peaks for each permutation was computed. The fold enrichment of the significant subfamilies was then plotted on a heatmap using R heatmap2 function for each cell type.Single-cell ATAC-seq peaks data were downloaded from the Atlas Of Chromatin Accessibility During Development . Figures p-value using a two-sided t test. Fig. p-value using two-sided t test with correction for multiple testing using the Benjamini–Hochberg’s method.No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized The Investigators were partly blinded to allocation during experiments and outcome assessment. All experiments contain at least 2 biologically independent replicates (for RNA-seq) and more than 3 for ChIP/RT-qPCR and FACS analysis. Figures Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Caenorhabditis nematodes led to extensive rewiring of germline transcriptional regulation. We find that about one-third of Caenorhabditis elegans germline-specific promoters have been co-opted from two related miniature inverted repeat transposable elements (TEs), CERP2 and CELE2. These promoters are regulated by HIM-17, a THAP domain–containing transcription factor related to a transposase. Expansion of CERP2 occurred before radiation of the Caenorhabditis genus, as did fixation of mutations in HIM-17 through positive selection, whereas CELE2 expanded only in C. elegans. Through comparative analyses in Caenorhabditis briggsae, we find not only evolutionary conservation of most CERP2 co-opted promoters but also a substantial fraction that are species-specific. Our work reveals the emergence and evolutionary conservation of a novel transcriptional network driven by TE co-option with a major impact on regulatory evolution.The movement of selfish DNA elements can lead to widespread genomic alterations with potential to create novel functions. We show that transposon expansions in  One third of germline-specific promoters in Caenorhabditis derive from the co-option of two transposon families. Caenorhabditis that two independent TE expansions gave rise to promoters that control the expression of a large fraction of germline-specific genes.Cis-regulatory elements play fundamental roles in gene expression yet can undergo remarkably rapid evolutionary turnover  based on the presence of a strong Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) signal in wild-type young adults but not in glp-1 mutants lacking a germ line (fig. S1A) (C. elegans genes with germline expression (To investigate transcription regulation in the ig. S1A)  that do pression . Genes aC. elegans genome in both divergent (n = 1458) and tandem  orientations. These sequences predominantly corresponded to the positions of CERP2 (n = 68) and/or CELE2 (n = 61), and annotated CERP2 and CELE2 elements are enriched at germline-specific promoters at genome-wide scale . We note that a previous study did not find m1m2 pairs in repetitive elements because repeat sequences were masked in the analyses  . The motif-containing promoters were found among nonpromoter MITE elements in both trees, supporting the view that the motif-containing promoters were derived from the MITE elements (fig. S2). On the basis of these findings and functional analysis described below, we will hereafter refer to m1m2-containing promoters as co-opted elements. Notably, both co-opted promoters and nonpromoter CERP2 elements, and to some extent the CELE2 family, contain a region of 10–base pair (bp) periodic TT bias. This feature was recently shown to be associated with strong nucleosome positioning in T05F1.2 promoter and found that this reduced expression by 5.9-fold (fig. S1H). These results show that co-opted promoters drive germline expression and that the motif pairs are needed for promoter activity. Notably, as for T05F1.2, around 60% (171 of 284) of germline-specific co-opted promoters are the only promoters for the associated genes. Thus, the co-opted promoters are required for the expression of hundreds of germline-specific genes.To functionally test the activity of MITE-derived promoters and to understand the relevance of the m1 and m2 motifs for germline transcription, we generated transgenes containing wild-type or mutant motifs. CELE2- and CERP2-derived promoters containing wild-type m1m2 sequences drove germline-specific expression of a histone–green fluorescent protein (GFP) reporter  modENCODE data were from a mutant background, we generated new HIM-17 ChIP-seq data from wild-type adults, which identified 3539 HIM-17 peaks . Overallhim-17(me24), which identified 1311 up-regulated and 1640 down-regulated genes (fig. S3E and table S1). We then defined 304 direct targets of HIM-17 as misregulated genes with HIM-17 binding in wild-type adults. Direct targets were largely down-regulated in the mutant, suggesting that HIM-17 is a transcriptional activator  mutant (table S1) suggests that additional transcription factors may also contribute to the expression of these genes.To determine whether HIM-17 plays a role in co-opted promoter activity, we analyzed gene expression in young adults of the strong loss-of-function mutant him-17 mutant phenotypes (him-17(me24) mutants are him-5 and rec-1 (fig. S3H), two paralogs that promote DSB formation during meiosis (him-17 mutants (fig. S3E) . We also observed a high number of tandem m2+m1+ pairs in Caenorhabditis becei and Caenorhabditis monodelphis with different spacing between m1 and m2 sequences compared to CELE2, suggesting that other related TEs likely underwent expansion in these Caenorhabditis species (fig. S4B). These data indicate that the CERP2 and CELE2 expansions took place at different times in the Caenorhabditis clade, seeding thousands of m1m2 sequences and generating a large reservoir of potential regulatory elements.To gain further insights into CERP2 and CELE2 co-option and their regulation by HIM-17, we investigated their evolution through comparative analyses in nematodes. We first sought to determine the timing of the co-option by dating the TE expansion events. CERP2 elements are abundant in the genomes of all ematodes . In contt family . The earCaenorhabditis-specific expansions of CERP2 and CELE2, as orthologs could be identified not only in Caenorhabditis genomes but also in other Eurhabditis nematodes, with the exception of Diploscapter species  in the same period in which the expansion of the CERP2 sequence also took place. Fourteen of the 34 sites under positive selection are located within its six THAP domains .A large fraction of CERP2-derived promoters showed evidence of evolutionary conservation across multiple species, as indicated by peaks of phyloP scores . In partC. elegans, we observed that C. briggsae germline–specific promoters are enriched for m1m2 pairs (C. elegans (n = 327) or in C. briggsae , including both germline-specific and nongermline-specific promoters. We found that 53% of the orthologs in each species were regulated by an evolutionary conserved co-opted promoter, and a further 22 to 27% had some evidence of conservation, indicated either by a promoter with only m1 or m2 or by an m1m2 pair not annotated as a promoter . Thus, 53 to 80% of CERP2 co-option events are conserved in C. elegans and C. briggsae  strain. Injections were performed in the wild-type (AF16) C. briggsae strain using guide RNA–Cas9 ribonucleoprotein complexes preassembled in vitro with in-house made Cas9 protein  and G1036E (GGA → GAA), to mimic the C. elegans temperature-sensitive e2141 and q231 alleles, respectively. Single mutants did not display germline defects, but each produced some dead eggs at 27°C. We thus generated a double mutant, glp-1(we58), carrying both R955C and G1036E. Double mutants were maintained at 16°C and failed to develop a germ line when grown from starved stage 1 larvae (L1s) at 27°C.CRISPR-Cas9 genome editing was used to generate the C. briggsae or glp-1(we58) mutants were grown in liquid culture from the starved L1 to the young adult stage using standard S-basal medium with HB101 bacteria , frozen in liquid nitrogen, and stored at −80°C until use. Nuclei were isolated, and ATAC-seq and nuclear RNA-seq libraries were generated from wild-type and glp-1(we58) C. briggsae young adults as in (C. briggsae strain.Wild-type AF16 +) and then resuspended in 1 ml of FA+ buffer per 1 ml of ground worm powder. The extract was sonicated to an average size of ~250 bp using a Bioruptor Pico (Diagenode), and 20 μg of DNA was used per ChIP reaction. ChIP DNA was blunt ended, A-tailed, ligated to adaptors, amplified by PCR, and then size-selected using AMPure beads. Sequencing was performed on an Illlumina HiSeq 1500 machine using TruSeq adaptors.HIM-17 ChIP-seq libraries were prepared from two biological replicates following the protocol described in (him-17(me24)  young adults grown at 20°C from the starved L1 stage were collected. him-17(me24) was derived from him-17(me24)/tmC12 [tmIs1194] mothers. Total RNA was extracted using TRIzol. Poly(A) RNA was isolated using the NEBNext Poly(A) mRNA Isolation Kit, and libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (E7760S).For each of two replicates, approximately 100 wild-type or https://github.com/FelixKrueger/TrimGalore) and mapped using bwa mem (version 0.7.17) (https://github.com/jurgjn/yapc)  accession: GSE100651]  (C. elegans and C. briggsae were identified using ATAC-seq data generated from wild-type and glp-1 mutant strains. Single-end ATAC-seq reads were mapped on the respective genome assembly (WS275 for both C. elegans and C. briggsae) using bwa-backtrack (glp-1 data using DiffBind (version 2.10.0) (glp-1 versus wild-type log2 fold change (LFC) < −2 and the adjusted P value (p.adj) < 0.01.Accessible sites in C. elegans or the C. briggsae genomes, using a slightly modified version of the annotation pipeline from . Enriched motifs were mapped on the genomes of different species with FIMO (P < 0.0005). We annotated all occurrences of m1 and m2 motifs separated by 10 to 30 bp—a range including the most frequently observed m1-m2 spacings—as m1m2 motif pairs and distinguished them based on the relative motif orientation into four arrangements: convergent m1m2, divergent m1m2, tandem m1+m2+, and tandem m2+m1+ (table S1).We used the MEME suite (version 5.0.5) (n = 1000). Random repeat locations were defined using shuffleBed from BEDTools  and tandem m2+m1+ pairs (intermotif spacing: 23 to 28 bp) plus 50-bp up-/downstream of each pair (orientation defined by m1) and then subjected the divergent m1m2 and tandem m2+m1+ sequences to separate multiple sequence alignments using MAFFT   were generated using Mos-1–Mediated single-copy insertion (MosSCI) (his-58 in site two (plasmid pJA357), and gfp-tbb-2 3′UTR in site three (pJA256) (Transgenes containing the annotated CERP2-associated promoter of T05F1.2. T05F1.2 expression in the wild-type and mutant strains (we59) was quantified by qPCR using two different sets of primers and compared to cdc-42 expression. Primer sequences used are available in table S1.We used CRISPR-Cas9 to scramble the m1 and m2 sequences in the endogenous CERP2-associated promoter of C. elegans TFs were downloaded as aggregated peaks from the modERN website (https://epic.gs.washington.edu/modERN/)  (C. elegans to refine Hidden Markov Models (HMMs) of these repeats using the HMMER3 suite (http://hmmer.org/). Fasta sequences of all repeats from each family were aligned against the CERP2 or CELE2 Dfam HMM using hmmalign (with parameter –trim). The resulting alignment was used to define new HMMs using hmmbuild. The HMMs were then used to annotate CERP2 and CELE2 repeats in nematodes with chromosome-level genome annotations (table S1) using nhmmer and requiring a minimal E value of 0.001.We extracted sequences from all CERP2 and CELE2 elements in E value < 0.00001) on the protein annotation from a number of nematode species (listed in table S1). To test the him-17 sequence for positive selection, HIM-17 orthologs were aligned using MAFFT  regulated by a co-opted promoter in at least one species, i.e., associated to at least one promoter containing an m1m2 pair in divergent orientation and spaced by 12 to 16 bp (CERP2-like arrangement). Co-opted promoters were defined as conserved when both orthologs were associated with a co-opted promoter. We considered co-opted promoters as potentially conserved when the ortholog in the other species was either (i) associated with a promoter containing at least m1 or m2 or (ii) when an m1m2 pair was located in the putative promoter region (−1000 bp/+200 bp) of the orthologs’ transcription start site but was not in an annotated promoter. When none of the criteria were met, we defined the co-opted promoter as species specific.Sequence conservation of m1m2 pairs located in CERP2-derived germline-specific promoters was assessed using phyloP scores from 26 nematodes (phyloP26way from the cell release) available from the UCSC genome browser. To evaluate the conservation of individual CERP2 promoters in


CD79A and CD79B and the downstream genes BLNK, CD22, CD72, NFATC1, PIK3CG and RASGRP3. Studying repression of CD79B, we show that EBNA2 decreases transcription by disrupting binding of Early B cell Factor-1 to the CD79B promoter. Consistent with repression of BCR signalling, we demonstrate that EBNA2 and EBNA3 proteins suppress the basal or active BCR signalling that culminates in NFAT activation. Additionally, we show that EBNA2, EBNA3A and EBNA3C expression can result in reductions in the active serine 473 phosphorylated form of Akt in certain cell contexts, consistent with transcriptional repression of the PI3K-Akt BCR signalling arm. Overall, we identify EBNA2, EBNA3A and EBNA3C-mediated transcription control of BCR signalling as an additional strategy through which EBV may control the growth and survival of infected B cells and maintain viral latency.The cancer-associated Epstein–Barr virus (EBV) latently infects and immortalises B lymphocytes. EBV latent membrane protein 2A and EBV-encoded microRNAs are known to manipulate B cell receptor signalling to control cell growth and survival and suppress lytic replication. Here, we show that the EBV transcription factors EBNA2, 3A, 3B and 3C bind to genomic sites around multiple B cell receptor (BCR) pathway genes, regulate their expression and affect BCR signalling. EBNA2 regulates the majority of BCR pathway genes associated with binding sites, where EBNA3 proteins regulate only 42% of targets predicted by binding. Both EBNA2 and 3 proteins predominantly repress BCR pathway gene expression and target some common genes. EBNA2 and at least one EBNA3 protein repress the central BCR components  Like all herpes viruses, EBV has two phases in its life cycle: a latent phase and an acute productive lytic phase. When EBV infects naïve B cells it expresses its full repertoire of viral latent genes including six EBV nuclear antigens , three latent membrane proteins  and several functional small encoded RNAs (EBERs) and microRNAs (miRNAs) . Ag. AgCD79A41 cells  [17]. Upll lines .CD79A and CD79B, we examined the location of EBNA2 binding sites at CD79B and CD79A mapped by our original ChIP-sequencing analysis in the EBV positive Mutu III BL cell line [CD79A TSS and at an additional site ∼1.5 kb upstream (CD79B EBNA2 binds a single site close to the TSS (CD79A and CD79B transcription at these promoter regions [CD79A and CD79B EBNA2 binding sites identified overlapping sequences with similarity to EBF-1 and RBP-J consensus motifs (CD79A and CD79B promoters in EBV-infected cells (not shown) and the known dependency of these genes on EBF-1 activity [To investigate the mechanism of EBNA2-mediated repression of ell line . EBNA2 bupstream . At CD79 the TSS . Consist the TSS . Since E regions . We founs motifs . The absactivity , led us CD79B promoter, since repression of CD79B was most robust and reproducible in different cell backgrounds and EBNA2 bound a single site at high level  knock-out cell line. We observed an increase in transcriptional activation by EBF-1 in RBP-J KO cells (14-fold compared with 8-fold), consistent with RBP-J acting as a repressor of CD79B transcription through inhibition of EBF-1 DNA binding [CD79B promoter by EBNA2 indicating that the small activating effect of EBNA2 is not dependent on its binding via RBP-J    and we oression) . This readdition . Since Eaddition . These laddition . Althoug binding . It is lepressed . In contepressed . It is cNFATC1 (NFAT2) was identified as the closest gene to a top significant EBNA2 peak and a significant EBNA3 binding peak   . We foun31 cells . This isAB cells  and EBNAAB cells .NFATC2 was identified as a gene closest to a significant EBNA2 binding peak and contains multiple significant EBNA2 binding peaks throughout the gene  was identified as the closest gene to significant EBNA2 and EBNA3 binding peaks. EBNA3 bound at one site within the gene and at a site upstream in the DUS2 gene where an EBNA2 peak was also present (NFATC3 showed evidence of repression by EBNA3 proteins in BL31 cells (NFATC4 (NFAT3) is targeted by coincident promoter-proximal binding of EBNA2 and EBNA3, with EBNA2 binding at the highest level observed across all NFAT genes  by EBNA2 and EBNA3 proteins affected BCR signalling through the Ca2+ arm in B cells using NFAT activity as a read out. For these experiments, we used Burkitt lymphoma cell lines where BCR activation could be induced by cross-linking with anti-IgM antibodies. We first examined the impact of EBNA2 on BCR signalling using EBV-negative BL41 cells expressing the conditionally active (ER)-EBNA2 fusion protein. Cells were transiently transfected with an NFAT luciferase reporter promoter plasmid containing 3 NFAT binding motifs upstream of a luciferase promoter [We next examined whether the regulation of BCR pathway genes  and NFATC1 were not significantly repressed at the mRNA level in our BL41 cell line samples (NFATC1 (but not the other EBNA2 target gene NFATC4) was identified as a repressed gene by Maier et al. [SYK and LYN are also not reported as EBNA2 repressed genes in BL41 cells [CD79B expression resulting in reduced signalling from the membrane. In BL31 cells, the impact of EBNA3 proteins on basal and induced NFAT activity could be mediated through their repression of CD79A and CD79B in addition to multiple NFATs .In summary our data indicate that EBNA2 and EBNA3 proteins can suppress the basal or active BCR signalling that culminates in NFAT activation. The effects of EBNA2 and EBNA3 proteins on NFAT activity could stem from reduced expression of the BCR components CD79B or CD79A combined with repression of other calcium pathway genes including the NFATs themselves. In BL41 cells, EBNA2 represses  samples , althougr et al. . The ada41 cells . It is tCD79A and CD79B multiple genes involved in this pathway are regulated targets. For EBNA2 our data showed the majority of target genes involved in PI3K-Akt signalling were repressed  consistent with suppression of BCR signalling, although repression of the inhibitor INPP5D and increased expression of PIK3R1 expression may be expected to elicit positive effects  by western blotting . ExpressWe next examined the effect of EBNA3 proteins using the BL31 cell line series. We observed that levels of Ser473 and Thr308 phosphorylated forms of Akt were reduced in BL31 cells infected with wild-type EBV (BL31 wtBAC3 and wtBAC2) and most revertant viruses, with variation in levels observed across the cell lines examined . ConsistOur study demonstrates that multiple genes in signalling pathways downstream of the BCR and the BCR itself are subject to down-regulation by EBNA2 and EBNA3 proteins, with the binding of gene regulatory elements and transcription repression by these EBNAs likely playing an important role. EBV manipulates various aspects of B cell activation to induce cell proliferation and differentiation . EvidencCD79A and CD79B. Since CD79A and CD79B form the signal transducing subunit of the BCR receptor and are expressed in virtually all immature and mature B cells [. We confirmed that EBNA2 was a negative regulator of CD79A and CD79B in both LCL and lymphoma cell backgrounds and we also showed reduced CD79A and CD79B protein levels in response to EBNA2 activation in LCLs. The chromatin landscape surrounding the promotor proximal EBNA2 binding sites at CD79A and CD79B corresponded with regions of H3K27 acetylation in LCLs indicating that the EBNA2 bound-promoter regions retained active chromatin marks in cells that express EBNA2 despite their reduced expression. Transcription factor binding site analysis of the CD79A and CD79B EBNA2 binding sites revealed overlapping binding sites for EBF-1 and RPB-J, although the EBF-1 binding sites were a better match than the RPB-J sites. Luciferase reporter assays using the EBNA2 bound-promoter regions of CD79B, in EBV-negative DG75 cells and DG75-RBP-J-KO cells showed that EBNA2 alone was able to activate the promoter, independently of RBP-J. This is interesting as EBNA2 activation of CD79B is not observed in vivo. This discrepancy may be because other repressive factors present at the CD79B promoter preclude EBNA2-mediated activation of these genes in vivo and the chromatin context at the promoter in EBV-infected is also likely to differ from that in transfected plasmids. Nonetheless, we observed that co-expression of EBNA2 and EBF-1 led to a decrease in activation of the CD79B promoter by EBF-1, independently of RBP-J. This is consistent with a previous study that identified CD79B as an RBP-J independent EBNA2 repressed gene and showed EBNA2 and EBF-1 can bind in vitro [CD79B.We identified large promoter-proximal EBNA2 binding sites at  B cells , this plCD79B promoter in vivo using ChIP-QPCR we found that EBNA2 and EBF-1 both bound the CD79B promoter and their binding patterns follow similar kinetics, with an initial increase then reduction in binding of both factors until 24 hrs when EBNA2 binding reduced and EBF-1 binding seemed to stabilise, despite continued repression of CD79B mRNA levels. There is clearly a complex series of events happening at the promoter that are hard to disentangle using population average ChIP assays, but we propose that EBNA2 is destabilising EBF-1 binding at CD79B and interfering with EBF-1 mediated transcriptional activation. This is consistent with EBNA2 functioning as a passive rather than active repressor.When we further explored the interplay between EBF-1 and EBNA2 at the NFATC1, NFATC2, NFATC3 and NFATC4), key effectors of the calcium signalling arm of the pathway, as repressed target genes of both EBNA2 and EBNA3s. Although we did not explore the mechanisms of transcriptional repression of the NFATs, multiple binding peaks were present in promoter-proximal and more distal intragenic locations, with the extent of binding of the EBNAs being a good indicator of extent of regulation. Thus, regulation at the beginning and end of the BCR pathway by the EBNAs can contribute to BCR signalling suppression.Focusing on other genes downstream of the BCR, we identified multiple members of the NFAT family  supplemented with 10% Fetal Bovine Serum, Penicillin and Streptomycin . The EBV-negative Burkitt lymphoma cell line BL31 infected with wild-type recombinant EBV bacmids or EBNA3A, 3B and 3C-exon 2 knock-out or EBNA 3A, 3B and 3C combined knock-out viruses  and their revertant bacmids  were provided by Prof M. Allday . Cells wLCLs established by infecting primary B cells with either wild-type EBV (LCL3A wt) or mutant virus lacking the coding sequence for EBNA3A (LCL 3A mutB) were provided by Prof Kempkes . LCL EBNThe EBV-immortalised LCL (ER-EB 2.5) expresses a conditionally active oestrogen receptor (ER)-EBNA2 fusion protein and was cultured in the presence of 1 μM β-estradiol (Sigma). For inactivation and reactivation of EBNA2, cells were maintained in the absence of β-estradiol for 4 days and 1 μM β-estradiol was re-added for either 4, 8, 17 or 24 h prior to cell harvest. The EBV-negative Burkitt lymphoma cell lines BL41K3 and BJABK3) (kindly provided by Prof B. Kempkes)  also exCD79B promoter (∼–765 to +176 relative to the predicted transcription start site) was amplified from genomic DNA extracted from a B cell line using primers to create NheI and SacI sites. The digested PCR product was then cloned into the Nhel and SacI sites of pGL3-Basic (Promega).To create pGL3 CD79B, a 0.94 kb fragment of the SDS–PAGE and western blotting was performed as described previously  using thoC/1 min)) for 40 cycles provided by the manufacturer. The data were analysed with the cloud suite software Symphoni (Life Technologies) using the Relative Quantification Module.Total RNA was isolated from BL31 cell lines, LCL stable cell lines and ER-EB 2.5 cells using Tri Reagent (Sigma) according to the manufacturer's instructions. cDNA was synthesised from 1 μg of RNA using the ImProm-II Reverse Transcription System (Promega). Ninety-six-well Custom TaqMan array cards (Thermo Fisher) were designed and ordered and arrived pre-loaded with gene expression assays for selected BCR signalling genes associated with EBNA2 or EBNA3 binding sites. Array cards also contained four reference gene expression assays  for relative quantification. Two-hundred nanograms of cDNA was added to TaqMan Universal PCR Master Mix (2×) with UNG in a final volume of 100 μl. Samples were subsequently loaded into each fill reservoir through ports on TaqMan array cards. Real-time PCR was performed on Applied Biosystems 7900HT Real-time PCR system with a TaqMan Array Microfluidic Card Thermal Cycling block using cycling conditions . QPCR was carried out in duplicate using the standard curve absolute quantification method on an Applied Biosystems StepOne Plus Real-time PCR system as described previously using cDNA specific primers for nd GAPDH , with th control .7 cross-linked cells as described previously [For the ER-EB 2.5 cell line, cells were cultured in medium without β-estradiol for 4 days. After 4 days of withdrawal, a control sample (time 0) was taken and β-estradiol (1 μM) was then added and chromatin prepared 4, 8, 17 and 24 h later. Chromatin was prepared from 1 × 107 DG75 and DG75-RBP-J KO cells were transfected using the Bio-Rad Gen Pulser II Electroporation system as described previously [6 BL41K3 or BL31 cells were transfected using the Neon Transfection System (Invitrogen). Cells were transfected with 5 μg of pGL3 NFAT reporter containing 3 NFAT binding motifs upstream of a luciferase promoter  and pRL


Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were activated by HNF-1β, whereas Efna1, Epha3, Epha4, Epha7, Ntn4, Plxna2, Sema3a, Sema4b, Slit3, Srgap1, Unc5c and Unc5d were repressed by HNF-1β. RNAscope in situ hybridization showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were expressed in wild-type UB and were dysregulated in HNF-1β mutant UB. These studies show that HNF-1β directly regulates the expression of multiple axon guidance genes in the developing mouse kidney. Dysregulation of axon guidance genes may underlie kidney defects in HNF-1β mutant mice.The transcription factor hepatocyte nuclear factor 1β (HNF-1β) is essential for normal development of the kidney and other epithelial organs. In the developing mouse kidney, HNF-1β is required for the differentiation and patterning of immature nephrons and branching morphogenesis of the ureteric bud (UB). Here, we used ChIP-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to identify genes that are regulated by HNF-1β in embryonic mouse kidneys. ChIP-seq revealed that HNF-1β binds to 8284 sites in chromatin from E14.5 mouse kidneys. Comparison with previous ATAC-seq and histone modification studies showed that HNF-1β binding peaks colocalized with open chromatin and epigenetic marks of transcriptional activation , indicating that the binding sites were functional. To investigate the relationship between HNF-1β binding and HNF-1β-dependent gene regulation, RNA-seq was performed on UB cells purified from wild-type and HNF-1β mutant embryonic kidneys. A total of 1632 genes showed reduced expression in HNF-1β-deficient UB cells, and 485 genes contained nearby HNF-1β binding sites indicating that they were directly activated by HNF-1β. Conversely, HNF-1β directly repressed the expression of 526 genes in the UB. Comparison with snATAC-seq analysis of UB-derived cells showed that both HNF-1β-dependent activation and repression correlated with chromatin accessibility. Pathway analysis revealed that HNF-1β binds near 68 axon guidance genes in the developing kidney. RNA-seq analysis showed that  The definitive kidney in mammals, the metanephros, originates from two distinct primordia: the ureteric bud (UB) is an outgrowth from the posterior Wolffian duct that elongates and undergoes branching morphogenesis to produce the renal collecting system. The adjacent metanephric mesenchyme (MM) contains stem cells that are the progenitors of nephrons, the functional units of the kidney. Kidney development is driven by reciprocal interactions between the UB and MM. The MM secretes factors such as glial cell line-derived neurotrophic factor (GDNF) that signal to receptors located on the UB and induce UB elongation and branching2. Conversely, the tips of the UB secrete factors, such as Wnt9b, that induce the adjacent MM to undergo mesenchymal-epithelial transition forming immature nephrons3. With further development, the nephrons become patterned into proximal and distal segments and fuse with the collecting ducts forming a continuous lumen.The kidneys are paired epithelial organs that are embryologically derived from intermediate mesoderm4. HNF-1β recognizes the consensus sequence 5′-GGTTAATNATTAAC-3′ that is often found in promoter or enhancer regions of HNF-1β target genes6. HNF-1β activates gene transcription by recruiting the histone acetyltransferases CREB-binding protein (CBP) and P300/CBP-associated factor (PCAF)8. HNF-1β can also repress gene transcription by recruiting histone deactylase-17. HNF-1β is expressed in all tubular epithelial cells in the kidney. In the developing mouse kidney, HNF-1β is detected in the Wolffian duct at embryonic day (E) 9.5 and is subsequently expressed in the branching UB4. HNF-1β is absent in the MM, but its expression is induced coincident with the formation of epithelia10. Expression of HNF-1β is maintained in adult nephrons and collecting ducts9. Gene targeting studies have revealed that HNF-1β is required at multiple steps of kidney development. Inactivation of HNF-1β in the UB significantly reduces branching morphogenesis and the induction of new nephrons11. Ablation of HNF-1β in nephron progenitors in the MM impairs nephron differentiation and inhibits the formation of the proximal and distal tubules and loops of Henle10.Hepatocyte nuclear factor 1β (HNF-1β) is a POU-homeodomain transcription factor that regulates tissue-specific gene expression in epithelial organs, including the kidneyHNF1B in humans are among the most common monogenic causes of congenital kidney abnormalities12. Mutations of HNF1B account for 10–30% of cases of congenital anomalies of the kidney and urinary tract (CAKUT)13. Over 100 pathogenic mutations in HNF1B have been reported14, including whole-gene deletions, point mutations and small insertions16. HNF1B mutations have an autosomal dominant inheritance, though there are also sporadic mutations13. Renal manifestations of HNF1B mutations include kidney agenesis, hypoplasia, dysplasia, duplicated ureters, and multicystic dysplastic kidneys18. Extrarenal phenotypes associated with HNF-1β-related CAKUT include hypomagnesemia19, diabetes20, female genital tract malformations21, and neurodevelopmental disorders22. To gain mechanistic insights into the functions of HNF-1β in kidney development, we conducted a genomewide analysis of HNF-1β gene targets in the embryonic mouse kidney. We specifically focused on genes expressed in the UB that may underlie branching defects in HNF-1β mutant animals.Heterozygous mutations of Cdh16 and Pcbd1 .To identify HNF-1β binding sites in the developing kidney, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Kidneys (metanephroi) were dissected from wild-type embryos at E14.5 and pooled. Kidneys from two independent pooled litters were analyzed separately . Chromatin was extracted from the kidneys, cross-linked, and immunoprecipitated with an anti-HNF-1β antibody. Cross-linking was reversed, and DNA libraries were generated and sequenced. Read depth and ChIP-seq quality measures can be found in Supplementary Table Of the HNF-1β binding sites, 26% were located in gene promoters, which were defined as 3 kb upstream from the transcription start site (TSS); 37% were intragenic, defined as the 5′-UTR, 3′-UTR, introns, exons, and 300 bp downstream from the transcription end site (TES); and 37% were located in distal intergenic regions, defined as > 300 bp downstream of the TES or > 3 kb upstream from the TSS  or were intragenic (51%). Only 19% were distal intergenic. A total of 982 sites mapped to long non-coding RNAs, of which 17% were in the promoter region, 27% were intragenic, and 56% were distal intergenic. A total of 134 sites mapped to microRNAs, of which 29% were in the promoter region and 71% were distal intergenic. The remaining gene classifications of HNF-1β peaks can be found in Supplementary Table 25. Moreover, HNF-1β itself functions as a pioneer transcription factor that remains bound to DNA through mitosis and can activate transcription by promoting histone modifications that result in increased chromatin accessibility26. To determine whether HNF-1β binding in embryonic mouse kidneys is associated with increased chromatin accessibility, we compared the locations of HNF-1β ChIP-seq peaks and ATAC-seq peaks. Here, we took advantage of an existing ATAC-seq dataset from the ENCODE database28 obtained from wild-type E14.5 mouse kidney29, the same tissue source used for HNF-1β ChIP-seq. We identified 2263 HNF-1β ChIP-seq peaks that colocalized with ATAC-seq peaks, which represented 27% of the total HNF-1β peaks. To validate this colocalization with HNF-1β activity, binding sites at known HNF-1β targets were examined. The HNF-1β binding sites at the promoters of Cdh16 and Pcbd1 overlapped with open chromatin .To determine if the HNF-1β binding sites were functional, we examined the relationship between HNF-1β binding and chromatin accessibility. Transcriptional activation is associated with open chromatin, which can be measured using ATAC-seq (Assay for Transposase Accessible Chromatin)tin Fig. . The ass8. Therefore, we tested the relationship between HNF-1β binding and histone acetylation in chromatin from embryonic kidneys. Data from a ChIP-seq analysis of H3K27 acetylation in wild-type E14.5 mouse kidney were downloaded from the ENCODE database29. Peaks of H3K27ac enrichment were identified, and the locations were compared with HNF-1β binding peaks. We identified enrichment of H3K27ac at 1970 HNF-1β binding peaks, representing 24% of all HNF-1β binding peaks. HNF-1β binding sites at the promoters of Cdh16 and Pcbd1 overlapped with H3K27ac sites . Similar to the ATAC-seq results, H3K27ac enrichment was higher in promoters compared to intragenic and distal intergenic regions . Enrichment of H3K4me3 was highest at HNF-1β binding peaks in promoter regions and was not detected at HNF-1β binding sites in intragenic and distal intergenic regions . In contrast to H3K4me3, H3K4me1 colocalized with HNF-1β binding peaks in intragenic and distal intergenic regions in addition to promoter regions , an epigenetic mark associated with gene activationtes Fig. . The asstes Fig. . The disTaken together, HNF-1β binding sites in E14.5 kidneys are associated with regions of open chromatin and activating histone modifications. The colocalization of HNF-1β binding and H3K27ac correlated with ATAC-seq and showed highest enrichment in gene promoters and lower signal in intragenic and distal intergenic regions. Consistent with this finding, colocalization with H3K4me3 was also highest in gene promoters, indicating that HNF-1β binding is associated with promoter activation. In contrast, colocalization with H3K4me3 was absent in intragenic and distal intergenic regions. Instead, HNF-1β peaks in these regions colocalized with H3K4me1, indicating that they corresponded to active enhancers.Hnf1bfl/fl;tdT (HNF-1β mutant) mice in which Cre recombinase was expressed under the control of the Hoxb7 promoter resulting in inactivation of HNF-1β and activation of a tdTomato reporter gene exclusively in the ureteric bud. Hoxb7/Cre;Hnf1bfl/+;tdT mice were used as heterozygous controls, as the heterozygotes lack a kidney phenotype. Consistent with previous studies11, HNF-1β mutant kidneys were hypoplastic and had reduced UB branching compared to heterozygous control kidneys . We defined these genes as being repressed by HNF-1β. Of these genes, 526 (24.1%) contained nearby HNF-1β binding sites, indicating that they were likely directly repressed by HNF-1β . Similarly, the enrichment of H3K27ac was 37% higher at intragenic HNF-1β binding sites and 33% higher at distal intergenic sites in activated genes compared with repressed genes . The enrichment of H3K4me1 was 25% higher at intragenic sites and 15% higher at distal intergenic sites in activated genes compared with repressed genes . There were no significant differences between activated and repressed genes in ATAC-seq, H3K27ac, and H3K4me1 enrichment at HNF-1β binding sites in promotor regions  but had a lower odds ratio (1.97) and much less overlap than ATAC-seq and activating histone modifications. Twenty H3K9me3 peaks overlapped with HNF-1β peaks, which was not statistically significant . There were no differences in H3K27me3 and H3K9me3 enrichment at HNF-1β binding sites in activated and repressed genes compared to genes with no change in expression  and intercalated cells (IC) was compared with immune cells and nephron progenitors (NP), which do not endogenously express HNF-1β. The enrichment of open chromatin at HNF-1β binding sites in PC was 47% higher than immune cells  and 41% higher than NP   and 41% higher than NP  ATAC-seq analysis performed on postnatal day (P)0 mouse kidneyst) Fig. .Figure 3–26, Wilcoxon test). The enrichment of open chromatin at HNF-1β binding sites in IC was 43% higher in activated genes than repressed genes . Taken together, the association of ATAC-seq signal with HNF-1β binding sites seen in embryonic kidney was also observed in cells derived from the UB. Moreover, there was greater enrichment of open chromatin in genes that were activated by HNF-1β compared to repressed genes.We then compared the snATAC-seq signal at the HNF-1β binding sites in activated and repressed genes Fig. c. The en36 analysis on the genes that were in closest proximity to the HNF-1β binding sites in E14.5 kidneys . The related Rap1 signaling pathway, which is involved in intracellular signaling for plexin axon guidance receptors37, was also identified. Axon guidance is a repulsive and attractant chemotaxis pathway that was first described in axon growth cones. More recently, the axon guidance signaling pathway has been identified in tubular epithelial cells where it has been implicated in branching morphogenesis in epithelial organs, including the kidney38. We found that HNF-1β binds near 68 axon guidance genes in the developing kidney. Of these genes, 34 encode ligands or receptors in the Semaphorin/Neuropilin/Plexin, Ephrin/Ephrin receptor, Slit/Robo, and Netrin/Unc families. Pathway analysis of RNA-seq data showed that axon guidance was also highly represented among the genes that were directly activated or repressed by HNF-1β . Hnf1bfl/+ mice48 containing loxP sites flanking exon 1 of the HNF-1β gene were a generous gift from Dr. Marco Pontoglio (Cochin Institute). Gt(Rosa)26Sortm9(CAG-tdTomato)Hze mice49 (tdT mice) containing a tdTomato reporter gene that is activated by Cre/loxP recombination were obtained from the Jackson Laboratory (Stock #007909). The genetic backgrounds of the mice were CFW (Hoxb7/Cre) and C57BL/6 (Hnf1bfl/+ and tdT).Hoxb7/Cre miceHnf1bfl/+ mice were crossed with Hnf1bfl/fl;tdT mice to generate Hoxb7/Cre;Hnf1bfl/fl;tdT embryos in which HNF-1β was specifically inactivated in the UB. Hoxb7/Cre;Hnf1bfl/+;tdT littermates do not have a kidney phenotype and were used as controls. The morning of detection of the copulation plug was considered embryonic day (E)0.5. On E14.5, pregnant dams were euthanized via carbon dioxide according to the American Veterinary Medical Associat guidelines, and embryos were removed and placed in phosphate buffered saline (PBS) on ice. Embryonic kidneys were dissected using a Leica M420 microscope. Embryos of both sexes were used. All experiments involving animals were approved by the Institutional Animal Care and Use Committee at the University of Minnesota and complied with ARRIVE guidelines50. All methods were performed in accordance with the relevant guidelines and regulations.Hoxb7/Cre;g for 5 min and were transferred into SDS lysis buffer ). Samples were incubated on ice for 10 min, and the chromatin was sheared using a Covaris S220 focused-ultrasonicator for 7.5 min at 7.5 Watts, peak power 75, and duty factor 10. The chromatin was cleared by centrifugation at 15,000×g at 4 °C for 10 min, and the chromatin was diluted 1:10 into ChIP dilution buffer , 167 mM NaCl). A 1% input sample was removed, and HNF-1β was immunoprecipitated using 5 μg rabbit anti-HNF-1β antibody at 4 °C overnight with rotation.Kidneys were dissected from wild-type C57BL/6J embryos at E14.5, pooled, and cross-linked in 2% paraformaldehyde (PFA) in PBS for 10 min at room temperature followed by neutralization in 0.125 M glycine for 5 min at room temperature. Cross-linked kidneys were transferred into lysis buffer (0.05 M HEPES–NaOH (pH 7.5), 0.14 M NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), and nuclei were isolated using a 1 ml tight Dounce homogenizer. Chromatin shearing and immunoprecipitation (IP) were performed using buffers from the Millipore EZChIP kit (Sigma-Aldrich). Nuclei were pelleted at 2300×g for 1 min then were rinsed sequentially with a low salt immune complex wash buffer , 150 mM NaCl), high salt immune complex wash buffer , 500 mM NaCl), LiCl immune complex wash buffer ), and twice with TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). Each wash was performed at 4 °C with rotation for 5 min. Protein-chromatin complexes were eluted twice by incubation in 100 μl elution buffer  at 37 °C for 15 min with rotation. Beads were pelleted at 4000×g for 1 min at room temperature, and the eluate was collected. Cross-linking was reversed by overnight treatment with 8 μl 5 M NaCl at 55 °C, then samples were incubated with 4 μl 0.5 M EDTA, 1 μl RNase A (10 mg/ml), and 1 μl Proteinase K (10 mg/ml) for 2 h at 65 °C. Chromatin was purified using Qiagen MinElute PCR Purification Kits.Protein A/G beads  were added to the immunoprecipitates and incubated at 4 °C for 3 h with rotation. Beads were pelleted by centrifugation at 4000×t) for the final library amplification . Libraries were quantitated using Qubit and were selected for 200–400 base pair fragments using Invitrogen 2% agarose E-Gels. Indexed libraries were sequenced on an Illumina Next-seq 1 × 75-bp high-output run.Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Roche). Chromatin was subjected to an end-repair poly-A tail reaction and ligated to the xGen Stubby Adaptor (Integrated DNA Technologies) at 1:50 dilution. Ligated samples were purified with 0.8 × volume of KAPA Pure beads (Roche). To determine the number of amplification samples, a 5% test library amplification was carried out by quantitative PCR (qPCR) with the Illumina universal P2 primer and one Illumina index primer for 40 cycles. Three cycles were added to the threshold cycle number (Cg and incubated in 0.25% Trypsin–EDTA (Gibco) at 37 °C for 15 min with mixing every 5 min. After neutralizing with 20% fetal bovine serum and washing in PBS, single-cell suspensions were incubated with near-infrared live/dead dye (ThermoFisher) at 1:1000 dilution for 5 min. TdTomato-positive live cells were purified using a BD FACSAria cell sorter. Cells were sorted directly into Qiazol RNA lysis buffer. Total RNA was extracted with the Qiagen miRNeasy kit using NEB Monarch RNA Cleanup Columns to elute into a low volume of 8 µl in H2O.Kidneys were dissected from heterozygous control and HNF-1β mutant embryos collected from several pregnant dams at E14.5 and dissociated into single-cell suspensions by incubating in 125 U/ml type 1 collagenase (Sigma-Aldrich) in PBS at 37 °C for 15 min with mixing every 5 min. Samples were centrifuged at 845×cDNA libraries were prepared using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina. RNA concentration and RNA integrity (RIN) scores were determined using an Agilent 4200 Tapestation. All sample RIN scores were ≥ 8.0. Libraries were prepared in the same batch, and the nanogram amount of the least concentrated RNA sample was used in each reaction. Samples were reverse transcribed with a template switching reverse transcriptase, and cDNA was amplified for 12 PCR cycles. Samples were purified using Ampure XP SPRI beads (Beckman). cDNA samples were fragmented using the NEBNext Ultra II FS Enzyme Mix. The NEBNext Adaptor for Illumina was ligated onto fragmented cDNA at a 1:25 dilution. Samples were purified using SPRI beads, and libraries were amplified with Illumina sequencing primers. Samples were purified using SPRI beads and eluted into 0.1 × TE buffer. Libraries were sequenced on an Illumina NextSeq 2 × 75-bp mid-output run.2O2 for 10 min at room temperature. After rinsing in distilled water three times, slides were incubated in ACD heat retrieval buffer for 5 min at 95 °C. Slides were rinsed in distilled water three times and incubated in 100% ethanol for 3 min at room temperature. Slides were air dried and incubated with ACD protease for 30 min at 40 °C in a humidified slide moat. Samples were incubated with RNAscope probes for 2 h at 40 °C. Slides were washed twice in ACD wash buffer for 2 min at room temperature after probe incubation and between each amplification step. Samples were incubated sequentially with Amp1 for 30 min at 40 °C, Amp2 for 30 min at 40 °C, Amp3 for 15 min at 40 °C, HRP-C1 for 15 min at 40 °C, the Opal570 reagent (Akoya) diluted 1:1000 in TSA buffer (ACD) for 30 min at 40 °C, and HRP blocker for 15 min at 40 °C.Kidneys were dissected from heterozygous control and HNF-1β mutant embryos at E14.5 and fixed in 4% PFA in PBS for 15 min at room temperature. Kidneys were transferred to 30% sucrose in PBS solution, embedded in Optimal Cutting Temperature compound, frozen and stored at − 80 °C. 4 μm-thick sagittal cryosections were prepared and mounted on glass slides. RNAscope in situ hybridization was performed using the RNAscope Multiplex Fluorescent V2 Assay kit (ACD). Slides were washed in PBS for 5 min and baked at 60 °C for 20 min. Slides were incubated in 4% PFA in PBS for 15 min at 4 °C then dehydrated in 50% ethanol for 5 min, 70% ethanol for 5 min, and 100% ethanol for 5 min twice at room temperature. Slides were air-dried and incubated with 10% HKidney sections were blocked in Rodent Block M  for 1 h at room temperature. Samples were incubated with a rabbit anti-RFP (TdTomato) primary antibody (1:1000) overnight at 4 °C followed by a secondary antibody (1:1000) for 2 h at room temperature. DAPI stain solution (ACD) was applied, and samples were mounted with Prolong Gold Antifade Mountant (ThermoFisher). Photomicrographs were acquired with a Leica DM5500 B upright microscope equipped with a DFC7000 T camera. Brightness and contrast were adjusted equally across all images, including controls, using FIJI/ImageJ.The primary antibodies used in this study were rabbit anti-HNF-1β  and rabbit anti-RFP (TdTomato) . Alexa Fluor 647-conjugated donkey anti-rabbit IgG  was used as a secondary antibody.RNAscope was performed using the following probes: Efna1 , Nrp1 , Sema3c , Sema3d , Sema6a , and Slit2 .51 using the TruSeq3-SE.fa:2:30:10 leading:3 trailing:3 slidingwindow:4:5 minlen:25 parameters. ChIP-seq read mapping was performed via BWA MEM (0.7.17)52 using the mouse genome (GRCm38) as a reference. ChIP peaks were identified using MACS (2.1.1.20160309)53. Peak lists from two independent experiments were merged using R (4.1.2)54 to find high confidence peaks present in both datasets. ChIP-sequencing quality analysis was performed using Phantompeakqualtools56 and is included in Supplementary Table 57 was used to visualize ChIP-seq peaks. Meme Suite 5.0.158 was used to perform de novo motif analysis. ClusterProfiler59 was used to perform KEGG pathway analysis, and EnrichedHeatmap60 was used to make heatmaps. ggplot261 was used to create figures.ChIP-sequencing reads were trimmed using Trimmomatic (v 0.32)63. Read mapping was performed via Hisat2 (v2.1.0)64 using the mouse genome (GRCm38) as a reference. Gene quantification was performed using Feature Counts for raw read counts. Differentially expressed genes were identified using the edgeR67  feature in CLCGWB  with raw read counts. ClusterProfiler was used for KEGG pathway analysis, and the GSEA software69 was used to perform gene set enrichment analysis. Ingenuity Pathway Analysis was performed on differentially-expressed genes (GSE97770) with log2 fold-change > 1 or < -1 in HNF-1β-deficient mIMCD3 cells compared to wild-type mIMCD3 cells39.RNA-sequencing reads were trimmed using Trimmomatic (v 0.33) enabled with the optional “-q” option; 3 bp sliding-window trimming from 3′ end requiring minimum Q30. Quality control on raw sequence data for each sample was performed with FastQC28. The overlap of each histone mark and open chromatin with HNF-1β binding sites was tested using hypergeometric tests. Enrichment of each mark at HNF-1β binding sites was examined by counting the number of reads within 1 kb of each HNF-1β peak. Read counts were normalized to peak width and read depth to generate reads per kilobase of transcript (RPKM) values for each HNF-1β peak.H3K4me3, H3K27ac, H3K4me1, H3K27me3, and H3K9me3 ChIP-seq data and ATAC-seq data was obtained from ENCODEThe Mann–Whitney Wilcoxon nonparametric test was used for pairwise comparisons. p < 0.05 was considered significant. The hypergeometric test was used to test overlap between ChIP-seq datasets, where p < 0.05 was considered significant.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.