Importance of chromatin insulators
It has become increasingly apparent that proper control of gene expression requires complex organization of DNA at the level of chromatin. Chromatin insulators are DNA-protein complexes that influence gene expression by establishing chromatin domains subject to distinct transcriptional controls, likely through alteration of their spatial organization. Insulators enforce the strict specific and temporal expression of complex loci such as the Drosophila bithorax complex (BX-C), a master regulator of body segmentation, and the vertebrate beta-globin locus, which changes in expression during erythroid development. Therefore, studying the mechanisms and regulation of insulator function is essential to further understand how higher order chromatin structure influences the intricately orchestrated transcriptional programs needed for proper development and differentiation. We primarily utilize the biochemically and genetically tractable model system Drosophila, which harbors the largest diversity of known chromatin insulator complexes.
The gypsy chromatin insulator
Defined by the specific binding of the Su(Hw) zinc finger DNA-binding protein, gypsy insulator complexes tend to associate with gene-poor, transcriptionally inert regions of the genome. Within the nucleus, gypsy insulator complexes concentrate at approximately 200nm diameter ovoid structures termed insulator bodies, which are tethered stably to the nuclear matrix. The proper localization of insulator bodies is highly correlated with gypsy chromatin insulator function, but their precise function and spatial relationship with respect to the genome is not well understood. We are currently investigating the ultrastructure of insulator bodies within the surrounding chromatin environment.
We recently identified a novel, tissue-specific negative regulator of gypsy insulator function that affects both enhancer blocking and barrier activities. This factor can bind directly to Su(Hw) as well as another core component of the gypsy insulator complex, potentially competing with inter- or intra-insulator complex interactions and thereby neutralizing insulator activity. The factor is highly enriched in the CNS and may serve to negatively regulate insulator function in these cell types to promote CNS-specific gene expression programs. Intriguingly, this factor harbors two highly conserved RNA recognition motifs (RRMs), and genetic evidence points to a functional relationship between its RNA-binding capability and insulator function.
Previously, we identified a role for RNA silencing in regulating gypsy insulator function as well the formation of insulator bodies. This phenomenon appears to be independent of the role of RNA silencing in gypsy retrotransposon expression. Current efforts center on identifying gypsy insulator associated RNAs using deep sequencing and characterizing additional novel insulator interacting proteins using genetic and biochemical approaches.
The Fab-8 chromatin insulator
One of the best-studied chromatin insulator sites within the Drosophila genome is the CTCF-dependent Fab-8 insulator, which resides within the BX-C homeotic gene cluster. This specialized locus features a high concentration of long-range tissue-specific enhancers and Polycomb Response Elements needed to control proper expression of genes required for posterior development. The presence of insulators demarcating these cis-regulatory domains is critical in controlling their activities and physical interactions with their target promoters.
We demonstrated that the insulator proteins CP190 and CTCF, as well as the RNAi component Argonaute2 (AGO2), are required for looping interactions within the BX-C. Intriguingly, AGO2, but not its RNA interference (RNAi) catalytic activity or other RNA silencing components, is required for Fab-8 insulator activity. At Fab-8 and genome-wide, AGO2 chromatin association depends on CTCF and CP190. AGO2 binding throughout the genome correlates extensively with CTCF/CP190 insulator proteins but exhibits minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Our findings identify a novel nuclear role for AGO2 and suggest that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
Heterochromatin
Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin in order to prevent genome rearrangement and loss of genetic material. In fission yeast, a direct role for RNAi in the establishment of pericentromeric heterochromatin has been elegantly demonstrated. In all eukaryotic organisms examined to date, there is a marked relationship between both heterochromatic and RNA silencing dependent repression of repetitive sequences. However, it is unclear whether a direct role for RNA silencing in nucleating heterochromatin is conserved. Utilizing complementary genetic and biochemical approaches in Drosophila, we monitored the heterochromatin state at discrete genomic locations from which small RNAs originate in endo-siRNA or Piwi-associated RNA (piRNA) pathway mutants. Our results indicate that heterochromatin can form independently of these two small RNA silencing pathways in somatic tissue. Furthermore, genome-wide localization of AGO2 indicates specific localization to euchromatic sequences, particularly with chromatin insulator sites. Our results suggest the possibility that RNA silencing pathways may actually help demarcate domains of silent chromatin.
