A list of eight retina-specific enhancers. The 8 retina-specific enhancers selected from the VISTA Enhancer Browser are listed above. They are grouped into 3 sub-groups according to the reporter expression in mouse embryos derived from these enhancers and the expression pattern of their flanking genes. The enhancer IDs, their expression pattern and the flanking gene names were retrieved directly from VISTA Enhancer Browser, with an expression pattern described as ‘tissue type (positive sample number/total sample number)’. Flanking genes with expression in the retina are shown in bold.
The formation of the neatly layered retina during embryonic development is dictated by a series of complicated transcription factor interactions. Retina-specific expression of these transcription factors is an essential step in establishing retinal progenitor cells (RPCs) from embryonic stem cells. The transcriptional control of gene expression is largely mediated by the combinatorial interactions between
Currently, the prevailing method of studying TFBSs and transcriptional regulatory networks is to determine the function of tissue-specific trans-acting factors based on data from genome-wide gene expression profiling and chromatin immunoprecipitation (ChIP). ChIP is often used to investigate protein-DNA interactions in a cell. Coupled with massive parallel sequencing, ChIP-seq is capable of mapping the genome-wide protein-DNA interaction at a finer resolution (Valouev et al., 2008) to identify candidate enhancer sequences (Visel et al., 2009). Thus, a regulatory cascade can be recognized via consequential analysis of the factors involved.
Here, we present a new method for the computational analysis of TFBSs and transcriptional regulatory networks utilizing genome-wide sequencing, expression, and enhancer data. In contrast to the traditional method, which mainly focuses on factor expression analysis, we emphasize the sequence elements with tissue-specific enhancer activity. Our hypothesis is that enhancers, non-coding sequences that direct gene expression in a cell-/tissue-specific manner, contain common TFBSs that allow the key protein factors (important for the development of that cell/tissue type) to bind. Experimentally verified tissue-specific enhancer elements selected from enhancer databases were carefully screened for common trans-acting factor binding sites to predict potential sequence-factor interacting networks. DNA-binding protein factors that associate with multiple enhancers can be analyzed using experimental methods.
As proof-of-principle, simple transcriptional regulatory networks of embryonic retinal development were assembled based on common/key factors and their interacting genes, as determined by literature search. These resulting networks provide a general view of embryonic retinal development and a new hypothesis for further experimentation.
2. Methods and results
In this study, we aimed to develop a method for the identification of regulatory networks for cell/tissue-specific gene expression. To test our hypothesis of the existence of common TFBSs on the enhancers of cell/tissue specific genes, we employ the mouse developing retina as a model system. Enhancers that direct retina-specific gene expression were selected and their sequences were thoroughly screened for common TFBSs and
2.1. Retina-specific enhancers
To determine the transcriptional regulatory networks that govern retinal development, we identified and selected enhancer elements that direct gene expression in the retina by searching the VISTA Enhancer Browser. The VISTA Enhancer Browser is a central resource for experimentally validated human and mouse non-coding DNA fragments with enhancer activity as assessed in transgenic mice. Most of these non-coding elements were selected for testing based on their extreme conservation in other vertebrates or epigenomic evidence (ChIP-Seq) of putative enhancer marks. The results of this
|Reporter expression pattern derived from enhancer activity||Annotation of reporter expression||Flanking genes of enhancers and their endogenous expression in mouse embryos|
|1||1113||eye(4/6), limb(4/6)||Retina + non-CNS|
|1487||eye(3/5), limb,||Retina + non-CNS||Ccdc39: E14.5|
|1753||eye(7/7), limb, nose||Retina + non-CNS||Arrdc3: E14-19|
|2||775||eye(6/9), limb, nose, branchial arch||Retina + non-CNS||AA408296: unknown||Irf6: not in retina|
|926||eye(4/5), heart||Retina + non-CNS||Lao1: unknown||Slc2a1: not in retina|
|3||1218||eye(6/7)||Retina+ Spinal cord||Pah: not in retina|
|760||eye(5/5), heart, other||Retina+ Spinal cord|
Trans-acting factor binding sites on retina-specific enhancers
The binding of trans-acting factors (e.g., transcription factors) to non-coding regulatory DNA (e.g., promoters, enhancers, etc.) is an essential process in the control of gene expression. This protein-DNA interaction helps recruit the DNA polymerase complex and co-activators to form the transcription machinery. The binding of these protein factors can also act as repressors to prevent transcription. Identification of a TFBS in the enhancer and promoter for a gene may indicate the possibility that the corresponding factors play a role in the regulation of that gene. Importantly, the ability of an enhancer to direct cell/tissue-specific gene expression is achieved via the binding of tissue-specific
2.3. A motif containing Pou3f2 binding sites
Since all 8 enhancers possess the ability to direct retina-specific gene expression, there may be key TFBSs shared amongst these retina-specific sequence elements. To test this hypothesis, we sorted and screened the TFBSs of each of the 8 enhancers to identify common ones using a Matlab program that we developed for this study (Supporting data 6). This Matlab program for common TFBS selection was designed to compare the TFBSs on each of the retina-specific enhancer elements predicted by TESS. TFBSs for two or three different enhancers can be sequentially compared. A “model” character was used as the comparison category instead of the binding site name in both TESS and our Matlab program. As defined in TESS, a model is “the site string or weight matrix used to pick this site” (Schug, 2002), and thus describes the nature of a binding site. One factor may have multiple models, and one model may be shared by multiple factors. The model character is the only necessary parameter to characterize the transcription factors depending on their binding site property.
With this sorting/searching program, we identified a TFBS for Pou3f2 (also known as Brn2) that was present in all 8 retina-specific enhancers (Fig. 1A). Previous studies have demonstrated that the Pou3f2 transcription factor plays an important role in the development of neural progenitor cells (Catena et al., 2004; Kim et al., 2008b; McEvilly et al., 2002; Sugitani et al., 2002). Furthermore, the literature reports that this motif was first discovered as a
We thus speculate that this Pou3f2 binding site may exist in regulatory sequences among genes important for the development of neural retinal progenitor cells (RPCs). Therefore, the
trans-acting factors involved in transcriptional regulatory networks of retinal development
It is unlikely that only one factor (i.e., Pou3f2) is involved in regulating retina-specific gene expression.
Key TFBSs should be common to all or a subset of retina-specific enhancer elements;
The flanking genes of these enhancer elements should be expressed in the retina during early retinal development, because an enhancer often regulates the expression of its flanking gene(s);
The binding factors should have a known function in retinal development, or
If the binding factors have an unknown function in retinal development, they should be at least expressed in the retina during retinal development. In this case, the expression of the factor provides novel hypothesis for their function in retinal development, which needs to be tested by functional studies.
Based on the above assumptions, the information on the expression of the flanking genes of enhancer elements in the developing retina is necessary for the TFBS analysis. Five databases of gene expression (see Table 2) were searched. The information on the expression of these flanking genes were retrieved from these databases (Tables 1, 4 and supporting data 3). The factors that do not express in the retina during embryonic retinal development, e.g., around E11.5 (when enhancer elements were active), were set aside for further analysis of retinal transcriptional regulatory networks. We then searched common TFBSs among subsets of the 8 retina-specific enhancers. The TFBSs common to individual different sub-groups were combined. In addition to Pou3f2, five other factors (i.e., Crx, Hes1, Meis1, Pbx2, and Tcf3) were identified (Table 3). Four of the 6 factors have known functions in retinal development, which is consistent with our hypothesis. The last two factors do not have known functions in the retina. However, the prediction of their binding with groups of enhancers suggests they play a role during retinogenesis. As the binding sites of these 6 factors were shared among a subset of retina-specific enhancer elements, these 6 binding
|Gene expression database (emage) of Edinburg Mouse Atlas Project (EMAP, v5.0_3.3)||http://www.emouseatlas.org/emage/||(Richardson et al., 2010)|
|Gene Expression Database in Mouse Genome Informatics (MGI, version 4.4)||http://www.informatics.jax.org||(Finger et al., 2011)|
|Eurexpress||http://www.eurexpress.org||(Diez-Roux et al., 2011)|
|VisiGene Image Browser||http://genome.ucsc.edu/cgi-bin/hgVisiGene||(Kent et al., 2002)|
|Genome-scale mouse brain transcription factor expression analysis||Supplementary data S4 and S6||(Gray et al., 2004)|
|Factor||Binding site||Known function||Expression pattern||Presence in enhancer element|
|Pou3f2||ATTTGCAT||Induce Bipolar cells||E10.5-14.5 in retina; diencephalon, future midbrain, future SC, rhombencephalon||All 8 elements|
|Crx||tgaggGGATCAAcagact||Induce Photoreceptors||E11-adult in retina||hs27, hs258, hs546, hs1170|
|Hes1||CTTGTG||Repress Amacrine, Horizontal, and Ganglion cells;|
|E11-13 in retina, thalamus, hypothalamus, striatum, olfactory epithelial||hs27, hs258, hs1170|
|Meis1||CTGTCActaagatgaca||retinal cell fate determination||E10.5-14.5 in retina, lens vesicle, diencephalon, future sc, hindbrain||hs27, hs258, hs546, hs1170, hs932, mm165|
|Pbx-2||cacctgagagTGACAGaaggaaggcagggag||No function known in retina||E10.5-14.5 in retina, thalamus, midbrain, hindbrain, sc, ear||hs27, hs258, hs546, hs1170, hs932, mm165|
|Tcf3||ccaccagCACCTGtc||No function known in retina||E13.5 in retina (MGI)||hs27, hs258, hs546, hs1170|
|Gene||Function (related to retina development) and reference|
|Ascl1||With Mash3, regulate the neuron/glia fate determination (Hatakeyama et al., 2001); with Mahs3 and Chx10, specify Biopolar cell identity (Satow et al., 2001).|
|Irx5||Off circuit subsets of bipolar interneuron (Cheng et al., 2005; Cohen et al., 2000; Kerschensteiner et al., 2008).|
|Irx6||No known clear function in retina. But It expresses in the in the area lining the lumen of the otic vesicle including the region giving rise to ganglion complex of CN VII/VIII at E11.5 through E16.5 and overlaps with Mash1 (Cohen et al., 2000; Mummenhoff et al., 2001)|
|Fxr1||Retina pigmentation(de Diego Otero et al., 2000); other function not known.|
|Nr2f1||Amacrine development, may involve in cone differentiation; express in a unique gradient in retina along D/V axis (Inoue et al., 2010).|
|Zfand5||No known function in retina.|
Interestingly, three common TFBSs (i.e., Pou3f2, Crx, and Meis1) were present among enhancer elements hs27, hs258, and hs1170 (see Tables 1, 3). Sequence alignment of the three enhancer elements and
Meis1 together with Meis2, as members of the TALE-homeodomain protein Homothorax (Hth) related protein family, were known to be expressed in the RPCs of mouse and chick (Heine et al., 2008). Meis1 was expressed in RPCs throughout the entire neurogenesis period, and Meis2 was expressed more specifically in RPCs before the initiation of retina differentiation. Together, they function to maintain the RPCs in a rapid proliferating state and control the expression of other ocular genes, e.g., Pax6, CyclinD1, Six3 and Chx10 (Bessa et al., 2008; Heine et al., 2008). Since Meis1 binding sites are present in a subset of retina-specific enhancers, Meis1 may function as an RPC-specific factor. Since the onset of mouse retina neurogenesis is approximately at E10.5 when the ganglion cells first appear (Leo M. Chalupa, 2008). By E11.5, RPCs of all six cell types are highly active. Therefore, binding of Meis1 with enhancers might influence the cell fate of these RPCs.
The presence of common Pbx2 binding sites may indicate a novel functional role of Pbx2 in RPCs, since the function of Pbx2 in retinal development has not been documented. Previous studies have shown that Pbx2 is expressed in the zebrafish retina and tectum (French et al., 2007) together with Pbx1 and Meis1, and down-regulation in their expression caused by the deficiency of Prep, the prolyl endopeptidase will lead to eye anomalies (Deflorian et al., 2004; Ferretti et al., 2006). Pbx and Meis proteins are major DNA-binding partners that form abundant complexes (Chang et al., 1997). Thus, there is a possibility that Pbx2 may function in the development of RPCs via the interaction with Meis1 and also regulate other RPC-specific genes (e.g., Irx5, Nr2f1, etc) through enhancer binding (Table 1).
Tcf3 is not yet known to have a function in embryonic retinal development. However, since Tcf3 binding sites are present among the retina-specific enhancer elements, and Tcf3 is expressed in the retina during embryogenesis, its specific function in retinal development needs to be confirmed.
2.5. Generation of transcriptional regulatory network for early retinal development
Based on the available expression data from VISTA Enhancer Browser and gene expression databases (Table 2), it is known that these 8 enhancer elements and their common binding
Retina-specific gene expression is most likely determined by two kinds of interactions: (1) the enhancers with their binding protein factors, and (2) the protein factors with their interacting partners. The information about these interactions was used to generate the transcriptional regulatory networks important for retinal development. Therefore, transcriptional regulatory networks of embryonic retina were predicted based on these 6 common/key
To construct retinal transcriptional regulatory networks, a java-based software program named BioTapestry (Longabaugh et al., 2009) (http://www.biotapestry.org/, version 5.0.2) was used to organize the factors and their known interacting partners. BioTapestry is a network facilitating software program designed for dealing with systems that exhibit increasingly complex over time, such as genetic regulatory networks. Its unique annotation system allows the illustration of enhancer-regulated gene expression and connection between factors. Experimental evidence can also be added to network elements after the network was built, as a proof of particular interactions. We only used the presenting function of BioTapestry here to show the networks of retinal development during early neurogenesis. For better illustration, the network was mapped according to the 3-layer structure of the mouse retina.
Based on the published information on the interacting factors of these 6
|Factor name||Reference cited|
|Cabp5||(Kim et al., 2008a)|
|Chx10||(Hatakeyama et al., 2001)|
|CyclinD1||(Bessa et al., 2008; Heine et al., 2008)|
|Foxn4||(Shengguo Li, 2004)|
|Grk1||(Young and Young, 2007)|
|Grmb||(Kim et al., 2008a)|
|Hes6||(Bae et al., 2000)|
|Mash3||(Hatakeyama et al., 2001; Satow et al., 2001)|
|Math5||(Lee et al., 2005)|
|Nestin||(Rowan and Cepko, 2005)|
|NeuroD1||(Conte et al., 2010)|
|Nr2f1||(Inoue et al., 2010; Satoh et al., 2009)|
|Otx2||(Kim et al., 2008a; Young and Young, 2007)|
|Pax6||(Ferretti et al., 2006; Lee et al., 2005; Oliver et al., 1995)|
|Prep1||(Deflorian et al., 2004; Ferretti et al., 2006)|
|Rax||(Heine et al., 2008; Martinez-de Luna et al., 2010)|
|Six3||(Oliver et al., 1995)|
|Six6||(Conte et al., 2010)|
In summary, the computational method we developed in this study can be described as following. First, experimentally verified enhancer elements can be selected from enhancer databases, e.g., the Vista Enhancer Browser, based on tissue/cell-specific expression patterns derived from the enhancer element and its flanking gene (Fig. 4A-A). These tissue-specific enhancer elements can be located in the non-coding regions in inter- or intra- genetic sequences. Then, the
In this study we have explored a new way of using existing TFBS-finding methods to predict
This work is supported in part by grants (EY018738 and EY019094) from the National Institute of Health, the New Jersey Commission on Spinal Cord Research (10A-003-SCR1 and 08-3074-SCR-E-0), and Busch Biomedical Research Awards (6-49121). The authors thank the Cai lab members for helpful discussions and for proof-reading the manuscript.