Search

Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary in vitro studies with recombinant chromatin and purified proteins demonstrate that binding of the MLL3/4 complex and H3K4me1 deposition is enhanced by mutant p53 and p300-mediated acetylation, which in turn reflects a MLL3/4-dependent enhancement of mutant p53 and p300-dependent transcriptional activation. Collectively, our findings establish a mechanism in which mutant p53 cooperates with MLL4 to regulate aberrant enhancer activity and tumor-promoting gene expression in response to chronic immune signaling.

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein β (C/EBPβ), to stimulate transcription of the gene encoding peroxisome proliferator-activated receptor (PPARγ), a master regulator of adipogenesis. However, the mechanism of PPARγ gene regulation by glucocorticoids, the glucocorticoid receptor (GR), and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPARγ gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPARγ GBRs, which also contain binding sites for C/EBP and PPARγ proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPARγ gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPARγ gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes and decreased induction of PPARγ, C/EBPα, and C/EBPδ. Although CCAR1 was required for stimulation of several GR-regulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis.

The baculovirus expression vector system (BEVS) has been used as a preferred platform for the production of recombinant protein complexes and efficacious vaccines. However, limited protein yield hinders the application of BEVS. It is well accepted that transcription enhancers are capable of increasing translational efficiency of mRNAs, thereby achieving better protein production. In this study, the ability of LvYY1 as a transcription enhancer was assessed. LvYY1 could interact with the WSSV ie1 promoter via binding to special DNA sites in BEVS. The effects of LvYY1 on protein expression mediated by WSSV ie1 promoter of BEVS was investigated using eGFP as a reporter gene. Enhanced eGFP expression was observed in Sf-9 cells with LvYY1. On this basis, a modified vector combining ie1 promoter and LvYY1 was developed to express either secreting CSFV E2 or baculovirus surface displayed H5 HA of AIVs. Compared to control groups without LvYY1, E2 protein yield increases to 1.6-fold, while H5 production improves as revealed by an upregulated hemagglutination titer of 8-fold at least. Moreover, with LvYY1, H5 displaying baculovirus driven by WSSV ie1 promoter (BV-LvYY1-ie1-HA) sustains the transduction activity in CEF cells. In chicken, BV-LvYY1-ie1-HA elicits a robust immune response against H5 AIVs in the absence of adjuvant, as indicated by specific antibody and cytokine responses. The findings suggest its potential function as both a vectored and subunit vaccine. These results demonstrate that the coexpression with LvYY1 serves as a promising strategy to extensively improve the efficiency of BEVS for efficacious vaccine production.

Background: Precise nucleosome positioning is an increasingly recognized feature of promoters and enhancers, reflecting complex contributions of DNA sequence, nucleosome positioning, histone modification and transcription factor binding to enhancer activity and regulation of gene expression. Changes in nucleosome position and occupancy, histone variants and modifications, and chromatin remodeling are also critical elements of dynamic transcriptional regulation, but poorly understood at enhancers. We investigated glucocorticoid receptor-associated (GR) nucleosome dynamics at enhancers in acute lymphoblastic leukemia. Results: For the first time, we demonstrate functionally distinct modes of nucleosome remodeling upon chromatin binding by GR, which we term central, non-central, phased, and minimal. Central and non-central remodeling reflect nucleosome eviction by GR and cofactors, respectively. Phased remodeling involves nucleosome repositioning and is associated with rapidly activated enhancers and induction of gene expression. Minimal remodeling sites initially have low levels of enhancer-associated histone modification, but the majority of these regions gain H3K4me2 or H3K27Ac to become de novo enhancers. Minimal remodeling regions are associated with gene ontologies specific to decreased B cell number and mTOR inhibition and may make unique contributions to glucocorticoid-induced leukemia cell death. Conclusions: Our findings form a novel framework for understanding the dynamic interplay between transcription factor binding, nucleosome remodeling, enhancer function, and gene expression in the leukemia response to glucocorticoids. [ABSTRACT FROM AUTHOR]

Most of the mammalian genome consists of nucleotide sequences not coding for proteins. Exons of genes make up only 3% of the human genome, while the significance of most other sequences remains unknown. Recent genome studies with high-throughput methods demonstrate that the so-called noncoding part of the genome may perform important functions. This hypothesis is supported by three groups of experimental data: 1) approximately 10% of the sequences, most of which are located in noncoding parts of the genome, is evolutionarily conserved and thus can be of functional importance; 2) up to 99% of the mammalian genome is being transcribed forming short and long noncoding RNAs in addition to common mRNA; and 3) mutations in noncoding parts of the genome can be accompanied by progression of pathological states of the organism. In the light of these data, in the review we consider the functional role of numerous known sequences of noncoding parts of the genome including introns, DNA methylation regions, enhancers and locus control regions, insulators, S/MAR sequences, pseudogenes, and genes of noncoding RNAs, as well as transposons and simple repeats of centromeric and telomeric regions of chromosomes. The assumption is made that the intergenic noncoding sequences without definite/clear functions can be involved in spatial organization of genetic loci in interphase nuclei. [ABSTRACT FROM AUTHOR]

In mammalian organisms, enhancers can regulate transcription from great genomic distances. How enhancers affect distal gene expression has been a major question in the field of gene regulation. One model to explain how enhancers communicate with their target promoters, the chromatin looping model, posits that enhancers and promoters come in close spatial proximity to mediate communication. Chromatin looping has been broadly accepted as a means for enhancer-promoter communication, driven by accumulating in vitro and in vivo evidence. The genome is now known to be folded into a complex 3D arrangement, created and maintained in part by the interplay of the Cohesin complex and the DNA-binding protein CTCF. In the last few years, however, doubt over the relationship between looping and transcriptional activation has emerged, driven by studies finding that only a modest number of genes are perturbed with acute degradation of looping machinery components. In parallel, newer models describing distal enhancer action have also come to prominence. In this article, we explore the emergence and development of the looping model as a means for enhancer-promoter communication and review the contrasting evidence between historical gene-specific and current global data for the role of chromatin looping in transcriptional regulation. We also discuss evidence for alternative models to chromatin looping and their support in the literature. We suggest that, while there is abundant evidence for chromatin looping as a major mechanism for enhancer function, enhancer-promoter communication is likely mediated by more than one mechanism in an enhancer- and context-dependent manner., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VD-induced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt (162 up, 210 down), villi (199 up, 63 down), or colon (102 up, 28 down), but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor-binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

IFNs λ1, λ2, and λ3, or type Ill IFNs, are recently identified cytokines distantly related to type I IFN5. Despite an early evolutionary divergence, the 2 types of IFN5 display similar antiviral activities, and both are produced primarily in dendritic cells. Although virus induction of the type I IFN-β gene had served as a paradigm of gene regulation, relatively little is known about the regulation of IFN-λ gene expression. Studies of virus induction of IFN-λ1 identified an essential role of IFN regulatory factors (IRF) 3 and 7, which bind to a regulatory DNA sequence near the start site of transcription. Here, we report that the proximal promoter region of the IFN-λ1 regulatory region is not sufficient for maximal gene induction in response to bacterial IPS. and we identify an essential cluster of homotypic NF-κB binding sites. Remarkably, these sites, which bind efficiently to NF-κB and function independently of the IRF3/7 binding sites, originate as transposable elements of the Alu and LTR families. We also show that depletion of the NF-κB RelA protein significantly reduces the level of the IFN-λ1 gene expression. We conclude that IFN-λ1 gene expression requires NF-κB, and we propose a model for IFN-λ1 gene regulation, in which IRF and NF-κB activate gene expression independently via spatially separated promoter elements. These observations provide insights into the independent evolution of the IFN-λ1 and IFN-β promoters and directly implicate transposable elements in the regulation of the IFN-λV;1 gene by NF-κB. [ABSTRACT FROM AUTHOR]

The clustered protocadherins (Pcdh) are encoded by three closely linked gene clusters (Pcdh-α, -β, and -γ) that span nearly 1 million base pairs of DNA. The Pcdh-α gene cluster encodes a family of 14 distinct cadherin-like cell surface proteins that are expressed in neurons and are present at synaptic junctions. Individual Pcdh-α mRNAs are assembled from one of 14 "variable" (V) exons and three "constant" exons in a process that involves both differential promoter activation and alternative pre-mRNA splicing. In individual neurons, only one (and rarely two) of the Pcdh α1-12 promoters is independently and randomly activated on each chromosome. Thus, in most cells, this unusual form of monoallelic expression leads to the expression of two different Pcdh-α 1-12 V exons, one from each chromosome. The two remaining V exons in the cluster (Pcdh-αC1 and αC2) are expressed biallelically in every neuron. The mechanisms that underlie promoter choice and monoallelic expression in the Pcdh-α gene cluster are not understood. Here we report the identification of two long-range cis-regulatory elements in the Pcdh-α gene cluster, HS5-1 and HS7. We show that HS5-1 is required for maximal levels of expression from the Pcdh α1-12 and ad promoters, but not the Pcdh-αC2 promoter. The nearly clusterwide requirement of the HS5-1 element is consistent with the possibility that the monoallelic expression of Pcdh-α V exons is a consequence of competition between individual V exon promoters for the two regulatory elements. [ABSTRACT FROM AUTHOR]

Past studies of the channel catfish immunoglobulin heavy-chain (IgH) locus indicates that it lacks an Eµ enhancer in theJ H-Cµ1intron but does have an enhancer, termed Eµ3', in the µ-d intergenic region. The positioning of the catfish enhancer downstream of the µ-chain exons is predicted to be unfavorable for antibody-affinity maturation in catfish, and would also have been an impediment to the evolution of class switch recombination, had it existed in early tetrapods. To determine if this downstream enhancer is a general feature of teleost fish, we have identified the location of the transcriptional enhancer in the zebrafishIgHlocus. We find that zebrafish, like catfish, only have an Eµ3'-like enhancer that has cross-species activity, but which is B-cell-specific in its activity. A 300-bp region of the zebrafish enhancer shares sequence homology with the core of the catfish Eµ3', although there has been loss and gain of specific octamer enhancer motifs. Mutagenesis studies demonstrate that the zebrafishIgHenhancer depends on a pair of E-box motifs that are found in the enhancer core. Similarly spaced E-box motifs appear to exist in the Eµ3' enhancer regions of other teleost fish, suggesting this is a common feature among fishIgHenhancers. We discuss how this distal positioning of the enhancer may influence affinity maturation in extant teleosts as well as the evolution of this process in the early tetrapods. [ABSTRACT FROM AUTHOR]

Vitamin D receptor (VDR) levels are highest in the intestine where it mediates 1,25 dihydroxyvitamin D-induced gene expression. However, the mechanisms controlling high intestinal VDR gene expression are unknown. Here, we used Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to identify the regulatory sites controlling intestine-specific Vdr gene expression in the small intestine (villi and crypts) and colon of developing, adult, and aged mice. We identified 17 ATAC peaks in a 125 kb region from intron 3 to -55.8 kb from exon 1 of the Vdr gene. Interestingly, many of these peaks were missing/reduced in the developing intestine. Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq) peaks for intestinal transcription factors (TFs) were present within the ATAC peaks and at HiChIP looping attachments that connected the ATAC/TF ChIP peaks to the transcription start site and CCCTF-binding factor sites at the borders of the Vdr gene regulatory domain. Intestine-specific regulatory sites were identified by comparing ATAC peaks to DNAse-Seq data from other tissues that revealed tissue-specific, evolutionary conserved, and species-specific peaks. Bioinformatics analysis of human DNAse-Seq peaks revealed polymorphisms that disrupt TF-binding sites. Our analysis shows that mouse intestinal Vdr gene regulation requires a complex interaction of multiple distal regulatory regions and is controlled by a combination of intestinal TFs. These intestinal regulatory sites are well conserved in humans suggesting that they may be key components of VDR regulation in both mouse and human intestines., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The baculovirus expression vector system (BEVS) has been used as a preferred platform for the production of recombinant protein complexes and efficacious vaccines. However, limited protein yield hinders the application of BEVS. It is well accepted that transcription enhancers are capable of increasing translational efficiency of mRNAs, thereby achieving better protein production. In this study, the ability of LvYY1 as a transcription enhancer was assessed. LvYY1 could interact with the WSSV ie1 promoter via binding to special DNA sites in BEVS. The effects of LvYY1 on protein expression mediated by WSSV ie1 promoter of BEVS was investigated using eGFP as a reporter gene. Enhanced eGFP expression was observed in Sf-9 cells with LvYY1. On this basis, a modified vector combining ie1 promoter and LvYY1 was developed to express either secreting CSFV E2 or baculovirus surface displayed H5 HA of AIVs. Compared to control groups without LvYY1, E2 protein yield increases to 1.6-fold, while H5 production improves as revealed by an upregulated hemagglutination titer of 8-fold at least. Moreover, with LvYY1, H5 displaying baculovirus driven by WSSV ie1 promoter (BV-LvYY1-ie1-HA) sustains the transduction activity in CEF cells. In chicken, BV-LvYY1-ie1-HA elicits a robust immune response against H5 AIVs in the absence of adjuvant, as indicated by specific antibody and cytokine responses. The findings suggest its potential function as both a vectored and subunit vaccine. These results demonstrate that the coexpression with LvYY1 serves as a promising strategy to extensively improve the efficiency of BEVS for efficacious vaccine production. [ABSTRACT FROM AUTHOR]

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans- regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis -region ( cis- REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans- REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4 ) in their native context., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Canver et al.)

The baculovirus expression vector system (BEVS) has been used as a preferred platform for the production of recombinant protein complexes and efficacious vaccines. However, limited protein yield hinders the application of BEVS. It is well accepted that transcription enhancers are capable of increasing translational efficiency of mRNAs, thereby achieving better protein production. In this study, the ability of Lv YY1 as a transcription enhancer was assessed. Lv YY1 could interact with the WSSV ie1 promoter via binding to special DNA sites in BEVS. The effects of Lv YY1 on protein expression mediated by WSSV ie1 promoter of BEVS was investigated using eGFP as a reporter gene. Enhanced eGFP expression was observed in Sf-9 cells with Lv YY1. On this basis, a modified vector combining ie1 promoter and Lv YY1 was developed to express either secreting CSFV E2 or baculovirus surface displayed H5 HA of AIVs. Compared to control groups without Lv YY1, E2 protein yield increases to 1.6-fold, while H5 production improves as revealed by an upregulated hemagglutination titer of 8-fold at least. Moreover, with Lv YY1, H5 displaying baculovirus driven by WSSV ie1 promoter (BV- Lv YY1- ie1 -HA) sustains the transduction activity in CEF cells. In chicken, BV- Lv YY1- ie1 -HA elicits a robust immune response against H5 AIVs in the absence of adjuvant, as indicated by specific antibody and cytokine responses. The findings suggest its potential function as both a vectored and subunit vaccine. These results demonstrate that the coexpression with Lv YY1 serves as a promising strategy to extensively improve the efficiency of BEVS for efficacious vaccine production.

The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP ( Hedgehog-interacting protein ) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Safgren et al.)

Over the past decade, thousands of long noncoding RNAs (lncRNAs) have been identified, many of which play crucial roles in normal physiology and human disease. LncRNAs can interact with chromatin and then recruit protein complexes to remodel chromatin states, thus regulating gene expression. However, how lncRNA-chromatin interactions contribute to their biological functions is largely unknown. Here, we collected and constructed an atlas of 188,647 lncRNA-chromatin interactions in human and mouse. All lncRNAs showed diverse epigenetic modification patterns at their binding sites, especially the marks of enhancer activity. Functional analysis of lncRNA target genes further revealed that lncRNAs could exert their functions by binding to both promoter and distal regulatory elements, especially the distal regulatory elements. Intriguingly, many important pathways were observed to be widely regulated by lncRNAs through distal binding. For example, NEAT1 , a cancer lncRNA, controls 13.3% of genes in the PI3K-AKT signaling pathway by interacting with distal regulatory elements. In addition, "two-gene" signatures composed of a lncRNA and its distal target genes, such as HOTAIR-CRIM1 , provided significant clinical benefits relative to the lncRNA alone. In summary, our findings underscored that lncRNA-distal interactions were essential for lncRNA functions, which would provide new clues to understand the molecular mechanisms of lncRNAs in complex disease., (© 2019 Zhang et al.)

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers., (© 2019 Huang et al.)

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.

Initiation and regulation of transcription by RNA polymerase II (RNAPII) in eukaryotes rely on the transcriptional regulatory elements. Promoters and enhancers share similar architectures and functions, and the prevailing view is that they can initiate bidirectional transcription. We summarize functional roles of enhancer transcription and possible mechanisms in enhancer-promoter communication. We discuss the potential roles of enhancer RNAs (eRNAs) in early elongation and highlight that transcriptional enhancers might modulate the release of paused RNAPII via 3D chromatin looping. Emerging evidence suggests that transcriptional enhancers regulate the promoter-proximal pausing of RNAPII, a key rate-limiting step required for productive elongation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Transcriptional activators bind DNA and recruit cofactors to modify chromatin. The extent to which these two events are separable is unclear. Here, using a custom ChIP tiling array to map chromatin modifications, we show that interferon-γ-induced DNA binding of signal transducer and activator of transcription 1 (STAT1), typically associated with the transcription factor interferon regulatory factor 1 (IRF1), causes histone acetylation (H3ac, H4ac). In contrast, among IRF1 sites lacking concomitant STAT1 recruitment, only 25% underwent inducible histone acetylation, 31% exhibited constitutive histone acetylation, and 44% had no histone acetylation. These latter "orphan sites" also lacked other activating modifications ( e.g. H3K4me1, H3K4me2) and were typically remote from transcription start sites. In these cases the closest gene was typically an IFNγ-inducible locus that did not respond to IFNγ in this setting. Orphan sites were detected in different cell types, suggesting broad relevance. Despite an atypical downstream response ( i.e. no histone modifications), IRF1 binding depended on SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4 or BRG1), as is typical of active IRF1 enhancers. Although SMARCA4 permitted IRF1 access to the orphan sites, there was no corecruitment of the histone acetyltransferases CREB-binding protein (CBP) and p300. Orphan sites were constitutively unacetylated, and several were marked with repressive chromatin modifications ( e.g. H3K27me3). In conclusion, although IRF1 can trigger enhanceosome formation independently of STAT1, its ability to do so depends on local chromatin cues., (© 2018 Abou El Hassan et al.)

Cell-autonomous oscillation in clock gene expression drives circadian rhythms. The development of comprehensive analytical techniques, such as bioinformatics and ChIP-sequencing, has enabled the genome-wide identification of potential circadian transcriptional elements that regulate the transcriptional oscillation of clock genes. However, detailed analyses using traditional biochemical and molecular-biological approaches, such as binding and reporter assays, are still necessary to determine whether these potential circadian transcriptional elements are actually functional and how significantly they contribute to driving transcriptional oscillation. Here, we focused on the molecular mechanism of transcriptional oscillations in the mammalian clock gene Period3 ( Per3 ). The PER3 protein is essential for robust peripheral clocks and is a key component in circadian output processes. We found three E box-like elements located upstream of human Per3 transcription start sites that additively contributed to cell-autonomous transcriptional oscillation. However, we also found that Per3 is still expressed in a circadian manner when all three E box-like elements are functionally impaired. We noted that Per3 transcription was activated by the synergistic actions of two D box-like elements and the three E box-like elements, leading to a drastic increase in circadian amplitude. Interestingly, circadian expression of Per3 was completely disrupted only when all five transcriptional elements were functionally impaired. These results indicate that three E box-like and two D box-like elements cooperatively and redundantly regulate cell-autonomous transcriptional oscillation of Per3 ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Patients with advanced congestive heart failure (CHF) or chronic kidney disease often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II, via its type 1 receptor, causes muscle protein breakdown and apoptosis and inhibits satellite cell (SC) proliferation and muscle regeneration, likely contributing to cachexia in CHF and chronic kidney disease. In contrast, Ang II, via its type 2 receptor (AT2R) expression, is robustly induced during SC differentiation, and it potentiates muscle regeneration. To understand the mechanisms regulating AT2R expression and its potential role in muscle regeneration in chronic diseases, we used a mouse model of CHF and found that muscle regeneration was markedly reduced and that this was accompanied by blunted increase of AT2R expression. We performed AT2R promoter reporter analysis during satellite cell differentiation and found that the 70 bp upstream of the AT2R transcription start site contain a core promoter region, and regions upstream of 70 bp to 3 kbp are dispensable for AT2R induction. Instead, AT2R intron 2 acts as a transcriptional enhancer during SC differentiation. Further deletion/mutation analysis revealed that multiple transcription factor binding sites in the +286/+690 region within intron 2 coordinately regulate AT2R transcription. Importantly, +286/+690 enhancer activity was suppressed in CHF mouse skeletal muscle, suggesting that AT2R expression is suppressed in CHF via inhibition of AT2R intronic enhancer activity, leading to lowered muscle regeneration. Thus targeting intron 2 enhancer element could lead to the development of a novel intervention to increase AT2R expression in SCs and potentiate skeletal muscle regenerative capacity in chronic diseases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Antagonism of pro-inflammatory transcription factors by monomeric glucocorticoid receptor (GR) has long been viewed as central to glucocorticoid (GC) efficacy. However, the mechanisms and targets through which GCs exert therapeutic effects in diseases such as asthma remain incompletely understood. We previously defined a surprising cooperative interaction between GR and NF-κB that enhanced expression of A20 (TNFAIP3), a potent inhibitor of NF-κB. Here we extend this observation to establish that A20 is required for maximal cytokine repression by GCs. To ascertain the global extent of GR and NF-κB cooperation, we determined genome-wide occupancy of GR, the p65 subunit of NF-κB, and RNA polymerase II in airway epithelial cells treated with dexamethasone, TNF, or both using chromatin immunoprecipitation followed by deep sequencing. We found that GR recruits p65 to dimeric GR binding sites across the genome and discovered additional regulatory elements in which GR-p65 cooperation augments gene expression. GR targets regulated by this mechanism include key anti-inflammatory and injury response genes such as SERPINA1, which encodes α1 antitrypsin, and FOXP4, an inhibitor of mucus production. Although dexamethasone treatment reduced RNA polymerase II occupancy of TNF targets such as IL8 and TNFAIP2, we were unable to correlate specific binding sequences for GR or occupancy patterns with repressive effects on transcription. Our results suggest that cooperative anti-inflammatory gene regulation by GR and p65 contributes to GC efficacy, whereas tethering interactions between GR and p65 are not universally required for GC-based gene repression., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

FGF21 is an atypical member of the FGF family that functions as a hormone to regulate carbohydrate and lipid metabolism. Here we demonstrate that the actions of FGF21 in mouse adipose tissue, but not in liver, are modulated by the nuclear receptor Rev-erbα, a potent transcriptional repressor. Interrogation of genes induced in the absence of Rev-erbα for Rev-erbα-binding sites identified βKlotho, an essential coreceptor for FGF21, as a direct target gene of Rev-erbα in white adipose tissue but not liver. Rev-erbα ablation led to the robust elevated expression of βKlotho. Consequently, the effects of FGF21 were markedly enhanced in the white adipose tissue of mice lacking Rev-erbα. A major Rev-erbα-controlled enhancer at the Klb locus was also bound by the adipocytic transcription factor peroxisome proliferator-activated receptor (PPAR) γ, which regulates its activity in the opposite direction. These findings establish Rev-erbα as a specific modulator of FGF21 signaling in adipose tissue., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many pathogenic microbes often release toxins that subvert the host's immune responses to render the environment suitable for their survival and proliferation. LeTx is one of the toxins causing immune paralysis by cleaving and inactivating the mitogen-activated protein kinase (MAPK) kinases (MEKs). Here, we show that inhibition of the histone deacetylase 8 (HDAC8) by either the HDAC8-specific inhibitor PCI-34051 or small interference (si)RNAs rendered LeTx-exposed murine macrophages responsive to LPS in pro-IL-1β production. HDAC8 selectively targeted acetylated histone H3 lysine 27 (H3K27Ac), which is known to associate with active enhancers. LeTx induced HDAC8 expression, in part through inhibiting p38 MAPK, which resulted in a decrease of H3K27Ac levels. Inhibition of HDAC8 increased H3K27Ac levels and enhanced NF-κB-mediated pro-IL-1β enhancer and messenger RNA production in LeTx-exposed macrophages. Collectively, this study demonstrates a novel role of HDAC8 in LeTx immunotoxicity and regulation of pro-IL-1β production likely through eRNAs. Targeting HDAC8 could be a strategy for enhancing immune responses in macrophages exposed to LeTx or other toxins that inhibit MAPKs., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8&#95;enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8&#95;enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/β-catenin signaling pathway. We demonstrated in vitro that WNT/β-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8&#95;enh1 was a direct target of the WNT/β-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The acquisition of skeletal muscle-specific function and terminal cell cycle arrest represent two important features of the myogenic differentiation program. These cellular processes are distinct and can be separated genetically. The lineage-specific transcription factor MyoD and the retinoblastoma protein pRb participate in both of these cellular events. Whether and how MyoD and pRb work together to effect terminal cell cycle arrest is uncertain. To address this question, we focused on cyclin D1, whose stable repression is required for terminal cell cycle arrest and execution of myogenesis. MyoD and pRb are both required for the repression of cyclin D1; their actions, however, were found not to be direct. Rather, they operate to regulate the immediate early gene Fra-1, a critical player in mitogen-dependent induction of cyclin D1. Two conserved MyoD-binding sites were identified in an intronic enhancer of Fra-1 and shown to be required for the stable repression of Fra-1 and, in turn, cyclin D1. Localization of MyoD alone to the intronic enhancer of Fra-1 in the absence of pRb was not sufficient to elicit a block to Fra-1 induction; pRb was also recruited to the intronic enhancer in a MyoD-dependent manner. These observations suggest that MyoD and pRb work together cooperatively at the level of the intronic enhancer of Fra-1 during terminal cell cycle arrest. This work reveals a previously unappreciated link between a lineage-specific transcription factor, a tumor suppressor, and a proto-oncogene in the control of an important facet of myogenic differentiation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2'-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The biological effects of 1α,25-dihydroxyvitamin D3 (1,25 (OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP sequence analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR-binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler CCAAT/enhancer-binding protein β. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures that were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)