REVIEW

Roles of chromatin remodeling BAF complex in neuraldifferentiation and reprogramming

Ramanathan Narayanan & Tran Cong Tuoc

Received: 30 October 2013 /Accepted: 19 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract ATP-dependent BAF chromatin remodeling com-plexes play an essential role in the maintenance of the geneexpression program by regulating the structure of chromatin.There is increasing evidence that BAF complexes based onthe alternative ATPase subunits, Brg1 and Brm, control thedifferentiation of neural stem cells (NSCs) to generate distinctneural cell types and modulate trans-differentiation betweencell types. The BAF complexes have dedicated functions atdifferent stages of neural differentiation that appear to arise bycombinatorial assembly of their subunits. Furthermore, thedifferentiation of NSCs is regulated by the tight interactionsbetween the BAF chromatin remodeling complex and thetranscriptional machinery. Here, we review recent insightsinto the functional interaction between BAF complexes andvarious transcription factors (TFs) in neural differentiation andcellular reprogramming.

Keywords BAF complex . Chromatin regulation . Neuralstem cells . Neural differentiation . Reprogramming

Introduction

The generation of neural subtypes involves activation and/orrepression of distinct transcriptional programs in progenitors,a mechanism in which epigenetic and chromatin regulationsare believed to play a decisive role (Wen et al. 2009; MuhChyiet al. 2013; Ronan et al. 2013). Epigenetic modifications andchromatin regulation display dual mode of actions: exertingdirect effects on gene transcription and serving as platformsfor the recruitment of TFs, resulting in persistent changes in

the chromatin state (Wen et al. 2009; MuhChyi et al. 2013;Ronan et al. 2013). Eventually, these changes activate orrepress transcriptional programs either globally or specifically,which ultimately affect developmental events. Epigeneticmechanism and chromatin regulation influence the accessibil-ity of consensus regulatory elements to TFs by three mainmechanisms: DNA methylation, covalent histone modifica-tions and non-covalent, energy-dependent chromatin modifi-cations involving ATP-dependent chromatin remodeling com-plexes, such as SWI/SNF (switch/sucrose nonfermentable)(Wen et al. 2009; MuhChyi et al. 2013; Ronan et al. 2013).

Vertebrate SWI/SNF, also known as the BAF (Brg1/Brm-associated factor) complex, contains at least 15 different sub-units including: two interchangeable core ATPase subunits(Brg1 or Brm), invariant core subunits (BAF47, BAF155,BAF170) and a variety of lineage-restricted subunits (Lessardet al. 2007; Ho et al. 2009; Kadoch et al. 2013; Ronan et al.2013). Biochemical experiments have reported that the ATPasesubunits Brg1 or Brm, together with other core components(BAF155, BAF170 and BAF47), can remodel the nucleosomein vitro at a rate that is comparable with the entire complex(Phelan et al. 1999) (Fig. 1). However, hundreds of distinct BAFcomplexes are predicted to form in vivo by the combinatorialassembly of at least 15 identified BAF subunits (Ronan et al.2013). Studies have revealed unique assemblies and biologicalspecificities of BAF complexes in neural cell types, e.g., BAFcomplexes in neural progenitors (npBAF), in neurons (nBAF),in oligodendrocytes (olBAF) and in schwann cells (scBAF)(Fig. 2). Composite surfaces of the integrated subunits withinBAF complexes are believed to be responsible for their func-tional specificity in targeting specific genomic areas andinteracting with specific TFs (Zhan et al. 2011; Weider et al.2012; Li et al. 2013; Limpert et al. 2013; Ninkovic et al. 2013;Ronan et al. 2013; Tuoc et al. 2013a; Yu et al. 2013). DistinctBAF complexes bind to and coordinate with tissue-specific TFsin regulating gene expression thereby explaining the widespread

R. Narayanan : T. C. Tuoc (*)Institute of Neuroanatomy, Universitätsmedizin Göttingen,Göttingen, Germanye-mail: tran.tuoc@med.uni-goettingen.de

Cell Tissue ResDOI 10.1007/s00441-013-1791-7

role of BAF complexes in the development of numerous organs,including the nervous system (Zhan et al. 2011; Weider et al.2012; Li et al. 2013; Limpert et al. 2013; Ninkovic et al. 2013;Tuoc et al. 2013a; Yu et al. 2013).

Neural subtype-specific BAF complexes

It is evident that BAF complexes have crucial roles in thedevelopment of different cell types. Although most knownBAF complexes contain some common BAF subunits, eachcomplex has its own specific subunit(s). It becomes increas-ingly clear that the tissue and cell type-specific expression andcombinatorial assembly of its subunits confer the functionalspecificity to BAF complexes (Fig. 2) (Lessard et al. 2007; Hoet al. 2009; Kadoch et al. 2013; Ronan et al. 2013).

Embryonic stem cells (ESCs) are characterized by a uniqueesBAF complex with multiple subunits, including Brg1,BAF155 and BAF250a subunits. These subunits are impor-tant for the maintenance of pluripotency, proliferation andself-renewal of ES cells (Yan et al. 2008; Ho et al. 2009;Schaniel et al. 2009). It is interesting to note that the commit-ment of ES cells to neuronal precursors coincides with theinitiation of transcription of Brm and BAF170 subunits, whichthen replaces Brg1 and one of the two BAF155 subunits in theesBAF, respectively (Ho et al. 2009).

Similarly, the neural stem/progenitor cell-specific BAFcomplex (npBAF), comprising BAF45a and BAF53a stoichio-metric subunits, is necessary for the self-renewal and prolifer-ative properties of these cells (Lessard et al. 2007). On the otherhand, post-mitotic neurons lack BAF45a/d, BAF53a and SS18but express the paralogous BAF45b/c, BAF53b and SS18l1subunits that confer neuronal properties (Lessard et al. 2007;

Kadoch et al. 2013; Ronan et al. 2013). Interestingly, the switchin the subunit composition of these complexes is crucial for thetransition from proliferating neural stem/progenitor cells topost-mitotic neurons (Lessard et al. 2007).

The BAF subunits Brg1, BAF60a and BAF45b/d areexpressed at low levels in oligodendrocyte precursors (OLPs)and are up-regulated during differentiation (Yu et al. 2013).This implicated the existence of an oligodendrocyte (OL)-specific BAF complex (olBAF). Similarly, through immuno-histochemical analysis, Brg1 and BAF60a were found to beco-expressed with the schwann cell markers, Sox10, Oct6 andKrox20. This suggested the existence of a schwann cell-specific scBAF complex that contains at least Brg1 andBAF60a subunits (Weider et al. 2012).

It should be noted that the neural progenitor population inthe brain is very heterogeneous, e.g., distinct types of corticalneural progenitors have been identified in the developingmouse cortex such as: neuroepithelial cells (NEs), ventricularradial glial cells (vRGs) (Gotz and Huttner 2005), intermediateprogenitor cells (IPs), short neural precursors (SNPs) (Pontiouset al. 2008) and, most recently, outer radial glial cells (oRGs)(Shitamukai et al. 2011; Wang et al. 2011). In addition, earlyand late cortical progenitors have also been shown to harbormolecular differences through the differential expression ofTFs at distinct developmental time windows (Molyneauxet al. 2007). It is thus conceivable that both the early and latemolecular programs lie intrinsically within the progenitors,which are regulated by sequentially activated or repressedTFs. Such specific TFs are one of the determining factors onhow cortical progenitors choose between the lower layer (LL)and upper layer (UL) neuronal fate (Schuurmans et al. 2004;Molyneaux et al. 2007). Interestingly, our comparative expres-sion analysis of the BAF170 and BAF155 subunits in mousecortical progenitors indicated that, while both subunits exist inthe early cortical progenitors (between E10.5 and E14.5), theBAF170 is substituted by a BAF155 subunit in the late corticalprogenitors (Tuoc et al. 2013a).

Likewise, the mammalian cortex contains numerous neu-ronal subtypes organized in six layers that are different in theircytoarchitecture and functional properties. After gaining thelaminar identity, post-mitotic neurons acquire projection sub-type specification programs. The specification of projectionneurons also follows a coordinated temporal order. DuringE10.5–16.5, neuronal specification is characterized by wavesof transition that flow from sub-plate (SP)/L1 neurons,corticothalamic L6 neurons, subcerebral projection L5 neu-rons and finally to callosal projection L2–6 neurons(Molyneaux et al. 2007; Leone et al. 2008). Using a co-immunoprecipitation/mass spectrometry (coIP/MS) approach,an earlier study has identified brain-specific BAF subunits thatare associated with Brg1/Brm. In addition, through gene ex-pression pattern analysis, the identified BAF subunits werefurther catalogued into either the npBAF or the nBAF complex

Fig. 1 Chromatin remodeling activity of a minimal BAF complex in vitro.The schema demonstrates the in vitro chromatin remodeling activity of aminimal BAF complex containing the core subunits (Brg1, Brm, BAF47,BAF155, BAF170). The ATP-dependent chromatin remodeling complexuses the energy derived from ATP hydrolysis to alter histone-DNA con-tacts, which allows nucleosomes to slide to another position and therebymaking the DNA more accessible to the transcriptional machinery. Theschema is adapted from Phelan et al. (1999)

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(Lessard et al. 2007). Nevertheless, each subtype of neuralprogenitors (e.g., the early and late cortical progenitors asdiscussed above) and post-mitotic neurons may have its ownBAF subunits. Accordingly, Ctip2, a recently identified BAFsubunit (Kadoch et al. 2013; Ronan et al. 2013), is known to beexpressed specifically in the subcerebral projection L5 neuronsof the cortex (Arlotta et al. 2005). Co-expression analysisperformed in our laboratory indicated that other BAF subunits,such as Brg1, Brm, BAF155, BAF170 and BAF47, areexpressed in the Ctip2+ cortical neurons. The results suggestedthat the L5 subcerebral projection neuron subtype has its

specific BAF complex, which is distinguishable from the BAFcomplexes of other neuronal subtypes by the Ctip2 subunit.

Thus, one of the fundamental studies in the future will be tosystematically identify the compositions of neural subtype-specific BAF complexes.

Importance of BAF complexes in neural differentiation

The role of epigenetic and chromatin remodeling factors inneural development is broadly assumed based in part on the

Fig. 2 Neural subtype-specific composition of BAF complexes. Sche-matic diagrams depicting known subunits of BAF complexes in neuralprogenitors (npBAF), neurons (nBAF), layer 5 neurons (l5-nBAF), oli-godendrocytes (olBAF) and schwann cells (scBAF). While the BAFcomplexes contain some common subunits, there are specific andunique subunits depending on the cellular context e.g., BAF complexesin the early and late cortical progenitors have either both BAF155 andBAF170 or only BAF155, respectively (Tuoc et al. 2013a). There is aswitch in subunit composition between the npBAF complex (withBAF53a, BAF45a/d and SS18 subunits) and the nBAF complex (with

BAF53b, BAF45b/c and SS18l1) during neuronal differentiation(Lessard et al. 2007; Ronan et al. 2013). In addition, during theacquisition of L5 subcerebral projection neuronal identity, a subunitexchange occurs between Ctip1 in the nBAF complex and Ctip2 in thel5-nBAF complex (Arlotta et al. 2005; Ronan et al. 2013). Note that thesubunits of npBAF, nBAF (Lessard et al. 2007; Ronan et al. 2013) wereidentified by co-immunoprecipitation and mass spectrometry(coIP/MS), whereas compositions of the olBAF (Yu et al. 2013),scBAF (Weider et al. 2012) and l5-nBAF (Tuoc et al., unpublisheddata) complexes were examined by immunohistochemistry (IHC)

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rising number of neurodevelopmental disorders caused bymutations in genes encoding epigenetic factors and chromatinremodeling proteins (van Bokhoven and Kramer 2010; Ronanet al. 2013). Several studies indicate the importance of BAFcomplexes in various stages of neural development (refer toTable 1 for the list of mutants and their phenotypes).

The core ATPase Brg1 subunit has essential roles in theself-renewal and maintenance of murine NSCs. The lossof Brg1 in embryonic NSCs results in precocious neuronaldifferentiation, including severe defects in neurogenesisand gliogenesis (Matsumoto et al. 2006; Lessard et al.2007). While the NSCs-specific homozygous Brg1 knock-out mice had reduced brain size with severely malformed

cerebral cortex, including thinning of the midbrain alongwith near complete absence of cerebellum, the heterozy-gous mutants displayed exencephaly, indicating the non-redundant and dosage-sensitive roles of Brg1 in neuraldevelopment (Matsumoto et al. 2006; Lessard et al. 2007). Arecent study also indicated that the loss of Brg1 in adult NSCsresults in the enhancement of gliogenesis, thereby compromis-ing the number of neuronal cells (Ninkovic et al. 2013).Similar to its role in NSCs, Brg1 promotes proliferation ofneural crest cells (NCCs), thereby maintaining a multipotentcell pool at the neural crest (Li et al. 2013). In addition,conditional knockout (cKO) mutants lacking Brg1 in NCCswere predisposed to bleeding in the forebrain and death

Table 1 BAF subunits with their role in NSC differentiation and neural development

BAF subunit Mutants Phenotype Interacting partners References

Type of mutant In neural lineage

Brg1 Nestin-Cre;Brgf/f Brg1 deletion in NSCs Defect in self-renewal andmaintenance of murine NSC

Pax6, Gli1/3 Matsumoto et al. 2006;Lessard et al. 2007;Zhan et al. 2011

Wnt1-Cre;Brg1f/f Brg1 deletion in NCCs Defect in proliferation of NCCs Li et al. 2013

Olig1-Cre;Brg1f/f Brg1 deletion in OLs Impairment of OL differentiationand maturation

Olig2 Yu et al. 2013

Dhh-Cre;Brg1f/f Brg1 deletion in schwanncells

Defect in differentiation andmyelination of schwann cells

Sox10, NF-kB Weider et al. 2012;Limpert et al. 2013

Glast-Cre;Brg1f/f Brg1 deletion in aNSCand astrocytes

Defect in gliogenesis andneurogenesis of aNSC in SVZ

Pax6 Ninkovic et al. 2013

BAF155 BAF155-/- Null mutation Abnormal proliferation anddifferentiation in heterozygotes

Kim et al. 2001

BAF170 Emx1-Cre;BAF170f/f BAF170 deletion incortical progenitors

Increased genesis of IPs, enhancedcortical volume, surface areaand thickness

Pax6 Tuoc et al. 2013a

Emx1-Cre;BAF170OE BAF170 overexpressionin cortical progenitors

Decreased genesis of IPs,diminished cortical volume,surface area and thickness

Tuoc et al. 2013a

BAF53b BAF53b-/- Null mutation Defective dendritic outgrowth andsynapse formation

Wu et al. 2007

Camk2a promoter;BAF53bΔHD

Expression of dominantnegative BAF53b inforebrain excitatoryneurons

Abnormal spine structure andfunction; decline in cognitivefunctions

Vogel-Ciernia et al. 2013

SS18 Ss18-/- Ss18kd Null mutation; SS18 KD Defect in closure of neural tube,NSC proliferation, dendriticoutgrowth

Pparbp/PBP de Bruijn et al. 2006;Staahl et al. 2013

Crest/Ss18l1 Crest-/- Null mutation Defects in dendrite development CBP Aizawa et al. 2004; Qiuand Ghosh 2008

Ctip1/Bcl11a Brn4-Cre;Ctip1f/f Defect in neuronal morphogenesisin spinal cord

Tlx, CASK Kuo et al. 2010a, b;Estruch et al. 2012;John et al. 2012

Ctip2/Bcl11b Ctip2-/- Targeted deletion of Ctip2gene

Defect in the differentiation ofvomeronasal sensory neurons,medium spiny neuronshippocampal neurogenesis,specification of subcerebralprojection neurons

Arlotta et al. 2005, 2008;Enomoto et al. 2011;Simon et al. 2012

NSC (adult) neural stem cell, IPs intermediate progenitors, NCC neural crest cell, OL oligodendrocyte, SVZ subventricular zone, KD knock-down

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between E11.5 and E12.5, due to immature cerebral vessels,defective pharyngeal arch arteries (PAA) patterning and short-ened cardiac outflow tract (OFT) (Li et al. 2013). Brg1 hasalso been found to be important for the differentiation andmaturation of glial cells in the central and peripheral nervoussystem (Weider et al. 2012; Limpert et al. 2013; Yu et al.2013). The oligodendrocyte-specific Brg1 mutant micedisplayed generalized tremors, hindlimb paralysis and sei-zures, and died at postnatal weeks 3–4. Further observationsrevealed a translucent myelinating optic nerve tract and severemyelination defect in the optic nerve and spinal cord of thesemutants (Yu et al. 2013). Mutant mice lacking Brg1 inschwann cells displayed motor deficits from postnatal week3 that progressed to complete hindlimb paralysis along withminor forelimb paralysis. Similar to the oligodendrocyte-specific Brg1mutants, a severe myelination defect was evidentfrom translucent peripheral nerves (Weider et al. 2012;Limpert et al. 2013).

The loss of other BAF subunits also has severe conse-quences highlighting the necessity of an intact BAF complexin developmental processes. Deficiency of BAF155 causesdefective inner cell mass and endoderm developmentresulting in peri-implantation lethality (Kim et al. 2001).Similar to Brg1 mutant mice, BAF155 heterozygotes arealso predisposed to exencephaly.

In our recent study, cortex-specific BAF170 cKO andoverexpression (cOE) indicated that the BAF complexes havean essential role in controlling the differentiation of vRGsand the genesis of IPs. Consequently, significant changes incerebral cortical volume and thickness were observed (Tuocet al. 2013a). While BAF170cKO mice had enhancedcortical volume, surface area and thickness, BAF170cOEmice had the opposite phenotypes (Tuoc et al. 2013a).

Besides its significant role in the differentiation of vRGsand IPs, the BAF complex also controls late events of corticaldevelopment such as the acquisition of projection neuronalsubtype identity. Ctip2 is not only required for the specifica-tion of subcerebral projection neurons but also controls thedifferentiation of medium spiny neurons (MSN) during theformation of the striatum. The loss of Ctip2 leads to a severedefect in the differentiation of MSN, as evidenced by thestriking downregulation of most known MSN markers(Arlotta et al. 2008). Similar to its paralogue, the Ctip1 subunithas been reported to be required for the differentiation ofdorsal spinal cord neurons (John et al. 2012).

Null mutation in the gene encoding SS18 subunit of theBAF complex results in embryonic lethality due to placentalfailure (de Bruijn et al. 2006). In addition, knockdown ofSs18 in NSC culture results in defective proliferation anddendritic outgrowth (Staahl et al. 2013). Similarly, loss ofthe homologous subunit Ss18l1 (Crest) causes defectivedendritic development in cortical and hippocampal neurons(Aizawa et al. 2004; Qiu and Ghosh 2008).

Homozygous mutants of mice lacking the neuron-specificBAF53b subunit display defective dendritic outgrowth andsynapse formation leading to death (Wu et al. 2007).Interestingly, deficiency of BAF53b also leads to a declinein cognitive functions due to abnormal spine structure andfunction (Vogel-Ciernia et al. 2013).

In summary, findings have indicated that, while the loss ofBAF subunits in BAF complexes in NSCs led to abnormalproliferation and differentiation of NSCs, defects in acquisi-tion of neural identity and neural maturation were observedwith the absence of BAF subunits in post-mitotic neural cells.

Interaction between transcription factors and BAFcomplex determines neural development

Cell proliferation and differentiation are regulated by theactivity of chromatin remodeling complexes and transcrip-tional programs. Studies in recent years have begun to identifylinks between chromatin changes and specific transcriptionalprograms that control proliferation and differentiation ofNSCs. Recent findings have demonstrated that BAF com-plexes control various aspects of neural development, likelyvia interactions with numerous tissue and cell type-specificTFs (Table 1).

A best characterized example of such an interaction isbetween BAF subunits and TF Pax6 in both embryonic andadult NSCs (aNSCs). In the developing cortex, the Brm-basedBAF complex with BAF170, BAF155 subunits associateswith TF Pax6, an intrinsic vRGs determinant that determinesthe direct versus indirect mode of cortical neurogenesis.Specifically, BAF170 was shown to repress several genesthat are activated by Pax6 by directly recruiting the REST(RE1-silencing transcription factor)-corepressor complex,thereby repressing indirect mode of neurogenesis and regu-lating cerebral cortical size (Tuoc et al. 2013a). A recentfunctional study revealed that Pax6 directly interacts withthe Brg1-containing BAF complex in aNSC and the neuro-genic activity of Pax6 is lost in the absence of ATPase Brg1subunit (Ninkovic et al. 2013).

Another study suggests that Brg1 is essential both forrepression of the basal expression and signal-induced tran-scriptional activation of sonic hedgehog (Shh) target genes(Zhan et al. 2011). While the activation functions of Brg1 aremediated by its interaction with TF Gli3, its repressive func-tions are possibly mediated by Gli co-repressor histonedeacetylase (HDAC) (Zhan et al. 2011).

The glial TF Sox10 recruits a Brg1-containing BAF com-plex, possibly through interaction with BAF60a subunit, toregulatory regions of specific genes that encode other TFs(Oct6 and Krox20) crucial for schwann cell differentiation(Weider et al. 2012). Similarly, a recent study has elucidatedthe importance of interaction between an oligodendrocyte-

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lineage determination factor, Olig2 and a BAF complex con-taining Brg1, BAF250, BAF60a and BAF45b/d in oligoden-drocyte differentiation. Further, the genome-wide study indi-cated that the TF Olig2 facilitates recruitment of the BAFcomplex to regulatory regions of myelination-associatedgenes (Yu et al. 2013).

In NCCs, Brg1 promotes cell proliferation by suppressingtwo factors: the apoptosis signal-regulating kinase 1 (Ask1)and p21cip1, a cell cycle inhibitor. Besides this, Brg1 alsointeracts with chromodomain-helicase-DNA binding protein7 (Chd7), thereby activating PlexinA2 and ultimately guidingmigration of NCCs (Li et al. 2013).

From these studies, it is clear that the interactions withcell type-specific TFs confer functional specificity to BAFcomplexes in initiating and establishing transcriptional pro-grams that promote differentiation of NSCs and develop-ment of the nervous system. Functional studies aimed atthe identification of new BAF complex interacting partnerswill add further insights into the mechanism of chromatinregulation mediated by BAF complexes that control neuraldifferentiation and development.

The dynamic combinatorial assembly of BAF complexesin neural differentiation: lessons from BAF155and BAF170 paralogous subunits

The progressive transition from ESCs to neural progenitors isaccompanied by subunit exchange within the BAF complexes(Lessard et al. 2007; Yan et al. 2008). Interestingly, thisprocess is accompanied by the exchange of one BAF155subunit in the esBAF complex for BAF170 in the npBAFcomplex (Lessard et al. 2007; Yan et al. 2008), suggesting thesignificance of BAF170 in cortical neurogenesis.

During cortical development, the projection neurons ariseexclusively from progenitors located within the dorsal wall ofthe telencephalon. Distinct types of cortical neuronal progen-itors have been identified within the developingmouse cortex,including two main populations: vRGs in the ventricular zone(VZ) (Gotz and Huttner 2005) and IPs in the sub-ventricularzone (SVZ) (Pontious et al. 2008). Recent findings indicatethat the vRG population is very heterogeneous. A fraction ofprominin + vRGs characterized by low activity of the hGFAPpromoter is largely restricted to the generation of neurons viathe direct mode of neurogenesis. In contrast, prominin + vRGswith a highly active hGFAP promoter produce neurons indi-rectly via IPs (Pinto et al. 2008).

A detailed expression pattern analysis has indicated thatBAF170 is only transiently expressed in the cortical VZpredominantly in non-neurogenic vRGs between E10.5 andE14.5, a period of extensive IP production (Tuoc et al. 2013a).In addition, BAF170 and BAF155 show complementary spa-tial and temporal expression patterns during corticogenesis

and embryogenesis (Tuoc et al. 2013a, b). One main findingof our analyses of the functional consequences of in vivoablation or overexpression of BAF170 is that BAF170 con-trols IP generation in the developing cortex. The loss ofBAF170 leads to a transient decrease in early-born neuronalprogenies (LL neuronal subsets) at E12.5–13.5 in conjunctionwith a surplus of generated IPs. At later stages, an enlargedpool of IPs produces a modest surplus of LL neurons andexcess neurons in the ULs. Compared to the loss of BAF170,the overexpression of BAF170 in cortical progenitors leadsto opposite cortical phenotypes. Thus, these findings indi-cate that BAF170 plays essential roles in controlling dis-tinct aspects of cortical development: neurogenic and non-neurogenic fates of vRGs, early and late cortical progenitorfates of vRGs, direct and indirect modes of corticalneurogenesis and composition of LL and UL laminae,cortical thickness and size. Mechanistically, we found that,during early cortical neurogenesis, the loss of BAF170leads to incorporation of an additional BAF155 subunit(s)into the BAF complex in cortical progenitors. Replacementof BAF170 by BAF155 results in the elimination of theREST–corepressor complex on Pax6 target gene promoters,inducing euchromatin state (including decreased DNAmethylation, H3K27Me3 and increased H3K9Ac marks),which enhances the accessibility of target promoters toPax6. The ultimate biological outcome of this BAF subunitexchange is an increase in the expression of IP-specificgenes and premature expression of late cortical progenitor(LP) genes, endowing indirect neurogenesis during earlycorticogenesis. In contrast, in BAF170cOE mice, BAF170competes with BAF155 for occupancy in the BAF complexand has opposite effects on chromatin status and expression ofIP/LP genes, thereby promoting the direct mode of corticalneurogenesis. Thus, the exchange between BAF170 andBAF155 is a primary event in determining layer-specificcell fates in direct versus indirect modes of neurogenesis,an event that modulates the size of the developing mam-malian cortex. Therefore, in contrast to previous modelssuggesting that the BAF complexes provide a chromatinniche that facilitates accessibility of genomic targets to TFs,our data show for the first time that the composition ofBAF complexes may exert differential and/or opposite ef-fects on chromatin status (active or repressive chromatin).

In addition, we extended our investigation to understandwhy the two paralogues, BAF170 and BAF155 subunits, havedifferent binding abilities to REST protein (Battaglioli et al.2002; Tuoc et al. 2013a). Comparing the amino acid se-quences of BAF170 and BAF155, we found that they areconserved not only within the three functional domains(Chromo, Swirm and SANT domains) but also outside thesedomains. However, they are less conserved in their C-terminaldomains (C170 and C155). We then mapped the region ofBAF170 that interacts with REST protein by using different

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BAF170 deletion constructs and examined their binding abil-ity with REST protein. Our data indicated that REST interactswith all truncated forms of BAF170 that contain C170 do-mains but not with those lacking this domain. Our findingtherefore suggests that the difference at the C-terminus ofBAF155 and BAF170 proteins likely determines their differ-ential roles in corticogenesis.

Our data indicated that the combinatorial assembly, celltype-specific composition of mutually exclusive BAF variantsin the BAF complex is not only essential for the differentiationof ESC to neural progenitors and ultimately towards neuronsbut also important for the gene expression program of sub-populations of neural progenitors.

BAF complexes in reprogramming and their perspectivesin neural trans-differentiation

During differentiation of stem cells/progenitors, cells undergochanges in their gene expression program in response toboth internal and external stimuli. In addition, each type ofdifferentiated cell lineage has a specific molecular andphysiological characteristic that contributes to their function.Nevertheless, studies in recent years have indicated thatmany cell types have high plasticity and can trans-differentiate from one type to another. This phenomenon,known as cellular reprogramming, is achieved by losing themolecular identities of the original cell and gaining a newmolecular program without any changes in the genomicsequence. Among various factors, TFs are the main effectorsare the main effectors that control gene expression changesduring differentiation and cellular trans-differentiation. Thecellular reprogramming ability of TFs in converting terminallydifferentiated cells into a stem cell-like state was first reportedin the leading study by Shinya Yamanaka’s laboratory(Takahashi and Yamanaka 2006). The addition of a TFcocktail in cell culture was sufficient to drive fibroblastsback to a pluripotent state. Subsequently, tissue-specificTFs have been successfully applied to convert numeroussomatic cell types to induced pluripotent stem (iPS) cells orother cell types. However, the reprogramming of somaticcells achieved by the combination of TFs has a very lowefficiency (Singhal et al. 2010).

The interaction between TFs and regulatory elements ofgenes determines the gene expression program. Importantly,the accessibility of regulatory elements to TFs is mainlyinfluenced by the chromatin status: euchromatin (open, activechromatin) or heterochromatin (closed, inactive chromatin)(Fig. 1). Therefore, the regulation of the gene expressionprogram is achieved by the coordination of TF activity andchromatin remodeling activity driven by chromatin modula-tors, such as the BAF chromatin remodeling complex.

In addition to the importance of epigenetic and chromatinregulatory mechanisms in differentiation, recent studies haveelucidated both permissive and instructive roles of the BAFchromatin remodeler in reprogramming, where they worktogether with TF partners to improve the conversion efficiencyof one cell type into another (Table 2). Addition of theBAF155 subunit, alone or together with Brg1 in the cocktailof Oct4, Sox2 and Klf4 even without c-Myc, facilitatesreprogramming of human fibroblasts into iPS cells (Singhalet al. 2010). Mechanistically, overexpression of these BAFsubunits promotes a euchromatic structure at the loci ofpluripotency genes, thereby modulating the binding efficiencyof Oct4 to the regulatory elements of Oct4 target genes, whichenhances the reprogramming efficiency (Singhal et al. 2010).Similarly, BAF155 and Brg1, when expressed together withKlf4 and c-Myc, promote reprogramming of mouse embryon-ic and adult liver progenitor cells to pluripotency by promotingan euchromatic state at the promoters of pluripotent genes(Kleger et al. 2012).

In mouse mesoderm, enrichment of the BAF complexsubunit BAF60c is sufficient to direct the differentiation intothe myocardium, through its interaction with TFs Gata4 andTbx5 (Takeuchi et al. 2007). It is interesting to note thatthe BAF60c subunit is an important constituent of thecardiac-specific BAF complex (cBAF) that is essential forheart development (Lickert et al. 2004). BAF60c is alsoexpressed in myoblasts. In addition, BAF60c interacts withthe muscle determination factor MyoD on the regulatoryelements of MyoD-target genes to promote the differentia-tion of myoblasts (Forcales et al. 2012). A recent studyindicated that BAF60c is essential for MyoD-dependentdifferentiation of human ESC towards myogenesis (Albiniet al. 2013). In the selective absence of the BAF60c sub-unit, ESCs resist MyoD-dependent myogenesis, whereas

Table 2 Role of BAF subunits inreprogramming

iPS induced pluripotent stem cell,ESC embryonic stem cell

BAF subunit TF partners Starting cells Induced cells References

Brg1/BAF155 Oct4, Sox2, Klf4 Fibroblasts iPS cells Singhal et al. 2010

Brg1/BAF155 Klf4 and c-Myc Liver progenitors/differentiated livercells

iPS cells Kleger et al. 2012

BAF60c Gata4 and Tbx5 Mesoderm Myocardium Takeuchi et al. 2007

BAF60c MyoD ESC Skeletal muscle cells Albini et al. 2013

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enhanced levels of BAF60c expression promotes myogenesis(Albini et al. 2013). Thus, the above pioneering works demon-strated an essential role of the BAF chromatin remodelingcomplex in cellular reprogramming.

The central nervous system (CNS) contains numerous sub-types of terminally differentiated neuronal and glial cells. Thedevelopmental plasticity of these cells, as demonstrated byvarious in vitro and in vivo studies discussed here, holdspromising applications in regenerating cells of the CNS. Inthe developing CNS, the acquisition of neural subtype identityis controlled by both cell-extrinsic and cell-intrinsic programs(Guillemot 2007). By using developmental morphogens ofsignaling pathways such as Shh, Wnt and TGF-β, definedprotocols for the differentiation of ESC into a number ofneural subtypes have been well established. More recently, anumber of in vitro and in vivo studies have demonstrated thepowerful roles of TFs in instructing changes in neural cellidentity (Rouaux et al. 2012). In addition, TFs-mediated neuralreprogramming has been revealed to be a very selective way togenerate specific neural subtypes (Rouaux et al. 2012).

Although no direct evidence has so far been reported thatthe BAF complex controls neural reprogramming, some of theBAF complex-interacting TFs such as Pax6 (Ninkovic et al.2013; Tuoc et al. 2013a) and Olig2 (Yu et al. 2013) have beenshown to mediate the trans-differentiation between neuralsubtypes. Accordingly, forced expression of Pax6 in astro-cytes purified from the juvenile mouse neocortex is sufficientto convert the astrocytes into immature neurons (Heins et al.2002). Similarly, after brain injury, overexpression of Pax6 ordown-regulation of Olig2 induces neuroblast differentiationfrom Olig2+ glial cells (Buffo et al. 2005).

Although the TFs-mediated reprogramming appears to be apreferable strategy to generate specific subtypes of neural cellsfrom complex structures of the CNS, the low efficiency ofthis technique is a serious drawback. For instance, immatureneurons that are converted from astrocytes are unable tofully differentiate and to form functional synapses(Berninger et al. 2007). Likewise, the reprogramming poten-tials of TFs are often decreased in aging animals. Therefore,the understanding of how BAF chromatin remodeling com-plexes influence TF activity and neural gene expression pro-gram is a promising strategy to improve TFs-mediatedreprogramming between neural subtypes.

Concluding remarks and future directions

One of themain challenges in the developmental neurobiologyfield is to understand how transcriptional programs and chro-matin regulation maintain gene expression in stem cells in astate that is permissive for subsequent activation during dif-ferentiation along different cell lineages. For instance, inESCs, complex networks of chromatin remodeling factors

and TFs act in a coordinated manner to determine events ofself-renewal and differentiation. In contrast, there is littleinformation on how transcriptional programs and chromatinregulation interact to control the fate of neural cells.

Recent findings, which indicated that the activation of BAFsubunits enable tissue-specific TFs to selectively promote celldifferentiation during development or to reprogram one celltype into another, support the model that a cell type-specificcomposition of BAF complexes is essential for the cell type-specific gene expression program. Earlier, the identification ofBAF subunit compositions for specific neural subtypes waslimited due to technical issues. However, the recent develop-ments in new culture systems for neural cells and transgenicmice with cell type-specific reporters allow us to collectspecific neural subtypes. In addition, the emergence of accu-rate proteomic approaches makes it possible to identify neuralsubtype-specific BAF compositions and BAF complex-interacting TFs. Understanding the interaction between BAFchromatin remodeling complexes and TFs that instruct neuralfates could improve cell replacement therapy for neurodegen-erative diseases through the generation of specific neural cells.

Acknowledgments We apologize to colleagues whose work we maynot have been able to include in this review due to space limitation. Wethank J. Staiger for his support and M. Ahmad and K. Shanmugarajan forproofreading of the manuscript. This work was supported by the Univer-sity Medicine Göttingen (UMG) and DFG grant (TU 432/1-1). Theauthors declare no competing financial interests

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