The epigenome of the human malaria vector Anopheles gambiae was characterized in midgut cells by mapping the distribution and levels of two post-translational histone modifications, H3K27ac and H3K27me3. These histone profiles were then correlated with levels of gene expression obtained by RNA-seq. Analysis of the transcriptome of A. gambiae midguts and salivary glands led to the discovery of 13,898 new transcripts not present in the most recent genome assembly. A subset of these transcripts is differentially expressed between midgut and salivary glands. The enrichment profiles of H3K27ac and H3K27me3 are mutually exclusive and associate with high and low levels of transcription, respectively. This distribution agrees with previous findings in Drosophila showing association of these two histone modifications with either active or inactive transcriptional states, including Polycomb-associated domains in silenced genes. This study provides a mosquito epigenomics platform for future comparative studies in other mosquito species, opening future investigations into the role of epigenetic processes in vector-borne systems of medical and economic importance.
Chromatin insulators are DNA-protein complexes with broad functions in nuclear biology. Based on the ability of insulator proteins to interact with each other, it was originally thought that insulators form loops that could constitute functional domains of co-regulated gene expression. Nevertheless, data from genome-wide localization studies indicate that insulator proteins can be present in intergenic regions as well as at the 5′, introns or 3′ of genes, suggesting a broader role in chromosome biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Recent results suggest that insulators mediate intra- and inter-chromosomal interactions to affect transcription, imprinting and recombination. It is possible that these interactions set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation. As a consequence, disruption of insulator activity could result in the development of cancer or other disease states.
Memory deficits in Drosophila nalyot mutants suggest that the Myb family transcription factor Adf-1 is an important regulator of developmental plasticity in the brain. However, the cellular functions for this transcription factor in neurons or molecular mechanisms by which it regulates plasticity remain unknown. Here, we use in vivo 3D reconstruction of identifiable larval motor neuron dendrites to show that Adf-1 is required cell autonomously for dendritic development and activity-dependent plasticity of motor neurons downstream of CaMKII. Adf-1 inhibition reduces dendrite growth and neuronal excitability, and results in motor deficits and altered transcriptional profiles. Surprisingly, analysis by comparative chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of Adf-1, RNA Polymerase II (Pol II), and histone modifications in Kc cells shows that Adf-1 binding correlates positively with high Pol II-pausing indices and negatively with active chromatin marks such as H3K4me3 and H3K27ac. Consistently, the expression of Adf-1 targets Staufen and Fasciclin II (FasII), identified through larval brain ChIP-Seq for Adf-1, is negatively regulated by Adf-1, and manipulations of these genes predictably modify dendrite growth. Our results imply mechanistic interactions between transcriptional and local translational machinery in neurons as well as conserved neuronal growth mechanisms mediated by cell adhesion molecules, and suggest that CaMKII, Adf-1, FasII, and Staufen influence crucial aspects of dendrite development and plasticity with potential implications for memory formation. Further, our experiments reveal molecular details underlying transcriptional regulation by Adf-1, and indicate active interaction between Adf-1 and epigenetic regulators of gene expression during activity-dependent neuronal plasticity.
Enhancer function underlies regulatory processes by which cells establish patterns of gene expression. Recent results suggest that enhancers are specified by particular chromatin marks in pluripotent cells, which may be modified later in development to alter patterns of gene expression and cell differentiation choices. These marks may contribute to the repertoire of epigenetic mechanisms responsible for cellular memory and determine the timing of transcription factor accessibility to the enhancer. Mechanistically, cohesin and non-coding RNAs are emerging as critical players responsible for facilitating enhancer-promoter interactions. Surprisingly, these interactions may be required not only to facilitate initiation of transcription but also to activate the release of RNAPII from promoter-proximal pausing.
Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.
The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.
Insulators are multi-protein-DNA complexes thought to affect gene expression by mediating inter- and intra-chromosomal interactions. Drosophila insulators contain specific DNA binding proteins plus common components, such as CP190, that facilitate these interactions. Here we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to the DNA in order to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.
Eukaryotic genomes are intricately arranged into highly organized yet dynamic structures that underlie patterns of gene expression and cellular identity. The recent adaptation of novel genomic strategies for assaying nuclear architecture has significantly extended and accelerated our ability to query the nature of genome organization and the players involved. In particular, recent explorations of physical arrangements and chromatin landscapes in higher eukaryotes have demonstrated that chromatin insulators, which mediate functional interactions between regulatory elements, appear to play an important role in these processes. Here we reflect on current findings and our rapidly expanding understanding of insulators and their role in nuclear architecture and genome function.
Summary
Insulators are DNA-protein complexes that can mediate interactions in cis or trans between different regions of the genome. Although originally defined on the basis of their ability to block enhancer–promoter communication or to serve as barriers against the spreading of heterochromatin in reporter systems, recent information suggests that their function is more nuanced and depends on the nature of the sequences brought together by contacts between specific insulator sites. Here we provide an overview of new evidence that has uncovered a wide range of functions for these sequences in addition to their two classical roles.
CTCF plays diverse roles in nuclear organization and transcriptional regulation. In this issue of Developmental Cell, Essafi et al. (2011) report a mechanism by which the repressive or active state of chromatin in a domain defined by CTCF can be switched by the Wt1 transcription factor to regulate gene expression.