Basic HTML Version
Poster Full Abstracts - Chromatin and Epigenetics
Poster board number is above title. The first author is the presenter
229
the interaction between SIN3 and methylation has led us to examine the connection of SIN3 to methionine metabolism. RNA interference (RNAi) induced
knockdown (KD) of SIN3 results in a low cell density in
Drosophila
cultured cells (S2 cells) and a curly wing phenotype in adult
Drosophila
wings. The
adult wings are also smaller and have fewer cells. These results suggest that reduction of SIN3 results in a reduction in cell proliferation. Moreover, RNAi
KD of some methionine pathway genes suppresses the SIN3 KD curly wing phenotype in adult
Drosophila
. These interesting data lead us to investigate the
potential changes of cell proliferation in S2 cells resulting from RNAi KD of some methionine metabolic enzymes. Our results show that in S2 cells, RNAi
KD of some methionine metabolic enzymes leads to loss of cell proliferation, similar to the effect seen in SIN3 KD cells. But RNAi KD of some methionine
pathway genes does not suppress the SIN3 KD phenotype in S2 cells. Given the finding that RNAi KD of some methionine pathway genes suppresses the
SIN3 KD phenotype in adult
Drosophila
but not in S2 cells, we are currently examining whether there are some specific genes regulated by both acetylation
and methylation during development. In addition, we are determining whether RNAi KD of some methionine pathway genes or SIN3 leads to potential
global histone methylation changes in S2 cells. Taken together, these data imply a connection between SIN3 and the regulation of biological methylation and
cellular proliferation.
361A
The role of Lid in mediating the cellular response to oxidative stress.
Xingyin Liu, Christina Greer, Julie Secombe. Department of Genetics, Albert
Einstein College of Medicine of Yeshiva University, Bronx, NY.
Drosophila Lid and its four human orthologs, KDM5A-D, are developmentally and clinically important: Lid is an essential gene in Drosophila,
overexpression of human KDM5A and KDM5B are associated with cancer, and loss of KDM5C causes mental retardation. Yet the basic mechanisms by
which this family of proteins function in vivo remain unknown. Lid/KDM5 proteins have multiple domains implicated in chromatin-mediated transcriptional
regulation, including a Jumonji C (JmjC) lysine demethylase domain and histone binding PHD fingers. Using these different domains, Lid can both read and
erase the histone code, suggesting that it regulates transcription by several mechanisms. To investigate the biological role of Lid/KDM5 proteins in vivo, we
are using Drosophila to take a whole genome (microarray) approach combined with bioinformatics assays to identify Lid target genes. Of the 18,500 genes
represented on the array, a total of 1049 genes were expressed differentially in lid homozygous mutants compared to wildtype, and that 345 genes were
expressed differentially in response to Lid overexpression. Gene ontology analysis revealed a significant enrichment of genes involved in the response to
oxidative stress and longevity. Consistent with this, we find that modulating Lid levels confers resistance or sensitivity to the oxidative stress agent paraquat
when overexpressed or reduced by RNAi, respectively. ChIP assays using larvae with Lid-overexpression demonstrate that Lid is recruited to the promoter
regions of the stress response genes Prx2540-2, Hsp22, Hsp67Bb and Glaz. In addition, we have shown a direct physical interaction between Lid and heat
shock factor (HSF), a key stress response transcription factor. Our current working model is that Lid is an essential component of the cellular response to
oxidative stress and that it is recruited to promoters via HSF to activate transcription.
362B
Epigenetic Regulation of Replication Origins.
Neha P. Paranjape, Jun Liu, Brian R. Calvi. Department of Biology, Indiana University, Bloomington, IN.
Origins of DNA replication are bound by a pre-Replicative complex (pre-RC) that is activated to initiate replication during S phase. It is not known,
however, how specific genomic loci are specified to be pre-RC binding sites; a DNA consensus sequence has not been found in metazoans. Moreover, the
genomic location of pre-RC binding sites and their activation during S phase changes during development. Evidence suggests that chromatin plays a key role
in specifying which origins are active in different cells. To investigate the role of chromatin in origin regulation, we are using the developmental gene
amplification in
Drosophila
ovary as a model system for origins. We showed that nucleosome acetylation influences the selection and activity of these
origins in follicle cells late in oogenesis. The mechanism by which nucleosome modification regulates origins remains unclear. Here, we present our new
high-resolution ChIP data, which reveals new aspects of the chromatin landscape at origins and its relationship to the pre-RC and origin activity. Histones
are highly acetylated on multiple lysines specifically when the origin is active, suggesting that multiple HATs regulate the origins. At the 3
rd
and X
chromosome amplicon origins, peak acetylation is adjacent to a preferred replication initiation site, suggesting that histone acetylation may facilitate pre-RC
binding or activation. Moreover, we have found that acetylation on some lysines is dependent on the pre-RC itself, suggesting that the pre-RC may recruit
multiple co-activators, analogous to transcription factor regulation of promoters. Immunofluorescent and ChIP data indicate that histone variants H3.3 and
H2Av, known components of hyperdynamic nucleosomes, are also enriched at the amplicon origins. We are using molecular and genetic methods to
determine whether H3.3 enrichment simply represents deposition of this variant behind replication forks or contributes to origin specification. Answers to
these questions are medically important because alterations of the epigenome and origin function cause genome instability and cancer.
363C
Interaction of the SIN3 histone deacetylase complex with the histone demethylase LID.
Lori A. Pile, Ambikai Gajan. Dept Biological Sci, Wayne State
Univ, Detroit, MI.
SIN3, which is conserved from yeast to mammals, is the key scaffold component of a histone deacetylase complex. SIN3 acts as a corepressor affecting
global transcription. Purification of
Drosophila
SIN3 isoform specific complexes in our laboratory identified the association of LID, a histone H3 lysine 4
demethylase, with the largest SIN3 isoform. Purification of a LID complex in
Drosophila
validates this interaction identifying many components of the SIN3
complex along with unique proteins that interact with LID. This suggests that the demethylase functions together with the deacetylase to co-regulate
transcription, possibly at a subset of their target genes. Supporting this, analysis of histone modification patterns at SIN3 target genes under SIN3
knockdown conditions show changes in both acetylation and methylation. Analysis of global histone modification changes upon LID knockdown in S2 cells
shows a specific increase in H3 lysine 4 trimethylation, with no significant effect on global levels of acetylation. It is thus of interest to look at possible gene
specific effects on acetylation levels upon loss of LID at target genes. We are currently analyzing global changes in gene expression upon loss of LID in S2
cells to identify potential genes co-regulated by LID and SIN3. Furthermore, LID may also be involved in regulating expression of SIN3 itself.
Overexpression of LID in S2 cells leads to a switch from the predominantly expressed SIN3 large isoform to the smaller isoform. Apart from the
biochemical interactions of LID with SIN3, interestingly, genetic analysis shows similarities in LID and SIN3 knockdown phenotypes. While ubiquitous
knockdown of SIN3 by RNAi in flies is lethal, LID RNAi leads to reduced viability. Further, both SIN3 and LID knockdown in Drosophila wing discs leads
to a curly wing phenotype. In S2 cells, both SIN3 and LID knockdown lead to a decrease in cell proliferation. These data suggest that the two enzymes may