Dissection of a Complex Enhancer Element: Maintenance of Keratinocyte Specificity but Loss of Differentiation Specificity

ABSTRACT In this report, we explored the mechanisms underlying keratinocyte-specific and differentiation-specific gene expression in the skin. We have identified five keratinocyte-specific, open chromatin regions that exist within the 6 kb of 5′ upstream regulatory sequence known to faithfully recapitulate the strong endogenous keratin 5 (K5) promoter and/or enhancer activity. One of these, DNase I-hypersensitive site (HSs) 4, was unique in that it acted independently to drive abundant and keratinocyte-specific reporter gene activity in culture and in transgenic mice, despite the fact that it was not essential for K5 enhancer activity. We have identified evolutionarily conserved regulatory elements and a number of their associated proteins that bind to this compact and complex enhancer element. The 125-bp 3′ half of this element (referred to as 4.2) is by far the smallest known strong enhancer element possessing keratinocyte-specific activity in vivo. Interestingly, its activity is restricted to a subset of progeny of K5-expressing cells located within the sebaceous gland. The other half of HSs 4 (termed 4.1) possesses activity to suppress sebocyte-specific expression and induce expression in the channel (inner root sheath) cells surrounding the hair shaft. Our findings lead us to a view of keratinocyte gene expression which is determined by multiple regulatory modules, many of which contain AP-2 and/or Sp1/Sp3 binding sites for enhancing expression in skin epithelium, but which also harbor one or more unique sites for the binding of factors which determine specificity. Through mixing and matching of these modules, additional levels of specificity are obtained, indicating that both transcriptional repressors and activators govern the specificity.

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Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.892 ◽

1995 ◽

Vol 15 (2) ◽

pp. 892-903 ◽

Cited By ~ 44

Author(s):

S Plaza ◽

C Dozier ◽

M C Langlois ◽

S Saule

Keyword(s):

Specific Activity ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Transcriptional Level ◽

Regulatory Sequences ◽

Independent Manner ◽

Specific Expression ◽

Enhancer Activity ◽

Enhancer Element

Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.

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Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices

eLife ◽

10.7554/elife.49921 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Claudia Racioppi ◽

Keira A Wiechecki ◽

Lionel Christiaen

Keyword(s):

Gene Expression ◽

Specific Activity ◽

Expression Profiles ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Specific Gene ◽

Specific Expression ◽

Pharyngeal Muscle ◽

Chromatin Dynamics ◽

Multipotent Progenitors

During embryogenesis, chromatin accessibility profiles control lineage-specific gene expression by modulating transcription, thus impacting multipotent progenitor states and subsequent fate choices. Subsets of cardiac and pharyngeal/head muscles share a common origin in the cardiopharyngeal mesoderm, but the chromatin landscapes that govern multipotent progenitors competence and early fate choices remain largely elusive. Here, we leveraged the simplicity of the chordate model Ciona to profile chromatin accessibility through stereotyped transitions from naive Mesp+ mesoderm to distinct fate-restricted heart and pharyngeal muscle precursors. An FGF-Foxf pathway acts in multipotent progenitors to establish cardiopharyngeal-specific patterns of accessibility, which govern later heart vs. pharyngeal muscle-specific expression profiles, demonstrating extensive spatiotemporal decoupling between early cardiopharyngeal enhancer accessibility and late cell-type-specific activity. We found that multiple cis-regulatory elements, with distinct chromatin accessibility profiles and motif compositions, are required to activate Ebf and Tbx1/10, two key determinants of cardiopharyngeal fate choices. We propose that these ‘combined enhancers’ foster spatially and temporally accurate fate choices, by increasing the repertoire of regulatory inputs that control gene expression, through either accessibility and/or activity.

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The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽

10.1182/blood.v85.2.319.319 ◽

1995 ◽

Vol 85 (2) ◽

pp. 319-329 ◽

Cited By ~ 66

Author(s):

S Dziennis ◽

RA Van Etten ◽

HL Pahl ◽

DL Morris ◽

TL Rothstein ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Transgene Expression ◽

Heterologous Gene Expression ◽

Myeloid Cells ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

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Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5206-5215.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5206-5215 ◽

Cited By ~ 1

Author(s):

Y Kamachi ◽

H Kondoh

Keyword(s):

Mutational Analysis ◽

Specific Activity ◽

Regulatory Elements ◽

Positive Element ◽

Specific Expression ◽

Enhancer Activity ◽

Negative Element ◽

Positive Elements ◽

Lens Cells ◽

Specific Regulation

Lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, and the 30-bp-long DC5 fragment was found to be responsible for eliciting the lens-specific activity. Mutational analysis of the DC5 fragment identified two contiguous, interdependent positive elements and a negative element which overlaps the 3'-located positive element. Previously identified ubiquitous factors delta EF1 bound to the negative element and repressed the enhancer activity in nonlens cells. Mutation and cotransfection analyses indicated the existence of an activator which counteracts the action of delta EF1 in lens cells, probably through binding site competition. We also found a group of nuclear factors, collectively called delta EF2, which bound to the 5'-located positive element. delta EF2a and -b were the major species in lens cells, whereas delta EF2c and -d predominated in nonlens cells. These delta EF2 proteins probably cooperate with factors bound to the 3'-located element in activation in lens cells and repression in nonlens cells. delta EF2 proteins also bound to a promoter sequence of the gamma F-crystallin gene, suggesting that delta EF2 proteins are involved in lens-specific regulation of various crystallin classes.

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Genetic influences on cell type specific gene expression and splicing during neurogenesis elucidate regulatory mechanisms of brain traits

10.1101/2020.10.21.349019 ◽

2020 ◽

Author(s):

Nil Aygün ◽

Angela L. Elwell ◽

Dan Liang ◽

Michael J. Lafferty ◽

Kerry E. Cheek ◽

...

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

Regulatory Mechanisms ◽

Specific Gene ◽

Cell Type ◽

Specific Expression ◽

Risk Variants ◽

Allele Specific ◽

Cell Type Specific ◽

Regulatory Effects

SummaryInterpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing is mainly performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements of cells present during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs and allele specific expression in primary human neural progenitors (n=85) and their sorted neuronal progeny (n=74). Using colocalization and TWAS, we uncover cell-type specific regulatory mechanisms underlying risk for these traits.

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Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer

Genome Medicine ◽

10.1186/s13073-021-00970-3 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Taotao Sheng ◽

Shamaine Wei Ting Ho ◽

Wen Fong Ooi ◽

Chang Xu ◽

Manjie Xing ◽

...

Keyword(s):

Gastric Cancer ◽

Histone Modification ◽

Cell Fate ◽

Copy Number ◽

Regulatory Region ◽

Regulatory Elements ◽

Specific Gene ◽

Functional Assay ◽

Enhancer Activity

Abstract Background Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity—however, most predicted enhancer regions remain to be functionally tested. Methods We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions. Results We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity. Conclusions Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

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Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development

Development ◽

10.1242/dev.124.24.4971 ◽

1997 ◽

Vol 124 (24) ◽

pp. 4971-4982 ◽

Cited By ~ 12

Author(s):

Z. Yin ◽

X.L. Xu ◽

M. Frasch

Keyword(s):

Homeobox Gene ◽

Regulatory Elements ◽

Bhlh Protein ◽

Expression Domain ◽

Enhancer Activity ◽

Enhancer Element ◽

Mesoderm Development ◽

Visceral Mesoderm

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.

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Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2777 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2777-2786 ◽

Cited By ~ 24

Author(s):

V Gailus-Durner ◽

J Xie ◽

C Chintamaneni ◽

A K Vershon

Keyword(s):

Transcriptional Activation ◽

Mutational Analysis ◽

Consensus Sequence ◽

Promoter Sequence ◽

Regulatory Elements ◽

Specific Gene ◽

Specific Expression ◽

Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

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Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3245 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3245-3254 ◽

Cited By ~ 31

Author(s):

V Ngô ◽

D Gourdji ◽

J N Laverrière

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Cell Lines ◽

Anterior Pituitary ◽

Regulatory Elements ◽

Specific Expression ◽

Site Specific ◽

Cpg Dinucleotides ◽

Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

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Imprinted gene expression at the Dlk1-Dio3 cluster is controlled by both maternal and paternal IG-DMRs in a tissue-specific fashion

10.1101/536102 ◽

2019 ◽

Cited By ~ 1

Author(s):

Katherine A. Alexander ◽

María J. García-García

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

Enhancer Activity ◽

Enhancer Element ◽

Tissue Specific ◽

Imprinted Gene ◽

Imprinting Control Region ◽

Regulatory Landscape ◽

Insight Into ◽

Different Tissues

ABSTRACTImprinting at the Dlk1-Dio3 cluster is controlled by the IG-DMR, an imprinting control region differentially methylated between maternal and paternal chromosomes. The maternal IG-DMR is essential for imprinting control, functioning as a cis enhancer element. Meanwhile, DNA methylation at the paternal IG-DMR is thought to prevent enhancer activity. To explore whether suppression of enhancer activity at the methylated IG-DMR requires the transcriptional repressor TRIM28, we analyzed Trim28chatwo embryos and performed epistatic experiments with IG-DMR deletion mutants. We found that while TRIM28 regulates the enhancer properties of the paternal IG-DMR, it also controls imprinting through other mechanisms. Additionally, we found that the paternal IG-DMR, previously deemed dispensable for imprinting, is required in certain tissues, demonstrating that imprinting is regulated in a tissue-specific manner. Using PRO-seq to analyze nascent transcription, we identified 30 novel transcribed regulatory elements, including 23 that are tissue-specific. These results demonstrate that different tissues have a distinctive regulatory landscape at the Dlk1-Dio3 cluster and provide insight into potential mechanisms of tissue-specific imprinting control. Together, our findings challenge the premise that Dlk1-Dio3 imprinting is regulated through a single mechanism and demonstrate that different tissues use distinct strategies to accomplish imprinted gene expression.

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