Prácticas Educativas Colegio San Bernardino (IED).

It has been previously shown, that stem cells have a chromatin structure, DNA methylation pattern and nuclear organization that is different from differentiated somatic cells (Mayer et al., 2005; Boyer et al., 2006a; Kobayakawa et al., 2007; Mikkelsen et al., 2007; Chen and Daley, 2008; Meissner et al., 2008; Gaspar-Maia et al., 2009; Lim et al., 2009; Sun et al., 2011; Yellajoshyula et al., 2011; Zhou et al., 2011; Armstrong, 2012). Additionally, alterations in chromatin structure are able to change cellular fate and to influence differentiation of stem cells into different cell types (Lin and Dent, 2006; Graf and Stadtfeld, 2008; Lim et al., 2009; Yellajoshyula et al., 2011; Hung et al., 2012). The control of such chromatin alterations is important to regulate amplification of stem cells versus their differentiation. Furthermore, heterogeneity plays a central role and is a hallmark in both embryonic and adult stem cells (Graf and Stadtfeld, 2008). In this context, for neural stem cells it has been previously suggested that there are subtypes of radial glia: some that mainly regenerate themselves and produce neurons directly and others that

generate basal progenitors (which in turn are prone to generate neurons) to amplify the neuronal output (Miyata et al., 2001; Noctor et al., 2001; 2004; Pinto and Götz, 2007; Stancik et al., 2010).

Epigenetic processes have previously been suggested to be involved in control of stem cell proliferation versus differentiation (Bibikova et al., 2008; Eilertsen et al., 2008; Sasaki and Matsui, 2008; Sauvageau and Sauvageau, 2010; Ben-David and Benvenisty, 2011; Cedar and Bergman, 2011; Su et al., 2011). In this context, several reports showed that differentiation-genes in ES cells are silenced by Polycomb Group (PcG) complexes (Azuara et al., 2006; Bernstein et al., 2006; Bracken et al., 2006; Lee et al., 2006; Loh et al., 2006; Boyer et al., 2006b). As another example, the NuRD (nucleosome remodeling and disruption) complex is necessary for lineage commitment of ES cells showing that nucleosome remodeling is crucial in this process (Kaji et al., 2006).

Analysis of genome-wide association was able to show that transcription factors occupy less than a few percent of their consensus target sites (Carr and Biggin, 1999; Iyer et al., 2001; Yang et al., 2006; Joseph et al., 2010; Kaplan et al., 2011). Recently, it has been suggested that there are three different levels of transcription factor dependent gene regulation: DNA-binding site recognition, chromatin accessibility (controlled by epigenetic modifications) and co-factor availability in different cell-types (Fong et al., 2012). Therefore, accessibility of distinct target sites is of key importance for regulation of gene expression. So-called pioneer transcription factors are the first ones in a cascade of events establishing competence for gene expression ultimately leading to determination of cell fate (Zaret and Carroll, 2011). These pioneer transcription factors initiate the sequential binding of a series of factors by independently accessing the first target sites and reducing the number of co-factors needed (Carroll et al., 2006; Eeckhoute et al., 2006; Zaret and Carroll, 2011). In addition these factors can actively open up local chromatin and thus make it competent for further transcription factor binding (Xu et al., 2009; Zaret and Carroll, 2011).

Trnp1 may act similar to UTF1 as a chromatin associated protein involved in gene expression but also in alteration of chromatin structure at the same time (Okuda et al., 1998; van den Boom et al., 2007; Kooistra et al., 2009). Upon down regulation of Trnp1 most regulated genes were down regulated whereas upon forced Trnp1 expression most

affected genes were up regulated. This suggested an activating rather than a repressing role of Trnp1 on gene expression. It is therefore conceivable that Trnp1 represents a pioneer factor initiating transcription directly but also concomitantly leading to changes in chromatin state (see also the genes affected upon down regulation of Trnp1 7.2.1) thereby influencing gene expression indirectly through other factors as well.

To further investigate the tight association of Trnp1 with chromatin, fluorescence recovery after photo bleaching was performed. However, fusion of GFP to the C-terminal end of Trnp1 inhibited its association with chromatin. FRAP experiments therefore where not able to show the strong interaction of Trnp1 with DNA. Trnp1-GFP exhibited much faster recovery rates than reported for core histones that are tightly associated with DNA and show slow recovery rates in FRAP experiments (Kanda et al., 1998; Kimura and Cook, 2001). However, Trnp1-GFP still showed much slower recovery rates as compared to GFP only. It has previously been suggested for Oct4-GFP (which recovered still faster as compared to Trnp1-GFP) that it is residing in a high molecular weight complex and therefore kinetics would be slower than expected for the size of the protein itself (van den Boom et al., 2007). The same may apply to Trnp1-GFP (although the protein does not interact anymore with DNA) as interactions with other protein complexes cannot be excluded (especially as Trnp1 has been reported to be endogenously expressed at low levels in the cell line used in this experiment (Volpe et al., 2006)). Additionally, as Trnp1 shows a highly predictable coiled coil domain, it may also interact with itself to form oligomers and therefore exhibit slower kinetics than GFP alone.

Towards an identification of Trnp1 interacting proteins, mass spectrometry analysis was performed. However, use of Trnp1-GFP for pull down in HEK cells showed no reproducible binding partner of Trnp1. This fusion construct later turned out to preclude chromatin association of Trnp1 during mitosis and it may therefore also inhibit interaction with binding partners. However, as HEK cells do not endogenously express Trnp1, it is also possible, that the natural binding partners of Trnp1 are not expressed in HEK cells. Thus use of a cell line that shows endogenous expression of Trnp1 may help to identify naturally Trnp1 associated proteins in the future.

The exact molecular mode of Trnp1 action remains partially unidentified and needs further investigation. Lacking any known DNA binding domain or protein motif, Trnp1 may represent the first member of a new class of molecules.

In summary, this work showed a key functional role of Trnp1 during mammalian forebrain development, which was further emphasized by its intriguing expression pattern in the developing human brain. Trnp1 regulates tangential versus radial expansion of the cerebral cortex and is able to control folding of the developing brain. Thus this work identified an entirely novel, previously unrecognized DNA associated protein as a molecular switch in neural stem cells.

6 Material and Methods