Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

“Cis-regulatory regions are associated with diverse histone modifications that include acetylations and methylations of histone tails (Bernstein et al. 2006; Roh et al. 2006; Barski et al. 2007; Heintzman et al. 2007; Robertson et al. 2008; Wang et al. 2008).”

“Lineage-specific combinations of histone marks at enhancers correlate with lineage-specific enhancer function (Heintzman et al. 2009), suggesting that specific histone modifications can be used to predict the functionality of cis-regulatory loci.”

“…loci are found within regions of the genome undergoing active transcription, are near expressed genes, and as they are typically associated with H3K4me1 and/or H3K4me3 (Barski et al. 2007; Wang et al. 2008), we suggest that both bimodal and monomodal sites generally occur within open chromatin.”

“PDX1 is essential for pancreas development (Jonsson et al. 1994) and is required for the expression of insulin and several other genes critical to pancreas development and function (Ohlsson et al. 1993; Ahlgren et al. 1998; Gerrish et al. 2001; Svensson et al. 2007).”

“Recent genome-wide analyses of transcription factor–DNA association in mammalian genomes show that most transcription factors bind thousands of sites (Robertson et al. 2007; Chen et al. 2008; Marson et al. 2008; Wederell et al. 2008; Hoffman and Jones 2009; Zheng et al. 2009).”

“Generation and analysis of Tag-seq and SAGE libraries Total RNA was isolated from hand-picked islets and liver using TRIzol, and RNA quality was assessed using a Bioanalyser (Agilent).”

“FoxA proteins, which share homology with histone H5, function as pioneer factors and can bind compact chromatin and open local chromatin structures (Cirillo et al. 2002; Sekiya et al. 2009).”

“HNF4A is not essential for the early phases of liver development, but controls many genes that are vital for adult liver functions, and is considered a central regulator of hepatocyte differentiation and function (Hayhurst et al. 2001; Battle et al. 2006).”

“…vivo (Adams and Workman 1995; Albert et al. 2007; Koerber et al. 2009), possibly due to binding at DNA sequences found within nucleosome exit or entry points, or at sequences exposed due to rotational phasing on the nucleosome surface (Albert et al. 2007; Jiang and Pugh 2009; Koerber et al. 2009).”

“Recent genome-wide analyses of transcription factor–DNA association in mammalian genomes show that most transcription factors bind thousands of sites (Robertson et al. 2007; Chen et al. 2008; Marson et al. 2008; Wederell et al. 2008; Hoffman and Jones 2009; Zheng et al. 2009).”

“PDX1 is essential for pancreas development (Jonsson et al. 1994) and is required for the expression of insulin and several other genes critical to pancreas development and function (Ohlsson et al. 1993; Ahlgren et al. 1998; Gerrish et al. 2001; Svensson et al. 2007).”

“PDX1 is essential for pancreas development (Jonsson et al. 1994) and is required for the expression of insulin and several other genes critical to pancreas development and function (Ohlsson et al. 1993; Ahlgren et al. 1998; Gerrish et al. 2001; Svensson et al. 2007).”

“3 is known to be enriched at transcription factor binding sites (Jin et al. 2009; He et al. 2010).”

“It is known that some yeast transcription factors can interact with nucleosomal DNA in vitro and at promoters in vivo (Adams and Workman 1995; Albert et al. 2007; Koerber et al. 2009), possibly due to binding at DNA sequences found within nucleosome exit or entry points, or at sequences exposed due…”

“of the transcription factors initially expressed within the endoderm remain expressed in both the liver and pancreas and function to maintain homeostasis in the adult tissues (Jensen 2004; Zaret 2008; Zaret and Grompe 2008).”

“For example, FOXA2 is essential for both pancreas and liver development (Lee et al. 2005; Gao et al. 2008).”

“Cis-regulatory regions are associated with diverse histone modifications that include acetylations and methylations of histone tails (Bernstein et al. 2006; Roh et al. 2006; Barski et al. 2007; Heintzman et al. 2007; Robertson et al. 2008; Wang et al. 2008).”

“3 is known to be enriched at transcription factor binding sites (Jin et al. 2009; He et al. 2010).”

“Arginine is a precursor of nitric oxide (NO), which plays critical roles in controlling insulin secretion from beta-cells, beta-cell apoptosis, and islet inflammation, as well as in controlling liver regeneration, inflammation, and blood flow (Schmidt et al. 1992; Shimabukuro et al. 1997; Wu and Morris 1998; Adeghate…”

“…recognized that the nucleosome occupancy of promoters and enhancers can be dynamically altered to control their function (Hogan et al. 2006; Shivaswamy et al. 2008; RamirezCarrozzi et al. 2009; He et al. 2010); however, the relevance of this to the control of transcription factor occupied…”

“Many of the transcription factors initially expressed within the endoderm remain expressed in both the liver and pancreas and function to maintain homeostasis in the adult tissues (Jensen 2004; Zaret 2008; Zaret and Grompe 2008).”

“However, in mammalian systems, the ability to interact with nucleosomal DNA has been considered one of the defining features of pioneer transcription factors, such as the FOXA proteins (Cirillo et al. 1998, 2002; Sekiya and Zaret 2007; Sekiya et al. 2009).”

“To further confirm these results, we compared the association of bimodal, monomodal, and low H3K4me1 sites with genes whose expression was significantly altered by adult-specific disruption of Foxa2, Pdx1, or Hnf4a.”

“chromatin (Berkes et al. 2004), and it is possible that these sites Hoffman et al.”

“Generation and analysis of Tag-seq and SAGE libraries Total RNA was isolated from hand-picked islets and liver using TRIzol, and RNA quality was assessed using a Bioanalyser (Agilent).”

“It is known that some yeast transcription factors can interact with nucleosomal DNA in vitro and at promoters in vivo (Adams and Workman 1995; Albert et al. 2007; Koerber et al. 2009), possibly due to binding at DNA sequences found within nucleosome exit or entry points, or at sequences exposed due…”

“Zaret KS. 2008.”

“Arginine is a precursor of nitric oxide (NO), which plays critical roles in controlling insulin secretion from beta-cells, beta-cell apoptosis, and islet inflammation, as well as in controlling liver regeneration, inflammation, and blood flow (Schmidt et al. 1992; Shimabukuro et al. 1997; Wu and Morris 1998; Adeghate…”

“FOXA2 is a wellestablished pioneer factor (Sekiya et al. 2009), while HNF4A and PDX1 are not.”

“1C), indicating the presence of H3K4me1-marked nucleosomes flanking these sites (Heintzman et al. 2007; Robertson et al. 2008).”

“PDX1 and/or PDX1:PBX1-like motifs were found in 62% of the PDX1-enriched sites.”

“PDX1 and/or PDX1:PBX1-like motifs were found in 62% of the PDX1-enriched sites.”

“Activation domains drive nucleosome eviction by SWI/SNF. EMBO J 26: 730– 740.”

“We scanned the reported PWMs in TF-enriched regions with a custom Java algorithm, largely as described in Wederell et al. (2008).”

“…vivo (Adams and Workman 1995; Albert et al. 2007; Koerber et al. 2009), possibly due to binding at DNA sequences found within nucleosome exit or entry points, or at sequences exposed due to rotational phasing on the nucleosome surface (Albert et al. 2007; Jiang and Pugh 2009; Koerber et al. 2009).”

“PDX1 is essential for pancreas development (Jonsson et al. 1994) and is required for the expression of insulin and several other genes critical to pancreas development and function (Ohlsson et al. 1993; Ahlgren et al. 1998; Gerrish et al. 2001; Svensson et al. 2007).”

“All SAGE libraries were generated as part of the Mouse Atlas of Gene Expression project (Siddiqui et al. 2005; Hoffman et al. 2008).”

“…analyses, indicate that tissue-specific gene expression can be dictated by tissue-specific transcription factor binding and by the tissue-specific recruitment of nucleosome-remodeling complexes (e.g., SWI/SNF) (Hogan et al. 2006; Schwabish and Struhl 2007) to transcription factor occupied loci.”

“Secondary co-occupying transcription factors ensure nucleosome displacement Previously, it was shown that differential recruitment of ESR1 and AR in breast and prostate cancer cell lines, respectively, correlates with the accessibility and functionality of FOXA1 sites (Eeckhoute et al. 2009).”

“Transcription factor binding (Gutierrez et al. 2007) and the presence of appropriate histone modifications (Hogan et al. 2006; Jiang and Pugh 2009) have been implicated as mechanisms for targeting nucleosome-remodeling complexes to specific sites in the genome.”

“FOXA1 is the only mammalian transcription factor that has previously been shown to interact directly with nucleosomal DNA in vivo (Chaya et al. 2001).”

“For this we used available microarray data from adult-specific knockout mouse models of Foxa2 in islets (Gao et al. 2007), Foxa2 in liver (Bochkis et al. 2009), and Hnf4a in liver (Holloway et al. 2008).”

“Recent genome-wide analyses of transcription factor–DNA association in mammalian genomes show that most transcription factors bind thousands of sites (Robertson et al. 2007; Chen et al. 2008; Marson et al. 2008; Wederell et al. 2008; Hoffman and Jones 2009; Zheng et al. 2009).”

“For this we used available microarray data from adult-specific knockout mouse models of Foxa2 in islets (Gao et al. 2007), Foxa2 in liver (Bochkis et al. 2009), and Hnf4a in liver (Holloway et al. 2008).”

“Arginine is a precursor of nitric oxide (NO), which plays critical roles in controlling insulin secretion from beta-cells, beta-cell apoptosis, and islet inflammation, as well as in controlling liver regeneration, inflammation, and blood flow (Schmidt et al. 1992; Shimabukuro et al. 1997; Wu and Morris 1998; Adeghate…”

“Recent genome-wide analyses of transcription factor–DNA association in mammalian genomes show that most transcription factors bind thousands of sites (Robertson et al. 2007; Chen et al. 2008; Marson et al. 2008; Wederell et al. 2008; Hoffman and Jones 2009; Zheng et al. 2009).”

“All SAGE libraries were generated as part of the Mouse Atlas of Gene Expression project (Siddiqui et al. 2005; Hoffman et al. 2008).”

“…roles in controlling insulin secretion from beta-cells, beta-cell apoptosis, and islet inflammation, as well as in controlling liver regeneration, inflammation, and blood flow (Schmidt et al. 1992; Shimabukuro et al. 1997; Wu and Morris 1998; Adeghate and Parvez 2000; Rockey and Shah 2004).”

“PDX1 is essential for pancreas development (Jonsson et al. 1994) and is required for the expression of insulin and several other genes critical to pancreas development and function (Ohlsson et al. 1993; Ahlgren et al. 1998; Gerrish et al. 2001; Svensson et al. 2007).”

“For this we used available microarray data from adult-specific knockout mouse models of Foxa2 in islets (Gao et al. 2007), Foxa2 in liver (Bochkis et al. 2009), and Hnf4a in liver (Holloway et al. 2008).”

“While the nature of this is discussed in detail below (and see also Hoffman et al. 2010), Tuteja et al. (2008) did find that medium- and lower-affinity, liver-specific targets were highly enriched for binding of other hepatic nuclear factors, suggesting a role for cooperative binding during the…”

“Therefore, the local distribution of chromatin modifications around transcription factor binding sites rather than the simple presence or absence of these marks may better reflect enhancer activity, with a bimodal distribution being indicative of an active enhancer, whereas a single peak indicates a switch to an inact…”

“…previously observed in distal genomic regions (Barski et al, 2007; Lupien et al, 2008; Robertson et al, 2008; Wang et al, 2008; Schmidl et al, 2009; Hoffman et al, 2010; Lin et al, 2010), this is the first time H3K4me3 enrich- ment have been directly correlated with enhancer activity in primary…”

“…some factors are detected bound to 10,000–60,000 euchromatic regions, including mammalian E2f1 (Bieda et al., 2006), Esrrb (Chen et al., 2008), FOXA2 (Hoffman et al., 2010), Klf4 (Chen et al., 2008), myc (Bieda et al., 2006), MyoD (Cao et al., 2010), SP1 (Cawley et al., 2004), and STAT1 (Robertson…”

“They also were enriched for histone H3 lysine 4 (H3K4) mono-methyl and tri-methyl signatures that mark transcriptionally active loci (Figure 6C) [45].”

“Bimodal distribution of H3K4me1 at Foxa2 binding sites has previously been correlated with tissue-specific gene activity and nucleosome displacement (Hoffman et al., 2010).”

“At enhancers, bimodal H3K4me1 peaks correlate with the binding of pioneering factors, including Pax7 and Foxa2 (Hoffman et al., 2010; Budry et al., 2012).”

“These combinedmotifs bear the cell-type-specific functions of enhancers and often allow for recruitment of TFs that respond to external stimuli such as nuclear receptors (Lupien et al. 2008; Heinz et al. 2010; Hoffman et al. 2010; Lefterova et al. 2010).”

“We integrated all these analyses with binding site maps of key transcription factors (TFs) essential for b cell identity and function (NeuroD1, Pdx1, and Foxa2) (Hoffman et al., 2010; Khoo et al., 2012; Tennant et al., 2013).”

“were suggestive of the pioneering role of PBX1 [36,70].”

“Other liver-expressed TFs, including HNF4A and HNF6, are also important for hepatic sex differences, as indicated by knockout studies and reporter gene analyses and by the enrichment of their motifs at GH-dependent regulatory sites (35–39).”

“We analyzed publicly available ChIP-Seq data (SRA008281) from Hoffman et al.22 to obtain an unbiased whole genome mapping of Hnf4a binding sites in mouse.”

“Another study of mouse liver and pancreas likewise found that H3K4me1 signal flanks active promoters, although the relationship to H3K4me3 was not examined (Hoffman et al., 2010).”

“In addition, bindingofNEUROD2atboth of these sets of sites induces a bimodal histone distribution that has been suggested to indicate a functional binding site (Hoffman et al., 2010) (Figure 3E).”

“Notably, we did not observe this enrichment among Foxa2 target genes in the mouse pancreatic islet, in which the Foxa2 regulatory network is rewired relative to the liver (30) (Fig.”

“The following NCBI data sets were used in this study: SRA008281, GSM751034, and GSM751035 (Hoffman et al., 2010; Tennant et al., 2013).”

“First, we compared HNF4a ChIP’ed from mouse liver (48) and RXRa ChIP’ed from mouse 3T3-L1 (pre-adipocyte) cells (49).”

“A previous study had shown that FOXA2 and HNF4a colocalize at a subset of sites in mouse liver [37], but that study did not examine the relationship of these sites with TCF7L2 binding.”

“We assessed HNF4α transcriptional activity by qPCR analysis of positive(12) and negative target genes of HNF4α(22) at the 0-, 6-, and 12-hours after PH.”

“Next, we took advantage of published mouse ChIP-seq data sets (Chen et al. 2008; Hoffman et al. 2010; Laudadio et al. 2012) to test if a panel of factors bound to A versus C regions”

“In agreement to previous results [42], we found 5976 sites to be co-”

“Specifically, the LDB-ISL1 common peaks are located in genomic regions devoid of inactive chromatin modifications (H3K27me3) and enriched for the histone modifications associated with active regulatory elements (H3K27ac and H3K4me1) but not active promoters (H3K4me3) (Figure 5C) (56, 58).”

“Nucleosome positions estimated from H3K4me1 ChIP-Seq data are unable to resolve closely spaced nucleosomes and also miss multiple nucleosomes detected by mapping of nucleosomes with micrococcal nuclease.”

“Previous analysis comparing Pdx1 and Foxa2 occupancy in mouse islets has been reported (58).”

“Superenhancers are thought of as hubs of functionally related transcription factors; thus, we incorporated key β cell factors Pdx1, MafA, NeuroD1, and FoxA2 into our analysis (17, 34).”

“A mouse islet tag sequencing library was analysed for RefSeq accessions with tag counts greater than five as described [ 30 , 31] (see also Electronic supplementary material [ESM] Methods).”

“ca/ data/histone-modification/histone-modification-data [41].”

“These loci reflect perhaps the ability of the TFs or that of their interacting partners, to bind to compacted chromatin (so-called “pioneer TF”) [56].”

“Our data also show that FOXA1/A2binding siteswere significantly enriched in sequence reads fromday 17 PDX1ChIPseq (Figure 2A), and FOXA2:PDX1 co-binding was observed frequently in nearly 2,000 loci in mouse islets (Hoffman et al., 2010).”

“5b) (Heintzman et al., 2007; Hoffman et al., 2010; Roudier et al., 2011; Bonn et al., 2012; Wang et al., 2014).”

“Thus, the same TF may exhibit completely different genome-wide binding patterns and regulate non-overlapping sets of target genes in different cell types (Hoffman et al , 2010; Pimkin et al , 2014).”

“5A, the mean enrichment of these two marks around ER BSs (as 386 defined by the sum of ER BSs detected in placebo- and E2-treated animals) exhibited biphasic curves 387 around the center of the ER BSs, reflecting the existence of modified nucleosomes surrounding 388 poised/activated genomic regions (69-72).”

“The hepatocyte nuclear factor 4α is a transcription factor found upstream in the regulation pathways of several hepatic genes (27).”

“In human prostate cancer LNCaP cells, transcriptional activation of prostate-specific antigen and kallikrein genes by the AR is associated with a cascade of distinct covalent histone modifications, such as acetylation of lysine 9 and 14, methylation of arginine 17, phosphorylation of serine 10, as well as H3K4me2 and…”

“As an independent approach for finding retrotransposons with promoter activity, we performed a series of database searches using genome-wide ChIP-Seq data, which were designed to survey active (H3K4me3) regions in different types of cells, including ES (Embryonic Stem), NP (Neural Precursor), and MEF (Mouse Embryonic…”

“RNA-seq [18]; SREBP-2 ChIP-seq peaks [22]; FOXA2 ChIP-seq peaks [32]; MNase-seq tag library (GEO: GSM717558) [16]; H3K4me1 and H3K4me3 ChIP-seq tag library (SRA008281) [15]; H3K27me2 ChIP-seq tag library (GEO: GSM751034).”

“HNF4AP1bindsto enhancers and promoters of > 50% of liver-speci fi c genes 6 , recruiting co-activators (e.g. P300, TET3) to establish active chromatin states associated with epigenetic modi fi cations such as histone H3K4me1, H3K27ac and 5-hydroxymethylation of cytosines 1,3,11 .”

“conducted ChIP-seq for FOXA2, HNF4A PDX1, and histone modifications in the mouse liver and pancreatic islets, and showed that both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment are involved in determining tissue-specific gene expression [96].”

“Some studies show a bimodal distribution of H3K4Me1 and/or H3K4Me3 signal at promoters (Barski et al., 2007; Kingsley et al., 2020) but this is not always observed (Hoffman et al., 2010; Bae and Lesch, 2020).”

“Several studies have shown PVP histone modification patterns at regulatory elements as a manifestation of their active state (5,6,49,50).”

“Next, we performed enrichment analyses with global histone modification profiling targeting H3K4me1 for primed enhancers, H3K27ac for active enhancers, and H3K27me3 for poised enhancers in pancreatic b-cells, as previously described (27).”

“For example, H3K4me1-marked nucleosomes have been suggested to locate at binding sites of the nuclear hormone receptor HNF4A in mouse liver [30].”

“5), exhibiting a monomodal distribution pattern of H3K4me1 at inactive enhancer regions (Alder et al., 2014; Hoffman et al., 2010).”

“Previous studies demonstrated that nucleosomes control tissue-specific transcription factor binding to gene promoters, and, thus, contribute to tissue-specific gene expression (Hoffman et al., 2010; Hu et al., 2011; Tsui et al., 2011; Yang et al., 2013; Zhang et al., 2015).”

“As a negative control, we show the same profiles generated from mouse adult liver, in which Pdx1 is not expressed, and in which Foxa2 binds & 25% of the sites identified in islets [19] (Figure 8 in ‘figure S1’).”

“Because these histone modifications are typically found at transcriptional enhancers and promoters, 17–20 these findings suggest that these accessible chromatin regions are B-ALL cis -regulatory elements implicated in gene regulation.”

“To bolster these transcriptional programmes in vivo, chronic regulation via the epigenome appears to step in to maintain beta cell identity in the long term [3–7].”

“Data were analyzed as previously described [2,28].”

“While uni-modal profiles for H3K4me1 have been observed previously in other conditions [HRZ ∗ 10], the unimodal pattern for H4ac is undocumented and warrants further investigation.”

“Motif analysis showed that all of the 5 top scored motifs were consistent with previously documented Foxa2 binding sites (Supplementary Figure S2C) (Hoffman et al., 2010; Tuteja et al., 2009).”

“In order to distinguish those genes specifically regulated by Hnf4a among the significantly perturbed genes obtained from microarray, we analyzed previously published chromatin immunoprecipitation (ChIP)-sequencing (ChIP-Seq) data for Hnf4a targets obtained from the National Center for Biotechnology Information Short…”

“DNA from 4 separate ChIP experiments was pooled for library construction and sequencing as previously described [29,30].”

“While a concurrence of HNF4A binding and H3K4me1 has been well documented 48 – 50, 52, 60 , we show that HNF4A is essential essential for maintaining H3K4me1 at HBRs, and consequently H3K27ac , in the adult mouse Previously, we determined that HNF4A interacts with TET proteins to maintain…”

“This marker indicates monomethylation of the fourth lysine residue of histone H3 protein and is commonly associated with gene enhancers (Hoffman et al., 2010).”

“[22] showed high nucleosome positioning at the center of bimodal H3K4me1 peaks compared to mono-modal in mouse islets and liver.”

“This study focused firstly on the regions around TSSs and stop codon sites, which are well-defined nucleosome distributions 23,24 .”

“Indeed, the mouse and human F11 loci are predicted to carry multiple functional HNF4α binding sites [15,16].”

“FOXA2 and PDX1 synergistically regulate the expression of genes involved in b-cell function, and FOXA2 and HNF4A collaborate to maintain the expression of genes involved in the developing pancreas and liver (Hoffman et al. 2010).”