Genetic and Epigenetic Regulatory Mechanisms in Mouse Ocular Development and Disease
Principal Investigator: Ales Cvekl, Ph.D.
Departments of Ophthalmology and Visual Sciences and Molecular Genetics
Summary of the research
Understanding the process of gene expression requires the elucidation of both genetic and epigenetic regulatory mechanisms. The genetic mechanisms include sequences of cis-acting regulatory elements in promoters and enhancers, and sequences of hundreds of genes encoding DNA-binding trans-acting transcription factors. These genes are expressed either ubiquitously or in specific cell types. The decision of which gene is expressed in specific cell types and at which level is epigenetically regulated. In addition to these primary DNA-binding factors, a number of proteins, the “co-factors” is required to make these regions functional in transcription. When all these molecules are properly assembled, RNA polymerase II holoenzyme and its auxillary proteins are recruited to the promoter regions, the transcription complex reaches the “maturate state” and the RNA synthesis is initiated (Hatzis and Talianidis, 2002). Thus, transcription requires a robust built up of multiple proteins assembled in the right place and right time.
The main reason for such an involved solution to gene activation is to overcome a generally repressive chromatin environment in which the DNA is organized in the eukaryotic cell. Thus, a significant portion of transcriptional co-activators possesses enzymatic activities that facilitate changes in chromatin to assist transcription. These enzymes covalently modify core histones and use ATP energy to change the positions of nucleosomes or to remove nucleosomes from specific regions of DNA (Narlikar et al. 2002).
Decisions about which genes are to be expressed or kept silent are often dependent on external stimuli that signal the information into the nucleus via a relatively small number of signal transduction pathways e.g. BMP/TGF-b, FGF, hedgehog (HH), Jak/Stat, Notch, Ras and Wnt. Their nuclear targets then share the responsibility of meditating transcription. The current developmental studies are aimed to dissect these regulatory processes in vivo by finding nuclear targets of these common signal transduction pathways (Barolo and Posakony, 2002).
Transcription also faces another challenge. During cellular division, the cell must decide either to maintain the “cellular” memory of its transcriptional status or to reset its transcriptional program by employing epigenetic mechanisms. During mitosis, most but not all transcription factors dissociate from DNA (Martinez-Balbas et al. 1995; Christova and Oelgeschlager, 2001). In contrast, the stable protein-DNA complexes in mitosis might help the rapid reactivation of transcription upon exit from mitosis as indicated by nuclease-hypersensitive sites that persist during mitosis (Martinez-Balbas et al. 1995). It is thought, that “chromatin” memory is important for lineage specification and commitment during development of multicellular organisms.
There is a small number of transcription factors whose activities can trigger formation of specific cell lineages or even an entire organ by epigenetic processes. These genes are called “stage-selector” or even “master” regulatory genes. One of the most widely studied such genes is Pax6/eyeless. Ectopic expression of Pax6/eyeless can trigger formation of the entire eye (see Gehring and Ikeo, 1999). Thus, expression of Pax6 is both essential and sufficient for determining distinct epigenetic states of the genome (Sheng et al. 1997; Pichaud et al. 2001; Cvekl et al. 2004). Studies of multiple roles of Pax6 in development, namely regulation of its expression and studies of its function in chromatin structure will reveal the details of transition from one developmental stage to another. “Master” regulatory role of Pax6 could be best explained if it can serve as a “pioneer” factor capable of initiating chromatin opening (Cirillo et al. 2002).
Goals
The long-term goal of our laboratory is to elucidate genetic and epigenetic mechanisms that regulate tissue-specific temporal and spatial gene regulation in the developing vertebrate lens. Lens development results from a series of inductive processes (Lang, 2004) and provides an excellent model system to study the link between tissue-restricted gene expression, lineage-specification, cellular proliferation, differentiation and signal transduction, migration and cell death, all of which are fundamental processes during organogenesis. Lens function depends on temporally and spatially controlled expression of crystallin genes. Crystallins encode structural proteins that accumulate to very high levels (approximately 450 mg/ml) while maintaining their water solubility to generate lens transparency and the refractive index. Expression of crystallins is initiated in lens precursor cells and is highly upregulated in terminally differentiated lens fiber cells.
We continue our work on studies of temporal and spatial regulation of crystallin genes. Specifically, we will focus on the elucidation of the function of a novel 220 bp long regulatory element DCR1, located 8 kb upstream from the mouse aA-crystallin promoter, that mediates activation via FGF signaling in lens fibers (Yang et al. 2005). aA-crystallin represents about 20% of all lens proteins. We will determine the nuclear targets of FGF-signaling that interact with DCR1. In addition, we will determine how DCR1 operates, focusing on the possibility of DNA-looping between the promoter and DCR1.
Lens-lineage originates from the surface ectoderm as a result of embryonic induction (see Grainger, 1992). Lens inducing tissues are thought to initially involve cardiac mesoderm and later the optic vesicle. Although the molecular identity of “lens-inducers” is not known, expression of Pax6 in the presumptive lens ectoderm is essential for the entire process of lens formation (see Cvekl and Piatigorsky, 1996; Lang, 2004). Expression of Pax6 is further required for the activation of expression of other lens lineage specific transcription factors Six3 and Sox2. Both Six3 and Sox2 are directly regulated by Pax6. It is moreover hypothesized that stabilization of Pax6 expression by Six3 and Sox2 is actually required for the formation of lens placode. Lens placode, comprised of lens progenitor cells, is the first morphological manifestation of lens formation. At the molecular level, it is characterized by the expression of N-cadherin, controlled by Pax6 (van Raamsdonk and Tilghman, 2000). In addition, Pax6 initiates expression of two transcription factors Mab21-like1 and FoxE3. Their function is to aid to the lens vesicle formation and its separation from the surface ectoderm. Pax6, Mab21-like1 and FoxE3 regulate expression of specific cell adhesion molecules (CAMs) including N-cadherin in the lens placode and lens vesicle. These CAMs appear to be critical for the rearrangement of the epithelial cells into the lens vesicle. Since the currently known targets of Pax6 only partially explain phenotypes associated with mutations in Pax6, we will continue our work to identify and characterize such morphoregulatory genes regulated by Pax6 during the initial stages of lens development.
During the late stages of lens placode formation, expression of two transcription factors, c-Maf and Prox1 is initiated. Although both of them are not essential for lens lineage formation, they control differentiation of lens fiber cells. Cell cycle arrest in lens is controlled by Prox1-regulated expression of p27KIP1 and p57KIP2 (Ring et al. 2000) and the activity of the retinoblastoma protein, pRb (Morgenbesser et al. 1994). Abnormal lens fiber cell differentiation in this model suggest that pRb also plays a unique role in this process. Thus, the identification of genes to which pRb binds should provide novel insight into its particular role in lens differentiation (see Harris, 2004; Nguyen and McCance, 2005). We will identify chromatin regions to which pRb binds in vivo in lens concomitant with the identification of the levels of histone acetylation and methylation in the pRb-occupied regions.
Lens vesicle, comprised of lens precursor cells, is a polarized structure. Its posterior cells, exposed to growth factors including FGFs from the prospective retina, withdraw from the cell cycle and undergo terminal differentiation to form the primary lens fibers. This process is accompanied by tenfold dowregulation of Pax6 expression and high upregulation of c-Maf (Yang and Cvekl, 2005) and Sox1 (Nishiguchi et al. 1998). The anterior cells maintain their proliferative capacity and will give rise to the anterior lens epithelium. When the cuboidal lens epithelial cells reach the lens equator, they also undergo terminal differentiation into secondary lens fiber cells. Thus, the lens grows throughout life by adding new layers of lens fibers. Nevertheless, this process never results in lens tumors. Concerning the role of Pax6 in lens development, the formation of lens progenitor cells requires high levels of its expression which is maintained in lens precursor cells. In contrast, differentiation of lens fiber cells requires downregulation of Pax6 expression. We hypothesize that pRb is involved in transcriptional silencing of Pax6. We will perform studies on the transcriptional regulation of Pax6 during eye development including a focus on several epigenetic mechanisms (e.g. histone modifications, incorporation of histone variants and its DNA methylation patterns).
Studies of temporal and spatial expression of lens differentiation markers, notably crystallins, are important for understanding the process of lineage commitment and terminal differentiation induced by growth factors. In our recent work (Yang et al. 2005), we investigated the molecular mechanism of activation of the mouse aA-crystallin locus by Pax6 and c-Maf. Our data suggest specific roles for chromatin remodeling complexes SWI/SNF (as inferred from the presence of the ATP-dependent enzyme Brg1) and ISWI (as inferred from the presence of the Snf2h enzyme). Using co-immunoprecipitations, we found that Pax6 binds to Brg1, and c-Maf associates with Snf2h (Yang et al. 2006). Our data suggest the following model of aA-crystallin gene activation: In lens precursor cells, Pax6 recruits moderate amounts of Brg1 to the locus resulting in initial chromatin remodeling of aA-crystallin locus followed by c-Maf binding to the aA-crystallin promoter. Promoter-bound c-Maf recruits Snf2h, which triggers moderate levels of aA-crystallin expression in the lens vesicle. In parallel, histone H3 K9 acetylations become evident in the promoter region. In differentiating lens fiber cells, expression of c-Maf is increased as a result of FGF signaling followed by additional recruitments of both Snf2h and Brg1 into the locus. The extensive chromatin remodeling and spreading of H3 K9 acetylation into a 14kb region promotes very high levels of expression of aA-crystallin gene. This model will be tested in vivo using conditional inactivation of Brg1 and Snf2h alleles.
Procedures
To identify novel and confirm known genes regulated by Pax6 during embryonic lens formation, we will use RNA microarray analysis of normal and Pax6 heterozygous lens tissues. Lens induction is delayed in Pax6 heterozygous embryos allowing the identification of Pax6-dosage sensitive genes. In addition, we will analyze differential gene expression in the surface ectoderm which failed to form lens precursor cells in Pax6 homozygous embryos. Expression of these Pax6-dependent genes will be characterized by immunohistochemistry and/or by in situ hybridizations using sections of the developing mouse embryo.
To identify the binding profiles of key transcription factors regulating lens lineage formation (such as Pax6 and Sox2) and differentiation (such as c-Maf, Prox1 and pRb), we will use high resolution tiled custom chicken and mouse arrays with approximately 200 genes/loci encoding protein critical for lens function using chromatin immunoprecipitation (ChIP) following chip analysis (ChIP on chip) using the Nimblegen platform. These genes will include all crystallin loci, other structural genes and all known regulatory genes including the key regulators of cell division and components of signal transduction pathways regulating lens development. We will use embryonic chicken lenses (stages E6, E12 and E18), as they are readily available in large quantities. We will also use newborn mouse (P1) lenses as our studies have shown distinct interactions of transcription factors and core histone modifications within the 16 kb mouse aA-crystallin locus (with high level of aA-crystallin expression) compared to the analysis of lens epithelial cells (with moderate level of aA-crystallin expression) (Yang et al. 2006).
To determine chromatin structure of the selected 200 genes, we will also use ChIP on chip technology described above. Specifically, we will map histone H3 K9 acetylation, histone H3 methylations and presence of histone variants H3.3 and H2A.Z; each of which indicate that a gene has been activated. In addition, using HpaII tiny fragment enrichment by ligation mediated PCR (HELP) developed by Dr. J. Greally, we will identify global changes in CpG methylation in these 200 loci during lens development. This integrative approach will lay the foundation for detailed studies of transcriptional regulation of the selected loci. For example, studies using the 450 kb long Pax6 locus will reveal which of its many distal enhancers are active in lens followed by their functional characterization in transgenic mice.
To determine the roles of ATP-dependent chromatin remodeling enzymes during lens development, we will use an available Brg1 flox/flox model (provided by Drs. D. Metzger and P. Chambon) and a similar Snf2h model currently being developed by Drs. T. Stopka and A. Skoultchi. We will delete these genes during three critical stages of lens development: 1. Using the Pax6EE/cre mouse (see Lang, 2004), we will delete Brg1 and Snf2h and from the presumptive lens ectoderm. 2. Using the DCR1/cre mouse currently being developed in the laboratory, we will inactivate these genes in the lens vesicle. 3. Using the aA-crystallin(-366)/cre mouse, we are currently performing analysis of Brg1 function in postmitotic lens fiber cells. Our data indeed demonstrate that Brg1 plays an important role in lens fiber cell differentiation.
Significance
Lens development is an excellent model system to study genetic and epigenetic regulatory mechanisms, because lens is formed from a single cell type. In addition, lens morphology and pattern of crystallin gene expression mark different steps of the differentiation process. The collective outcome from these studies will greatly expand the current models of the regulatory networks governing vertebrate lens development. The data gathered here will aid in the experimental design and analysis of lens lineage-formation in vitro using embryonic stem cells. Understanding of congenital ocular defects caused by mutations in genes that are the subject of this study (e.g. PAX6, c-MAF, and SOX2) will be expanded.
The known biology of Pax6 suggests that its distinct expression levels regulate two epigenetic states: formation of lens precursor cells and terminal differentiation of lens fibers. However, the currently known Pax6 ectodermal and 3’-SIMO enhancers do not promote its expression in the prospective lens ectoderm. We also do not know which distal enhancers of the Pax6 locus and which proteins are involved in Pax6-downregulation in postmitotic lens fibers. High-resolution studies of the 450 kb Pax6 locus using ChIP on chips will reveal these distal enhancers as they should be marked by increased or reduced histone H3 K9 hyperacetylations and other core histone modifications associated with transcriptional activation or repression.
The integrative analysis of gene expression with a particular focus on specific DNA-binding proteins, histone modifications, histone replacements and DNA methylation during lens development offers a unique opportunity to study this process in vivo. Analysis of the functions of Brg1 and Snf2h ATP-dependent chromatin remodeling enzymes at various stages of lens development will shed new light into the function of these enzymes during organogenesis.
Identification of genes occupied with the retinoblastoma protein in various stages of lens development will unmask the roles of this protein in lens terminal differentiation. This information has the potential value of determining how mutations in pRb cause formation of retinal tumors.
Dissection of the FGF-signal transduction pathway during lens fiber cell differentiation using the FGF-responsive regulatory element DCR1 in the mouse aA-crystallin locus will provide novel insight into the regulation of cell cycle exit and upregulation of genes, e.g. crystallins, that mark differentiated lens fibers.
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