n1084

1 kb egl-1

CGAGTGGTC n2248

CGGATGGTC
n986 n987 n2164

CGGGTGGTC
9-base-pair motif is conserved in other Caenorhabditis species
CGGGTGATC
n487 n1084 n1796

This conservation suggests that the motif has functional importance,

especially because it is located between transcription units.
(It is probably an important cis-regulatory element!)
 n1084
A

1 kb egl-1

TRA-1 TRA-1

CGGGTGGTC CGGGTGGTC
CGGGTGGTC
CGGGTGATC
9-base-pair motif is conserved in other Caenorhabditis species n1084

This conservation suggests that the motif has functional importance,

especially because it is located between transcription units.
(It is probably an important cis-regulatory element!)
 n1084
A

1 kb egl-1

TRA-1 TRA-1

CGGGTGGTC CGGGTGGTC
CGGGTGATC
n1084

TRA-1 is a zinc-finger DNA binding protein

It is the terminal and global regulator of somatic sexual fate
 TRA-1 suppresses the transcription of genes required for male development
promotes the transcription of genes required for female development

Hermaphrodite Male

TRA-1
XX XO
TRA-1 activity high TRA-1 activity low

The HSNs are sexually dimorphic...

IT MADE PERFECT SENSE!
 Regulation of egl-1 by TRA-1
•  Confirmed: TRA-1 can bind to the 9-base-pair motif in vitro
–  binds only weakly (or not at all) to the mutated motifs identified

TRA-1
CGGGTGGTC
TRA-1 TRA-1
TRA-1
CGGGT CGGGT
GGTC
CGAGTGGTC GGTC
CGGATGGTC CGGGTGGTC
CGGGTGATC
n2248 n986 n987 n2164 n487 n1084 n1796
 Regulation of egl-1 by TRA-1
•  Confirmed: TRA-1 can bind to the 9-base-pair motif in vitro
–  binds only weakly (or not at all) to the mutated motifs identified
–  binds more strongly to semi-dominant mutations than fully-dominant
TRA-1 binds the motif strongly = HSNs survive
TRA-1 binds the motif weakly = fewer HSNs survive
TRA-1 does not bind the motif at all = HSNs die TRA-1
CGGGTGGTC
TRA-1 TRA-1
TRA-1
CGGGT CGGGT
GGTC
CGAGTGGTC GGTC
CGGATGGTC CGGGTGGTC
CGGGTGATC
fully-dominant fully-dominant semi-dominant
 Regulation of egl-1 by TRA-1
A model…
TRA-1 acts to block
transcription of the
egl-1 gene
R L in the HSNs
CGGGTGGTC

Hermaphrodite HSNs survive

TRA-1 egl-1
binding site
anti-apoptotic
activity

CGGGTGGTC

TRA-1 Male HSNs die

egl-1
binding site
 Regulation of egl-1 by TRA-1

CGGGTGATC
n1084
TRA-1 egl-1(n1084) HSNs die
egl-1
binding site

A
Having only one chromosome
with a mutated TRA-1 binding
site is sufficient to get egl-1
expression, killing the HSNs
 Regulation of egl-1 by TRA-1

CGGGTGATC
n1084
TRA-1 egl-1(n1084) HSNs die
egl-1
binding site

n3082
CGGGTGATC
R L
n1084
TRA-1 egl-1(n1084 n3082) egl-1 HSNs survive
binding site
 Regulation of egl-1 by TRA-1

•  A egl-1 reporter is expressed in the HSNs of males, not hermaphrodites

•  Same reporter with n1084 mutation is expressed in both
This supports the model that TRA-1 controls egl-1 transcription in the HSN neurons

Males Hermaphrodites

egl-1 reporter expressed?

✔
egl-1 + n1084
reporter expressed? ✔ ✔
 cis-regulation of egl-1

•  There are many conserved motifs scattered about the egl-1 locus
–  both upstream and downstream of the egl-1 transcription unit
•  Likely binding sites for regulatory proteins (cis-regulatory elements)
potentially
60 – 70
transcriptional regulators

CGGGTGGTC

TRA-1 egl-1
binding site
 cis-regulation of egl-1

•  There are many conserved motifs scattered about the egl-1 locus
–  both upstream and downstream of the egl-1 transcription unit
•  Likely binding sites for regulatory proteins (cis-regulatory elements)
potentially
60 – 70
transcriptional regulators

CGGGTGGTC

TRA-1 egl-1
binding site
 trans-regulation of egl-1

•  TRA-1 was the first trans-acting factor identified for egl-1 locus
•  Each trans-acting factor has a homolog in humans
•  …that has been implicated in tumorigenesis!

TRA-1
CGGGTGGTC
 trans-regulation of egl-1

•  Gli is the human homolog of TRA-1

–  associated with glioma (cancer that arises from glial cells)
•  Gli is transcriptionally up-regulated in many other types of tumors

Like Bcl-2, Gli is a proto-oncogene

block in BH3-only
block in apoptosis
Gli TUMORIGENESIS

BH3-only
(pro-apoptotic)
Transcriptional Transcriptional
repressors of activators of
pro-apoptotic genes, pro-apoptotic genes
such as BH3-only are potential
genes, are potential tumor suppressors
proto-oncogenes
CEP-1

Bcl-2
Gli BH3-only
(pro-apoptotic)
 inactivated in more than

p53 85%
of all human cancers
(mammalian homolog of CEP-1)

CEP-1

egl-1 transcription
cannot be activated
in response to CEP-1
DNA damage required to activate
egl-1 transcription
in germ cells
in response to
inappropriate survival DNA damage
of germ cells with
damaged DNA
 inactivated in more than

p53 85%
of all human cancers
(mammalian homolog of CEP-1)
other mammalian BH3-only genes:

Noxa
HRK
BIM

PUMA regulated predominantly at the transcriptional level

(BH3-only)