SWItching On Epidermal Cell Fate

Abstract

Chromatin regulatory complexes are well known regulators of stem cell fate; however, the mechanisms regulating their activity are not well understood. In this issue of Cell Stem Cell, Bao et al. (2013) show that ACTL6a inhibits targeting of the SWI/SNF complex to differentiation genes, thereby preserving the epidermal progenitor state.

Overwhelming evidence collected over the last decade has pointed to a critical role for chromatin regulators, such as Polycomb Repressive Complex 1/2, Nucleosome Remodeling and Deacety-lase Complex, and the SWI/SNF complex, in controlling stem cell fate (Hu and Wade, 2012; Lessard and Crabtree, 2010; Surface et al., 2010; Wu et al., 2009). In general, chromatin regulators are thought to alter chromatin structure either by modulating DNA, histone modifications, or both, or by remodeling nucleosomes, which changes the accessibility of gene regulatory elements to the transcriptional machinery and leads to either gene activation or silencing. Most chromatin regulators are multisubunit complexes that consist of a few proteins possessing enzymatic activities and a large number of regulatory subunits that are thought to have a modulatory function in regulating affinity to DNA sites, stabilizing the complex, and enhancing or inhibiting the complex’s activity (Hu and Wade, 2012; Surface et al., 2010; Wu et al., 2009). Thus far, the majority of insights into their function have been gained from loss-of-function studies where enzymatic components of chromatin regulators were ablated, revealing phenotypes ranging from loss of stem cell self-renewal and aberrant differentiation to apoptosis (Hu and Wade, 2012; Surface et al., 2010; Wu et al., 2009). However, despite the proposed role for the nonenzymatic regulatory subunits of chromatin complexes in performing a fine-tuning function, our knowledge of the relative importance of their activities in controlling stem cell function is very limited. In work by Bao et al. (2013) published in this issue of Cell Stem Cell, the authors address this issue by uncovering the role for the SWI/SNF interacting protein Actin-like 6a (ACTL6a) in promoting the maintenance of epidermal progenitor cell state.

The SWI/SNF complex is a chromatin remodeling complex that functions to destabilize histone-DNA interactions, resulting in an open chromatin state and leading to transcriptional activation (Hu and Wade, 2012; Wu et al., 2009). It consists of the catalytic ATPase subunits Brg1 and Brm as well as 11 regulatory subunits. Interestingly, these subunits can be encoded by at least 20 different genes, resulting in 288 possible combinations (Wu et al., 2009). It has been speculated that different combinatorial assemblies of the SWI/SNF complexes have different DNA target affinities and functions (Wu et al., 2009). Studies of neuronal stem cells have confirmed that the composition of the SWI/SNF complex changes between stem cells and postmitotic neurons, and, importantly, showed that this switch is required for proper differentiation (Lessard et al., 2007). The SWI/SNF complex has also been shown to be essential for proper execution of the differentiation program in the epidermis, where it regulates the continuous transition of epidermal progenitor cells to the stratified layers (Indra et al., 2005). Previous studies showed that loss of expression of Brg1 and Brm leads to aberrant expression of differentiation genes and results in defective formation of the differentiated layers (Indra et al., 2005). However, how SWI/SNF activity is modulated during epidermal differentiation was unknown up to this point, and has been addressed in the current study by Bao et al. (2013).

Khavari and colleagues focused on ACTL6a because it is expressed in epidermal progenitor cells and is downre-gulated during differentiation. To dissect the role of ACTL6a in control of the epidermis, loss-of-function studies were performed where ACTL6a was conditionally ablatedin embryonic and adult mouse skin. These studies revealed thinning and erosion of the epidermis, and showed that the phenotype was due to loss of proliferation of the progenitor cells and premature expression of differentiation markers. The authors expanded this analysis to human cells and showed that RNAi-induced ablation of ACTL6a led to proliferation arrest and induced differentiation, while ACTL6a overexpression suppressed the epidermal differentiation program, indicating that the role of ACTL6a in the maintenance of epidermal progenitor state is conserved between mammalian species.

To gain insight into the mechanism of ACTL6a function in controlling epidermal differentiation, the authors performed bio-informatic analysis and found that the majority of the ACTL6a-regulated genes contained a DNA binding site for KLF4, the Kruppel-like factor 4 transcription factor, a prominent activator of differentiation in the epidermis (Segre et al., 1999). Consistent with this analysis, loss of ACTL6a expression from both human organotypic cultures and mouse skin resulted in the upregulation of KLF4 and its target genes, and further genetic epistasis analysis confirmed that KLF4 is indeed required for the expression of differentiation genes upon loss of ACTL6a in vitro.

ACLT6a has been described to be part of several epigenetic regulator complexes (Wu et al., 2009). Loss-of-function analysis of the key components of ACTL6a-containing complexes, however, revealed that reduced proliferation and precocious expression of differentiation genes in epidermal progenitors was unique to loss of BAF250a/ARID1A, the largest SWI/SNF subunit. Biochemical purifications confirmed interaction between ACTL6A and the SWI/SNF complex and genetic studies showed that a mutant form of ACTL6a that cannot bind the catalytic Brg1 subunit of SWI/SNF failed to reverse the differentiation phenotype observed upon loss of ACTL6a in vitro. These data thus clearly indicate that ACTL6a works together with SWI/SNF to control the epidermal progenitor cell state.

Removal of Brg1 and Brm from the epidermis results in differentiation defects (Indra et al., 2005), while removal of ACTL6a leads to precocious differentiation (Bao et al., 2013), suggesting that ACTL6a functions to inhibit SWI/SNF activity. This model was confirmed by the observation that SWI/SNF binding to the target differentiation genes is increased in differentiated keratinocytes as well as in keratinocytes where ACTL6a had been removed (Bao et al., 2013). These data indicate that the downregulation of ACTL6a in keratinocytes is critical to allow SWI/SNF to bind and promote expression of differentiation genes, including KLF4, to execute proper epidermal differentiation.

Importantly, ACTL6a appears to maintain epidermal progenitor state by preventing premature exit from the cell cycle and downregulating the expression of differentiation genes (Bao et al., 2013). Recent studies showed that ACTL6a/BAF53a activity is required for progenitor proliferation and maintenance in hematopoietic and neuronal stem cells (Krasteva et al., 2012; Lessard et al., 2007). It is thus tempting to speculate that ACTL6a might be a general regulator of stem cell maintenance.

In the future it will be important to understand the different mechanistic roles that the actin-like proteins are carrying out in the SWI/SNF complexes and how these roles are modulated in specific cell types. How does ACTL6a inhibit DNA targeting of the SWI/SNF complex? Also, how is ACTL6a downregulated upon differentiation? In neuronal stem cells, microRNAs are required for repression of ACTL6a/BAF53a (Yoo et al., 2009); however, the mechanism of ACTL6a downregulation in the epidermis remains to be determined. Studies in neuronal stem cells and embryonic stem cells showed that there are changes in the composition of the SWI/SNF complex between stem cell and committed progenies (reviewed in Lessard and Crabtree, 2010). While Bao et al. (2013) clearly showed that ACTL6a and BAF250a are required for epidermal progenitor cell maintenance, it will be important to investigate SWI/SNF composition in epidermal differentiated cells and to uncover subunits that are required for differentiation. It will also be important to define how the different combinations of SWI/SNF subunits confer functional diversity and uncover other cell-specific factors that may be implicated in the recruitment of this complex to tissue-specific target genes.

Finally, understanding this complexity will be critical for uncovering the role that the SWI/SNF complex plays in cancer. Loss of expression of different subunits of SWI/SNF, such as Brg1, Brm, BAF47, BAF57, BAF45, and BAF155 (Wu et al., 2009), has been reported in human tumor cell lines. Due to the identified role of SWI/SNF in promoting progenitor/stem cell proliferation in the epidermis, brain, and blood, it is plausible to hypothesize that changes in expression of SWI/SNF components might also provide proliferative advantages to other cell types, contributing to tumorigenesis (Wu et al., 2009). This paper provides an interesting opening into studying the functions of different combinatorial assemblies, as well as the precise mechanisms of cell-specific recruitment to target genes, and we look forward to future research into this area.