Worm Breeder's Gazette 14(1): 93 (October 1, 1995)

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.

Does the cell-death specification gene ces-1 encode an autoregulatory transcription factor?

Mark Metzstein, Bob Horvitz

HHMI, Dept. Biology, MIT, Cambridge MA 02139

We are interested in how individual cells decide to activate the cell
death program during development.  To analyze this process we, in
collaboration with other members of the laboratory, have been studying two
genes thought to be involved in this process, ces-1 and ces-2(1).  As
reported at the last worm meeting(2), both ces-1 and ces-2 encode putative
transcription factors, a zinc-finger protein similar to members of the
family defined by the snail gene of Drosophila in the case of ces-1, and
a protein of the bZip family in the case of ces-2.  These findings suggest
that programmed cell death, like many other cell fates, is regulated at
the level of differential gene transcription.

There exist three dominant, gain-of-function alleles of ces-1, n703, n1895
and n18961, which behave similarly, causing survival of four pharyngeal
cells that normally undergo programmed cell death.  To identify the
molecular lesions in these alleles, we determined the sequences of ces-1
genomic DNA.  For all three alleles the coding sequence, 3' UTR, and all
introns examined (four of five) were identical to wild-type.  We then used
a mutation-detection technique (Chemical Cleavage of Mismatch; thanks to
Giovanni Lesa and Paul Sternberg for their version of this protocol) to
scan larger regions around the ces-1 coding sequence.  We identified a
lesion 5' to the start of the coding sequence in the allele n703.
Subsequent studies of this region in all three gain-of-function alleles
showed that they contain identical changes: a G-to-A transition ~600 bp
before the translation start site. The most interesting feature about this
change is that it alters the sequence TAGGTA (mutated sequence TAGATA),
which contains the core of the binding consensus of the Snail protein,
CAGGTG(3), suggesting that ces-1 might regulate its own transcription.
This hypothesis is consistent with ces-1 genetics, since snail family
members have been shown to function primarily as repressors and a mutation
in regulatory DNA that leads to a gain-of-function results from disrupted
negative regulatory sites.

To determine whether this region is involved in ces-1 regulation, we have
been conducting two sets of experiments. First, we have cloned the C.
briggsae homolog of ces-1 (thanks to David Baillie for his library) and
are comparing the regions upstream of coding sequences for conservation.
We have found that the upstream regions between the two species are
entirely unconserved except for a 130  bp region that is 82% identical
(107/130 bases).  This region surrounds the site of the gain-of-function
mutations.   This comparison has also revealed that there are five sites
in this region close or identical to snail consensus binding sites (see

Second, we are using bacterially produced CES-1 protein in electrophoretic
mobility-shift assays to determine the binding-site specificity of CES-1.
Preliminary results show that CES-1 is capable of binding to snail
consensus binding sites.  We hope to use this assay in the future to
determine the in vivo targets of ces-1 and whether any of the ced genes
are transcriptionally regulated by CES-1 to control programmed cell death.

{See WBG for Figure.}
Figure: Alignment between the regions of C. elegans and C. briggsae DNA
around the site of the ces-1 gain-of-function mutations. Shaded residues
indicate identities.  Boxed regions conform or are close to snail
consensus binding sites.  The arrow indicates the base altered in all
three ces-1 gain-of-function mutations.

1. Ellis, R.E. & Horvitz, H.R.  Development 112, 591-603 (1991).
2. Metzstein, M.M., Tsung, N., Hengartner, M.O., Ellis, R.E. & Horvitz,
   H.R. 10th International C. elegans Meeting, p374 (1995) .
3. Mauhin, V., Lutz, Y., Dennefeld, C. & Alberga, A.  Nucleic Acids Res
   21, 3951-7 (1993).