Biology

Psychology 110

First and Last Name: _________________________

Instructions: For this assignment, you will download and read a scientific article, and then answer the questions below relating to the theory, methodology, variables and operational definitions in the article. Focus your responses on the information from experiments 1a and 1b only.

The scientific article is in the lab assignments module under course content:

Harris, J. L., Bargh, J. A., & Browneel., K.D. (2009). Priming effects of television food advertising on eating behaviour. Health Psychology, 28(4), 404-413.

Questions:

1. What was the purpose of the study? What was the hypothesis?

2. What was the independent variable?

3. What was the dependent variable? How did the researchers operationally define this variable?

4. Very briefly, what was the procedure?

6. In relation to the hypothesis, what were the results? Was the hypothesis supported?

7. What conclusions did the researchers make?

9/19/2020 Insulators are fundamental components of the eukaryotic genomes | Heredity

https://www.nature.com/articles/6800669 1/22

Published: 06 April 2005

Insulators are fundamental components of the eukaryotic genomes

E Brasset & C Vaury

Heredity  94, 571–576(2005)

629 Accesses 47 Citations 3 Altmetric Metrics

Abstract

The properties of cis-regulatory elements able to influence gene transcription over large

distances have led to the hypothesis that elements called insulators should exist to limit

the action of enhancers and silencers. During the last decades, insulators have been

identified in many eukaryotes from yeast to human. Insulators possess two main

properties: (i) they can block enhancer–promoter communication (‘enhancer blocker

activityʼ), and (ii) they can prevent the spread of repressive chromatin (‘barrier activityʼ).

This review focuses on recent studies designed to elucidate the molecular mechanisms

of the insulator function, and gives an overview of the critical role of insulators in

nuclear organization and functional identity of chromatin.

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Introduction

Precise control over the expression of a gene is exerted through interactions between

the basic transcriptional machinery at the gene promoter and specific protein

complexes at enhancer or silencer elements. Enhancers and silencers exert long-

distance effects independently of their position and orientation. Nevertheless,

neighbouring genes potentially influenced by the presence of the same enhancer within

a defined chromosomal locus may display independent transcription profiles. A

fundamental question is then how to explain the limited range of the enhancer action.

The formation of independent domains of gene function may depend upon a class of

regulatory elements able to block the inappropriate action of enhancers or silencers.

Such regulatory elements are called insulators (Kuhn and Geyer, 2003). Insulators are

defined by two functional properties illustrated in Figure 1. First, an insulator is able to

block interaction between an enhancer and a promoter when positioned in-between

(Conte et al, 2002; Geyer and Corces, 1992; Kellum and Schedl, 1992). Second, an

insulator (also called barrier) prevents the advance of nearby condensed chromatin and

protects gene expression from positive or negative chromatin effects (Kellum and

Schedl, 1991; Roseman et al, 1993; Saitoh et al, 2000). In this review, we discuss recent

advances in our knowledge of the complexity of the mechanism underlying the insulator

function and its role in gene regulation.

Figure 1

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Insulators possess two main properties: (a) they can block enhancer–promoter

communication (enhancer blocker activity), and (b) they can prevent the spread of

repressive chromatin (barrier activity).

What are the mechanisms of action of insulators?

Insulators are regulatory elements that can shelter genes from inappropriate regulatory

interactions. Transgenic assays have helped to dissect the exact sequences required for

insulation and have shown that short sequences – if multimerized – can reconstitute the

insulator effect (Scott et al, 1999). They have also helped to define the general

properties of insulators such as their enhancer-blocker and/or barrier functions.

However, we are at present unable to understand the molecular mechanisms underlying

these functions or to integrate into a general scheme additional observations such as:

(i) the enhancer-blocker and barrier activities are separable (Recillas-Targa et al, 2002);

(ii) insulator effectiveness is influenced by its structure, and by the nature of the

enhancer, promoter and genomic context (Scott et al, 1999; Walters et al, 1999); and

(iii) insulators are not permanent and impassable elements (Cai and Shen, 2001;

Muravyova et al, 2001). Two nonexclusive models are currently proposed: one of them

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is established according to a series of data reporting links between insulators and the

higher-order chromatin structures, and the other integrate data reporting connections

between the insulator properties and gene transcription.

Insulators and higher-order chromatin structures

A structural model proposes that the properties of the insulators result from their

relationship with the organization of higher-order chromatin structures (Labrador and

Corces, 2002). Experiments performed on a Drosophila insulator identified in a

retroelement called gypsy help to illustrate this model. This insulator was identified just

3′ of the 5′ long terminal repeat of gypsy. This is a 340-bp fragment, which contains a

cluster of 12 degenerate binding sites for a zinc-finger DNA protein, Su(Hw). This

insulator is able to block the interaction between enhancers and promoters, and to

protect a gene from nearby chromatin effects (van der Vlag et al, 2000). Both

properties depend on Su(Hw), which recruits the Mod(mdg4) protein. The gypsy

insulator is not specific to a single enhancer, but has been shown to act as enhancer-

blocker to more than 20 enhancers. Even so, this insulator does not establish an

impassable barrier. In certain conditions, the insulator is bypassed, the enhancer-

blocking effect is neutralized and enhancer–promoter communication is restored. Such

a bypass is observed when two gypsy insulators are placed between an enhancer and a

promoter. This loss of insulator activity has been proposed to result from

intrachromosomal pairing between the two gypsy insulators, causing chromatin to fold

and allowing the distal enhancer to contact the promoter. By extension, a single

intervening gypsy insulator would block enhancer–promoter communication by

interacting either with other insulators located at distant loci or at specific nuclear sites

(Cai and Shen, 2001; Muravyova et al, 2001). Evidence that the gypsy insulator

establishes chromatin domains is strengthened by the fact that Su(Hw) and Mod(mdg)4

associate with 500 sites in the Drosophila genome, but coalesce into only 25 large

structures. These structures, named insulator bodies, are proposed to establish

separate loop domains within the genome. The gypsy insulator sequences could then

be genomic sites where such interactions are favoured, and thus be responsible for the

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generation of such loops. According to this model, Gerasimova et al (2000) have shown

that the nuclear positioning of a sequence can be altered. If tethered to the gypsy

insulator, this sequence is targeted to the nuclear periphery where the insulator bodies

are mostly detected.

Furthermore, recent experiments have shown that pairing between two heterologous

insulators such as the binding sites for the GAGA factor and the gypsy insulator may

also occur in the genome and be a possible means to bypass the insulator activity

(Melnikova et al, 2004).

Almost all vertebrate insulators described require binding of the regulatory protein

CTCF for their activity. Some recent results show that CTCF is copurified with a

nucleolar protein present at the nucleolar periphery, suggesting that it helps to displace

insulators to the periphery of the nucleole. These interactions may generate similar

loops described for the gypsy insulator element in Drosophila (Yusufzai et al, 2004).

Taken together with the fact that CTCF is also associated with the nuclear matrix, these

results suggest a functional connection between insulators, the nuclear matrix and

nuclear organization.

A connection between insulator activities and their interaction with some nuclear

structures is further supported by data obtained through a genetic screen performed in

yeast and specifically addressed to isolate genes involved in a possible link between

nuclear order and chromatin boundaries. Various proteins involved in nuclear-

cytoplasmic traffic, such as the exportins Cse1p or Mex67p, have been identified in this

screen and appear to block the propagation of heterochromatin by direct or indirect

tethering of the insulator element to the nuclear pore (Ishii et al, 2002) (Figure 2).

Figure 2

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Boundaries interact with nuclear pore proteins by the nuclear pore complex (from

Ishii et al, 2002). Triangles S represent silencer elements, the white circles B

represent the boundary elements and the grey squares presumed unidentified

proteins. The boundary elements interact with nuclear pore proteins via the nuclear

pore complex (NPC). The authors propose that this nuclear organization allows the

gene located between both boundaries to be isolated from the silencing effect. Its

transcription is ON. The gene located outside the loop is not protected from the

silencer effect, and its transcription is OFF.

Faswb, a notch mutation in Drosophila, disrupts a boundary element, which results in an

alteration of the structural organization of the chromosome visualized by the elimination

of a band observed in the giant larval polytene chromosomes (Vazquez and Schedl,

2000).

Although all these examples implicate 3D loops in the insulator function, some results

do not fit well with a structural model as a unique model for insulation. As an example,

the first insulators identified, the Drosophila specialized chromatin structures, scs and

scs′ (Kellum and Schedl, 1991, 1992), are boundaries surrounding the 87A7 locus where

two hsp70 genes reside. As proposed above for the gypsy elements, interaction

between scs and scs′ could explain their insulator function; however, this interaction

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fails to explain why interaction between scs and scs′ is not a general property of these

elements but depends on sequences located outside the specific domain bearing the

insulator function (Kuhn et al, 2004). Additionally, Majumder and Cai have tested the

effect of pairing on enhancer-blocking activity of 11 homologous and heterologous

insulator combinations. The results have shown that, unlike the homologous pairing of

gypsy insulator or heterologous pairing of gypsy and binding sites for the GAGA factor

(Melnikova et al, 2004), heterologous combinations of gypsy and other insulators, as

well as homologous pairing with other boundary elements such as scs or SF1, do not

always reduce their enhancer-blocking activity (Majumder and Cai, 2003). Further,

some paired insulators exhibit a higher level of enhancer-blocking activity than either

single insulator alone, suggesting that they can function independently or additively

(Majumder and Cai, 2003).

Overall, the structural model proposes that insulators separate the chromatin fibre into

loops attached to a fixed perinuclear substrate, perhaps the nuclear lamin, which serves

as a scaffold to maintain the nuclear organization. However, if such 3D loops provoke

special localizations inside the nucleus, they can also be a means to prevent cis-

diffusion of some molecules necessary for the transcription machinery. Formation of

loops could then act as the primary step of the transcriptional model.

Insulators and gene transcription

The transcriptional model advances that insulators have direct consequences on

transcription (Geyer, 1997; Bell and Felsenfeld, 1999; Dorsett, 1999). Thus, this

transcriptional model depends on the prevailing models of enhancer function and may

be summarized in two different mechanisms. If it is assumed that a signal is propagated

along the chromatin fibre from the enhancer to the promoter, then insulators assembled

in nucleoprotein complexes might block the propagation of the enhancer signal along

the DNA. In this case, they act as physical barriers able to stop the activation of a gene

by its enhancer. Experiments performed on the transcription factor GAGA from

Drosophila melanogaster illustrate this model. GAGA can stimulate transcription by

linking an enhancer to its cognate promoter. It facilitates long-range activation by

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providing a protein bridge that mediates enhancer–promoter communication. Insulators

could interfere with this property of GAGA, and restrict the recruitment of this factor to

the promoter (Mahmoudi et al, 2002).

If it is assumed that the enhancer advances as an obligatory propagated signal toward

the promoter, then an insulator could compete with the promoter for the enhancer, and

trap it into a nonproductive liaison (Geyer, 1997). Supporting this model is the fact that

a promoter has been detected within the scs and scs′ elements (Glover et al, 1995;

Avramova and Tikhonov, 1999), suggesting that these elements may not only be neutral

structural elements as proposed by the structural model, but rather their promoter may

titrate the enhancer function and keep it from activating transcription. A limit to the

transcriptional model is that it fails to explain why boundary elements have to be

between the enhancer and the promoter to function as enhancer blockers. In any case,

it fails to explain how an enhancer blocked on one side by an insulator can activate a

promoter on the other side. Thus, an alternative model involving proteins named

facilitators that bring the enhancer and the promoter close to each other can be

considered. Among these facilitators, the Drosophila Chip protein has been found to

interact with Su(Hw) (Morcillo et al, 1997). Genetic evidence has shown that Su(Hw)

becomes a more effective insulator when enhancer–promoter communication is

weakened by mutations in Chip. It is proposed that formation of Chip–Su(Hw)

complexes breaks the chain of interaction between Chip and homeodomain proteins,

interfering with the process that brings the enhancer towards the promoter.

Recent analyses have shown that barrier elements might play a role in preserving the

separation between a silenced and an active chromatin state. Repressive chromatin has

been characterized by several molecular marks such as enrichment in methylation of

histone H3 lysine 9, hypoacetylation of histones H3 and H4 as well as the binding of

heterochromatin protein 1. On the other hand, transcriptionally active chromatin is

associated with hyperacetylation of H3 lysine 9 and 14. Several observations suggest

that barriers break the code of histone modifications necessary for the propagation of

silencing along the chromatin fibre. For example, methylated nucleosomes around the

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HS4 insulator of the chicken β-globin locus have been proposed to recruit Suv39H1 and

allow methylation of the adjacent nucleosomes. The 5′HS4 insulator of the β-globin

locus would acetylate the adjacent upstream nucleosomes, which prevents methylation

and thus terminates the propagation of the condensation signal (Burgess-Beusse et al,

2002). This modification state of nucleosomes within an insulated transgene suggests

that another model may account for the position effect protection of insulators.

Insulators might directly facilitate nucleosome acetylation. The resulting open chromatin

structure would bind factors protecting the gene against DNA methylation (Recillas-

Targa et al, 2002).

In conclusion, separate data obviously support one and/or the other of the structural

and transcriptional models. It is then possible that insulators may utilize several of these

mechanisms, although this remains to be demonstrated.

Role of insulators in nuclear function

From all the data reported so far, several roles can be attributed to insulators within the

cell.

Partition of distinct chromosomal regions

In addition, to play a structural role in the organization of DNA within the nucleus,

chromatin is also intimately involved in the regulation of eukaryotic gene expression

(Felsenfeld et al, 1996). Barriers are fundamental actors, keeping adjacent domains of

active and inactive chromatin distinct and preventing these regions from inappropriate

interactions (Figure 3).

Figure 3

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Schematic model of the insulator function in the nuclear organization of chromatin.

Proteins (spheres) associated to insulators coalesce within the nucleus. These

structures named insulator bodies establish separate loop domains. Located within

such a loop, the enhancer E1 can activate transcription of a promoter located within

the same loop. However, it is unable to activate a promoter located outside in

another domain.

In the yeast Saccharomyces cerevisiae, a barrier is described at the junction between a

heterochromatic region with hypoacetylated lysines of all core histones and an active

euchromatic region with numerous acetylated histones (Kimura et al, 2002; Suka et al,

2002).

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