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CONTROL OF GENE EXPRESSION DURING DEVELOPMENT
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Judith
A. Kassis, PhD, Head, Section on Gene
Expression J.
Lesley Brown, PhD, Staff Scientist Sarah Kelly, MS, Technician Alayne Brown, BS, Postbaccalaureate Fellow Amanda
Gordon, BS, Postbaccalaureate Fellow |
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During
development and differentiation, genes either become competent to be expressed
or are stably silenced in an epigenetically heritable manner. This selective
activation/repression of genes leads to the differentiation of tissue types.
Our group is interested in the molecular mechanisms that lead to the
heritable transmission of the silenced state. In particular, we are studying
the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila.
The PcG genes encode a diverse group of proteins known to be important for
silencing of homeotic genes during development in organisms as diverse as Drosophila
and man. Many PcG genes encode chromatin-associated proteins that are thought
to silence transcription through modification of histones and formation of
inactive chromatin. PcG proteins act through cis-acting DNA sequences
called Polycomb group Response Elements (PREs). PREs contain binding sites
for multiple proteins that act together to recruit PcG protein complexes to
the DNA. We are working to identify the DNA-binding proteins necessary to
recruit PcG protein complexes. Interestingly, PREs also mediate interactions
between distant DNA elements, suggesting that they may bring distant
silencers and enhancers together with promoters. We are investigating this
possibility at the Drosophila engrailed gene. DNA-binding
protein requirement of the Polycomb group response elements Brown JL, Kassis We
have completed a detailed study of one PRE from the segmentation gene engrailed
and, within a 139-bp minimal PRE, have identified eight protein-binding
sites. Our functional data have shown that at least five of the sites are
required for PRE activity. One of the proteins that binds to this PRE is the
product of the pleiohomeotic (pho) gene, a known PcG gene. The pho
gene encodes a Drosophila homolog of the mammalian zinc-finger
transcription factor Yin Yang 1 (YY1). Another site is the sequence GAGAG,
known to bind to GAGA factor and pipsqueak, two Drosophila proteins.
There are two Pho binding sites and two GAGAG sequences within the engrailed
PRE. The proteins that bind to the other four sites have not been identified.
Isolation of these proteins is a major focus of our laboratory. We are using
a yeast one-hybrid screen and biochemical methods to isolate the other
DNA-binding proteins involved in this important process. We have identified a
family of zinc-finger proteins that binds to one of the sequences important
for PRE activity. By depleting the proteins with the use of RNAi in tissue
culture cells and determining whether PRE function is compromised, we are
investigating which of these family members may be involved in Polycomb group
repression. Americo J, Whiteley M, Brown JL, Fujioka M, Jaynes JB, Kassis
JA. A complex array of DNA binding proteins required for pairing-sensitive silencing
by a Polycomb-group response element from the Drosophila engrailed
gene. Genetics 2002;160:1561-1571. The
role of Pho and Pho-like in Polycomb group repression Brown JL, Kassis; in
collaboration with Jones, Wang The
identification of Pho binding sites in many different PREs suggests that Pho
is an integral component for the recruitment of PcG protein complexes to the
DNA. If this were the case, then the phenotype of pho mutants should
be severe derepression of homeotic genes. Curiously, in pho mutants,
homeotic genes are only mildly derepressed. With the sequence of the Drosophila
genome known, we identified a possible explanation for this apparent paradox:
another YY1 homolog exists in Drosophila. We are studying the function
of this gene, which we call pho-like, and have generated mutants in
the gene. Flies mutant for pho-like are viable, but the females are
sterile; homozygous pho-like mothers lay eggs that are fertilized but
undergo no nuclear divisions. Thus, Pho-like must be deposited in the
egg by the mother for development to occur. Organisms that are double mutant
for pho and pho-like (from mothers and fathers heterozygous for
these two mutations) develop into larvae and have much more severe
derepression of homeotic genes than pho mutants alone, showing that pho-like
enhances the pho phenotype. In fact, the derepression of homeotic
genes seen in imaginal disks from pho-like/pho double mutants
is comparable to that seen in many other PcG mutants, suggesting that Pho and
Pho-like play redundant but highly important roles in PcG repression. Pho-like
binds to Pho binding sites in vitro, and pho-like/pho double
mutants show more severe misexpression of homeotic genes than single mutants.
The question arises as to how these two proteins function. Undertaking
chromatin-immunoprecipitation (ChIP) experiments, we used two methods to
demonstrate that Pho and Pho-like are required for the binding of the PcG
proteins Enhancer of zeste (E(z)) and Polycomb (Pc) to a PRE from the
homeotic gene Ultrabithorax (Ubx). With the first method,
treatment of Drosophila tissue culture cells with RNAi to Pho
(to deplete Pho protein levels) led to a loss of E(z) and Pc from the Ubx
PRE; with the second method, which involved pho-like/pho double
mutants, neither E(z) nor Pc bound to the Ubx PRE. Consistent with our
previous experiments on the redundancy of pho-like and pho,
E(z) and Pc were still bound to the Ubx PRE in either single mutant.
Furthermore, our data suggest an order of recruitment of Pc complexes such
that Pho and Pho-like directly recruit an E(z) protein complex, which helps
to recruit Pc to the DNA. Experiments are now in progress to see what other
DNA-binding proteins may be responsible for the recruitment of PcG protein complexes. Brown JL, Fritsch C, Müller J, Kassis JA. The Drosophila pho-like
gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic
in homeotic gene silencing. Development 2003;130:285-294. Wang L, Brown JL, Cao R, Zhang Y, Kassis JA, Jones RS.
Hierarchical recruitment of Polycomb group silencing complexes. Mol Cell
2004;14:637-646. Interaction
of distant regulatory elements with a promoter Kelly, Gordon, Brown
A, Kassis Regulatory
sequences in eukaryotic genes can be located many tens of kilobases away from
the promoter, but it is not known how these distant regulatory sequences
activate or repress the promoter. According to one model, proteins bound to
the distant enhancer interact with proteins bound near the promoter, causing a
loop of the intervening DNA. One of the fragments of DNA from the Drosophila
engrailed gene that has PRE activity also can cause interactions
between distant DNA fragments. We are currently trying to understand the
function of this DNA in vivo by deleting it from the endogenous engrailed
gene. Our results suggest that the DNA fragment facilitates interactions
between a distant enhancer involved in positively regulating engrailed
expression and the engrailed promoter. Thus, the fragment of DNA,
which acts as a negative regulatory element (a PRE) in some cases, can act as
a positive regulatory element in other contexts. The results are consistent
with a model in which the role of this element is to facilitate the
interactions between distant enhancers or silencers with promoter elements. The engrailed
gene is regulated in a complex manner, and regulatory sequences seem to
stretch throughout a 70 kb region. We have been working to identify all the
enhancer sequences and all the PREs within this region. We have used two
algorithms to identify potential PREs within the engrailed DNA
sequence and found at least seven PREs located throughout the engrailed
region. We have been using reporter constructs to identify enhancer elements
that control engrailed’s complex expression pattern and have
found regulatory elements located 40 kb upstream of the engrailed
promoter. These regulatory sequences have nearby PREs. Our current
experiments are designed to test the role of these PREs at engrailed. Kassis JA. Pairing-sensitive silencing, Polycomb group response
elements, and transposon homing in Drosophila. Adv Genet
2002;46:421-438. COLLABORATORS Richard S. Jones, PhD, Southern Liangjun Wang, PhD, Southern
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