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. Loss of insulator activity can result in substantial positive or negative changes in gene expression, culminating in disease, defects in development, and/or lethality. For example, deletion of insulator binding sites at the H19/IGF2 imprinting center have been implicated in Beckwith-Wiedemann syndrome and Wilms’ Tumor.
The gypsy chromatin insulator
We primarily utilize the biochemically and genetically tractable model system Drosophila, which harbors the largest diversity of known chromatin insulator complexes. 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 investigating the ultrastructure of insulator bodies within the surrounding chromatin environment.
We recently identified two novel, tissue-specific negative regulators of gypsy insulator function that affect both enhancer blocking and barrier activities. Shep 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. Shep 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. Shep harbors two highly conserved RNA recognition motifs (RRMs), and genetic evidence points to a functional relationship between its RNA-binding capability and insulator function. In contrast, Rump, which contains 3 RRMs, antagonizes gypsy insulator activity in tissues outside of the CNS. Given that Rump is ubiquitously expressed, we are examining potential mechanisms for its specificity of action.
Using RNA immunoprecipitation followed by deep sequencing (RIP-seq), we made the striking finding that certain mRNAs, including that encoding Su(Hw) itself, associate stably with gypsy insulator complexes. Expression of untranslatable versions of these mRNAs alters insulator body localization and promotes insulator activity. We speculate that these, and possibly other mRNAs, also harbor a noncoding function, such as acting as a scaffold for insulator complexes at specific subnuclear locations. We continue to delve into the mechanisms by which these mRNAs function and the roles of RNA-binding proteins in insulator regulation.
(This work was highlighted in Editors' Choice, Riddihough G. Noncoding mRNAs. Science, (341)6149: 938, 2013.)
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. We are currently probing the mechanisms by which AGO2 regulates insulator activity and transcription.