BACKGROUND
studies over the last two decades have revealed that cis-regulatory elements, i.e.
enhancers, contain multiple dna-binding sites for different transcription factors  that cooperatively function to direct the tissue specific expression of their associated genes  <cit> .
dna sequence comparisons of different co-regulating enhancers suggest that many of these enhancers rely on different combinations of tfs to achieve coordinate gene regulation  <cit> .
for example, during early drosophila neural development, combinatorial interaction of proneural basic helix-loop-helix  tfs with homeodomain proteins, regulate commitment and patterning of neural precursors  <cit> .
cross-species analysis of individual drosophila enhancers, using evoprinter or conventional alignment based phylogenetic comparative analysis  <cit>  and the twelve sequenced drosophila genomes, representing over  <dig> million years of collective evolutionary divergence, reveals that these enhancers are made up of clusters of highly conserved sequence blocks , separated by less conserved sequences of variable length  <cit> .
csbs that are longer than 8– <dig> bp are likely to be made up of adjacent or overlapping dna-binding sites for different tfs.
for example, the drosophila krüppel central domain enhancer contains overlapping highly conserved binding sites for its known regulators  <cit> .
specifically, work from the jäckle laboratory  <cit>  has shown that one csb of the central domain enhancer,  <dig> base pairs in length, contains overlapping binding sites for the antagonistic bicoid activator and the knirps repressor tfs.
in order to initiate the functional dissection of csbs that make up neural precursor gene enhancers and to gain a better understanding of their architecture in terms of the substructure of their constituent sequence elements, we have developed a multi-step protocol  that allows for the rapid identification of short  <dig> to  <dig> bp dna elements, termed cis-decoder tags , within enhancer csbs; these cdts are shared between csbs of two or more enhancers with either related or divergent functions  <cit> .
to discover enhancer type-specific elements that regulate gene expression in neural precursor cells – including genes expressed in early delaminating cns neuroblasts  and the proneural clusters and sensory organ precursors of the pns – we have performed cis-decoder analysis of csbs from in vivo characterized enhancers.
for early cns development, we have selected the previously described enhancers of six genes that activate expression in early delaminating cns nbs: deadpan , hunchback , nerfin- <dig>  scratch , snail  and worniu    <cit> .
for the cis-regulatory regions that drive expression in the proneural clusters  and sensory organ precursors  of the pns we selected the in vivo characterized enhancers for bearded , deadpan , rhomboid , scrt and sna   <cit> .
*relative to transcription start site
our analysis of the csbs from these characterized enhancers has identified known tf dna-binding sites and novel sequences of as yet unknown function.
enhancer type-specific sequence elements within csbs appear in different combinations and contexts in enhancers of co-regulated genes.
the information gained from cis-decoder analysis of the neural precursor cell enhancer csbs was used to discover a novel co-regulating enhancer that directs drosophila nervy expression.
our studies indicate that although specific core dna-binding sites  are enriched in enhancers of co-regulated genes, enhancer-binding specificity is most likely conferred through sequences that flank the consensus core docking sites.
the fact that shared sequence elements of co-regulated enhancers reside in different combinations and positional ordering within each of the enhancers, suggests that their combined presence but not necessarily their relative positions is required for cis-regulatory function.
RESULTS
neural precursor cell enhancers share highly conserved core sequence elements
to determine the extent to which neural precursor cell enhancers share highly conserved sequence elements, we performed cis-decoder analysis of in vivo characterized enhancers   <cit> .
our analysis revealed the presence of both novel elements and sequences that contained consensus dna-binding sites for known regulators of early neurogenesis.
table  <dig> lists cdts shared by multiple cns or pns neural precursor cell enhancers.
none of the elements shown were present in our collection of  <dig> csbs from in vivo characterized mesodermal enhancers, thus ensuring their enrichment in neural enhancers.
highlighted are consensus binding sites for known tfs; basic helix-loop helix  factors and suppressor of hairless , respectively acting in proneural and neurogenic pathways  <cit> ; antennapedia class homeodomain proteins  <cit> , identified by their core atta binding sequence, and the ubiquitously expressed pbx-  class homeodomain protein extradenticle, a cofactor of many tfs  <cit> , identified by the core binding sequence of atca.
more than half the conserved cdts were novel, without identified interacting proteins.
many of the csbs consisted of  <dig> or more bp, and often contained core sequences identical to binding sites for known factors as well as other core sequences that aligned with shorter novel cdts, suggesting that the longer cdts may contain core recognition sequences for two or more tfs.
consensus binding sites for bhlh transcription factors , antp class homeodomain proteins , pbx/extradenticle , tramtrack  and su  are bold.
most cdts discovered in this analysis represent elements that are shared pairwise, i.e., by only two of the nb enhancers examined .
the fact that the majority of cdts are shared two ways, with only a small subset of sequences being shared three or more ways, suggests that the cis-regulation of early neural precursor genes is carried out by a large number of factors acting combinatorially and/or that many of the identified cdts may in fact represent interlocking sites for multiple factors, and the exact orientation and spacing of these sites may differ among enhancers.
neural specific cdts that contain bhlh tf dna-binding sites
during drosophila neurogenesis, bhlh proteins function as proneural tfs to initiate neurogenesis in both the central and peripheral nervous system.
tfs encoded by the achaete-scute complex function in both systems, while the related atonal bhlh protein functions exclusively in the pns  <cit> .
different proneural bhlh tfs, acting together with the ubiquitous dimerization partner daughterless, bind to distinct e-boxes that contain different core sequences  <cit> .
in addition to the core recognition sequence, flanking bases are important to the dna binding specificity of bhlh factors  <cit> .
one of the principle observations of this study was that the core central two bases of the hexameric e-box dna-binding site  were conserved in all the species used to generate the evoprint.
all of the enhancers included in this study contained one or more conserved bhlh-binding sites , with nb and pns enhancers averaging  <dig>  and  <dig>  binding sites respectively.
more than a third of the core bases in nb bhlh sites contained a core gc sequence, and more than a third of the core bases in pns bhlh sites contained either a core gc or a gg sequence.
the most common e-box among the nb csbs was cagctg with  <dig> sites in four of the six enhancers.
the cagctg and caggtg e-boxes are high-affinity sites for achaete/scute bhlh proteins  <cit> .
however the cagctg site itself is not specific to nb enhancers, as evidenced by its presence in four of the mesodermal enhancer csbs characterized previously  <cit> .
the most common bhlh-binding site among pns enhancers was also the cagctg e-box with  <dig> occurrences in six of the  <dig> enhancers.
in contrast, the most common bhlh motif in enhancers of the e-complex  <cit>  was caagtg , with  <dig> occurrences in  <dig> of the  <dig> enhancers.
caggtg, previously shown to be an atonal dna-binding site  <cit> , was also common in e enhancers, with  <dig> occurrences in  <dig> of the  <dig> enhancers, but was less prevalent among nb enhancers.
the caggtg box was also overrepresented in pns and e enhancers relative to its appearance in nb enhancers, and it was also present in four of the characterized mesodermal enhancer csbs.
the cagatg box was present six times among pns enhancers but not at all among nb enhancers.
thus there appears to be some specificity of e-boxes in the different enhancer types.
the fact that each of these e-boxes is conserved in all the species in the analysis, suggests that there is a high degree of specificity conferred by the e-box core sequence.
our analysis also reveals that not only are the core bases of e-boxes shared between similarly regulated enhancers, but bases flanking the e-box were also found to be highly conserved and are also frequently shared by these enhancers.
among the e-boxes found in csbs of nb enhancers  aacagctg  is repeated three times in nerfin- <dig> and once in scrt; gcacttg is repeated three times in scrt; cagctgca is repeated twice in wor, and cagctgctg is repeated twice in scrt .
in the dpn cns nb enhancer, the e-box cagctg is found twice, separated by a single base .
none of these sequences were present in mesodermal enhancers examined, but each is found in pns enhancers; cagctgca is repeated multiple times among pns enhancers.
among the conserved pns enhancer e-boxes  each, including flanking bases, is found in two or three pns enhancers, and these are distributed among all  <dig> enhancers.
of these, only agcacgtgcc, cagctgccg, ccaggtga were found once in our sample of neuroblast enhancers and none were found in our sample of mesodermal enhancers.
the sequence aacaagtg is found in  <dig> e complex enhancers, those for em <dig>  mγ, hlhmδ and m <dig>  and the sequence acagctgc is found twice in em <dig> and once in m <dig> and m6; neither sequence was found in our mesodermal enhancers.
therefore, although a given hexameric sequence may often be shared by all three types of enhancers, nb, pns and e, when flanking bases are taken into account there appears to be enhancer type-specific enrichment for different e-boxes.
neural specific cdts that contain antennapedia class homeodomain dna-binding sites
antennapedia class homeodomain proteins play essential roles in multiple aspects of neural development including cell proliferation and cell identity  <cit> .
the segmental identity of drosophila nbs is conferred by input from tfs encoded by homeotic loci of the antennapedia and bithorax complexes  <cit> .
for example, ectopic expression of abd-a, which specifies the nb6-4a lineage, down-regulates levels of the g <dig> cyclin, cyce  <cit> .
loss of polycomb group factors has been shown to lead to aberrant derepression of posterior hox gene expression in postembryonic nbs, which causes nb death and termination of proliferation in the mutant clones  <cit> .
we have examined the enhancer-type specificity of sequences flanking the antennapedia class core dna-binding sequence, atta  <cit> .
nearly 25% of the nb and pns csbs examined in this study contain this core recognition sequence.
atta-containing sites were found multiple times in selected nb and pns enhancers .
the cis-decoder analysis identified  <dig> different neural specific atta containing cdts that were exclusively shared by two or more pns enhancers or cns enhancers and  <dig> were found to be shared between pns and cns.
the most common cdt, attagca, was shared by two cns and two pns enhancers .
in addition,  <dig> homeodomain-binding site cdts were found twice in wor csbs, aattaccg, tttgaatta, aatcaatta, attaatctt and aaacaaattag, but not in other cns or pns enhancer csbs.
in some cases these cdts were found repeated in given enhancer csbs.
only one of these cdts aligned with csbs of enhancers of the e complex.
given that 2/ <dig> of the occurrences of hox sites in these promoters can be accounted for by cdts whose flanking sequences are shared between enhancers, it is unlikely that the appearance of these shared sequences occurs by chance.
in summary, the appearance of hox sites in the context of conserved sequences shared by functionally related enhancers suggests that the specificity of consensus homeodomain-binding sites is conferred by adjacent bases, either through recognition of adjacent bases by the tf itself or in conjunction with one or more co-factors.
neural specific cdts that contain pbx/extradenticle sites
examination of the cdts from drosophila nb and pns enhancers revealed that many contained the core pbx/extradenticle docking site atga  <cit> .
in drosophila, extradenticle has been shown to have hox-dependent and independent functions  <cit> .
studies have also shown that pbx factors provide dna-binding specificity for homeodomain tfs, facilitating specification of distinct structures along the body axis  <cit> .
in the cns enhancers of drosophila, most predicted pbx/extradenticle sites are not, however, found adjacent to hox sites.
our analysis revealed that  <dig> of the pbx motifs were shared between cns and pns enhancer types, and  <dig> were shared between similarly expressed enhancers , thus indicating that there appears to be some degree of specificity to pbx site function when flanking bases are taken into account.
three of the pbx binding-site containing elements also exhibit atta hox sites: 1) the dodecamer gatgattaatct  shared by the pns enhancers edl and amos , contains a homeodomain atta site that overlaps the pbx site by a single base, and 2) the smaller heptamer atgatta, shared by pfe and ato, likewise contains a homeodomain atta site  that overlaps atga pbx site by a single base.
adjacent hox and pbx sites have been documented to facilitate synergy between the two factors  <cit> .
taken together our findings suggest that, as with homeodomain-binding sites, the conserved bases flanking putative pbx sites are functionally important.
these flanking bases are likely to confer different dna-binding affinities for pbx factors or are required for binding of other tfs.
neural specific cdts that contain suppressor of hairless binding sites
also indicating a degree of biological specificity of enhancer types is the distribution of suppressor of hairless su binding sites among neural enhancers.
su is the notch pathway effector tf of drosophila  <cit> .
the members of the e complex, both the multiple basic helix-loop-helix  repressor genes and the bearded family members, have been shown to be su dependent  <cit> .
the consensus in vitro dna binding site for su is rtgrgar   <cit> .
notch signaling via su occurs through conserved single or paired sites  <cit>  and the presence of conserved sites for other transcription regulators associated with csbs containing su binding sites has been documented  <cit> .
within the csbs of the six nb enhancers examined, only two, dpn and wor, contained conserved putative su-binding sites; two dpn sites matched one of the su consensus sites  and two wor sites match the sequence atgggaa.
only one of the two dpn sites contained flanking bases conforming to the widely distributed cgtgggaa site of e su binding sites and none of the nb enhancers contained paired su sites typical of the e enhancers  <cit> .
of the  <dig> pns cis-regulatory regions examined, only four enhancers contained putative su-binding sites  and dpn .
dpn also contained a pair of sites that conforms to the sps configuration frequently found in su enhancers  sites in bold, pbx site is atca).
the lack of su sites in pns enhancers was noted by reeves and posakony  <cit> , who suggested that these enhancers are directly regulated by the proneural proteins but not activated in response to notch-mediated lateral inhibitory signaling.
among the conserved sequences of e gene enhancers there is an average of  <dig>  consensus su binding sites per enhancer, with most enhancers containing both types of sites, i.e., those with either a or g in the central position .
we offer three insights with respect to su binding sites.
first, although in vitro dna-binding studies suggest there is a flexibility in the su binding site, like the bhlh e-box, comparative analysis shows that within any one the su sites there is no sequence flexibility.
except for the pair of su sites in the dpn pns enhancer, none of the cns or pns sites contained a central a; less that a quarter of the e sites consisted of a central a, and all these were conserved across all species examined.
in light of the high conservation in these regions the invariant core and flanking sequences are important for the unique su function at any particular site.
a second finding was the extensive conservation of bases flanking the consensus su sequence in the e complex genes .
for example, the cdt gtgggaaacacacgac  was present in hlhm <dig> and hlhm <dig> enhancer csbs, and accgtgggaaac was conserved in hlhm <dig> and hlhmβ enhancers.
the conservation of bases flanking the consensus su binding site suggests that the su site may be flanked by additional binding sites for co-operative or competitive factors, or else, that su contacts additional bases besides the consensus heptamer.
a third observation is that in most cases su binding sites are imbedded in larger csbs, suggesting that csb function is regulated by the integrated function of multiple tfs.
for example the dpn nb enhancer su site is imbedded in a csb of  <dig> bases, and the atonal pns enhancer su site is imbedded in a csb of  <dig> bases.
in the e complex, csb # <dig> of hlhmγ, consisting of  <dig> bases and csb# <dig> of m <dig>  consisting of  <dig> bases  site, a cacgag element, conforming to a hairy n-box consensus cacnag  <cit> , and an agga tramtrack  dna-binding core recognition sequence  <cit> , but the order and context of these three sites is different for each enhancer).
although su binding sites were present in only a minority of nb and pns enhancers, the conservation of core bases, as well as the complexity of their flanking conserved sequences points to a diversity of su function and interaction with other factors.
neural specific cdts that contain core dna-binding sites for other known tfs
two of these elements, one exclusively present in nb enhancers  and a second exclusively present in pns enhancers , contained consensus core agga dna-binding sites for ttk  <cit> , a btb domain tf that has been shown to regulate pair rule genes during segmentation and to repress neural cell fates  <cit> .
another site , shared by both nb and pns enhancers, conforms to the consensus binding site of ia- <dig> , the vertebrate homolog of nerfin- <dig>  <cit> .
most of the cdts of table  <dig> do not contain sequences corresponding to consensus binding-sites of known regulators of nb expression.
the fact that they are represented multiple times in nb csb sequences suggests that they contain binding sites for unknown regulators of neurogenesis in drosophila.
neural-enriched cdts
neural enriched cdts that are shared between multiple nb enhancers and also exhibit a low frequency in the sample of mesodermal enhancers examined in this study serve as a resource for understanding enhancer elements that may not have an exclusive neural function .
notable here is the presence of cagctg bhlh dna binding sites  and antennapedia class homeobox  core dna binding site atta  <cit> , as well as additional ttk and pbx/extradenticle sites.
present in this list are portions of sequences conforming to su binding sites described above.
of particular interest in this table are sequences that are also enriched in the pns ; these sites may bind factors that play similar developmental roles in different tissues.
for example, the presumptive ttk site, aaagga  is highly enriched in segmental enhancers.
thus, some of these sites can be identified as targets of known tfs, but the identity of most are as yet unknown.
these elements shared by multiple enhancers may be useful in identifying other enhancers driving expression in nbs.
cis-decoder analysis reveals a complex sub-structure of enhancer csbs
evoprint analysis revealed that all of the enhancer regions examined in this study contained multiple csbs that were greater that  <dig> to  <dig> bases in length.
the occurrence of overlapping dna-binding sites for different tfs is currently the best explanation for the maintenance of intact csb sequences across ~ <dig> millions of years of collective species divergence.
our analysis has revealed that the sequence context, order and orientation of shared cdts can differ between co-regulating enhancers.
two examples are given here of the complex contextual appearance of cdts that appear frequently in cns and pns enhancers .
each of the eight csbs shown was nearly fully 'covered' by cdts of the nb library , suggesting that each contains multiple overlapping binding sites for a number of tfs.
first, examination of the distribution of cdt gctgca reveals that it overlaps, by one and two bases, adjacent but different consensus bhlh sites in scrt csb# <dig>  while in scrt csb# <dig> it overlaps a third consensus bhlh sequence by two bases.
in the pns enhancer char, in csb# <dig>  gctgca overlaps a bhlh site, but in a different configuration  than found in the two cns enhancers illustrated in figure 3a.
in amos csb# <dig>  gctgca appears adjacent to a hox site and does not overlap a bhlh site.
second, examination of the distribution of the cdt ggcacg reveals that it overlaps different consensus bhlh sites in scrt csb# <dig> and wor csb# <dig>  overlapping the bhlh site in the former by one base and in the latter by four bases.
ggcacg overlaps a cagctg bhlh-binding site in rho csb# <dig>  but in a different configuration than the overlap with cagctg in the wor csb.
in the pns enhancer scrt, ggcacg in csb# <dig> overlaps a hairy site n-box   <cit> .
n-boxes were most common in e csbs, but were also present in nb and pns enhancer csbs.
in these two examples, and others we have examined, there is no consistent spatial constraints to the association of known tf-binding sites  with novel cdts; a picture that emerges is one of combinatorial complexity, in which known or novel cdts are associated with each other in different contexts on different csbs.
as an initial step toward determining if different tfs interacted with one another or competed for flanking dna-binding sites, we examined the proximity of known binding sites to one another in csbs for bhlh, hox, pbx and su.
the results of this analysis for nb csbs are shown in table 4; data for other enhancer types is summarized here.
most striking was the presence of multiple adjacent hox atta sites  and combinations of hox and pbx sites .
a typical example is the association of one pbx site, a bhlh site and two hox sites on a wor nb enhancer csb .
associations of hox and pbx sites was also apparent in pns enhancer csbs, and in addition there was a high level of combined hox and bhlh sites , but in e enhancers only a higher level of the combination of hox and pbx sites  was apparent.
an example of the association of hox and bhlh sites in a pns enhancer is found in an achaete-scute dorso-central enhancer csb .
there was also a distinctly higher level of pbx sites on the same csbs as bhlh sites in nbs csbs , but this combination was not apparent for pns or e csbs.
association of bhlh sites with su binding sites was apparent in e enhancer csbs, especially when presence on adjacent csbs  was taken into account.
only in one of the  <dig> instances of paired su sites on e enhancers were these sites on the same csbs, while in four other instances they were on adjacent csbs.
although we often find sites in close proximity, both known and functionally uncharacterized sites are, with a few exceptions, not present in fixed uniform orientation in similarly regulated enhancers.
this highlights the complex combinatorial arrangement and position flexibility of tf-binding sites within enhancer csbs.
*first and second numbers represent appearance on same and adjacent csbs, respectively.
the use of cis-decoder, flyenhancer and evoprinter to identify novel enhancers
we have used the information derived from cis-decoder analysis of neural precursor cell enhancers to search for other genomic sequences with similar cis-regulatory properties.
having identified cdts found multiple times among nb enhancers, we used the genomic search tool flyenhancer  <cit>  to identify drosophila melanogaster genomic sequences that contained clusters of the following cdts : ggcacg , ggaatc , tgacag , tggggt , cagctg , tgattt  caagtg , catattt , tgatcc  and ctaagc .
as a lower limit, a minimum of three cagctg bhlh sites was set for this search, because of the prevalence of this site in nerfin- <dig> and deadpan nb enhancers.
each sequence detected by this search was subjected to evoprinter analysis to determine the extent of its sequence conservation.
among the cdt clusters identified, our search identified a 5' region adjacent to the nervy gene .
nervy, originally identified as a target of homeotic gene regulation, is expressed in a subset of early cns nbs, as well as in pns sop cells  <cit> .
later studies have implicated nervy, along with cyclic adenosine monophosphate -dependent protein kinase  in antagonizing sema-1a-plexa-mediated axonal repulsion  <cit> , and nervy has been shown to promote mechanosensory organ development by enhancing notch signaling  <cit> .
evoprinter analysis revealed that the cluster of neural precursor cell enhancer cdts positioned  <dig> bp upstream from the nervy transcribed sequence contains highly conserved sequences .
this region contains  <dig> csbs that include six conserved e-boxes, three of which conform to the cagctg sequence that was prominent in nerfin- <dig> and deadpan promoters.
to determine if this region functions as a neural precursor cell enhancer, we generated transformant lines containing the nervy csb cluster linked to a minimal promoter/gfp reporter transgene .
our analysis of the reporter expression driven by the nervy upstream fragment revealed a pattern indistinguishable from early nervy mrna expression  <cit>  .
specifically, we detected expression in a large subset of early delaminating nbs and in sops and secondary precursor cells of the pns.
significantly, the nervy enhancer, unlike nerfin- <dig> and deadpan nb enhancers, activates reporter expression in then pns and not just in early nbs.
a new cdt-library was generated combining the nervy enhancer csbs and the nb and pns enhancer csbs used to generate the libraries described above.
the new cdts, along with the previously defined cdts were aligned back to nervy csbs .
most cdts were found only once in previously examined nb or pns csbs, but  <dig> cdts appeared in our original analysis, described above, that did not include the nervy enhancer.
the addition of this new enhancer to our analysis resulted in the discovery of a significant number of cdts that had not been found previously.
three cdts that were identified in the previous analysis, tcagctgc, cagcagctg and aacagctg, contain bhlh dna-binding sites .
aligning cdts that are specific to the cns or pns may indicate sequences required to specifically drive expression in either the cns or pns.
CONCLUSIONS
the major finding of this study is that enhancers of co-regulated genes in neural precursor cells possess complex combinatorial arrangements of highly conserved cdt elements.
comparisons between nb and pns enhancers identified cns and pns type-specific cdts and cdts that were enriched in one or another enhancer type.
cis-decoder analysis also revealed that many of the conserved sequences contain dna-binding sites for classical regulators of neurogenesis, including bhlh, hox, pbx, and su factors.
although in vitro dna-binding studies have shown that many of these factors have a certain degree of flexibility in the sequences to which they bind, defined in terms of a position weight matrix  <cit> , our studies show that for any given appearance these sites are actually highly conserved across all species of the drosophila genus.
the genus invariant conservation in many of these characterized binding sites indicates that there are distinct constraints to that sequence in terms of its function.
the high degree of conservation displayed in the enhancer csbs could derive from unique sequence requirements of individual tfs, or the intertwined nature of multiple dna-binding sites for different tfs.
thus there is a higher degree of biological specificity to these sites than the flexibility that is detected using in vitro dna-binding studies.
as an example, the requirement for a specific core for the bhlh binding site, i.e., for a cagctg e-box for nerfin- <dig>  deadpan and nervy, suggests that it is the tf itself that demands sequence conservation; however, the requirement for conserved flanking sequences suggests that additional specific factors may be involved.
although the inter-species conservation of core and flanking sites has been noted by others  <cit> , the extent of this conservation is rather surprising.
to what extent and how evolutionary changes in enhancer function take place, given the conservation of core enhancer sequences, remains a question for future investigation.
in addition to classic regulators of neurogenesis, cis-decoder reveals additional conserved novel elements that are widely distributed or only detected in pairs of enhancers.
many of these novel elements flank known transcription binding motifs in one csb, but appear independent of known motifs in another.
the appearance of novel elements in multiple contexts suggests that they may represent dna-binding sites for additional factors that are essential for enhancer function.
only through discovery of the factors binding these sequences will it become clear what role they play in enhancer function.
preliminary functional analysis of csbs within the nerfin- <dig> neuroblast enhancer reveals that csbs carry out different regulatory roles .
altering cdt sequences within the nerfin- <dig> csbs reveals that most are required for cell-specific activation or repression or for normal enhancer expression levels.
csb swapping studies reveals that, for the most part, the order and arrangement of a number of tested csbs was not important for enhancer function in reporter studies.
the discovery of the nervy neural enhancer by searching the genome with commonly occurring nb cdts underscores the potential use of evoprinter and cis-decoder analysis for the identification of additional neural enhancers.
by starting with known enhancers and building cdt libraries from their csbs, one now has the ability to search for other genes expressed during any biological event.

 studies over the last two decades have revealed that cis-regulatory elements, i.e. enhancers, contain multiple dna-binding sites for different transcription factors that cooperatively function to direct the tissue specific expression of their associated genes. dna sequence comparisons of different co-regulating enhancers suggest that many of these enhancers rely on different combinations of tfs to achieve coordinate gene regulation. for example, during early drosophila neural development, combinatorial interaction of proneural basic helix-loop-helix tfs with homeodomain proteins, regulate commitment and patterning of neural precursors. cross-species analysis of individual drosophila enhancers, using evoprinter or conventional alignment based phylogenetic comparative analysis and the twelve sequenced drosophila genomes, representing over million years of collective evolutionary divergence, reveals that these enhancers are made up of clusters of highly conserved sequence blocks, separated by less conserved sequences of variable length. csbs that are longer than 8– bp are likely to be made up of adjacent or overlapping dna-binding sites for different tfs. for example, the drosophila krüppel central domain enhancer contains overlapping highly conserved binding sites for its known regulators. specifically, work from the jäckle laboratory has shown that one csb of the central domain enhancer, base pairs in length, contains overlapping binding sites for the antagonistic bicoid activator and the knirps repressor tfs. in order to initiate the functional dissection of csbs that make up neural precursor gene enhancers and to gain a better understanding of their architecture in terms of the substructure of their constituent sequence elements, we have developed a multi-step protocol that allows for the rapid identification of short to bp dna elements, termed cis-decoder tags, within enhancer csbs; these cdts are shared between csbs of two or more enhancers with either related or divergent functions. to discover enhancer type-specific elements that regulate gene expression in neural precursor cells – including genes expressed in early delaminating cns neuroblasts and the proneural clusters and sensory organ precursors of the pns – we have performed cis-decoder analysis of csbs from in vivo characterized enhancers. for early cns development, we have selected the previously described enhancers of six genes that activate expression in early delaminating cns nbs: deadpan, hunchback, nerfin- scratch, snail and worniu. for the cis-regulatory regions that drive expression in the proneural clusters and sensory organ precursors of the pns we selected the in vivo characterized enhancers for bearded, deadpan, rhomboid, scrt and sna. *relative to transcription start site our analysis of the csbs from these characterized enhancers has identified known tf dna-binding sites and novel sequences of as yet unknown function. enhancer type-specific sequence elements within csbs appear in different combinations and contexts in enhancers of co-regulated genes. the information gained from cis-decoder analysis of the neural precursor cell enhancer csbs was used to discover a novel co-regulating enhancer that directs drosophila nervy expression. our studies indicate that although specific core dna-binding sites are enriched in enhancers of co-regulated genes, enhancer-binding specificity is most likely conferred through sequences that flank the consensus core docking sites. the fact that shared sequence elements of co-regulated enhancers reside in different combinations and positional ordering within each of the enhancers, suggests that their combined presence but not necessarily their relative positions is required for cis-regulatory function.  neural precursor cell enhancers share highly conserved core sequence elements to determine the extent to which neural precursor cell enhancers share highly conserved sequence elements, we performed cis-decoder analysis of in vivo characterized enhancers. our analysis revealed the presence of both novel elements and sequences that contained consensus dna-binding sites for known regulators of early neurogenesis. table lists cdts shared by multiple cns or pns neural precursor cell enhancers. none of the elements shown were present in our collection of csbs from in vivo characterized mesodermal enhancers, thus ensuring their enrichment in neural enhancers. highlighted are consensus binding sites for known tfs; basic helix-loop helix factors and suppressor of hairless, respectively acting in proneural and neurogenic pathways; antennapedia class homeodomain proteins, identified by their core atta binding sequence, and the ubiquitously expressed pbx- class homeodomain protein extradenticle, a cofactor of many tfs, identified by the core binding sequence of atca. more than half the conserved cdts were novel, without identified interacting proteins. many of the csbs consisted of or more bp, and often contained core sequences identical to binding sites for known factors as well as other core sequences that aligned with shorter novel cdts, suggesting that the longer cdts may contain core recognition sequences for two or more tfs. consensus binding sites for bhlh transcription factors, antp class homeodomain proteins, pbx/extradenticle, tramtrack and su are bold. most cdts discovered in this analysis represent elements that are shared pairwise, i.e., by only two of the nb enhancers examined. the fact that the majority of cdts are shared two ways, with only a small subset of sequences being shared three or more ways, suggests that the cis-regulation of early neural precursor genes is carried out by a large number of factors acting combinatorially and/or that many of the identified cdts may in fact represent interlocking sites for multiple factors, and the exact orientation and spacing of these sites may differ among enhancers. neural specific cdts that contain bhlh tf dna-binding sites during drosophila neurogenesis, bhlh proteins function as proneural tfs to initiate neurogenesis in both the central and peripheral nervous system. tfs encoded by the achaete-scute complex function in both systems, while the related atonal bhlh protein functions exclusively in the pns. different proneural bhlh tfs, acting together with the ubiquitous dimerization partner daughterless, bind to distinct e-boxes that contain different core sequences. in addition to the core recognition sequence, flanking bases are important to the dna binding specificity of bhlh factors. one of the principle observations of this study was that the core central two bases of the hexameric e-box dna-binding site were conserved in all the species used to generate the evoprint. all of the enhancers included in this study contained one or more conserved bhlh-binding sites, with nb and pns enhancers averaging and binding sites respectively. more than a third of the core bases in nb bhlh sites contained a core gc sequence, and more than a third of the core bases in pns bhlh sites contained either a core gc or a gg sequence. the most common e-box among the nb csbs was cagctg with sites in four of the six enhancers. the cagctg and caggtg e-boxes are high-affinity sites for achaete/scute bhlh proteins. however the cagctg site itself is not specific to nb enhancers, as evidenced by its presence in four of the mesodermal enhancer csbs characterized previously. the most common bhlh-binding site among pns enhancers was also the cagctg e-box with occurrences in six of the enhancers. in contrast, the most common bhlh motif in enhancers of the e-complex was caagtg, with occurrences in of the enhancers. caggtg, previously shown to be an atonal dna-binding site, was also common in e enhancers, with occurrences in of the enhancers, but was less prevalent among nb enhancers. the caggtg box was also overrepresented in pns and e enhancers relative to its appearance in nb enhancers, and it was also present in four of the characterized mesodermal enhancer csbs. the cagatg box was present six times among pns enhancers but not at all among nb enhancers. thus there appears to be some specificity of e-boxes in the different enhancer types. the fact that each of these e-boxes is conserved in all the species in the analysis, suggests that there is a high degree of specificity conferred by the e-box core sequence. our analysis also reveals that not only are the core bases of e-boxes shared between similarly regulated enhancers, but bases flanking the e-box were also found to be highly conserved and are also frequently shared by these enhancers. among the e-boxes found in csbs of nb enhancers aacagctg is repeated three times in nerfin- and once in scrt; gcacttg is repeated three times in scrt; cagctgca is repeated twice in wor, and cagctgctg is repeated twice in scrt. in the dpn cns nb enhancer, the e-box cagctg is found twice, separated by a single base. none of these sequences were present in mesodermal enhancers examined, but each is found in pns enhancers; cagctgca is repeated multiple times among pns enhancers. among the conserved pns enhancer e-boxes each, including flanking bases, is found in two or three pns enhancers, and these are distributed among all enhancers. of these, only agcacgtgcc, cagctgccg, ccaggtga were found once in our sample of neuroblast enhancers and none were found in our sample of mesodermal enhancers. the sequence aacaagtg is found in e complex enhancers, those for em mγ, hlhmδ and m and the sequence acagctgc is found twice in em and once in m and m6; neither sequence was found in our mesodermal enhancers. therefore, although a given hexameric sequence may often be shared by all three types of enhancers, nb, pns and e, when flanking bases are taken into account there appears to be enhancer type-specific enrichment for different e-boxes. neural specific cdts that contain antennapedia class homeodomain dna-binding sites antennapedia class homeodomain proteins play essential roles in multiple aspects of neural development including cell proliferation and cell identity. the segmental identity of drosophila nbs is conferred by input from tfs encoded by homeotic loci of the antennapedia and bithorax complexes. for example, ectopic expression of abd-a, which specifies the nb6-4a lineage, down-regulates levels of the g cyclin, cyce. loss of polycomb group factors has been shown to lead to aberrant derepression of posterior hox gene expression in postembryonic nbs, which causes nb death and termination of proliferation in the mutant clones. we have examined the enhancer-type specificity of sequences flanking the antennapedia class core dna-binding sequence, atta. nearly 25% of the nb and pns csbs examined in this study contain this core recognition sequence. atta-containing sites were found multiple times in selected nb and pns enhancers. the cis-decoder analysis identified different neural specific atta containing cdts that were exclusively shared by two or more pns enhancers or cns enhancers and were found to be shared between pns and cns. the most common cdt, attagca, was shared by two cns and two pns enhancers. in addition, homeodomain-binding site cdts were found twice in wor csbs, aattaccg, tttgaatta, aatcaatta, attaatctt and aaacaaattag, but not in other cns or pns enhancer csbs. in some cases these cdts were found repeated in given enhancer csbs. only one of these cdts aligned with csbs of enhancers of the e complex. given that 2/ of the occurrences of hox sites in these promoters can be accounted for by cdts whose flanking sequences are shared between enhancers, it is unlikely that the appearance of these shared sequences occurs by chance. in summary, the appearance of hox sites in the context of conserved sequences shared by functionally related enhancers suggests that the specificity of consensus homeodomain-binding sites is conferred by adjacent bases, either through recognition of adjacent bases by the tf itself or in conjunction with one or more co-factors. neural specific cdts that contain pbx/extradenticle sites examination of the cdts from drosophila nb and pns enhancers revealed that many contained the core pbx/extradenticle docking site atga. in drosophila, extradenticle has been shown to have hox-dependent and independent functions. studies have also shown that pbx factors provide dna-binding specificity for homeodomain tfs, facilitating specification of distinct structures along the body axis. in the cns enhancers of drosophila, most predicted pbx/extradenticle sites are not, however, found adjacent to hox sites. our analysis revealed that of the pbx motifs were shared between cns and pns enhancer types, and were shared between similarly expressed enhancers, thus indicating that there appears to be some degree of specificity to pbx site function when flanking bases are taken into account. three of the pbx binding-site containing elements also exhibit atta hox sites: 1) the dodecamer gatgattaatct shared by the pns enhancers edl and amos, contains a homeodomain atta site that overlaps the pbx site by a single base, and 2) the smaller heptamer atgatta, shared by pfe and ato, likewise contains a homeodomain atta site that overlaps atga pbx site by a single base. adjacent hox and pbx sites have been documented to facilitate synergy between the two factors. taken together our findings suggest that, as with homeodomain-binding sites, the conserved bases flanking putative pbx sites are functionally important. these flanking bases are likely to confer different dna-binding affinities for pbx factors or are required for binding of other tfs. neural specific cdts that contain suppressor of hairless binding sites also indicating a degree of biological specificity of enhancer types is the distribution of suppressor of hairless su binding sites among neural enhancers. su is the notch pathway effector tf of drosophila. the members of the e complex, both the multiple basic helix-loop-helix repressor genes and the bearded family members, have been shown to be su dependent. the consensus in vitro dna binding site for su is rtgrgar. notch signaling via su occurs through conserved single or paired sites and the presence of conserved sites for other transcription regulators associated with csbs containing su binding sites has been documented. within the csbs of the six nb enhancers examined, only two, dpn and wor, contained conserved putative su-binding sites; two dpn sites matched one of the su consensus sites and two wor sites match the sequence atgggaa. only one of the two dpn sites contained flanking bases conforming to the widely distributed cgtgggaa site of e su binding sites and none of the nb enhancers contained paired su sites typical of the e enhancers. of the pns cis-regulatory regions examined, only four enhancers contained putative su-binding sites and dpn. dpn also contained a pair of sites that conforms to the sps configuration frequently found in su enhancers sites in bold, pbx site is atca). the lack of su sites in pns enhancers was noted by reeves and posakony, who suggested that these enhancers are directly regulated by the proneural proteins but not activated in response to notch-mediated lateral inhibitory signaling. among the conserved sequences of e gene enhancers there is an average of consensus su binding sites per enhancer, with most enhancers containing both types of sites, i.e., those with either a or g in the central position. we offer three insights with respect to su binding sites. first, although in vitro dna-binding studies suggest there is a flexibility in the su binding site, like the bhlh e-box, comparative analysis shows that within any one the su sites there is no sequence flexibility. except for the pair of su sites in the dpn pns enhancer, none of the cns or pns sites contained a central a; less that a quarter of the e sites consisted of a central a, and all these were conserved across all species examined. in light of the high conservation in these regions the invariant core and flanking sequences are important for the unique su function at any particular site. a second finding was the extensive conservation of bases flanking the consensus su sequence in the e complex genes. for example, the cdt gtgggaaacacacgac was present in hlhm and hlhm enhancer csbs, and accgtgggaaac was conserved in hlhm and hlhmβ enhancers. the conservation of bases flanking the consensus su binding site suggests that the su site may be flanked by additional binding sites for co-operative or competitive factors, or else, that su contacts additional bases besides the consensus heptamer. a third observation is that in most cases su binding sites are imbedded in larger csbs, suggesting that csb function is regulated by the integrated function of multiple tfs. for example the dpn nb enhancer su site is imbedded in a csb of bases, and the atonal pns enhancer su site is imbedded in a csb of bases. in the e complex, csb # of hlhmγ, consisting of bases and csb# of m consisting of bases site, a cacgag element, conforming to a hairy n-box consensus cacnag, and an agga tramtrack dna-binding core recognition sequence, but the order and context of these three sites is different for each enhancer). although su binding sites were present in only a minority of nb and pns enhancers, the conservation of core bases, as well as the complexity of their flanking conserved sequences points to a diversity of su function and interaction with other factors. neural specific cdts that contain core dna-binding sites for other known tfs two of these elements, one exclusively present in nb enhancers and a second exclusively present in pns enhancers, contained consensus core agga dna-binding sites for ttk, a btb domain tf that has been shown to regulate pair rule genes during segmentation and to repress neural cell fates. another site, shared by both nb and pns enhancers, conforms to the consensus binding site of ia-, the vertebrate homolog of nerfin-. most of the cdts of table do not contain sequences corresponding to consensus binding-sites of known regulators of nb expression. the fact that they are represented multiple times in nb csb sequences suggests that they contain binding sites for unknown regulators of neurogenesis in drosophila. neural-enriched cdts neural enriched cdts that are shared between multiple nb enhancers and also exhibit a low frequency in the sample of mesodermal enhancers examined in this study serve as a resource for understanding enhancer elements that may not have an exclusive neural function. notable here is the presence of cagctg bhlh dna binding sites and antennapedia class homeobox core dna binding site atta, as well as additional ttk and pbx/extradenticle sites. present in this list are portions of sequences conforming to su binding sites described above. of particular interest in this table are sequences that are also enriched in the pns; these sites may bind factors that play similar developmental roles in different tissues. for example, the presumptive ttk site, aaagga is highly enriched in segmental enhancers. thus, some of these sites can be identified as targets of known tfs, but the identity of most are as yet unknown. these elements shared by multiple enhancers may be useful in identifying other enhancers driving expression in nbs. cis-decoder analysis reveals a complex sub-structure of enhancer csbs evoprint analysis revealed that all of the enhancer regions examined in this study contained multiple csbs that were greater that to bases in length. the occurrence of overlapping dna-binding sites for different tfs is currently the best explanation for the maintenance of intact csb sequences across ~ millions of years of collective species divergence. our analysis has revealed that the sequence context, order and orientation of shared cdts can differ between co-regulating enhancers. two examples are given here of the complex contextual appearance of cdts that appear frequently in cns and pns enhancers. each of the eight csbs shown was nearly fully'covered' by cdts of the nb library, suggesting that each contains multiple overlapping binding sites for a number of tfs. first, examination of the distribution of cdt gctgca reveals that it overlaps, by one and two bases, adjacent but different consensus bhlh sites in scrt csb# while in scrt csb# it overlaps a third consensus bhlh sequence by two bases. in the pns enhancer char, in csb# gctgca overlaps a bhlh site, but in a different configuration than found in the two cns enhancers illustrated in figure 3a. in amos csb# gctgca appears adjacent to a hox site and does not overlap a bhlh site. second, examination of the distribution of the cdt ggcacg reveals that it overlaps different consensus bhlh sites in scrt csb# and wor csb# overlapping the bhlh site in the former by one base and in the latter by four bases. ggcacg overlaps a cagctg bhlh-binding site in rho csb# but in a different configuration than the overlap with cagctg in the wor csb. in the pns enhancer scrt, ggcacg in csb# overlaps a hairy site n-box. n-boxes were most common in e csbs, but were also present in nb and pns enhancer csbs. in these two examples, and others we have examined, there is no consistent spatial constraints to the association of known tf-binding sites with novel cdts; a picture that emerges is one of combinatorial complexity, in which known or novel cdts are associated with each other in different contexts on different csbs. as an initial step toward determining if different tfs interacted with one another or competed for flanking dna-binding sites, we examined the proximity of known binding sites to one another in csbs for bhlh, hox, pbx and su. the results of this analysis for nb csbs are shown in table 4; data for other enhancer types is summarized here. most striking was the presence of multiple adjacent hox atta sites and combinations of hox and pbx sites. a typical example is the association of one pbx site, a bhlh site and two hox sites on a wor nb enhancer csb. associations of hox and pbx sites was also apparent in pns enhancer csbs, and in addition there was a high level of combined hox and bhlh sites, but in e enhancers only a higher level of the combination of hox and pbx sites was apparent. an example of the association of hox and bhlh sites in a pns enhancer is found in an achaete-scute dorso-central enhancer csb. there was also a distinctly higher level of pbx sites on the same csbs as bhlh sites in nbs csbs, but this combination was not apparent for pns or e csbs. association of bhlh sites with su binding sites was apparent in e enhancer csbs, especially when presence on adjacent csbs was taken into account. only in one of the instances of paired su sites on e enhancers were these sites on the same csbs, while in four other instances they were on adjacent csbs. although we often find sites in close proximity, both known and functionally uncharacterized sites are, with a few exceptions, not present in fixed uniform orientation in similarly regulated enhancers. this highlights the complex combinatorial arrangement and position flexibility of tf-binding sites within enhancer csbs. *first and second numbers represent appearance on same and adjacent csbs, respectively. the use of cis-decoder, flyenhancer and evoprinter to identify novel enhancers we have used the information derived from cis-decoder analysis of neural precursor cell enhancers to search for other genomic sequences with similar cis-regulatory properties. having identified cdts found multiple times among nb enhancers, we used the genomic search tool flyenhancer to identify drosophila melanogaster genomic sequences that contained clusters of the following cdts: ggcacg, ggaatc, tgacag, tggggt, cagctg, tgattt caagtg, catattt, tgatcc and ctaagc. as a lower limit, a minimum of three cagctg bhlh sites was set for this search, because of the prevalence of this site in nerfin- and deadpan nb enhancers. each sequence detected by this search was subjected to evoprinter analysis to determine the extent of its sequence conservation. among the cdt clusters identified, our search identified a 5' region adjacent to the nervy gene. nervy, originally identified as a target of homeotic gene regulation, is expressed in a subset of early cns nbs, as well as in pns sop cells. later studies have implicated nervy, along with cyclic adenosine monophosphate -dependent protein kinase in antagonizing sema-1a-plexa-mediated axonal repulsion, and nervy has been shown to promote mechanosensory organ development by enhancing notch signaling. evoprinter analysis revealed that the cluster of neural precursor cell enhancer cdts positioned bp upstream from the nervy transcribed sequence contains highly conserved sequences. this region contains csbs that include six conserved e-boxes, three of which conform to the cagctg sequence that was prominent in nerfin- and deadpan promoters. to determine if this region functions as a neural precursor cell enhancer, we generated transformant lines containing the nervy csb cluster linked to a minimal promoter/gfp reporter transgene. our analysis of the reporter expression driven by the nervy upstream fragment revealed a pattern indistinguishable from early nervy mrna expression. specifically, we detected expression in a large subset of early delaminating nbs and in sops and secondary precursor cells of the pns. significantly, the nervy enhancer, unlike nerfin- and deadpan nb enhancers, activates reporter expression in then pns and not just in early nbs. a new cdt-library was generated combining the nervy enhancer csbs and the nb and pns enhancer csbs used to generate the libraries described above. the new cdts, along with the previously defined cdts were aligned back to nervy csbs. most cdts were found only once in previously examined nb or pns csbs, but cdts appeared in our original analysis, described above, that did not include the nervy enhancer. the addition of this new enhancer to our analysis resulted in the discovery of a significant number of cdts that had not been found previously. three cdts that were identified in the previous analysis, tcagctgc, cagcagctg and aacagctg, contain bhlh dna-binding sites. aligning cdts that are specific to the cns or pns may indicate sequences required to specifically drive expression in either the cns or pns.  the major finding of this study is that enhancers of co-regulated genes in neural precursor cells possess complex combinatorial arrangements of highly conserved cdt elements. comparisons between nb and pns enhancers identified cns and pns type-specific cdts and cdts that were enriched in one or another enhancer type. cis-decoder analysis also revealed that many of the conserved sequences contain dna-binding sites for classical regulators of neurogenesis, including bhlh, hox, pbx, and su factors. although in vitro dna-binding studies have shown that many of these factors have a certain degree of flexibility in the sequences to which they bind, defined in terms of a position weight matrix, our studies show that for any given appearance these sites are actually highly conserved across all species of the drosophila genus. the genus invariant conservation in many of these characterized binding sites indicates that there are distinct constraints to that sequence in terms of its function. the high degree of conservation displayed in the enhancer csbs could derive from unique sequence requirements of individual tfs, or the intertwined nature of multiple dna-binding sites for different tfs. thus there is a higher degree of biological specificity to these sites than the flexibility that is detected using in vitro dna-binding studies. as an example, the requirement for a specific core for the bhlh binding site, i.e., for a cagctg e-box for nerfin- deadpan and nervy, suggests that it is the tf itself that demands sequence conservation; however, the requirement for conserved flanking sequences suggests that additional specific factors may be involved. although the inter-species conservation of core and flanking sites has been noted by others, the extent of this conservation is rather surprising. to what extent and how evolutionary changes in enhancer function take place, given the conservation of core enhancer sequences, remains a question for future investigation. in addition to classic regulators of neurogenesis, cis-decoder reveals additional conserved novel elements that are widely distributed or only detected in pairs of enhancers. many of these novel elements flank known transcription binding motifs in one csb, but appear independent of known motifs in another. the appearance of novel elements in multiple contexts suggests that they may represent dna-binding sites for additional factors that are essential for enhancer function. only through discovery of the factors binding these sequences will it become clear what role they play in enhancer function. preliminary functional analysis of csbs within the nerfin- neuroblast enhancer reveals that csbs carry out different regulatory roles. altering cdt sequences within the nerfin- csbs reveals that most are required for cell-specific activation or repression or for normal enhancer expression levels. csb swapping studies reveals that, for the most part, the order and arrangement of a number of tested csbs was not important for enhancer function in reporter studies. the discovery of the nervy neural enhancer by searching the genome with commonly occurring nb cdts underscores the potential use of evoprinter and cis-decoder analysis for the identification of additional neural enhancers. by starting with known enhancers and building cdt libraries from their csbs, one now has the ability to search for other genes expressed during any biological event. 
analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. we have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the cns and pns. the csbs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. the combined use evoprinter and cis-decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. the presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. these data establish a basis for further analysis and understanding of neural enhancer function. this study focuses on regulation of gene expression during the early events of drosophila neural development.
 the presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. this study focuses on regulation of gene expression during the early events of drosophila neural development. we describe the use of evoprinter and cis-decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers.  analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. the csbs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. we have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the cns and pns.  the combined use evoprinter and cis-decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. each of the enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. these data establish a basis for further analysis and understanding of neural enhancer function. 