Tissue-specific Transcriptional Enhancer Research Articles

The nuclear periphery is a scaffold for tissue-specific enhancers.

Nuclear architecture influences gene regulation and cell identity by controlling the three-dimensional organization of genes and their distal regulatory sequences, which may be far apart in linear space. The genome is functionally and spatially segregated in the eukaryotic nucleus with transcriptionally active regions in the nuclear interior separated from repressive regions, including those at the nuclear periphery. Here, we describe the identification of a novel type of nuclear peripheral chromatin domain that is enriched for tissue-specific transcriptional enhancers. Like other chromatin at the nuclear periphery, these regions are marked by H3K9me2. But unlike the nuclear peripheral Lamina-Associated Domains (LADs), these novel, enhancer-rich domains have limited Lamin B interaction. We therefore refer to them as H3K9me2-Only Domains (KODs). In mouse embryonic stem cells, KODs are found in Hi-C-defined A compartments and feature relatively accessible chromatin. KODs are characterized by low gene expression and enhancers located in these domains bear the histone marks of an inactive or poised state. These results indicate that KODs organize a subset of inactive, tissue-specific enhancers at the nuclear periphery. We hypothesize that KODs may play a role in facilitating and perhaps constraining the enhancer-promoter interactions underlying spatiotemporal regulation of gene expression programs in differentiation and development.

Drosophila Distal-less and Rotund Bind a Single Enhancer Ensuring Reliable and Robust bric-a-brac2 Expression in Distinct Limb Morphogenetic Fields

Most identified Drosophila appendage-patterning genes encode DNA-binding proteins, whose cross-regulatory interactions remain to be better characterized at the molecular level, notably by studying their direct binding to tissue-specific transcriptional enhancers. A fine-tuned spatio-temporal expression of bric-a-brac2 (bab2) along concentric rings is essential for proper proximo-distal (P-D) differentiation of legs and antennae. However, within the genetic interaction landscape governing limb development, no transcription factor directly controlling bab2 expression has been identified to date. Using site-targeted GFP reporter assay and BAC recombineering, we show here that restricted bab2 expression in leg and antennal imaginal discs relies on a single 567-bp-long cis-regulatory module (CRM), termed LAE (for leg and antennal enhancer). We show that this CRM (i) is necessary and sufficient to ensure normal bab2 activity in developing leg and antenna, and (ii) is structurally and functionally conserved among Drosophilidae. Through deletion and site-directed mutagenesis approaches, we identified within the LAE essential sequence motifs required in both leg and antennal tissues. Using genetic and biochemical tests, we establish that in the LAE (i) a key TAAT-rich activator motif interacts with the homeodomain P-D protein Distal-less (Dll) and (ii) a single T-rich activator motif binds the C2H2 zinc-finger P-D protein Rotund (Rn), leading to bab2 up-regulation respectively in all or specifically in the proximal-most ring(s), both in leg and antenna. Joint ectopic expression of Dll and Rn is sufficient to cell-autonomously activate endogenous bab2 and LAE-driven reporter expression in wing and haltere cells. Our findings indicate that accuracy, reliability and robustness of developmental gene expression do not necessarily require cis-regulatory information redundancy.

Using zebrafish transgenesis to test human genomic sequences for specific enhancer activity

We detail an approach for the identification of human tissue-specific transcriptional enhancers involving three steps: delineation of search space around a locus or target gene, in silico identification and size definition of putative candidate sequences, and testing through several independent genomic insertions in a transgenic zebrafish reporter assay. Candidate sequences are defined through evolutionary conservation, transcription factor binding and chromatin marks (e.g. ENCODE data) and are amplified from genomic DNA, cloned into basal promoter:fluorescent protein reporter vectors based on the Tol2 transposon system and are microinjected into fertilized zebrafish eggs. After raising injected founders to sexual maturity, fluorescent screening identifies positive founder fish whose offspring undergo a detailed expression analysis to determine tissue specificity and reproducibility of specific enhancers.

Transcription Factors and DNA Regulatory Elements

The interactions between sequence‐specific transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. My group developed highly parallel in vitro microarray technology, termed protein binding microarrays (PBMs), for the characterization of the sequence specificities of DNA‐protein interactions at high resolution. Using PBMs, we have determined the DNA binding specificities of hundreds of TFs from a wide range of species. More recently we have used the PBM technology to investigate TF heterodimers and higher order complexes. The PBM data have permitted us to identify novel TFs and their DNA binding sequence preferences, predict the target genes and condition‐specific regulatory roles of TFs, predict and analyze tissue‐specific transcriptional enhancers, investigate functional divergence of paralogous TFs within a TF family, investigate the molecular determinants of TF‐DNA recognition specificity, and distinguish direct versus indirect TF‐DNA interactions in vivo. Notably, not all DNA binding sites of a TF function equally. Further analyses of TFs and cis regulatory elements are likely to reveal features of cis regulatory sequences that are important in gene regulation.

Large-scale identification of tissue-specific enhancers in vivo

Regulatory sequences that control gene expression in time and space play important roles in evolution, phenotypic variation and human disease, but their systematic identification and functional characterization remains a largely unresolved quest. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non- coding portion of the genome. We and others have previously used extreme evolutionary conservation to identify human candidate enhancer sequences. Large-scale transgenic mouse assays revealed that many extremely constrained non-coding sequences are tissue-specific transcriptional enhancers active during embryonic development. However, comparative genomic methods fail to predict the spatiotemporal activity patterns of individual enhancers. To address this limitation, we used chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively parallel sequencing (ChIP-seq) to identify more than 17,000 in vivo binding sites of p300 in eight different embryonic mouse tissues. Using a transgenic mouse reporter assay, we determined the embryonic in vivo activities of more than 130 of these sequences and show that tissue-specific p300 binding accurately predicts not only the genomic location of tissue-specific enhancers, but also the tissues in which they are active. Our high-confidence predictions of tissue-specific distant-acting enhancers add an important layer of functional annotation to the non-coding portion of the human genome, and we expect them to enable genome-wide studies of the role of enhancers in vertebrate development, human disease and regulatory evolution.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation

A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Interpreting mammalian evolution using Fugu genome comparisons

Recently, it has been shown that a significant number of evolutionarily conserved human– Fugu noncoding elements function as tissue-specific transcriptional enhancers in vivo, suggesting that distant comparisons are capable of identifying a particular class of regulatory elements. We therefore hypothesized that by juxtaposing human/ Fugu and human/mouse conservation patterns we can define conservation criteria for discovering transcriptional regulatory elements specific to mammals. Genome-scale comparisons of noncoding human/ Fugu evolutionary conserved elements (ECRs) and their humans/mouse counterparts revealed a particular signature common to human/mouse ECRs (≥350 bp long, ≥77% identity) that are also conserved in fishes. This newly defined threshold identifies 90% of all human/ Fugu noncoding ECRs without the assistance of human– Fugu genome alignments and provides a very efficient filter for identifying functional human/mouse ECRs.

Identification of an Enhancer Sequence within the First Intron Required for Cartilage-specific Transcription of the α2(XI) Collagen Gene

Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.

The mouse Eb meiotic recombination hotspot contains a tissue-specific transcriptional enhancer.

A meiotic recombination hotspot exists within the second intron of the mouse major histocompatibility complex (MHC) gene, Eb. In the present study, a small fragment from the intron which contains two potential transcriptional regulatory elements was cloned into an expression vector and its effect on transcription was tested. This fragment was found to contain tissue-specific transcriptional enhancer activity. An octamer-like sequence and a B motif may contribute to this enhancer activity. Similar regulatory sequences with the same orientation and distance from one another are found in another mouse MHC recombination hotspot.

DNA position-specific repression of transcription by a Drosophila zinc finger protein.

Expression of the yellow (y) gene of Drosophila melanogaster is controlled by a series of tissue-specific transcriptional enhancers located in the 5' region and intron of the gene. Insertion of the gypsy retrotransposon in the y2 allele at -700 bp from the start of transcription results in a spatially restricted phenotype: Mutant tissues are those in which yellow expression is controlled by enhancers located upstream from the insertion site, but all other structures whose enhancers are downstream of the insertion site are normally pigmented. This observation can be reproduced by inserting just a 430-bp fragment containing the suppressor of Hairy-wing [su(Hw)]-binding region of gypsy into the same position where this element is inserted in y2, suggesting that the su(Hw)-binding region is sufficient to confer the mutant phenotype. Insertion of this sequence into various positions in the y gene gives rise to phenotypes that can be rationalized assuming that the presence of the su(Hw) protein inhibits the action of those tissue-specific enhancers that are located more distally from the su(Hw)-binding region with respect to the promoter. These results are discussed in light of current models that explain long-range effects of enhancers on gene expression.

Hepatocyte nuclear factor 1 and C/EBP are essential for the activity of the human apolipoprotein B gene second-intron enhancer.

The tissue-specific transcriptional enhancer of the human apolipoprotein B gene contains multiple protein-binding sites spanning 718 bp. Most of the enhancer activity is found in a 443-bp fragment (+621 to +1064) that is located entirely within the second intron of the gene. Within this fragment, a 147-bp region (+806 to +952) containing a single 97-bp DNase I footprint exhibits significant enhancer activity. We now report that this footprint contains four distinct protein-binding sites that have the potential to bind nine distinct liver nuclear proteins. One of these proteins was identified as hepatocyte nuclear factor 1 (HNF-1), which binds with relatively low affinity to the 5' half of a 20-bp palindrome located at the 5' end of the large footprint. A binding site for C/EBP (or one of the related proteins that recognize similar sequences) was identified in the center of the 97-bp footprint. This binding site is coincident or overlaps with the binding sites for five other proteins, two of which appear to be distinct from the C/EBP-related family of proteins. The binding site for a nuclear factor designated protein I is located between the HNF-1 and C/EBP binding sites. Finally, the 3'-most 15 bp of the footprinted sequence contain a binding site for another nuclear protein, which we have called protein II. Mutations that abolish the binding of either HNF-1, protein II, or the C/EBP-related proteins severely reduce enhancer activity. However, deletion experiments demonstrated that neither the HNF-1-binding site alone, nor the combination of binding sites for HNF-1, protein I, and C/EBP, nor the C/EBP-binding site plus the protein II-binding site is sufficient to enhance transcription from a strong apolipoprotein B promoter. Rather, HNF-1 and C/EBP act synergistically with protein II to enhance transcription of the apolipoprotein B gene.

Hepatocyte nuclear factor 1 and C/EBP are essential for the activity of the human apolipoprotein B gene second-intron enhancer.

The tissue-specific transcriptional enhancer of the human apolipoprotein B gene contains multiple protein-binding sites spanning 718 bp. Most of the enhancer activity is found in a 443-bp fragment (+621 to +1064) that is located entirely within the second intron of the gene. Within this fragment, a 147-bp region (+806 to +952) containing a single 97-bp DNase I footprint exhibits significant enhancer activity. We now report that this footprint contains four distinct protein-binding sites that have the potential to bind nine distinct liver nuclear proteins. One of these proteins was identified as hepatocyte nuclear factor 1 (HNF-1), which binds with relatively low affinity to the 5' half of a 20-bp palindrome located at the 5' end of the large footprint. A binding site for C/EBP (or one of the related proteins that recognize similar sequences) was identified in the center of the 97-bp footprint. This binding site is coincident or overlaps with the binding sites for five other proteins, two of which appear to be distinct from the C/EBP-related family of proteins. The binding site for a nuclear factor designated protein I is located between the HNF-1 and C/EBP binding sites. Finally, the 3'-most 15 bp of the footprinted sequence contain a binding site for another nuclear protein, which we have called protein II. Mutations that abolish the binding of either HNF-1, protein II, or the C/EBP-related proteins severely reduce enhancer activity. However, deletion experiments demonstrated that neither the HNF-1-binding site alone, nor the combination of binding sites for HNF-1, protein I, and C/EBP, nor the C/EBP-binding site plus the protein II-binding site is sufficient to enhance transcription from a strong apolipoprotein B promoter. Rather, HNF-1 and C/EBP act synergistically with protein II to enhance transcription of the apolipoprotein B gene.

The presence of sequence-specific protein binding sites correlate with replication activity and matrix binding in a 1.7 Kb-long DNA fragment of the chicken ∝-globin gene domain

Several recognition sites for novel sequence-specific DNA-binding proteins were found at the 5′-side of the chicken ∝-globin gene domain in a 1.7 Kbp DNA fragment. This fragment includes the replication origin, a non tissue-specific transcriptional enhancer, a DNAse I hypersensitive site and a permanent site of DNA attachment to the nuclear matrix. Most of the identified protein binding sites differ from previously known consensus sequences. Two sites coincide with MARs located at the 5′-end of the 1.7 kbp fragment. The proteins interacting with these two recognition sites were observed only in proliferating cells and were virtually absent in the extracts obtained from the non-replicating differentiated form of the same cells. One of them seems to belong to the GATA protein family, but its presence in nuclear extracts correlates with cell proliferation rather than expression of the domain.

Characterization of tissue-specific enhancer elements in the second intron of the human apolipoprotein B gene.

We report the identification and characterization of tissue-specific transcriptional enhancer elements that influence the expression of the human apolipoprotein B gene. A 704-base pair PstI fragment comprising sequences from the first and second introns of the human apolipoprotein B gene (positions +360 to +1064) possesses tissue-specific transcriptional enhancer elements when assayed in transient transfection experiments using either the apolipoprotein B or thymidine kinase promoter. The majority of the enhancer activity, which was observed in transcriptionally active HepG2 and CaCo-2 cells, but not in transcriptionally inactive Chinese hamster ovary or HeLa cells, was subsequently localized to a 443-base pair SmaI-PvuII fragment (positions +621 to +1064) within the second intron of the apolipoprotein B gene. Gel retention experiments demonstrated that sequence motifs within this region interact with a number of nuclear proteins from HepG2, CaCo-2, and HeLa cells. The actual sequence elements that bound to nuclear proteins from HepG2 cells were identified by DNase I footprinting. Deletion experiments were performed to distinguish those protein-binding regions involved in the enhancer effect. Our data demonstrate that sequences between positions +806 and +940 are essential for this enhancer activity. This segment contains one large 97-base pair footprint, whose sequence has been conserved between the human and mouse genes. Binding sites for the liver-specific transcription factors HNF-1 and HNF-3 are present within this footprint.

Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene.

Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells. We have studied several overlapping genomic clones containing the 5'-flanking region of the gene. These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence. The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined. DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene. For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells. Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed. Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells. However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells. This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters. Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.

Tissue-specific transcriptional enhancers may act in trans on the gene located in the homologous chromosome: the molecular basis of transvection in Drosophila.

The y2 mutation resulted from the insertion of the gypsy element into the X-linked yellow locus of Drosophila melanogaster. As a consequence of this insertion, transcriptional enhancers that control the expression of the yellow gene in the wings and body cuticle of adult flies are unable to act on the yellow promoter, resulting in a tissue-specific phenotype characterized by mutant coloration in these structures. Some yellow null alleles (yn) are able to complement the y2 phenotype giving rise to near wild type y2/yn females. The molecular structure of the yellow locus in complementing and noncomplementing mutations was determined by cloning and sequencing the various alleles examined. From the information obtained in these studies, we propose a model suggesting that the complementing wild type phenotype of y2/yn flies might be due to the ability of functional wing and body cuticle transcriptional enhancers located in the yn locus to act in trans on the promoter of the yellow gene found in the y2-containing chromosome. Furthermore, this transactivation is abolished by the presence of an intact promoter in cis, suggesting that promoter competition between the yellow genes located on each homolog precludes the activation in trans by transcriptional enhancers in favour of cis effects on their own promoter.

Binding of a liver-specific factor to the human albumin gene promoter and enhancer.

A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-B1-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms.

Binding of a Liver-Specific Factor to the Human Albumin Gene Promoter and Enhancer

A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-B1-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms.

The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers.

We have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta-globin gene and the murine H-2Kb gene. S1 nuclease and transcriptional run-off analysis showed that the human beta-globin gene contains at least three globin specific transcriptional control elements. One is a promoter element and is located 160 bp upstream from the transcription initiation site; it increases the efficiency of the promoter after MEL cells are induced to differentiate. The other two elements are tissue-specific transcriptional enhancers and are located downstream from the transcription initiation site, the first in the structural beta-globin gene and the second in the 3' flanking sequences.