Distinct Regulatory Regions Research Articles

Paired related homeobox protein-like 1 (Prrxl1) controls its own expression by a transcriptional autorepression mechanism

The homeodomain factor paired related homeobox protein-like 1 (Prrxl1) is crucial for proper assembly of dorsal root ganglia (DRG)–dorsal spinal cord (SC) pain-sensing circuit. By performing chromatin immunoprecipitation with either embryonic DRG or dorsal SC, we identified two evolutionarily conserved regions (i.e. proximal promoter and intron 4) of Prrxl1 locus that show tissue-specific binding of Prrxl1. Transcriptional assays confirm the identified regions can mediate repression by Prrxl1, while gain-of-function studies in Prrxl1 expressing ND7/23 cells indicate Prrxl1 can down-regulate its own expression. Altogether, our results suggest that Prrxl1 uses distinct regulatory regions to repress its own expression in DRG and dorsal SC.

Features of the RAP1A and RAP1B core promoters: recombinant approaches to testing regulatory element function in expression control (946.10)

The RAP1 genes, closely related to the RAS oncogenes, have been shown to have numerous functions, yet little is known about the expression regulation of these genes in human cells. Our lab has constructed plasmids containing putative promoter regions of human RAP1A and RAP1B, driving a gene for green fluorescent protein (GFP). We have transiently transfected these plasmids into a wide‐range of human cell types, and analyzed GFP expression via fluorescence microscopy, qRT‐PCR, and western blotting. By comparing the efficacy of each putative core promoter segment, some distinct regulatory regions have been revealed. Additionally, comparing promoter‐driven expression to the actual native expression of the RAP1B genes in specific cell types has suggested an important regulatory role for enhancer elements not included in the core promoter regions. Various sub‐cloning techniques, including inverse fusion PCR cloning (IFPC), have been employed to add putative regulatory regions to existing GFP core promoter plasmids. These should allow us to further characterize the expression control of the RAP1 genes, and to understand the cell‐type differences observed in native RAP1 expression.

Stepwise acetyltransferase association and histone acetylation at the Myod1 locus during myogenic differentiation

While chromatin modifications can offer a useful readout for enhancer activities, it is less clear whether these modification marks are a cause or consequence of transcription factor occupancy and enhancer activation. We have examined in details the temporal events of acetyltransferase associations and histone acetylations at different regulatory regions of the Myod1 locus. Our studies demonstrate that the histone acetyltransferase (HAT) p300 is stepwise enriched at distinct Myod1 regulatory regions during myogenic differentiation. This enrichment of p300 is associated with increased histone acetylation in a discrete pattern. Inhibition of p300 HAT activity impedes myogenic differentiation, which is coupled with decreased histone acetylation at specific Myod1 regulatory regions. We show for the first time that p300 is directly involved in the early regulation of Myod1 enhancer, and provide molecular insights into how p300 HAT activity and histone acetylation are related to enhancer activation and, consequently, gene transcription.

Multiple activating and repressive cis-promoter regions regulate TNFSF15 expression in human primary mononuclear cells

TL1A/TNFSF15 has been associated with IBD (inflammatory bowel disease) in GWAS (genome-wide association study) and plays a role mediating mucosal inflammation in IBD. Higher TL1A expression is associated with disease severity in both patients and mouse models. Although TL1A has been studied extensively for IBD-associated SNPs, the cis/trans-regulatory regions are poorly defined. Herein we identify response elements regulating TNFSF15 in primary human myeloid cells. Peripheral mononuclear cells transfected with TNFSF15 promoter constructs displayed 30-fold enhanced promoter activity in a minimal −74bp region. Transactivation was mediated partly by AP-1, since mutation of the AP-1 site resulting in loss of promoter activity. Monocytes transfected with c-Jun siRNA or treated with TAT-TI-JIP (JNK Inhibitor VII TAT-TI-JIP) demonstrated reduced TL1A mRNA and protein levels. Surprisingly, constructs larger than −74bp did not increase promoter expression (expression of −1275bp construct was 25% of −74bp activity), suggesting the presence of both activating and repressing TL1A promoter elements. In fact, mutation of the −210bp NFκB site enhanced promoter activity (60-fold) suggesting a repressive role for this site. DNA–protein binding to the TL1A AP-1 and NFκB elements was inhibited by excess consensus or TL1A oligonucleotides and binding and confirmed by chromatin immuno-precipitation analysis. Yet, despite the fact that the −210bp NFκB site acts as a suppressor element, overall mRNA and protein expression were inhibited in monocytes treated with MG132 (NFκB/proteasome inhibitor) or SN50 (NFκB-p50 blocking peptide), suggesting that NFκB acts as both an activator and silencer of TL1A expression. These data suggest that modulation of TL1A expression involves a complex interplay between positive and negative signals, binding to distinct regulatory regions.

The Border Between the Ultrabithorax and abdominal-A Regulatory Domains in the Drosophila Bithorax Complex

The bithorax complex in Drosophila melanogaster includes three homeobox-containing genes--Ultrabithorax (Ubx), abdominal--A (abd-A), and Abdominal-B (Abd-B)-which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the bithorax complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain.

Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3

BackgroundThe TCF7L2 transcription factor is linked to a variety of human diseases, including type 2 diabetes and cancer. One mechanism by which TCF7L2 could influence expression of genes involved in diverse diseases is by binding to distinct regulatory regions in different tissues. To test this hypothesis, we performed ChIP-seq for TCF7L2 in six human cell lines.ResultsWe identified 116,000 non-redundant TCF7L2 binding sites, with only 1,864 sites common to the six cell lines. Using ChIP-seq, we showed that many genomic regions that are marked by both H3K4me1 and H3K27Ac are also bound by TCF7L2, suggesting that TCF7L2 plays a critical role in enhancer activity. Bioinformatic analysis of the cell type-specific TCF7L2 binding sites revealed enrichment for multiple transcription factors, including HNF4alpha and FOXA2 motifs in HepG2 cells and the GATA3 motif in MCF7 cells. ChIP-seq analysis revealed that TCF7L2 co-localizes with HNF4alpha and FOXA2 in HepG2 cells and with GATA3 in MCF7 cells. Interestingly, in MCF7 cells the TCF7L2 motif is enriched in most TCF7L2 sites but is not enriched in the sites bound by both GATA3 and TCF7L2. This analysis suggested that GATA3 might tether TCF7L2 to the genome at these sites. To test this hypothesis, we depleted GATA3 in MCF7 cells and showed that TCF7L2 binding was lost at a subset of sites. RNA-seq analysis suggested that TCF7L2 represses transcription when tethered to the genome via GATA3.ConclusionsOur studies demonstrate a novel relationship between GATA3 and TCF7L2, and reveal important insights into TCF7L2-mediated gene regulation.

Expression of a desaturase gene,desat1, in neural and nonneural tissues separately affects perception and emission of sex pheromones inDrosophila

Animals often use sex pheromones for mate choice and reproduction. As for other signals, the genetic control of the emission and perception of sex pheromones must be tightly coadapted, and yet we still have no worked-out example of how these two aspects interact. Most models suggest that emission and perception rely on separate genetic control. We have identified a Drosophila melanogaster gene, desat1, that is involved in both the emission and the perception of sex pheromones. To explore the mechanism whereby these two aspects of communication interact, we investigated the relationship between the molecular structure, tissue-specific expression, and pheromonal phenotypes of desat1. We characterized the five desat1 transcripts-all of which yielded the same desaturase protein-and constructed transgenes with the different desat1 putative regulatory regions. Each region was used to target reporter transgenes with either (i) the fluorescent GFP marker to reveal desat1 tissue expression, or (ii) the desat1 RNAi sequence to determine the effects of genetic down-regulation on pheromonal phenotypes. We found that desat1 is expressed in a variety of neural and nonneural tissues, most of which are involved in reproductive functions. Our results suggest that distinct desat1 putative regulatory regions independently drive the expression in nonneural and in neural cells, such that the emission and perception of sex pheromones are precisely coordinated in this species.

A Regulatory Archipelago Controls Hox Genes Transcription in Digits

The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.

Gfi1 Expression Is Controlled by Five Distinct Regulatory Regions Spread over 100 Kilobases, with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 Acting as Upstream Regulators in Early Hematopoietic Cells

The growth factor independence 1 (Gfi1) gene was originally discovered in the hematopoietic system, where it functions as a key regulator of stem cell homeostasis, as well as neutrophil and T-cell development. Outside the blood system, Gfi1 is essential for inner-ear hair and intestinal secretory cell differentiation. To understand the regulatory hierarchies within which Gfi1 operates to control these diverse biological functions, we used a combination of comparative genomics, locus-wide chromatin immunoprecipitation assays, functional validation in cell lines, and extensive transgenic mouse assays to identify and characterize the complete ensemble of Gfi1 regulatory elements. This concerted effort identified five distinct regulatory elements spread over 100kb each driving expression in transgenic mice to a subdomain of endogenous Gfi1. Detailed characterization of an enhancer 35 kb upstream of Gfi1 demonstrated activity in the dorsal aorta region and fetal liver in transgenic mice, which was bound by key stem cell transcription factors Scl/Tal1, PU.1/Sfpi1, Runx1, Erg, Meis1, and Gata2. Taken together, our results reveal the regulatory regions responsible for Gfi1 expression and importantly establish that Gfi1 expression at the sites of hematopoietic stem cell (HSC) emergence is controlled by key HSC regulators, thus integrating Gfi1 into the wider HSC regulatory networks.

Transcriptional regulation of Elf-1 : locus-wide analysis reveals four distinct promoters, a tissue-specific enhancer, control by PU.1 and the importance of Elf-1 downregulation for erythroid maturation

Ets transcription factors play important roles during the development and maintenance of the haematopoietic system. One such factor, Elf-1 (E74-like factor 1) controls the expression of multiple essential haematopoietic regulators including Scl/Tal1, Lmo2 and PU.1. However, to integrate Elf-1 into the wider regulatory hierarchies controlling haematopoietic development and differentiation, regulatory elements as well as upstream regulators of Elf-1 need to be identified. Here, we have used locus-wide comparative genomic analysis coupled with chromatin immunoprecipitation (ChIP-chip) assays which resulted in the identification of five distinct regulatory regions directing expression of Elf-1. Further, ChIP-chip assays followed by functional validation demonstrated that the key haematopoietic transcription factor PU.1 is a major upstream regulator of Elf-1. Finally, overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated that Elf-1 downregulation is necessary for terminal erythroid differentiation. Given the known activation of PU.1 by Elf-1 and our newly identified reciprocal activation of Elf-1 by PU.1, identification of an inhibitory role for Elf-1 has significant implications for our understanding of how PU.1 controls myeloid–erythroid differentiation. Our findings therefore not only represent the first report of Elf-1 regulation but also enhance our understanding of the wider regulatory networks that control haematopoiesis.

TRESK Background K+ Channel Is Inhibited by Phosphorylation via Two Distinct Pathways

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel, KCNK18) is directly regulated by the calcium/calmodulin-dependent phosphatase calcineurin and 14-3-3 adaptor proteins. The calcium signal robustly activates the channel via calcineurin, whereas the anchoring of 14-3-3 interferes with the return of the current to the resting state after the activation in Xenopus oocytes. In the present study, we report that the phosphorylation of TRESK at two distinct regulatory regions, the 14-3-3 binding site (Ser-264) and the cluster of three adjacent serine residues (Ser-274, Ser-276, and Ser-279), are responsible for channel inhibition. The phosphorylation of Ser-264 by protein kinase A accelerated the return of the current of S276E mutant TRESK to the resting state after the calcineurin-dependent activation. In the presence of 14-3-3, the basal current of the S276E mutant was reduced, and its calcineurin-dependent activation was augmented, suggesting that the direct binding of the adaptor protein to TRESK contributed to the basal inhibition of the channel under resting conditions. Unexpectedly, we found that 14-3-3 impeded the recovery of the current of S264E mutant TRESK to the resting state after the calcineurin-dependent activation, despite of the mutated 14-3-3 binding site. This suggests that 14-3-3 inhibited the kinase phosphorylating the regulatory cluster of Ser-274, Ser-276, and Ser-279, independently of the direct interaction between TRESK and 14-3-3. In conclusion, two distinct inhibitory kinase pathways converge on TRESK, and their effect on the calcineurin-dependent regulation is differentially modulated by the functional availability of 14-3-3.

CREB‐dependent Nur77 induction following depolarization in PC12 cells and neurons is modulated by MEF2 transcription factors

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

The importance of altered gene promoter methylation and transcription factor binding in developmental programming of central appetitive drive

The importance of altered gene promoter methylation and transcription factor binding in developmental programming of central appetitive drive

Quantitative and cell type-specific transcriptional regulation of A-type cyclin-dependent kinase in Arabidopsis thaliana

A-type cyclin-dependent kinase (CDKA) is an ortholog of yeast Cdc2/Cdc28p, and is assumed to have an essential function in plant growth and organogenesis. Previous studies revealed that its kinase activity is controlled by post-translational modifications, such as binding to cyclins and phosphorylations, but its transcriptional regulation is poorly understood. Here, we generated a promoter dissection series of Arabidopsis ( Arabidopsis thaliana) CDKA;1, and used β-glucuronidase ( GUS) gene-fused reporter constructs for expression analyses in planta. The results revealed two types of transcriptional control in shoots: general quantitative regulation and cell type-specific regulation. We identified a promoter region that promotes CDKA;1 expression in the leaf epidermis, but not in the L1 layer of the shoot apical meristem. This region also directed abaxial side-biased expression, which may be linked to the adaxial/abaxial side specification. Another reporter construct showed that CDKA;1 expression in the inner layers of leaves is controlled by a distinct regulatory region in the promoter. These results suggest that the transcriptional regulation of CDKA;1 may play a key role in proper development of leaves by coordinating cell division and differentiation of different cell types.

T Helper Cell-specific Regulation of Inducible Costimulator Expression via Distinct Mechanisms Mediated by T-bet and GATA-3

Inducible costimulator (ICOS) expression is critical for T cell-mediated immunity. We showed previously that T cell receptor and CD28 co-engagement up-regulate ICOS expression in activated T cells via the induction of NFATc2. Here, we examined the regulation of ICOS expression by Th-specific transcription factors T-bet and GATA-3. Overexpression of T-bet or GATA-3 alone could enhance, and NFATc2 could further synergize with either of them to increase, icos transcription. Although T-bet acted on the icos promoter, GATA-3 operated via an icos 3'-un-translated region element. Interestingly, NFATc2 was found to bind promiscuously the icos promoter in developing Th0, Th1, and Th2 cells but became selectively associated with T-bet at the promoter and with GATA-3 at the 3'-untranslated region in fully differentiated Th1 and Th2 cells, respectively. Collectively, our results reveal a temporally evolving circuit in which the non-selectively expressed NFATc2 cooperates with Th-restricted T-bet or GATA-3 to direct transcription of a costimulatory gene via distinct regulatory elements in different Th cells undergoing differentiation.

OTK18, a zinc-finger protein, regulates human immunodeficiency virus type 1 long terminal repeat through two distinct regulatory regions

It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA-transcription factor ELISA system; the negative-regulatory element (NRE) at -255/-238 and the Ets-binding site (EBS) at -150/-139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.

Conserved regulatory elements establish the dynamic expression of Rpx/HesxI in early vertebrate development

TheRpx/Hesx1 homeobox gene is expressed during gastrulation in the anterior visceral and definitive endoderm and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland. This expression pattern suggests the presence of at least two distinct regulatory regions that control early and late Rpx transcription. Using transgenic mice, we have demonstrated that regulatory sequences in the 5′ upstream region of Rpx are important for early expression in the anterior endoderm and neural plate and regulatory elements in the 3′ region are required for late expression in Rathke's pouch. We have found that the genetically required LIM homeodomain-containing proteins Lim1/Lhx1 and Lhx3 are directly involved in the regulation of Rpx transcription. They bind two LIM protein-binding sites in the 5′ upstream region of Rpx, which are required for Rpx promoter activity in both mice and Xenopus. Furthermore, we have found that a conserved enhancer in the 3′ regulatory sequences of Rpx is not only required, but is also sufficient for the expression of Rpx transgenes in the developing Rathke's pouch.

In Drosophila, don juan and don juan like encode proteins of the spermatid nucleus and the flagellum and both are regulated at the transcriptional level by the TAFII80 cannonball while translational repression is achieved by distinct elements

The genes don juan (dj) and don juan like (djl) encode basic proteins expressed in the male germline. Both proteins show a similar expression pattern being localized in the sperm heads during chromatin condensation and along the flagella. Prematurely expressed Don Juan-eGFP and Myc-Don Juan Like localize to the cytoplasm of spermatocytes and in mitochondrial derivatives from the nebenkern stage onward suggesting that both proteins associate with the mitochondria along the flagella in elongated spermatids. Premature expression of Myc-Don Juan Like does not impair spermatogenesis where-as Don Juan-eGFP when prematurely expressed causes male sterility as spermatids fail to individualize. In spite of the sequence identity of 72% on the nucleotide level and 42% on the protein level, the presumptive promoter regions and the untranslated regions of the mRNA are diverged. Our in vivo analysis revealed that don juan and don juan like are transcriptionally and translationally controlled by distinct short cis regulatory regions. Transcription of don juan and don juan like depends on the male germ line specific TAF(II)80, Cannonball (Can). Translational repression elements for both mRNAs are localized in the 5' UTR and are capable to form distinct secondary structures in close proximity to the translational initiation codon.

The Distal and Proximal Regulatory Regions of the Involucrin Gene Promoter have Distinct Functions and Are Required for In Vivo Involucrin Expression

Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions - the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia.

MtENOD11 Gene Activation During Rhizobial Infection and Mycorrhizal Arbuscule Development Requires a Common AT-Rich-Containing Regulatory Sequence

The MtENOD11 gene from the model legume Medicago truncatula is transcriptionally activated both in response to Sinorhizobium meliloti Nod factors and throughout infection of root tissues by the nitrogen-fixing microsymbiont. To identify the regulatory sequences involved in symbiosis-related MtENOD11 expression, a series of promoter deletions driving the beta-glucuronidase reporter gene were analyzed in transgenic M. truncatula roots. These studies have revealed that distinct regulatory regions are involved in infection-related MtENOD11 expression compared with preinfection (Nod factor-mediated) expression. In particular, the 257-bp promoter sequence immediately upstream from the start codon is sufficient for infection-related expression, but is unable to drive gene transcription in response to the Nod factor elicitor. This truncated promoter is also sufficient to confer MtENOD11 expression during both the arbuscular mycorrhizal (AM) association and the parasitic interaction with root-knot nematodes. Site-directed mutagenesis further showed that a previously identified nodule-specific AT-rich motif is required for high-level MtENOD11 expression during S. meliloti infection as well as during AM colonization. However, mutation of this motif does not affect gene expression associated with nematode-feeding sites. Taken together, these results suggest a close link between regulatory mechanisms controlling transcriptional early nodulin gene activation during both rhizobial and AM root endosymbioses.