Analysis and function of transcriptional regulatory elements: insights from Drosophila.

Abstract

Analysis of gene expression is assuming an increasingly important role in elucidating the molecular basis of insect biology. Transcriptional regulation of gene expression is directed by a variety of cis-acting DNA elements that control spatial and temporal patterns of expression. This review summarizes current knowledge about properties of transcriptional regulatory elements, based largely on research in Drosophila melanogaster, and outlines ways that new technologies are providing tools to facilitate the study of transcriptional regulatory elements in other insects.

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Annu. Rev. Entomol. 2003. 48:579–602
doi: 10.1146/annurev.ento.48.091801.112749
Copyright c ? 2003 by Annual Reviews. All rights reserved
First published online as a Review in Advance on September 25, 2002
ANALYSIS AND FUNCTION OF TRANSCRIPTIONAL
REGULATORY ELEMENTS: Insights from Drosophila
David N. Arnosti
Department of Biochemistry and Molecular Biology and Program in Genetics, Michigan
State University, East Lansing, Michigan 48824-1319; e-mail: arnosti@msu.edu
Key Words
transcription, promoter, enhancer, transcriptional regulation
I Abstract
role in elucidating the molecular basis of insect biology. Transcriptional regulation
of gene expression is directed by a variety of cis-acting DNA elements that con-
trol spatial and temporal patterns of expression. This review summarizes current
knowledge about properties of transcriptional regulatory elements, based largely on
research in Drosophila melanogaster, and outlines ways that new technologies are
providing tools to facilitate the study of transcriptional regulatory elements in other
insects.
Analysis of gene expression is assuming an increasingly important
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
FEATURES OF TRANSCRIPTIONAL REGULATORY REGIONS . . . . . . . . . . . . . 581
BASAL PROMOTER ELEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
BOUNDARY ELEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
ENHANCERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
Definition of the Term Enhancer and Functional Analysis . . . . . . . . . . . . . . . . . . . . 585
Models of Enhancer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
Pattern Generation by Enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Shared or Exclusive Enhancer-Promoter Interactions . . . . . . . . . . . . . . . . . . . . . . . . 588
Enhancer Redundancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
SOME SPECIAL PROMOTERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
Modular Enhancers and Short-Range Repression . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
Compact Testis-Specific Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
Promoters in Heterochromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
runt: A Disperse Regulatory Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
EVOLUTION OF CIS-REGULATORY ELEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . 592
Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
NEW TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
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Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Transgenesis in Other Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
INTRODUCTION
Transcriptional regulation is woven into the entire fabric of biology; thus, knowl-
edge of gene expression is a critical component of understanding many issues
relevant to entomology, including pesticide and disease resistance, behavior, evo-
lution,anddevelopment.Theseandothertopicshavebeenstudiedwiththetoolsof
molecularbiology,allowingtheidentificationofrelevantstructuralgenes;however,
the control of these genes is often poorly understood. Transcriptional regulation
is effected by cis-acting DNA sequences that direct the assembly of the protein
machinery responsible for transcription. Alterations in these cis-acting regulatory
sequences are thought to play driving roles in morphological diversification and
evolution of developmental mechanisms (106). In addition, quantitative trait loci
that contribute to adaptations often correspond to genetic alterations in putative
cis-acting regulatory elements rather than protein-coding regions of genes (68).
Often the entomologist will be presented with sequences of genomic DNA that
may contain transcriptional regulatory sequences, but functional understanding
of these DNA elements can be hampered by the lack of genetic and molecular
genetic tools. Functional analysis of insect transcriptional regulatory regions is
based largely on work in Drosophila melanogaster. Fortunately, general proper-
tiesofeukaryotictranscriptionalregulationarehighlyconserved,especiallyamong
metazoans; thus, lessons learned from Drosophila can often be directly applied
to other insects. The extensive analysis of Drosophila transcriptional “wiring dia-
grams” should provide the basis for accelerated analysis of cis-acting elements in
other organisms.
This review focuses on the cis-acting DNA sequences, rather than the trans-
acting transcriptional machinery, to assist in answering questions about subjects
such as the size of a regulatory region, the possibility of coordinate regulation of
closely spaced genes, the nature and importance of basal promoter sequences, and
how to predict the transcriptional output of a given promoter. We are still some
way from answering all these questions without the use of empirical tests, but
consideration of general properties of transcriptional control regions identified in
Drosophila will allow investigators to identify features that might be important
for their system. In addition, new bioinformatic approaches promise to directly
“read” transcriptional regulatory information from the genome, at least partly
circumventing the need to experimentally determine the function of cis-acting
sequences. This possibility is especially important to those working in genetically
less tractable systems.
For more information on the transcriptional machinery, the reader is directed
to recent reviews on RNA polymerase II transcription (58,80,84,116), chro-
matin and chromatin remodeling machinery (90,115), transcriptional activators

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andrepressors(26,58),boundaryelements(4,36),more-specializedelementssuch
as molecular memory modules (4,69), and heterochromatin (42).
FEATURES OF TRANSCRIPTIONAL
REGULATORY REGIONS
Threegeneraltypesofcis-actingelementscontroltheactivityofRNApolymerase
II–transcribed(i.e.,proteincoding)genes(Figure1):(a)basalpromotersequences
near the transcriptional initiation site. These elements provide a binding site for
RNA polymerase II and the basal transcriptional machinery that acts on most pro-
moters. (b) Enhancer elements that contain binding sites for sequence-specific
transcription activators and repressors, which regulate levels of gene activity.
(c) Boundary elements or insulators, which can functionally separate regulatory
elements.Ratherthanactinginseparateroles,thethreetypesofelementsarefunc-
tionally interrelated, and these complexities contribute to the specificity of gene
regulation. The term “promoter” is often used to indicate all regulatory sequences
associated with a gene, including enhancer sequences and the basal promoter.
More specialized elements, such as those that interact with Polycomb and Tritho-
rax regulatory proteins, are not thought to be associated with most genes and are
discussed in recent reviews (31,69).
Transcriptional regulatory information can be located entirely within 100 bp
of the transcriptional initiation site, as with testis-specific promoters, or in some
Figure 1
∼100 bp of sequence flanking the initiation site, onto which general transcriptional
machinery loads. Enhancer sequences (ranging in size from 50 bp to several hundred
basepairs) are located at variable distances from +1; these bind to sequence-specific
transcriptional regulators that control output levels of basal promoter. Boundary ele-
ments, found near some genes, insulate the gene from local chromatin effects and can
screen the gene from the influence of distal enhancers. The inset shows the disposition
of three sequence elements of basal promoters (see text).
Cis-acting transcriptional control elements. Basal promoter includes

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cases distributed over a large region, as in the bithorax complex, which contains
three HOX genes in a 300-kbp region replete with regulatory elements (34,60).
In general, larger regulatory regions are required for diverse tissue-specific and
temporal patterns of expression because in most cases individual enhancers are
requiredforseparateportionsofanexpressionpattern.Genesdedicatedtoasingle
function, such as tissue-specific structural genes, often contain all information for
tissue and temporal specificity in a small regulatory element of a few hundred
basepairs.
When no other information is available, the region within a few kilobasepairs
5?of the initiation site is often the first place examined for regulatory information.
For many genes, such as those encoding heat shock proteins, glue proteins, some
immune-response proteins, cytochrome P450 enzymes, and chorion proteins, it
appears that all necessary regulatory information is present within 1 kbp of the
basal promoter (30,57,59,78,107). For a number of important regulatory genes,
however, essential transcriptional information is found tens of kilobasepairs from
the initiation site (33,60,75).
How representative are cis-regulatory elements in Drosophila for other insects
and for metazoans in general? Given the high degree of conservation of the tran-
scriptional machinery in all eukaryotes (2,58), the ability of regulatory elements
to function in heterologous systems (53,77,86), and the common occurrence of
long-distance regulatory elements in metazoans, it is clear that in most aspects
Drosophila is an appropriate model system. One area in which this system was
not believed to be representative was in DNA methylation. Until recently, D.
melanogaster was generally thought to lack 5-methylcytosine, an important modi-
fied nucleotide in mammalian genomes that functions in transcriptional regulatory
phenomena such as imprinting and certain types of repression. DNA methylation
in other insects such as aphid, cricket, and mosquito have been previously re-
ported, leading to the question of whether Drosophila is atypical in this respect
(37,109). Recent genomic analyses showing the presence of genes homologous
to DNA methyltransferase and methyl CpG binding proteins in D. melanogaster
have prompted a reexamination of this issue (47,109). Using new, more sensitive
techniques, low levels of 5-methylcytosine have been identified, primarily during
early stages of embryogenesis (37,67). The possible functional role of cytosine
methylation in Drosophila remains unknown, as in other insects; thus, we do not
know whether some types of transcriptional regulation via DNA methylation will
be accurately modeled in the fly.
BASAL PROMOTER ELEMENTS
A basal promoter can be defined as the ∼100 bp of sequence surrounding the
transcriptional initiation site that comes into close contact with the general tran-
scriptionalmachinery.ThisregioncontainsTATAsequences(consensusTATAAA)
centered at −30, Initiator (Inr) sequences at +1 (consensus TCAGT), and down-
stream promoter elements (DPE) at +30 (consensus A/GGA/TC/TGT) (Figure 1).

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Not all basal promoters contain all three elements. In a survey of 205 promoters
with accurately mapped start sites, approximately half had a recognizable TATA
sequence,almosthalfhadaDPE,andaboutonethirdhadneither(56).Initiator-like
sequences were found in approximately one quarter of arthropod promoters sur-
veyed (24). Identification of the transcriptional initiation site requires mapping the
5?endofatranscribedRNAbyprimerextension,S1nucleaseprotectionassays,or
cloning of cDNAs, especially those isolated based on the mRNA 5?cap structure
(104). Computer identification of basal promoters in genomic sequences has met
with limited success, but a current effort to map ∼2000 Drosophila transcription
startsitesfromoligo-cappedcDNAlibrariesshouldprovideamuchlargerbasisfor
development of bioinformatic methods [(81); U. Ohler, personal communication].
Theelementsofthebasalpromoterprovidenucleationsitesforbindingbybasal
transcriptional machinery. The TATA sequence interacts with the TATA binding
protein(TBP),acrucialpartofthebasaltranscriptionmachinerythathelpsanchor
the RNA polymerase and basal transcription factors at the promoter. TBP is a
subunit of the multicomponent TFIID general transcription factor, which contains
about 10 TBP-associated proteins (TAFs) (2). The initiator region contacts TAFs
and the RNA polymerase itself (23,87). The DPE was identified by its contacts
with TAF proteins and has been suggested to function as an alternative docking
site for the TFIID factor (19).
The functional role of these basal promoter elements appears to be strongly
context dependent. In vitro studies in mammalian systems indicated that TATA
and Inr elements can be functionally redundant (121), but in vivo analysis of the
hsp70 promoter indicated that TATA function was indispensable and was not re-
dundant with the Inr (117). On this promoter, Inr and DPE have important but
largely redundant activities, so mutation of either element individually has little
effect (117). In contrast to the hsp70 promoter, loss of the conserved Initiator el-
ement of the TATA-less testis-specific β tubulin promoter has a stronger effect,
decreasing transcription by one half (92). On the TATA-less vermillion promoter,
aTATAelementcansubstituteforaDPE(32),suggestingequivalenceoffunction,
but a number of embryonic enhancers tested can discriminate between otherwise
identical basal promoters containing either a TATA element or a DPE (20,83).
An additional indication of functional distinctions between basal promoters is that
TATA-containingpromotersarerepressedbytheNC2(Dr1/Drap1)basaltranscrip-
tion factor, whereas DPE-containing promoters are activated by the same factor in
in vitro assays (113).
Basal promoter regions are also sometimes bound by sequence-specific tran-
scription factors that play important roles in regulation of individual genes.
Binding of the Zn finger Ovo protein to the initiator region of the otu gene is
important for ovary-specific expression (64). The TATA-less dpp core promoter
extending from −22 to +6 is bound by a sequence-specific factor in the embryo,
and this core promoter is by itself sufficient to direct a qualitatively correct pattern
of expression (96). GAGA factor binding sites located within the even-skipped
basal promoter confer enhancer-blocking activities upon this element (82). The