Myocyte enhancer factor 2
Since nautilus is expressed later than Mef2, it does not appear to take a role in the activation of Mef2. Likewise, the initiation of nau expression does not require Mef2 (Bour, 1995). Mutants exhibit an absence of alpha PS2 integrin gene expression. It appears that Mef2 regulates inflated, the alpha PS2 integrin gene (see myospheroid) through a visceral muscle enhancer of that gene (Rangarayakulu, 1995). Despite the appearance of an apparently normal dorsal vessel in Mef2 mutants, including the expression of Beta 3 tubulin, individual myosin subunit genes (myosin heavy chain, myosin alkaline light chain, and myosin light chain-2) are not expressed in the mutant dorsal vessel (Ranganayakulu, 1995).
Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains two different muscle enhancers within the first intron of the gene. Both enhancers contain a Mef2 binding site, and mutation in either site reduces expression in somatic body wall muscles. For one enhancer, a high level of gene expression in somatic muscles requires the cooperative activity of Mef2 and a cis-acting muscle activator region located within the enhancer. There is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that Mef2 is expressed in these muscles, indicating that TmI is regulated differently in these muscles (Lin, 1996).
Mef2 is a positive regulator of Drosophila Tromomyosin I in the body-wall muscles of embryos, larvae, and adults. Ectopic expression of Mef2 in the epidermis and ventral midline cell in embryos activates the expression of TmI and other muscle genes in these tissues; this activation is stage-dependent, suggesting a requirement for additional factors. Among the genes activated by Mef2 are ß3-Tubulin, a cytoskeletal protein found in myoblasts, muscle cells and some nonmuscle cells and Muscle LIM protein at 60A, a Drosophila homolog of the vertebrate muscle LIM protein, which is a positive regulator of myogenic differentiation. Mesodermal nautilus expression is diminished, but there is no indication of ectopic Mef2 activating nautilus in the ectoderm or ventral midline. Ectopic expression of Mlp60A is observed only in the ventral midline cells but not in the epidermis of embryos that ectopically express Mef2 in the epidermis and ventral midline. Furthermore, ectopic expression of Mef2 in the epidermis results in a decrease in the expression of signaling molecules in the epidermis and a failure of the embryo to properly form body-wall muscles. Muscle VA1 of the ventral group body-wall muscles is missing and muscle VA2 is much shorter than that found in wild type embryos. Muscles VO 4 to 6 are also much shorter than those found in wild-type embryos and often fuse to one another. LT1 to LT4 of the lateral group body-wall muscles are disorganized. These results indicate that Mef2 can function out of context in the epidermis to induce the expression of muscle genes and interfere with a requirement for the epidermis in muscle development. The level of Mef2 in the mesoderm and/or muscles in embryos is critical to body-wall muscle formation; however, no effect is observed on the development of the visceral muscle or dorsal vessel (M.-H. Lin, 1997)
A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and
maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich
protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined.
Two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, have been characterized that show restricted expression in
differentiating muscle lineages. To extend this analysis of Drosophila Mlps, the expression of Mlps have been characterized in mutant backgrounds that disrupt specific
aspects of muscle development. A genetic requirement is found for the transcription factor dMEF2 in regulating Mlp expression, and dMEF2 can bind, in
vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within
the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation,
which are dedicated primarily to assembly of the contractile apparatus, the subcellular distribution of Mlp84B has been analyzed in detail. Immunofluorescent studies
reveal the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of
integrins and alpha-actinin, key components of these structures, also fail to perturb Mlp84B distribution. In conclusion, molecular epistasis
analysis has been used to position Mlp function downstream of events involving mesoderm specification and patterning, concomitant with terminal muscle differentiation.
Furthermore, these results are consistent with a structural role for Mlps as components of muscle cytoarchitecture (Stronach, 1999).
The Mlp60A gene
exhibits three exons interrupted by two small introns, one in the 5' untranslated region and another in the coding region. The Mlp84B gene contains one noncoding
and one coding exon separated by a single large intron. Analysis of noncoding DNA within and surrounding the Mlp genes reveals the presence of multiple A/T-rich
sequences matching exactly the reported MEF2 target binding consensus sequence. The Mlp60A gene contains three
potential dMEF2 binding sites; two of these sites are located in the region 5' to the start of gene, whereas the third is found 3' to the coding sequence. Six putative
dMEF2 binding sites are found in the Mlp84B gene. Four of the six are clustered in the intron, another is located 3' to the coding region of the gene, and another is
contained completely within the first exon (Stronach, 1999).
Knowledge of the subcellular distribution of a protein often contributes substantially to an understanding of its function. In embryonic somatic muscles, Mlp60A and
Mlp84B are found in both the nuclear and cytoplasmic compartments, consistent with either a regulatory or structural role in differentiating muscle. When expressed in rat embryo fibroblast cells, Drosophila Mlps showed a specific association with the actin cytoskeleton. To determine the precise localization of Mlps within mature myofibrils at higher resolution, whole, third instar larval midguts were double labelled using
antibodies directed against the Mlps and alpha-actinin, which marks Z-bands.
Surrounding the midgut, elongated visceral mesodermal cells form a lattice of transverse and longitudinal fibers. Although these cells do not undergo myoblast fusion,
they appear striated and display sarcomeric repeats. Within the midgut visceral mesoderm, alpha-actinin prominently localizes to Z-bands. Z-bands demarcate the ends of individual sarcomeres, where the barbed ends of
actin thin filaments terminate. In the same tissue, Mlp84B distributes as a doublet that flanks each Z-band. As seen in merged images, Alpha-actinin and Mlp84B are localized in adjacent regions. Mlp84B extends away from the periphery of the Z-band, whereas alpha-actinin is clearly
more restricted. The localization of Mlp84B to discrete sites within the muscle sarcomere provides evidence for a specific association of
Mlp84B with the microfilament cytoskeleton in vivo. Although
Western immunoblot analysis reveals that Mlp60A is present in isolated midgut preparations, the protein was not detected using immunofluorescent
methods. It is unclear why Mlp60A protein was not observed in situ, but perhaps within the mature myofibril, Mlp60A is complexed with protein partners such that
the epitopes recognized by the antibodies were masked (Stronach, 1999).
The beta 3 tubulin gene is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm, while enhancers localized in the first intron guide expression in the visceral mesoderm. In addition to this tissue-specific mode of transcriptional control, beta 3 tubulin is regulated in a distinct stage-specific manner. From stage 10 to stage 12, expression in the visceral mesoderm is regulated along the anterior-posterior axis by a first intron localized enhancer; this regulation is mediated by homeotic selectors genes like Ubx. After stage 12, an additional farther 3'-located enhancer mediates expression in the visceral and somatic mesoderm. In the epidermis, transcription of the beta 3 tubulin gene is repressed by Engrailed (Damm, 1998).
Deletion analysis carried out in transgenic flies reveals independent regulatory elements for the dorsal vessel and the somatic mesoderm. These elements are located upstream of the transcriptional start site. For expression in the
somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2). Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements; D-MEF2 cannot, on its own, direct beta 3 tubulin expression. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator (Damm, 1998).
muscleblind (mbl) is a recently described Drosophila gene involved in terminal differentiation of adult ommatidia. mbl is a nuclear protein expressed late in the embryo in pharyngeal, visceral, and somatic muscles, the ventral nerve cord, and the larval photoreceptor system. All three mbl alleles studied exhibit a lethal phenotype and die as stage 17 embryos or first instar larvae. These larvae are partially paralyzed, show a characteristically contracted abdomen, and lack striation of muscles. An analysis of the somatic musculature shows that the pattern of muscles is established correctly, and they form morphologically normal synapses. Ultrastructural analysis, however, reveals two defects in the terminal differentiation of the muscles: inability to differentiate Z-bands in the sarcomeric apparatus and reduction of extracellular tendon matrix at attachment sites to the epidermis. Failure to differentiate both structures could explain the partial paralysis and contracted abdomen phenotype. Analysis of mbl expression in embryos that are either mutant for Dmef2 or ectopically express Dmef2 places mbl downstream of Dmef2 function in the myogenic differentiation program. mbl, therefore, may act as a critical element in the execution of two Dmef2-dependent processes in the terminal differentiation of muscles (Artero, 1998).
It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell
differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this
differentiation process. A novel molecular screen has been undertaken in Drosophila in a systematic search for such
genes. The approach utilizes subtractive hybridization coupled to directional cDNA library construction. Libraries were
made from as little as 20 mg total RNA isolated from hand-picked embryos of defined stage of development and genotype.
In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic
mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA
libraries, before subtracting twi from wild-type, increases the subtraction efficiency. Subtraction results in a cDNA population
enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression
pattern of each clone in a high throughput procedure followed by DNA sequencing. The method, which is comprehensive and
does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10
embryos. Analysis of one clone, Dmeso18E, reveals a gene encoding a putative nuclear protein of 553 amino acids.
The protein is novel; its expression is mesoderm-specific, twi-dependent, and occurs early during somatic, visceral, and heart muscle
differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis
of Dmeso18E expression reveals new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier
than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral
muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The
tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and
genes like tin and Dmef2 or it lies in a parallel pathway of gene activation (Taylor, 2000).
Thus, there is tin-independent expression
of Dmeso18E in the cells that would go on to form the
visceral muscle. Dmeso18E expression can be compared to
that of bagpipe, which is expressed in the mesoderm from
stage 8, like Dmeso18E, and then in segmentally repeated
patches corresponding to the visceral muscle primordia as
they form during stages 10 and 11.
However, in contrast to Dmeso18E, expression of the bap
gene is not activated in the germ band in tin mutants and is
a candidate target gene for Tinman. Two possible explanations follow for the presence of
Dmeso18E mRNA in tin mutants in cells that would go on to
form visceral muscle: (1) there may be
a tin-independent route for activation of transcription in
prospective visceral muscle cells; (2) it could be mRNA
remaining from an earlier transcription activation event
before the visceral muscle progenitors form. However, this
would require that Dmeso18E RNA be quite stable -- however, the
dynamic nature of its embryonic expression pattern suggests
a relatively short half-life. Whatever the mechanism
that lies behind this expression, the main point is that a
muscle-specific gene that does not require tin is expressed
in the visceral muscle primordia. Together with the tin-independent
invagination of these cells, these results show
that some features of the cells that will form the visceral
muscle do not require tin (Taylor, 2000).
To identify regulatory events occurring during myogenesis, the
transcriptional regulation of a Drosophila melanogaster actin gene, Actin 57B, was characterized.
Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the end of embryogenesis. Through
deletion analysis a 595 bp promoter element has been identified that is sufficient for high levels of expression in all three muscle lineages. This fragment contains a Mef2 binding site conserved between D. melanogaster and Drosophila virilis that binds Mef2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduces promoter activity in embryos, and in Mef2 mutants
Act57B expression is severely decreased, demonstrating that Mef2 is an essential regulator of Act57B. Mef2 likely acts synergistically with
factors bound to additional sequences within the 595 bp element. These findings
underline the importance of Mef2 in controlling differentiation in all muscle
lineages. These experiments reveal a novel regulatory mechanism for a structural
gene where high levels of expression in all embryonic muscles is regulated
through a single transcription factor binding site (Kelly, 2002).
Mef2 function alone is not sufficient to induce Act57B
expression at high levels in muscle cells. LacZ reporter
constructs with the Mef2 site and flanking sequences, but
lacking region b (consisting of about half of the 595 bp promoter element), had reduced reporter activity. In addition, ectopic expression of Mef2 under
control of a heat shock promoter does not induce expression
of Act57B. Low levels of Act57B expression occur
in the absence of Mef2 function, either when the Mef2 site
is mutated in the minimal reporter construct or in a Mef2 mutant background. The simplest explanation for these results is that Mef2 functions
with additional factor(s) to regulate muscle gene expression
and that the presumptive cofactor is also restricted in its
activity to the musculature (Kelly, 2002).
However, the identity of this putative cofactor is far from
clear. PAR-domain protein 1 has been shown to be a Mef2 cofactor in Drosophila,
regulating TmI gene expression in the somatic mesoderm, however there are no PDP1 binding
sites contained within the minimal Act57B enhancer fragment.
In vertebrate systems the myogenic bHLHs (MyoD,
myogenin, Myf5, MRF4) have been shown to regulate gene
expression with Mef2 through E-boxes. Again, there are no E-boxes
contained within the minimal Act57B enhancer. Since the
upstream region required for high promoter activity contains
areas of strong sequence conservation between D. melanogaster
and D. virilis, it is likely that these might represent
binding sites for the putative cofactors. It is unclear at this
time exactly what factors may bind to this region (Kelly, 2002).
All of the data presented in this paper indicate that the
Act57B locus is under the positive control of Mef2 and
potential cofactors. Moreover, none of the deletion
constructs show inappropriate expression, suggesting the
absence of cis-acting negative regulatory sequence within
the enhancer. However, there is a time during early embryogenesis
when Mef2 is present, but Act57B is not
expressed. This could simply be due to the absence of positively
acting cofactors at this stage in development. Alternatively,
this could be the result of negative regulators
interacting directly with factors bound to the Act57B promoter (Kelly, 2002).
The Drosophila C2-H2-type zinc-finger transcription factor CF2 has been shown to regulate follicular cell fate determination during
oogenesis. CF2 is also expressed in the developing muscles of the embryo where it first appears at stage 12 at the time of
skeletal myoblast fusion. Later it is expressed in all muscle lineages including skeletal, visceral and cardiac. Epistatic analysis has shown that
CF2 expression is dependent on the myogenic factor MEF2 (Bagni, 2002).
The distinct muscles of an organism accumulate different quantities of structural proteins, but always maintaining their stoichiometry. However, the mechanisms that control the levels of these proteins and that co-ordinate muscle gene expression remain to be defined. The paramyosin/miniparamyosin gene encodes two thick filament proteins transcribed from two different promoters. The regulatory regions were analyzed that control expression of this gene and that are situated in the two promoters, the 5' and the internal promoters, both in vivo and in silico. A distal muscle enhancer containing three conserved MEF2 motifs is essential to drive high levels of paramyosin expression in all the major embryonic, larval and adult muscles. This enhancer shares sequence motifs, as well as its structure and organisation, with at least four co-regulated muscle enhancers that direct similar patterns of expression. However, other elements located downstream of the enhancer are also required for correct gene expression. Other muscle genes with different patterns of expression, such as miniparamyosin, are regulated by other basic mechanisms. The expression of miniparamyosin is controlled by two enhancers, AB and TX, but a BF modulator is required to ensure the correct levels of expression in each particular muscle. A mechanism of transcriptional regulation is proposed in which similar enhancers are responsible for the spatio-temporal expression of co-regulated genes. However, it is the interaction between enhancers that ensures that the correct amounts of protein are expressed at any particular time in a cell, adapting these levels to their specific needs. These mechanisms may not be exclusive to neural or muscle tissue and might represent a general mechanism for genes that are spatially and temporally co-regulated (Marco-Ferreres, 2005).
Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. A chromatin immunoprecipitation (ChIP)-chip strategy has been developed for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. Use of ChEST in Drosophila is described to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. The approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome (Junion, 2005).
To predict Dmef2-dependent CRMs, the Drosophila genome was scanned for modules containing one of the three previously described in vivo-acting Dmef2 binding sites. Because muscle differentiation events are controlled by the synergistic action of MADS-box (Mef2 family) and E-box (Twist and MyoD bHLH family) factors modules were sought containing Mef2- and E-box-binding sites. This scanning procedure led to the identification of ~1,243 potential Dmef2-binding CRMs, from which 99 modules were selected, amplified, and spotted to produce a computed Dmef2-CRM array. Three of four previously described Dmef2-CRMs, located in the vicinity of Paramyosin, Act57B, and Dmef2, were identified during genome scanning and selected for spotting as positive control. The fourth in vivo-acting Dmef2-CRM located close to b3-tubulin did not come out from the screen because of the high number of E-box sites used as the scanning criterion. The CRMs located in the vicinity of genes that are not expressed in the mesoderm or genes of unknown expression and function (some of CGs) were rejected. In parallel, X-ChIP was used to isolate DNA fragments to which Dmef2 binds in vivo. ChIP-DNA immunoprecipitated either with Dmef2 antibody or with nonimmune serum was then labeled and used to probe the Dmef2-CRM array. The three previously described in vivo-acting CRMs were found enriched in ChIP-DNA immunoprecipitated with Dmef2 antibody. Importantly, numerous other in silico-identified Dmef2-CRMs were enriched in ChIP-DNA, thus demonstrating the efficacy of the ChEST method (Junion, 2005).
Of the 99 in silico-predicted Dmef2-CRMs, 62 were enriched in the DNA immunoprecipitated with anti-Dmef2 antibody. The CRM-associated Dmef2 targets included genes expressed in all muscle cell types, in which Dmef2 has previously been reported to function. In addition to expected candidates encoding fusion (Lmd, Hibris) and structural (Ket, Pod1) muscle proteins, a large number of CRM-associated Dmef2 target genes coded for TFs and signal transduction proteins. For example, CRMs upstream of Fz2 and within the introns of Ci and Pan indicate a potential role of Dmef2 in transcriptional regulation of genes transducing to the mesodermal cells ectodermal Wg and Hh signals, whereas CRMs within the introns of If and Pka-C3 suggest that by regulating transcription of these genes Dmef2 is involved in the attachment of muscle fibers and in fiber contraction, respectively. In some cases (e.g., Kettin, NetB, N-cad), several Dmef2-dependent CRMs were mapped in the vicinity of the same gene, highlighting the complexity of transcriptional regulation in which Dmef2 is involved. Analysis of the position of CRMs in relation to adjacent genes revealed that the majority of ChIP-enriched modules are located upstream (42%) or within the introns (39%) of target genes. In these two categories, the most frequent positions of Dmef2-CRMs appear between 1 and 5 kb upstream of the gene and within the first intron (Junion, 2005).
To determine whether the ChEST-identified DNA fragments are able to act as regulatory modules in vivo, ten Dmef2-CRMs by reporter gene transgenesis were tested. Nine of 10 CRMs were found to drive reporter gene expression in Dmef2-positive muscle cells. In all transgenic Drosophila lines, lacZ reporter expression at least partially reproduced endogenous gene expression, indicating that the identified CRMs were bona fide enhancers of adjacent genes. For example, the Ket-1, Ket-2, and Ket-3 CRMs laying in the vicinity of the kettin gene, which encodes a giant muscle protein required for the formation and maintenance of normal sarcomere structure, were found to drive lacZ expression in distinct subsets of differentiating body wall muscles. These data indicate that the pan-muscular expression of kettin is regulated in a muscle-type-specific manner, and by multiple Dmef2-binding enhancers. Interestingly, four other analyzed CRMs located within the introns of N-cad and acon and upstream of fz2, sfl, and Meso18E also drive lacZ expression in discrete subsets of somatic muscle precursors. The muscle-type-restricted activity of these modules suggests that both CRM regulators (Dmef2, E-box factors) and their target genes are involved in different aspects of muscle precursors diversification, including muscle fiber shape and axial positioning. Alternatively, the observed muscle-type-specific expression of lacZ may result from the limited size (250-300 bp) of genomic sequences tested. In embryos carrying a Dmef2-dependent CRM found in the intron of If, lacZ expression is detected in a group of ventrolateral muscles. This lacZ pattern correlates with distribution of endogenous If, which accumulates at the extremities of ventrolateral muscle fibers and is required for their correct epidermal insertion. The reduced level of target gene expression in Dmef2 mutant embryos provides an additional support for Dmef2-dependent in vivo regulation of ChEST-identified CRMs (Junion, 2005).
Compared with other genomewide ChIP-chip approaches, ChEST does not require access to the whole-genome tiling arrays. It is dedicated to the identification of a subset of factor-specific CRMs that can be predicted by using computer-assisted methods. Depending on the TF and the organism concerned, the predictability of CRMs may represent a limiting factor. This study used ChEST to identify Dmef2-regulated CRMs in Drosophila. Because Dmef2 binds to long, well characterized DNA sequences and the Drosophila genome is well adapted to CRM prediction (rich in annotations), this approach created an attractive framework for analysis and contributed to a high percentage (63%) of ChIP-enriched CRMs. The fact that 37% of in silico-selected Dmef2-CRMs have not been found as significantly enriched in ChIP material results from one of the following reasons or from a combination of these reasons: (1) inefficient ChIP due to restricted expression of target genes (13 CRMs listed lay close to genes expressed in a short time window or in a subset of mesodermal cells); (2) inefficient ChIP due to chromatin conformation, and/or (3) false positives generated during the computer-assisted genome scanning (Junion, 2005).
To test ChEST in more challenging conditions, it was applied for mapping CRMs to which bind two other myogenic factors, Lame duck (Lmd) and Ladybird early (Lbe). Compared with Dmef2, Lmd and Lbe are expressed in a progressively more restricted subset of cells in the embryo, and their in vivo binding sites have not yet been identified, meaning only the in vitro consensus binding sequences can be used for CRM prediction. In such suboptimal conditions, the efficacy of ChEST dropped from 63% (Dmef2) to 34% of ChIP-enriched CRMs for Lmd and <20% for Lbe. Thus, in Drosophila, ChEST can also be used (with a lower efficacy) for mapping CRMs to which bind transcription factors recognizing short and/or in vitro-only characterized DNA sequences (Junion, 2005).
Furthermore, because the genome annotations for several invertebrate and vertebrate organisms are rapidly progressing, ChEST is expected to be easily adapted to other systems. Otherwise, targets for the evolutionarily conserved TFs can be identified in Drosophila and then validated in other organisms. One possible application of ChEST in the future is to use it in cell- or tissue-culture systems to follow the repertoire of target CRMs for TFs involved in important biological processes, including human pathologies (Junion, 2005).
Drosophila Dmef2 is required for the terminal differentiation of skeletal, visceral, and cardiac musculature but seems not to be involved in early myogenic events such as muscle specification and diversification. It directs mesodermal cell differentiation programs by regulating transcriptional activity of genes associated with these processes. Here, among the identified Dmef2-binding CRMs, those were found that map in the vicinity of ket, Tm1, Act87E, and Pod1 genes encoding muscle structural proteins and a few others located close to Lmd, N-cad, NetB, Hibris, Sema-5c, If, dei, and Flw genes implicated in cell adhesion/fusion or muscle attachment, thus supporting previously described muscle differentiation functions of Dmef2. The identification of Lmd, Hibris, and N-cad CRMs is consistent with the involvement of Dmef2 in myoblast fusion processes, which are affected in Dmef2 mutant embryos. Furthermore, the presence of Dmef2-dependent CRMs close to NetB, which is involved in the attraction of motoneurons, suggests that the Dmef2-dependent formation of presynaptic active zones may involve NetB. However, in addition to these novel but anticipated Dmef2 targets, the data indicate that Dmef2 also contributes to the transcriptional regulation of genes implicated in muscle specification and diversification. Candidates that indicate an early myogenic role of Dmef2 include components of the Wg, Hh, and RTK signaling pathways (Ci, Pan, GATAe, AbdA, Jeb, Sfl, Fz2, ttk, argos, stumps, Concertina, and Src42A) that play key roles in the specification of myogenic lineages and their subsequent diversification (Junion, 2005).
The role of Dmef2 in the initial steps of myogenesis is in agreement with the early, twist-dependent phase of Dmef2 expression, and is further supported by the previously described regulation of early expression of Dmeso18E. Another unexpected group of potential transcriptional targets of Dmef2 corresponds to genes encoding proteins involved in ion transport, channel activity, and metabolism (e.g., Slo, Itp-r83A, NheII, and Acon). The regulation of this class of genes may reflect a role for Dmef2 in the transmission of neural stimulation and muscle contraction. The maintenance of Dmef2 expression in fully differentiated adult muscles is in agreement with such a function. In conclusion, the ChEST approach presented here has led to the identification of a set of previously unknown Dmef2-dependent regulatory modules whose activity and adjacent genes indicate novel myogenic functions for Dmef2 (Junion, 2005).
Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).
Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present
study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).
The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).
Ectopic expression of a constitutively active Pnt variant (Pnt.P1) mediates strong activation of Rst expression in neuroectodermal cells. Since Pnt.P1 recognizes the same target sequences as its splice variant Pnt.P2, the nuclear effector of the Ras-MAPK pathway, ectopic activation of Rst expression by Pnt.P1 is consistent with a regulation by the Ras-MAPK pathway. Similarly, the ectoptic activation of Rst expression in neuroectodermal cells by Su(H)-VP16 points to a regulation of rst by the Notch pathway (Apitz, 2005).
The Ras-MAPK and the Notch pathways display significant crosstalk during developmental processes in Drosophila, e.g., in cell fate specification of the eye disc. It is difficult to elucidate a possible regulation of a candidate gene by mutant analysis if it is activated by both pathways. In mutants, for one of the pathways, the activity of the other pathway will ensure residual expression of the candidate gene under scrutiny. rst expression is activated by both pathways and single mutant analysis did not reveal a significant loss of expression. Both pathways converge on regulatory elements contained within F6 and not in F5. Furthermore, a regulatory module is present in the nonoverlapping part of F6 that is activated in IOC before apoptotic decisions are made in these cells (Apitz, 2004). This module is located within an approximately 600-bp sequence and is active during several apoptotic decisions (Apitz, 2004). Consensus binding sites for Su(H) and Pnt in this module are conserved between D. melanogaster and D. pseudoobscura. Both the Ras-MAPK and Notch pathways are involved in apoptotic processes of IOC cells. Together, these data suggest that rst transcription is regulated by these pathways in the context of apoptotic decisions (Apitz, 2005).
The transactivation screen shows that Dmef2 acts on rst cis-regulatory sequences: the overlapping reporter gene constructs rstF5 -lacZ and rstF6 -lacZ are both ectopically activated by Dmef2 in the scabrous expression domain. Regions F5 and F6 contain partially overlapping sequences and it is likely that Dmef2 acts on a sequence interval bracketed by this overlap. This region contains a regulatory module for expression in the mesoderm (Apitz, 2004). This sequence contains a putative Dmef2 binding site that is also conserved between D. melanogaster and D. pseudoobscura. The sequence of this putative element exactly matches a Dmef2 binding site found in the enhancer of β3 tubulin. Therefore, it is argued that rst may be a direct target of Dmef2. This is consistent with similar mesodermal expression patterns and mutant phenotypes of Dmef2 and rst. In contrast, Rst is expressed in the mesoderm of Dmef2 loss-of-function mutants. An explanation for this result is provided by analysis of rst cis-regulatory sequences. rst is regulated in the mesoderm by at least two independent regulatory modules that mediate a differential expression pattern (Apitz, 2004). One is contained in F6p, the other in F5p. This points to a mesodermal regulation of rst by at least two factors, one of which is still active in Dmef2 mutants (Apitz, 2005).
MicroRNAs (miRNAs) are genomically encoded small RNAs that hybridize with messenger RNAs, resulting in degradation or translational inhibition of targeted transcripts. The potential for miRNAs to regulate cell-lineage determination or differentiation from pluripotent progenitor or stem cells is unknown. MicroRNA1 (miR-1) is an ancient muscle-specific gene conserved in sequence and expression in Drosophila. Drosophila miR-1 (dmiR-1) is regulated through a serum response factor-like binding site in cardiac progenitor cells. Loss- and gain-of-function studies demonstrated a role for dmiR-1 in modulating cardiogenesis and in maintenance of muscle-gene expression. In vivo evidence is provided that dmiR-1 targets transcripts encoding the Notch ligand Delta, providing a potential mechanism for the expansion of cardiac and muscle progenitor cells and failure of progenitor cell differentiation in some dmiR-1 mutants. These findings demonstrate that dmiR-1 may 'fine-tune' critical steps involved in differentiation of cardiac and somatic muscle progenitors and targets a pathway required for progenitor cell specification and asymmetric cell division (Kwon, 2005).
The single orthologue of miR-1 in Drosophila, dmiR-1, is nearly identical in sequence to mouse and human miR-1. In situ hybridization revealed dmiR-1 transcripts in presumptive mesodermal cells as early as stage 5 (2.2-2.8 h) of Drosophila development. This pattern changed dynamically throughout gastrulation, but dmiR-1 consistently marked mesodermal cells. Transcripts persisted in later stages of cardiac and somatic (body wall) muscle differentiation, as in mice, and were also found in visceral muscles of the gut. dmiR-1 expression overlaps, but precedes, that of dmef2, a transcriptional regulator of muscle precursors (Kwon, 2005).
To determine whether transcriptional regulation of miR-1 is evolutionarily conserved, ten kb of genomic DNA surrounding Drosophila melanogaster and Drosophila pseudoobscura miR-1 genes were aligned to find regions of sequence conservation. Transgenic flies containing conserved islands 4.6 kb upstream of dmiR-1 adjacent to the gene encoding nuclear GFP (nGFP) recapitulates the endogenous dmiR-1 expression in all muscle types. Cardiac nGFP expression coincided with dmef2 expression in cardial cells and was present in pericardial cells, which do not express dmef2. The basic helix–loop–helix transcription factor Twist is essential for mesoderm specification and regulates dmiR-1 in certain domains, so the expression of nGFP driven by the dmiR-1 enhancer was directly compared with Twist expression. Whereas there was considerable overlap, the dmiR-1 enhancer directs expression in many areas of low Twist expression, including cardiac and visceral muscle progenitors, suggesting twist-independent regulation in these domains (Kwon, 2005).
Deletion analyses indicated that a 2.5-kb region was sufficient for expression in all domains of dmiR-1 expression except pericardial cells. Within this domain, a 720-bp genomic region containing a highly conserved SRF-like binding site recapitulates the expression directed by the 2.5-kb fragment. SRF, which is closely related to MEF2, controls the expression of genes involved in muscle differentiation, cell migration and proliferation. Prior studies showed that SRF is an obligate activator of miR-1 expression during cardiac development in the mouse. Mutation of the SRF-like site in flies abolishes nuclear GFP expression in cardiac and visceral muscle cells but not somatic muscle. In vitro, Drosophila SRF (DSRF) weakly activates transcription of a luciferase reporter through the SRF-like binding site. Addition of the potent cardiac and smooth muscle-specific SRF cofactor myocardin-related transcription factor robustly activated luciferase activity dependent on an intact SRF-like binding site. This observation is consistent with regulation of miR-1 in mice, but the possibility cannot be ruled out that Dmef2 also regulates cardial expression of dmiR-1 through this site independently or cooperatively with SRF (Kwon, 2005).
To begin to define the functions of dmiR-1 in vivo, two Exelixis lines of Drosophila containing FRT sites surrounding the dmiR-1 gene were used to generate a FRT-FLP-based deletion of the dmiR-1 locus. Successful excision of dmiR-1, the only known or predicted gene in the 31-kb deleted interval, was confirmed by sequence analysis and RT-PCR. Homozygous dmiR-1 deletion was 100% lethal, but a spectrum of severity was observed, with approximately one-third dying at embryonic stages, one-third around hatching, and the remaining at larval stages. Homozygous mutant larvae were abnormally lethargic compared with their heterozygous siblings before death. The embryonic and larval lethality was fully rescued by overexpression of dmiR-1 by using a mesoderm specific twi-Gal4 driver or by a 5.1-kb transgene encompassing the dmiR-1 genomic locus including the 4.6-kb enhancer and the sequence encoding dmiR-1, consistent with dmiR-1 being the sole gene within the deleted region responsible for the lethal phenotype. The variability in phenotype may be related to previously described maternal dmiR-1 transcripts, redundancy with other miRNAs or may simply reflect the role of dmiR-1 in 'fine-tuning' whether cells achieve the thresholds of critical proteins to initiate critical developmental events (Kwon, 2005).
Because one-third of all dmiR-1 mutants died around the time of hatching and another one-third at larval stages with poor mobility, whether there might be a discernable muscle defect was investigated. Nearly half of all dmiR-1 mutant embryos displayed severe defects in muscle gene expression with down-regulation of sarcomeric genes such as myosin heavy chain (MHC), indicating a late requirement for miR-1 to maintain muscle differentiation. This phenotype was also uniformly rescued by dmiR-1 under the control of the twist driver, indicating that the muscle differentiation defect was due to loss of dmiR-1 and not other sequences within the deleted region (Kwon, 2005).
Because miRNAs typically target numerous mRNAs, the phenotype of dmiR-1 mutants is likely due to down-regulation of multiple critical proteins. Despite the likely complexity of targets, attempts were made to identify mRNA targets of miR-1 in flies that might be involved in dmiR-1-dependent lineage determination and differentiation decisions. Although mouse miR-1 targets transcripts encoding the cardiac-enriched basic helix-loop-helix transcription factor Hand2, no miR-1-binding sites were identified in the 3'-UTR of Drosophila hand, suggesting alternative targets in flies. Because the more severe dmiR-1 gain- and loss-of-function phenotypes were reminiscent of progenitor defects induced by altering Notch signaling, the 3'-UTRs of genes involved in the Notch pathway were examined for potential sequence matching and accessibility to dmiR-1 (Kwon, 2005).
Several conserved putative miR-1-binding sites were found in the 3'-UTR of the gene encoding Delta, a membrane-bound ligand for Notch. Upon interaction with Delta, Notch is cleaved, allowing the Notch intracellular domain to translocate into the nucleus and regulate gene expression. Signaling between neighboring Delta- and Notch-expressing cells is necessary for lateral inhibition and asymmetric cell fates during lineage determination and involves repression of Delta in Notch-expressing cells and similar repression of Notch in adjacent Delta-expressing cells. Notch signaling also later regulates differentiation of numerous cell types, including cardial cells (Kwon, 2005).
Introduction of one of the putative dmiR-1-binding sites from the Delta 3'-UTR into the 3'-UTR of luciferase resulted in dose-dependent and specific down-regulation of luciferase activity in the presence of dmiR-1 in cultured fly S2 cells. Although the in vitro data supported Delta as a dmiR-1 target, attempts were made to determine whether dmiR-1 affected Delta protein levels in vivo. Available Delta antibodies are not sensitive enough to distinguish levels of Delta protein in embryonic muscle precursors. Therefore, an in vivo assay was used involving the well described role of Delta-Notch signaling in the developing wing disc, where disruption of Delta results in thickening of fly wing veins. Delta protein is normally detectable and expressed in two perpendicular stripes in the wing pouch. dmiR-1 was overexpressed along one of the two stripes using a dpp-Gal4 driver and the effects on Delta protein expression were assayed. Delta protein was markedly reduced exclusively in the domain of dmiR-1 expression, providing in vivo support for Delta as a target of miR-1 in flies. dmiR-1-induced loss of Delta in this specific subdomain of the wing resulted in thickening of wing veins, recapitulating the loss-of-Delta phenotype. The shortened-leg phenotype upon dmiR-1 overexpression provided further evidence of dmiR-1's effects on the Notch pathway, because this, too, was similar to the phenotype of flies lacking Delta. Together, the in vivo experiments provided compelling evidence that dmiR-1 can regulate Delta protein levels, providing a potential means to fine-tune cellular responses to Notch signaling. Given the recognized role of Notch signaling in asymmetric cell division of muscle progenitors, dmiR-1 regulation of Delta, along with other dmiR-1 targets, may be important in cardiac lineage determination events (Kwon, 2005).
Embryogenesis is controlled by large gene-regulatory networks, which generate spatially and temporally refined patterns of gene expression. This study reports the characteristics of the regulatory network orchestrating early mesodermal development in the fruitfly, where the transcription factor Twist is both necessary and sufficient to drive development. Through the integration of chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) experiments during discrete time periods with computational approaches, >2000 Twist-bound cis-regulatory modules (CRMs) were identified and almost 500 direct target genes. Unexpectedly, Twist regulates an almost complete cassette of genes required for cell proliferation in addition to genes essential for morophogenesis and cell migration. Twist targets almost 25% of all annotated Drosophila transcription factors, which may represent the entire set of regulators necessary for the early development of this system. By combining in vivo binding data from Twist, Mef2, Tinman, and Dorsal an initial transcriptional network was constructed of early mesoderm development. The network topology reveals extensive combinatorial binding, feed-forward regulation, and complex logical outputs as prevalent features. In addition to binary activation and repression, it is suggested that Twist binds to almost all mesodermal CRMs to provide the competence to integrate inputs from more specialized transcription factors (Sandmann, 2007).
ChIP-on-chip was performed at two consecutive developmental time periods: 2-4 h (stages 5-7) and 4-6 h (stages 8-9), covering the stages of gastrulation, mesoderm expansion, migration, and early subdivision into different primordia. For each time period, four independent ChIPs were performed using two different anti-Twist antibodies to reduce possible off-target effects (Sandmann, 2007).
To systematically identify Twist-bound regions in an unbiased, global manner, a high-density microarray tiling across the Drosophila melanogaster genome was designed with ~380,000 60mer oligonucleotide probes. Twist binds to E-box motifs: As a degenerate E-box (CANNTG) is expected to occur every ~256 base pairs (bp) in the Drosophila genome, a 60mer oligonucleotide was designed for each E-box motif within the nonrepetitive, noncoding regions of the genome. This design made no assumptions about the specificity of the E-box bound by Twist, yet ensured all putative E-boxes were covered and that each Twist-bound sequence was detected by at least two neighboring 60mers (Sandmann, 2007).
These experiments identified 2096 nonoverlapping genomic regions significantly bound by Twist within one or both developmental time periods. This set includes all known Twist-bound enhancers tested, except the eve-cardiac enhancer that is regulated outside the period of development assayed. The majority of Twist-bound regions are found within introns of gene loci, rather than noncoding 5' and 3' regions. A similar positional bias was also observed for p53 and Krüppel, suggesting that introns close to the transcriptional start site represent hotspots for active CRMs. Intronic binding of Twist correlates significantly with the misregulation of these genes' expression in twist loss-of-function mutant embryos and their expression within the ventral blastoderm and mesoderm (Sandmann, 2007).
One of the major challenges for ChIP-on-chip studies is to accurately link the TF-bound enhancers to their appropriate target gene. Rather than simply taking the closest 5' or 3' gene, a more stringent approach was taken and a Twist-bound region was not assigned to a gene based on proximity alone. The results demonstrate that Twist binds more frequently to gene loci genetically downstream from the TF and/or expressed in the same cells as the TF. These criteria to systematically match all 2096 Twist-bound regions (intronic or intergenic) to their likely targets, leading to a high-confidence gene assignment for 854 Twist-bound sequences. This increased the number of Twist direct targets from the previously known 11 to 494 genes. All Twist-bound regions and surrounding genes can be visualized and searched at http://furlonglab.embl.de (Sandmann, 2007).
The RedFly database contains a comprehensive collection of previously described Drosophila enhancers, mainly characterized through single gene studies. Of the 2096 Twist-bound regions, 143 overlap with known enhancers for 62 genes, confirming that these regions have regulatory potential in vivo. Twist was not known to bind to many of these enhancers; this overlap therefore provides strong evidence for a regulatory link between Twist and the 62 target genes (e.g., Abd-A, Abd-B, aop, Brd, slp1, and bap). To further examine the regulatory potential of Twist-bound regions, reporter constructs of new putative enhancer sequences were tested in transgenic animals. Six Twist-bound regions within or close to the following gene loci were assayed: T48, trbl, retn, CG4221, CG8788, and CG32372. All regions proved sufficient to function as enhancers in vivo and could reproduce all or part of the endogenous spatio-temporal gene-expression pattern (Sandmann, 2007).
The T48, tribbles, retained, CG4221, and CG8788 enhancers initiate expression within the early blastoderm. The T48 module mirrors the expression of the endogenous gene within the presumptive mesoderm. The zygotic expression of tribbles is highly dynamic, which is reflected by the assayed CRM. This enhancer drives expression very transiently in the ventral blastoderm and quickly becomes ubiquitously expressed. The relatively small enhancer region for retained is activated in the anterior and posterior ventral blastoderm, where it is coexpressed with Twist, and its expression extends into the dorsal blastoderm. The CRMs for CG4221 and CG8788 initiate expression in the presumptive mesoderm, and continue to drive expression throughout the trunk mesoderm at later stages. The expression of the CG32372 module initiates after gastrulation in the head mesoderm, a domain that overlaps with twist expression. It is interesting to note that Twist binds to multiple enhancer regions for many of these genes. This feature is also evident more globally: Almost 50% of Twist target genes have two or more Twist-bound enhancers, reflecting the complexity of their regulation (Sandmann, 2007).
In summary, these results demonstrate that ChIP-on-chip experiments provide a sensitive and accurate global map of Twist-bound regulatory regions during key stages of early mesoderm development (Sandmann, 2007).
To assay the requirement of Twist function for target gene expression, the expression was examined of six novel direct targets in twist mutant embryos. These genes are expressed in the presumptive mesoderm prior to gastrultion, and therefore at stages when the role of twist function can be assessed. Mesodermal cells are absent in twist mutant embryos later in development due to a block in gastrulation. Triple-fluorescent in situ hybridization was performed using probes directed against twist (blue channel; while twist1 is a protein-null allele, twist RNA is still expressed), inflated (red channel; this gene is dependent on twist for its expression and was used as a marker to distinguish homozygous mutant embryos from their siblings), and a probe directed against one of the six direct target genes (green channel). The spatial expression of all six targets overlaps with twist within the presumptive mesoderm (Sandmann, 2007).
Importantly, twist activity is essential for the expression of five out of six genes examined. Note, for CG32982 and CG9005, residual expression remains outside the twist expression domain in the dorsal and posterior blastoderm, respectively. These results, in combination with in vivo binding data, indicate that Twist binding to a CRM is a prerequisite to activate target gene expression for a large percentage of its targets. The role of Twist binding to the NetA enhancer remains unclear. Twist may act redundantly with other TFs, or alternatively may function in a more subtle manner to modulate the levels of expression (Sandmann, 2007).
One of the earliest functions of Twist within the pregastrula embryo is the coregulation of D-V patterning with the NFkappaB ortholog Dorsal. Dorsal acts as a morphogen by regulating its target genes at (at least) three threshold concentrations along the D-V axis. Type I-regulated Dorsal enhancers receive high levels of Dorsal, contain low-affinity Dorsal sites and drive expression in ventral mesodermal domains (e.g., sna, htl, twi). Type II enhancers receive intermediate levels of Dorsal and drive expression in mediolateral domains of different sizes (e.g., sim, brk, vn), while Type III enhancers receive low levels of Dorsal, contain high-affinity Dorsal sites, and can be either activated (sog, ths) or repressed (dpp, tld, zen) by Dorsal. This system has been studied so intensively that the level of knowledge is sufficient for quantitative modeling of cis-regulatory interactions. It was therefore of interest to determine whether global analysis could reveal new insights into this process. The data identified in vivo binding of Twist to both Type I and II Dorsal enhancers, as expected. The boundaries of Twist binding are in remarkable agreement with the limits of characterized minimal enhancers (e.g., htl, rho, and ths). More importantly, new CRMs were identified for several of these well-characterized genes (Sandmann, 2007).
Seven novel enhancers for D-V patterning genes reveal the regulatory complexity of Twist-bound CRMs: The cactus, stumps, and wntD enhancers drive expression in a domain overlapping Twist within the ventral blastoderm and likely represent Type I enhancers. Cactus, an IkappaB ortholog, is expressed both maternally and zygotically and sequesters Dorsal within the cytoplasm. While the regulation of zygotic cactus expression was previously not understood, these data reveal a Twist-bound CRM that is sufficient to drive expression in the presumptive mesoderm. Twist also binds to a CRM of Toll. Although the function of cactus'and Toll's zygotic regulation remains unclear in Drosophila, positive feedback regulation of zygotic Toll-receptor expression is required to refine the Dorsal nuclear gradient in the flour beetle Tribolium castaneum (Sandmann, 2007).
The stumps CRM is expressed in a subset of Twist-expressing cells, yielding a salt and pepper pattern that may reflect the requirement for a second, partially redundant enhancer (e.g., the 'stumps_early' enhancer) to give robust expression. The wntD CRM is highly expressed at the anterior and posterior poles of the ventral blastoderm, but is very weakly expressed within the central region. This mirrors the transient expression of the endogenous gene at this stage of development. This single enhancer reflects the regulatory logic deduced from genetic studies: The inputs from Twist and Dorsal activate WntD, while Snail represses its transcription within the presumptive mesoderm. The CRM for crumbs reproduces the endogenous genes expression. This 480-bp region can function as an enhancer in the ectoderm while acting as a silencer within the ventral blastoderm. This ventral repression is most likely due to direct input from Snail on this CRM. Therefore, even at the same stage of development, these four Twist-bound CRMs drive expression in different spatial patterns within a small population of cells. This complexity is clearly mediated by context-dependent integration of additional inputs. Three additional CRMs for mir-1 (Type I), vn, and sim (Type II Dorsal targets) drive expression later in development, reproducing part of the endogenous gene’s expression (Sandmann, 2007).
Unexpectedly, Twist also binds to characterized Dorsal Type III enhancers known to regulate dpp, ind, and ths. Dorsal and its associated corepressors Cut, Retained, and Capicua recruit Groucho to repress dpp, confining its expression to the dorsal blastoderm. The cobinding of Twist and Dorsal to Type III CRMs suggests that these factors may also collaborate in transcriptional repression. Interestingly, Twist binds to regulatory regions of all three Dorsal corepressors, providing another level at which Twist may modulate Dorsal-mediated repression. Overall, this exhaustive map of new CRMs for D-V patterning genes greatly extends previous knowledge and will likely improve predictive models for this system (Sandmann, 2007).
Twist is not only required for D-V patterning. The 494 direct target genes are significantly enriched in functional groups of genes involved in cell communication, signal transduction, cell motility, and cell adhesion. Genes in these categories are essential for multiple aspects of development, including gastrulation and directed migration of mesodermal cells. Genetic studies have demonstrated a requirement for twist in these processes; however, the molecular mechanism remained ill-defined. These data reveals Twist binding to CRMs for entire functional modules necessary for both gastrulation and migration (the FGF pathway) (Sandmann, 2007).
The present study highlights a new direct connection between Twist and many key components involved in cell cycle progression and cell growth. Members of both the Cdk2/CyclinA/B and Cdk2/CyclinE complexes are targeted, as well as modifiers of their activity and genes involved in cytokinesis and replication. In many cases, Twist binds to several CRMs of these genes (e.g., cyclinE and E2f), revealing the complexity of their regulation. This surprising link between Twist and the cell cycle is highly likely to be of regulatory significance; twist mutant embryos have proliferative defects that can be genetically separated from the block in mesoderm gastrulation (Sandmann, 2007).
These three functional groups of target genes (involved in morphogenesis, migration, and cell proliferation) have been defined as essential developmental network 'plug-ins.' Twist orchestrates early mesoderm development by binding to CRMs of virtually all genes within functional groups essential for gastrulation, mesoderm proliferation, migration, and specification. In contrast, few CRMs for genes involved in terminal differentiation (e.g., sarcomere structure) are targeted by Twist (Sandmann, 2007).
This global map of Twist-bound CRMs provides a first glimpse of Twist’s connectivity to the rest of the regulatory genome. Remarkably, TFs represent the largest group of Twist targets: Twist binds to CRMs of a striking 25% (113/454) of all sequence-specific Drosophila TFs. Among these are TFs essential for mesoderm development, including gap (hb, hkb, kr, kni), pair rule (eve, slp, opa, odd, prd, run), and segmentation genes (en, hh, ptc, wg), as well as homeotic genes (pb, Scr, Antp, Abd-A, Abd-B, Ubx). These classes of target genes implicate a new role for Twist in the establishment or maintenance of anterior-posterior patterning within the mesoderm in addition to its known role in D-V axis formation. Although the function of many of the remaining TFs is unknown, this data links these regulators to mesoderm development. The sheer number of TFs regulated by Twist does not support a simple hierarchical network, where Twist regulates a small set of TFs, which in turn control another layer of regulators, and so forth. Rather, the data suggests a model for Twist contributing to the regulation of the majority of TFs involved in every aspect of early mesoderm development (Sandmann, 2007).
Although Twist is expressed during both developmental time periods assayed, it binds to CRMs in a temporally regulated manner. Approximately half of the enhancer regions are detected at both time periods, indicating continuous binding of Twist throughout these developmental stages. In contrast, 23% of Twist-CRMs are only bound in early development (2-4 h), while 26% are specific to later time periods (4-6 h). This dynamic occupancy reveals that the ability of Twist to bind to CRMs is tightly controlled beyond the mere presence of a suitable binding site, and is likely regulated by other TFs that aid or inhibit binding. To identify additional regulators that could differentiate between temporally bound CRMs, a search was performed for overrepresented sequence motifs, using two complementary computational approaches: statistical enrichment of position weight matrices (PWMs) for characterized TFs, and the de novo detection of overrepresented motifs (Sandmann, 2007).
Twist and Snail consensus motifs are significantly overrepresented in all three groups of CRMs, indicating a potential for extensive cobinding between these two TFs. In contrast, Dorsal motifs are exclusively enriched in the early-bound CRMs, and not in the late group. While Tinman motifs are specifically overrepresented in the continuous and late-bound CRMs. A number of other motifs were also uncovered, including sites for potential Twist/Daughterless heterodimers, suggesting additional mechanisms to generate diverse outputs from Twist-CRMs (Sandmann, 2007).
These data reveals Twist binding to almost all previously characterized Dorsal enhancers. Twist and Dorsal are known to interact physically and to coregulate enhancers in the early, but not the late, time window of this experiment. It is therefore hypothesized that Dorsal may be coregulating many of the newly discovered Twist CRMs, in keeping with the specific enrichment of Dorsal consensus motifs within these enhancers. To experimentally test Dorsal's presence on predicted sites in vivo, ChIP experiments were performed at 2-4 h of development. Significant binding of Dorsal was detected by quantitative real-time PCR to all seven predicted sites tested. Similarly, since Tinman consensus sites were significantly enriched in 4-6-h CRMs, the in vivo occupancy of predicted sites by Tinman was tested at this stage of development. ChIP experiments detected significant binding of Tinman to 10 of 11 sites tested. Given the large number of early and late CRMs, the enrichment of these motifs highlights extensive combinatorial binding of Dorsal and Twist at 2-4 h, and Tinman and Twist at 4-6 h. A substantial part of Twist's temporal specificity likely stems from its association with these upstream and downstream coregulators (Sandmann, 2007).
To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).
The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).
Through the integration of ChIP-on-chip analysis with expression profiling data during early stages of Drosophila development, this study has identified >2000 Twist-bound regulatory regions and almost 500 direct target genes. This data, in combination with in vivo binding data for other TFs, lays the foundation of a transcriptional network describing early mesoderm development. The resulting network view reveals regulatory features that form the basis of Twist's functional versatility (Sandmann, 2007).
The data revealed extensive Twist binding to characterized Dorsal enhancers and also, surprisingly, to Dorsal-regulated silencers (e.g., dpp). Moreover, many of the new regulatory regions identified for D-V patterning genes can function either as enhancers or integrated enhancer-silencer modules (e.g., WntD and crumbs). This ability of Twist to act within the context of silencers, as well as enhancers, may partially explain the widespread recruitment of Twist to many regulatory regions and its ability to regulate diverse developmental processes (Sandmann, 2007).
An attractive molecular explanation for this bifunctionality is the potential of Twist to form both homodimers and heterodimers. Twist homodimers drive gene activation in Drosophila, while Twist-Daughterless heterodimers are associated with transcriptional repression. This model is supported by the significant overrepresentation of the Twist/Daughterless heterodimer consensus motif in both 2-4-h and 4-6-h CRMs. Direct protein-protein interactions between Twist and Dorsal is an alternative mechanism for Twist's incorporation into repressive complexes (Sandmann, 2007).
Although the network generated in this study is far from complete, it represents the largest set of combinatorial-bound CRMs during these stages of development described to date, and therefore provides a comprehensive resource to decipher general regulatory principles. The resulting network topology was surprising. Instead of Twist regulating a restricted group of TFs, which in turn regulate a successive wave of transcription in a relay model, Twist directly impinges on CRMs for the vast majority of genes expressed in the early mesoderm (Sandmann, 2007).
The extent of combinatorial binding was also unanticipated. There is extensive cobinding of Twist and Dorsal to early 2-4-h CRMs. In fact, the presence of Dorsal binding may be a general prerequisite for Twist binding to enhancers specific for early development. The cooperative binding of Dorsal and Twist to the rho and sim CRMs supports this model. At 4-6 h of development, the composition of TFs impinging on Twist-bound CRMs changes. Although genome wide ChIP-on-chip data is currently not available for Tinman, the significant overrepresentation of Tinman motifs in Twist-bound CRMs and the ability of Tinman to bind to the majority of sites tested indicates prevalent combinatorial binding between these two TFs during 4-6 h of development. Comparing Twist-bound CRMs with a previously generated data set for Mef2 revealed extensive cobinding to enhancers early in development. Converging regulatory connections through combinatorial binding can produce diverse logical outputs, depending on the nature of the TFs. The cobinding of several pan-mesodermal TFs (Twist, Tinman, and Mef2) may ensure robust gene expression. While in other contexts (for example, the WntD-enhancer) the combined inputs of Twist and Snail allow for spatial fine-tuning of gene expression (Sandmann, 2007).
The core network also revealed an abundance of feed-forward loops, providing directionality during early mesoderm development. This is prevalent with both upstream regulators of Twist (Dorsal and Twist) and downstream regulators (Tinman and Twist and Mef2 and Twist). This network motif will likely become even more widespread as additional ChIP-on-chip data becomes available. Twist targets an astounding number of TFs, which may represent an almost complete repertoire of TFs required for early mesoderm development. It is tempting to speculate that Twist participates in feed-forward regulation, with many of these factors through combinatorial binding to different subsets of the ~2000 Twist-bound CRMs (Sandmann, 2007).
Both the composition and connectivity of regulatory networks describing developmental progression will naturally change over time. To capture dynamic changes within the early mesodermal network, these experiments were performed at consecutive time periods. The data reveals temporally regulated binding of Twist to three classes of CRMs: early, continuous, and late. Similar temporally restricted enhancer occupancy has also been observed for other regulators with broad temporal expression, suggesting that this may be a general feature of developmental networks—e.g., MyoD, PHA-4, and Mef2 (Sandmann, 2007).
The temporal occupancy of specific CRMs by Twist reflects the development of this tissue. At 2-4 h of development, Twist and Dorsal coregulate genes essential for D-V patterning. Twist also targets an almost complete set of genes essential for gastrulation and is required to progress to the next phase of development, mesoderm maturation. During this developmental window, the predominant target genes are part of functional modules essential for the cell migration, proliferation, patterning, and specification events occurring within the mesoderm at these stages. As expected for a TF essential for early aspects of mesoderm development, Twist does not bind to significant numbers of CRMs for genes involved in terminal differentiation. This is in sharp contrast to Mef2, which first co-occupies CRMs involved in early mesoderm development with Twist, and later selectively regulates an alternative group of CRMs driving genes involved in later aspects of differentiation; e.g., sarcomere structure or muscle attachment (Sandmann, 2007).
Integrating these data with genetic evidence from other species suggests that the regulation of several functional gene cassettes by Twist is conserved throughout evolution, from flies to man. These include (1) the FGF signaling pathway: Mutations in human FGF receptors phenocopy mutants in human twist (Htwist). (2) Genes implicated in epithelial-mesenchymal transitions (EMTs): In mice and humans, Twist facilitates tumor metastasis through the promotion of EMTs. (3) Cell proliferation and apoptosis: Htwist has been classified as a potential oncogene, since it maintains tissue culture cells in a proliferative state. Interestingly, ectopic expression of Htwist in Drosophila also induces proliferation and inhibits p53-dependent apoptosis, indicating that the ability to regulate these processes is conserved. However, for each process, only a few Twist-regulated genes have been known. Extrapolating from the current findings in flies points toward a role for Twist in the direct regulation of entire gene modules required for each process in vertebrates (Sandmann, 2007).
The results provide an initial global view of the transcriptional network describing early mesoderm development within the metazoan Drosophila. Twist resides at the top of this network and binds to CRMs for the vast majority of genes that need to be expressed during these stages. In many cases, Twist is essential and sufficient to drive expression of the target gene. In other cases, however, the contribution of Twist remains unclear (e.g., crumbs and NetA) . Rather than acting as a binary switch, Twist may act redundantly with other TFs. Alternatively, Twist may provide the competence for more specific TFs to bind to these CRMs; for example, by acting as a pioneer TF to facilitate chromatin remodeling (Sandmann, 2007).
In species as diverse as flies, jellyfish, and mice, Twist is only expressed in mesodermal cells when they are in an immature state, and loss of twist expression correlates with the initiation of differentiation. Moreover, overexpression of Twist-1 in mice is sufficient to block osteoblast differentiation. It is suggested that Twist provides the mesoderm with the competence to be pluripotent: first, by providing these cells with the components necessary to respond to inductive cues directing further specification; and second, by providing an almost universal repertoire of mesodermal CRMs with the competence to respond to other TFs. Once bound by Twist, these regulatory regions may be primed for activation by more specialized TFs, and thereby allow rapid developmental progression at the appropriate time (Sandmann, 2007).
In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by
enhancers that direct the combinatorial binding and/or interaction of Mef2 and the bHLH MyoD
family of myogenic factors. Drosophila Mef2 plays a role similar to its vertebrate
counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic
muscles, however, unlike vertebrates, Drosophila Mef2 interacts with a muscle activator region that
does not have binding sites for myogenic bHLH-like factors or any other known Drosophila
transcription factors. This study describes the isolation and characterization of a component of the muscle
activator region named PDP1 (PAR-domain protein 1). PDP1 is a novel transcription
factor, highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF,
DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be
identified in Drosophila. PDP1 is involved in regulating expression of the Tropomyosin I
gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle
activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate
muscle activator function and severely reduce expression of a muscle activator plus Mef2
mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger
protein/DNA complex that interacts with Mef2 to regulate transcription of Drosophila muscle genes.
In addition to being expressed in the mesoderm that gives rise to the somatic muscles,
PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and
Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also
involved in the terminal differentiation of these tissues (S. C. Lin, 1997a).
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Myocyte enhancer factor 2:
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