Worm Breeder's Gazette 16(5): 34 (February 1, 2001)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

egl-5 promoter analysis provides clues toward understanding anterior/posterior addressing at the cis-regulatory level

Lisa Girard, Paul Sternberg

California Institute of Technology, Division of Biology 156-29, Pasadena, Ca 91125

The Hox genes encode a conserved set of transcription factors that are
essential for anterior/posterior patterning in many animals and are
expressed in a series of consecutive domains along this axis.  These
genes are almost always expressed in cells that are related by position
rather than lineage and mutations in these genes often result in the
transformation of one region to an adjacent one.  Hox genes are
typically found in clusters, with the order of the genes in the cluster
colinear with their order of expression along the anterior/posterior
axis, except for in C. elegans in which the order of two of the genes is
inverted.  In C. elegans, the Hox cluster contains a homolog of the
anterior group genes, ceh-13, which is most similar to labial in the
Drosophila HOM-C complex; two genes homologous to medial-group genes;
lin-39 (sex-combs reduced, deformed, proboscipedia); mab-5
(Antennapedia) and a homolog of the posterior group, egl-5
(Abdominal-B)(1).  Additionally, two more posterior homologs not
contained within the quasi-cluster, nob-1 and php-3, have been
characterized (2).  The expression of Hox genes in positionally-related
groups of cells raises interesting questions at the cis-regulatory
level.  Functional analysis of Hox gene promoters will help us
understand how positional cues are read and interpreted resulting in
correct anterior/posterior addressing along the body-length.  C. elegans
is ideal for such an analysis because fate-specification changes
consequential to small perturbations in these promoters can be observed
in individual cells, rather than the fields of cells as in many other
organisms.  To understand how cell-fate is specified in the posterior
region of the hermaphrodite by the egl-5 Hox gene, we have undertaken,
in a joint project with Yingqi Teng and Scott Emmons, a promoter
analysis of this gene.
egl-5 is expressed in the hermaphrodite specific neuron (HSN), body wall
muscle, posterior lateral microtubule neuron, PVC interneuron, M, V6,
the rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell
(3).  The experiments described here focus on our progress delineating
cis-regulatory elements which direct expression in K, F, B, U and P12.
egl-5 and its adjacent Hox gene mab-5 are divergently transcribed with
approximately 30 kb between them.  A gfp translational fusion containing
approximately 13 kb of promoter was sufficient to direct expression in
K, F, B, U, P12 while a second construct, containing the most proximal 7
kb of promoter was not, suggesting that the 6 kb differential fragment
between the two may be sufficient to direct expression in these
cell-types.  We tested this 6 kb region fused to a heterologous pes-10
basal promoter driving gfp, and found that indeed it was sufficient.  We
then examined a series of egl-5 promoter truncations, with this 6 kb
piece as a starting point and identified a 1.3 kb region within the 6 kb
that is sufficient for expression.  Within this 1.3 kb fragment we
identified a 469 bp sub-fragment sufficient to drive gfp expression in
B, but not K, F, U, P12 and a 446 bp piece sufficient to drive
expression in K, but not F, B, U or P12.  F, U and P12 elements have
proven more difficult to define.
        The 1.3 kb region sufficient to drive gfp expression in K, F, B,
U, and P12 contains six sites (denoted here in distal to proximal order
as 1-6) in the range of 20-30 bp each which are conserved between C.
elegans and the closely related species C. briggsae.  There are several
notable consensus sites for trans-acting factors within these conserved
regions including a binding site for SOX5 and FORKHEAD in site 3.  The
six sites are arranged in two quasi-clusters, with approximately 300 bp
between the clusters.  Specific deletions of either 1, 2 and 3 or 4, 5
and 6 abolishes expression in K, F, B, U and P12 suggesting that
sequences in both clusters may be important for expression in these
regions.  One caveat of these types of experiments is that these
deletions could alter spacing in the promoter, disturbing
enhancer-promoter interactions.  However, constructs which specifically
deleted 3, 4 and 5, removing a similar number of base pairs as the 1,2,3
and 4,5,6 constructs, and found that that expression in K, F, B, U and
P12 was not affected, suggesting that the spacing in this region is not
affected by deletions of this magnitude.  These conserved regions are
being further studied to assess their relative contributions to egl-5
expression in the rectal epithelial cells and P12.  The study of the
cis-regulation of egl-5 will reveal the natural taxonomy of differential
anterior/posterior addressing within C. elegans.
1. Salser, S.J. and Kenyon, C. (1994) Trends in Genetics 10, 159-164
2. Van Auken, K., Weaver, D.C., Edgar, L.G. and Wood, W.B. (2000) Proc.
Natl. Acad. Sci. 97(9), 4499-4503
3. Ferreira, H.B., Zhang, Y., Zhao, C. and Emmons, S.W. (1999) Dev.
Biol. 207, 215-228