A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.     

Cloning and characterization of cDNAs encoding putative CTCFs in the mosquitoes, <Emphasis Type="Italic">Aedes aegypti</Emphasis> and <Emphasis Type="Italic">Anopheles gambiae</Emphasis>

Background  

One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes.     

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.     

Several copies of insulator from MDG4 can determine the interaction between positively and negatively acting regulatory elements and promoter of the miniwhite gene of Drosophila melanogaster

Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated promoter. The best studied insulator of Drosophila melanogaster was found in the 5′ regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression, induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines, two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter and functionally opposite elements, responsible for either gene activation, or repression. Original Russian Text ? M.V. Kostyuchenko, E.E. Savitskaya, M.N. Krivega, P.G. Georgiev, 2008, published in Genetika, 2008, Vol. 44, No. 12, pp. 1693–1697.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

Ibf1 and Ibf2 are novel CP190‐interacting proteins required for insulator function

Insulators are DNA‐protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190‐binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190‐interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190‐binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA‐binding proteins that form hetero‐oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer‐blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.     

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3′ side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ^u1 mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.     

Gene expression reveals evidence for EGFR‐dependent proximal‐distal limb patterning in a myriapod

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR‐signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR‐signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that—in at least mandibulate arthropods—EGFR‐signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.     

CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species

Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.     

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3′UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari–Wari, Wari–gypsy or 1A2–Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.