A model system for investigating the interplay between chromosome conformation and gene expression. A. The eve locus is composed of individual regulatory modules (enhancers), which regulate expression throughout embryogenesis (left column). Each regulatory module can drive a reporter gene in a discrete part of the eve pattern. A Polycomb response element (PRE) is located just beyond the 3'-most enhancer. The locus is flanked by two insulators, nhomie at the 5' end and homie at the 3' end. B. Chromosome conformation capture (3C) shows that homie and nhomie interact with each other. The data are presented as % input, which is: ligated product, representing an interaction between the homie region (anchor primer, common to all reactions) and another part of the eve locus (numbered primer), divided by the total anchor fragment.
3 primary areas of research:
Chromosome Architecture and Gene Regulation by Chromatin Insulators, Epigenetic Gene Regulation by Polycomb-group Proteins, and Cooperative DNA binding by Sequence-specific Transcription Factors
Current work in the laboratory is focused on understanding two major aspects of nuclear genome regulation in eukaryotic organisms that affect genome organization in 3 dimensions. We and others have shown that these regulatory systems have a major impact on the packaging and utilization of the genome. The first is repression (and sometimes activation) of gene expression through structural changes in chromatin by the Polycomb group of chromatin regulators, which work in part through modification of histone side chains.
The eve locus is a Polycomb (Pc) domain, which keeps eve off in tissues where it must remain off for viability of the organism. This domain ends abruptly at each end of the locus, where the two insulators are located. When homie is replaced by non-insulator DNA, the Pc domain spreads into the adjacent domain, the expression of which is essential for survival. This is a major function of insulators in eukaryotic genomes, to stabilize boundaries between repressive and active chromatin.
The second aspect of genome regulation that we study is the organization of chromosomal loops by chromatin insulators, which impacts several chromosome functions. These include gene expression (by either facilitating or blocking interactions between enhancers and promoters), DNA recombination and repair (by influencing which linearly distant DNA sequences are accessible to each other and which are not), chromatin compaction during mitosis and meiosis, and epigenetic maintenance of gene expression. Insulators may facilitate epigenetic maintenance by helping to keep sister chromatids aligned following DNA replication, thereby allowing histone modifications, such as those propagated by the Polycomb group, to be faithfully templated from one cellular generation to the next.
homie-carrying transgenes pair with endogenous eve. A: eve-patterned lacZ RNA expressed from reporters with Homie is independent of transgene orientation. Embryonic stages with expression are shown. Top: diagram of chromosome ("hebe E": hebe CNS enhancer). Left and middle: red block arrows show Homie orientation. Right: control. B: Map of eve and flanking genes: restriction sites (MfeI, blue ticks; EcoRI, red ticks) and primers (numbered arrows) used in C, D. Green: stripe enhancers, blue: later-acting enhancers, yellow: PRE, orange: Homie and Nhomie. C: Endogenous Homie and Nhomie link with transgene Homie 142 kb away. Inset map: transgene Homie (red box), or DNA in the control, transgene-specific anchor primer, and EcoRI site. Main graph shows averages and standard deviations of 6 independent H3C assays, each in triplicate. Red lines, with Homie; green lines, with DNA; % input: ligated product % of anchor fragment, within each sample. Inset graph: data normalized to the average signal with DNA. D: Endogenous Homie and Nhomie interact with each other. Results of H3C, as in C, except with primer 9 (at endogenous Homie) as the anchor.
We are analyzing how long-range repression and activation occur over an entire genetic locus, even skipped (eve), and its genomic neighborhood, through the regulation of chromatin structure. The eve gene is flanked by insulators (called homie and nhomie) that functionally isolate it from neighboring genes. Along with Polycomb-group response elements, they maintain both the activated and repressed state within different developing lineages of cells. Both of these kinds of elements function in a variety of genes, and in mammals as well as in Drosophila, to regulate developmental processes such as stem cell maintenance and differentiation. Understanding the mechanisms will provide novel ways to attack cancer, which is caused in large part by mis-regulation of gene expression and chromatin structure.
Orientation-specific facilitation of long-range communication by homie. Dual reporter RNA expression 142 kb from eve. A and B have opposite orientations of homie, resulting in distinctly different topologies that give rise to different reporter gene activation patterns. Large green check marks indicate which of the two possible topologies is consistent with the observed activation pattern. Within the diagrams, magenta block arrows represent homie; smaller green and dotted green block arrows represent strong and weak activation by endogenous eve enhancers, respectively. Stages 5-6 (left panel in each quadrant) and 13 (right panel in each quadrant) are shown for each reporter. Within the micrographs, green arrows indicate aspects of eve-like expression; black arrows indicate elements of hebe-like expression (repeated in each segment).
Another focus of the laboratory has been to understand the biochemical basis of combinatorial control of gene transcription by DNA binding proteins. Embryos regulate their growth and development in many ways, but control of gene transcription is essential for directing cells along particular developmental pathways. In Drosophila, a cascade of nuclear regulatory events establishes very early differences in cell fates by producing intricate patterns of gene expression. Many of these pattern-forming genes encode DNA binding proteins that regulate each others expression, and subsequently instruct the rest of the genome in a manner appropriate to each position in the organism. These regulatory proteins are conserved across the evolutionary distance separating flies and humans. This applies to both their primary structure, implying similarity in mechanism, and often their developmental function. That is, the regulatory scheme in which they function solves a common problem of developing multi-cellular organisms.
Homie-Homie pairing facilitates transvection. Dual reporter RNA (lacZ or GFP, as indicated at the top) is seen in the APR (right end of embryos), and the mesoderm (seen in the middle panel for lacZ) from the diagrammed transgene combinations. Transgene reporters and enhancers (eve mesodermal, "Me", and anal plate ring, "APR") are in trans on homologous chromosomes. Block arrows show homie orientation. Stage 13 embryos are shown. A red "X" in a topology diagram indicates that this topology, predicted based on head-to-head insulator pairing, may not form because it tends to disrupt homolog pairing. Note that only lacZ expression is facilitated, due to the blocking action of the intervening pair of insulators in 3-D space (diagrammed at the bottom).
Our current studies revolve around understanding specific mechanisms of two types: first, which gene products interact directly with which genes and other gene products, and second, how this impinges on transcriptional regulation and, relatedly, the stability of the epigenome.
We study the regulation and function of two homeodomain-containing proteins. The homeodomain is a highly conserved sequence-specific DNA binding domain found in transcriptional regulators from yeast to humans. One of these, Engrailed (En), is a potent repressor of transcription that recruits the corepressor Groucho, a homolog of the TLE family of mammalian cofactors. We study interactions between En and the Pbx and Meis/PREP families of Hox protein cofactors, which serve to increase its DNA-binding specificity and thereby direct it to particular target genes. The interaction with En confers a novel activity on the Meis/PREP-Pbx complex (in Drosophila, Hth-Exd), that of transcriptional repression. Our analysis focuses on the biochemical interactions among these factors, and on the functional consequences of altering those interactions.
Even-skipped (Eve) is another homeodomain transcription factor that regulates developmental processes in a highly conserved fashion. Eve, like En, uses both Groucho-dependent and -independent mechanisms to repress transcription. The combinatorial regulation of gene expression by the homeodomain superfamily of transcription factors serves as a paradigm for understanding how cell-type specificity and intercellular signaling are integrated by DNA elements in all eukaryotic organisms.