The diversity of ATP-dependent chromatin remodelling complexes has probably evolved to accommodate the major changes in chromatin regulation that occurred during the evolution from unicellular eukaryotes to vertebrates.
The SWI/SNF family is one of the most-studied of chromatin remodelling complexes. It has exchanged and rearranged subunits during evolution from yeast to vertebrates. The increase in combinatorial diversity of complexes, which regulate chromatin structure could be a strategy to handle the developing 30-fold increase of genetic regulatory information from files to vertebrates, excluding protein coding genes (Wu et al, 2009).
It has been demonstrated that many vertebrate chromatin-regulatory complexes are assembled in a combinatorial manner. Consequently, this offers an expanding potential for diverse gene expression patterns compared to unicellular eukaryotes. This notion is underlined, as during the evolution of multicellularity and complex body
plans, the demand for tissue-specific and developmental-stage specific expression of genes coincides with an increase of complexity in chromatin organization and chromatin regulation.
Considering that the greatest demand for diverse patterns of gene expression occurs in the development and function of the brain, it is probably no co-incidence that a diversity of neural phenotypes is emerging from genetic studies of the subunits of chromatin remodelers in the nervous system (Yoo & Crabtree, 2009). The dynamic context with different regulatory levels from the integration of signalling events to the complexity of epigenetic histone modifications and chromatin remodelling can in turn influence the output of gene regulatory information. Therefore, further contemplations are necessary to evaluate the data of this work.
5.4.1 A dynamic remodelling complex with different functions
Aside from CHD4/NuRD it has recently been described that ATP-dependent chromatin remodelling complexes, based on the alternative DNA dependent ATPases, Brg1 and Brm, play essential roles during neural development in both vertebrates and invertebrates. For example Brg1 misexpression affects the development of neural tissue development, as Brg1 Morpholinos injection into zebrafish embryos expands the expression domain of the fore brain marker gene six3, a reduction of the mid-brain boundary marker engrailed2 and the hindbrain marker gene krox20. Neural crest cells derive from the neuroectoderm and migrate laterally to become pigmented skin cells, peripheral neurons, and glia. In addition, they form cartilage and bones of the facial structures. The lack of Brg1 function severely reduces the prospective neural crest cells and neural crest derived structures fail to express the neural crest marker gene snail2.
In the context of Xenopus neurogenesis, Brg1 is expressed ubiquitously in early development and becomes restricted to the neural tissue a later stages. In situ studies demonstrated distinctive expression pattern for the brg1 and brm paralogs. In 2004, our laboratory could demonstrate the specific expression pattern of Brg1 and Brm. At tailbud, stage brm is expressed in the hindbrain, spinal cord, pronephros and somites. Brg1 however is restricted to the branchial arches (Linder et al, 2004).
Xenopus Brg1 is important in the ß-catenin dependent determination of secondary body axis formation (Singhal, 2005). In addition, the transactivation of the bHLH transcription factors Neurogenin and NeuroD (see results) proves Brg1 to be required for neurogenesis (Seo et al, 2005a; Seo et al, 2005b). Loss of Brg1 function
in Xenopus results in a reduction in n-ß tubulin expression and a failure of Ngnr1 and NeuroD to promote neural differentiation of neurons from proneural cells (Seo et al, 2005a; Seo et al, 2005b). In mammals, Brg1 is expressed in neural stem cells, which give rise to neurons and glial cells (Matsumoto et al, 2006). Loss of Brg1 function reduces the expression of proteins associated to neural stem cell maintenance, e.g. Pax6 and Sox1. These remodelling complexes have dedicated functions at different stages of neural development that appear to arise by combinatorial assembly of its subunits (Yoo & Crabtree, 2009)
Considering the stage specific functions of CHD4 in its ATPase dependent and independent manner and the different potential interaction partners, forming a putative NuRD complex, my observations could also be influenced due to different interaction partners at different stages during development.
In mammals, NuRD includes the subunits CHD3/CHD4 and the histone deacetylases HDAC1 and HDAC2 to function as transcriptional repressors. Like BAF complexes, mammalian NuRD complexes achieve diversity in regulatory function through combinatorial assembly of its subunits. Beside the core ATPases CHD3 and CHD4, there are three main accessory subunits, which are encoded by different gene families. First, MTA (metastasis-associated), MBD (methyl-CpG-binding domain) and the RbBP (retinoblastoma-associated-binding protein) are part of NuRD as described in the introduction. In addition, each complex contains one MTA protein, MTA1, MTA2 or MTA3, which are mutually exclusive and nucleate complexes with different, and sometimes opposite, functions. The composition of the NuRD complexes varies with cell type and in response to signals within a tissue (for review see (Denslow & Wade, 2007)), which gives rise to a diversity of complexes with distinct functions. Thus, CHD4 misexpression during development and cellular differentiation must be seen under this premise. As an example for different compositions of the NuRD complex, which lead to different CHD4 functions, the subunit MBD2 or MBD3, are functionally distinct and contribute to different forms of the complex (Feng & Zhang, 2001). This is also true for the subunits RbBP4 and/or RbBP7.
Further studies of the different subunits of the mammalian NuRD complex have shown additional functions during development. Inactivation of mouse Mbd3 results in death during mid-gestation, stemming from the failure of the inner cell mass to form an epiblast and the subsequent failure of embryonic and extra-embryonic tissues to organize properly after implantation (Kaji et al, 2007).
A further role of the NuRD complex is crucial during differentiation. Loss of Mbd3 results in the failure to assemble NuRD complexes and probably reflects a loss of function for these complexes. Mbd3-null ESCs (embryonic stem cells) have been
shown to be viable and can initiate differentiation in culture, but they fail to commit to developmental lineages, due to impaired silencing of pluripotency genes (Kaji et al, 2006). Conditional inactivation of CHD4 in the haematopoietic cells of mice leads to impaired haematopoietic stem-cell homeostasis and impaired differentiation into myeloid cells with a defective thymocyte development and defective activation of the cd4 locus (Williams et al, 2004; Yoshida et al, 2008). Consequently, the NuRD complex is crucial for the correct silencing of genes during early development to allow proper patterning and cell lineage commitment.
In Drosophila, CHD4/Mi-2 also exists in a novel chromatin-remodelling complex, referred to as dMec that does not rely on histone deacetylation to affect transcriptional repression of proneural genes. Furthermore, the CHD4/Mi-2 related factor CHD3 acts as a monomer and does not associate with additional subunits in vivo. These results ad an additional complexity to the composition and function of CHD chromatin remodelling complexes (Kunert & Brehm, 2009). In relation to my work, these new insights in CHD4 function open the possibility that CHD4 gain-of- function could be mediated via additional functions, but HDAC activity.
In addition, our notion that CHD4/NuRD regulates signalling events to coordinate gene expression dynamically in a context dependent manner is underlined by very recent data that demonstrate that CHD4/NuRD orchestrates proper signalling in the context of DNA damage repair (Chou et al, 2010; Larsen et al, 2010; Polo et al, 2010; Smeenk et al, 2010).
5.4.2 Dynamic remodelling complexes to serve additional functions?
One essential question can be asked, why this diversity of different remodelers evolved and why the regulation of the genome requires functionally different ATP- dependent chromatin remodelers if they all function in an increase of nucleosome mobility?
One argument could be that beside their function as remodelers, additional roles and molecular functions have been discovered recently. For example, ISWI complexes have been shown to be required for maintaining the higher order structure of the Drosophila melanogaster male X chromosome 8, and INO80 complexes are involved in telomere regulation, chromosome segregation, checkpoint control, and DNA replication during cell division. For review see (Morrison & Shen, 2009).
Even within their traditional role of transcriptional regulation, ATP-dependent chromatin remodelers do not function in a consistent manner. For example, the
Brahma-associated factor (BAF) complexes, which belong to the SWI/SNF family, can function as both transcriptional activators, as well as repressors and can even switch between these two functions at the same gene (Chi et al, 2003). Moreover, tissue-specific BAF complexes have been reported to interact with a variety of transcription factors in different cell types. This enables the complexes to provide context dependent functions arising from their different interaction partners, as described above. This information-integrating network raises the question, how it can be entangled experimentally and which functions are crucial in different contexts? How can such a system be studied and which fundamental rules can be administered to a dynamic system, which evolved during development to flexibly provide a balanced system of information integration for the benefit of evolution? I approached these questions to entangle this complexity by interfering at two different time points during development and could demonstrate changing roles of the CHD4 chromatin remodelling ATPase function during neural development. Bearing in mind that we approach truth, as we apply ontological, epistemological and methodological reductionism to the complexity of a developing organism, this is the most adequate manner to generate new knowledge. A profound understanding of these fundamental biological processes achieved by experimental approaches in model organism as Xenopus will enable to dissect the complexity of epigenetic regulatory network and translate it to human biology. This understanding of biological processes will provide opportunities to understand disease development and putative solutions for problems in the medical field.