The following lines of evidence support our conclusion that SS18 and CREST are stable, dedicated, and functional subunits of the npBAF and nBAF complexes, respectively: (1) both proteins cosediment exclusively with Brm/Brg on glycerol gradients of whole-brain preparations, indicating that they are dedicated to these complexes; (2) they resist dissociation from BAF complexes even under partially denaturing conditions, indicating that they make stable, possibly cofolded interactions; (3) they do not co-IP each other, indicating that they are mutually exclusive subunits; and (4) SS18 cannot substitute for CREST in supporting dendritic growth. From these studies and their patterns of expression, we conclude that SS18 is a subunit of esBAF and npBAF complexes and that CREST is a subunit of nBAF complexes as illustrated in A
. Although we and others have detected SS18 and CREST in studies of BAF complexes, it was not apparent that they were dedicated, functional, nonexchangable subunits rather than simply being interacting proteins. From our proteomic analysis, we conclude that NS/progenitors contain at least 36 different npBAF complexes and that neurons contain at least 36 different nBAF complexes. We do not know if these complexes are present in subtypes of neural progenitors and neurons or if each neuron has all possible assemblies. From a limited analysis of different brain regions and inspection of the Allen Brain Atlas (http://www.brain-map.org/
), we favor multiple assemblies in each neural progenitor or neuron. These findings indicate that the spectrum of neurologic diseases due to mutations in npBAF and nBAF complexes must be interpreted in light of the emergent features of different combinatorial assemblies of these complexes.
Figure 9. Summary of ordered events in npBAF to nBAF complex switching. A, The npBAF to nBAF switch involves three subunits regulated by miR9/9* and miR-124. Combinatorial assembly of npBAF and nBAF subunits could create up to 36 distinct npBAF or nBAF complexes. (more ...)
SS18 and CREST appear to be functionally exclusive, because expression of SS18 in postmitotic neurons inhibits activity-dependent dendritic outgrowth, whereas expression of CREST does not. In fact, SS18 expression in neurons phenocopied the CREST loss-of-function phenotype that is indicative of a dominant-negative function (Aizawa et al., 2004
). We have recently identified two mutations in CREST in amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) patients (Chesi et al., 2013
). We find that these CREST mutants strongly block dendrite outgrowth even in the presence of endogenous WT CREST, suggesting a dominant-negative mode of action. One of the mutations terminates the final 9 aa of CREST, which has been shown to be necessary for interaction of CREST with CBP, a histone acetylase (Aizawa et al., 2004
). The finding that CREST is entirely bound to nBAF complexes and resists dissociation even under denaturing conditions indicates that CBP is targeted to sites of action by nBAF complexes in a way comparable to the targeting of STAT3 to its DNA binding sites by the prior action of esBAF in ES cells (Ho et al., 2011
Neither SS18 nor CREST have orthologs in yeast, flies, or worms. Although BAF complexes are frequently referred to as the mSWI/SNF complex, only six of the subunits have yeast homologs and other subunits have homologs in the RSC and SWR1 complexes. In most cases, the new BAF subunits appeared either in insects or fish. An SS18 homolog is present in the zebrafish and fugu genomes and is present in birds, Xenopus
, and mammals (UCSC Genome Browser; http://www.genome.ucsc.edu/
). However, CREST is present in neither the zebrafish nor fugu genomes but is present in the coelacanth genome. Coelacanths are lobe-finned fishes more closely related to tetrapods than to ray-finned fishes. Therefore, SS18 and CREST appear to have originated to deal with some aspect of chromatin regulation that has emerged relatively recently in tetrapod evolution. The exclusive expression of CREST in the nervous system and the extraordinary sensitivity of neural progenitors to reduction in SS18 levels indicates that this recently evolved chromatin function might be used in the development of the tetrapod motor nervous system. Neither SS18 nor CREST is required for nucleosome remodeling in vitro
, indicating they may play a role in genome targeting or some as-yet-unrecognized functions of these complexes.
The appearance of CREST in the lobe-finned coelacanth is interesting because it, like all tetrapods, has limb and finger bones covered with muscle and skin compared with ray-finned fishes, which have slender bony fin rays (lepidotrichs) covered with only skin. One hypothesis is that, during the tetrapod transition, CREST co-evolved with tetrapod limb development. Indeed, the majority of the putative regulatory elements located near CREST
appear to originate in the tetrapod ancestor (Lowe et al., 2011
). CREST may play a specific role in motor neuron chromatin regulation, where it pairs with motor neuron factors necessary for limb neuromuscular development in a way similar to the essential pairing of BAF60c with myoD to activate the skeletal muscle program (Albini et al., 2013
) or esBAF with Sox2 and Oct4 in ES cells (Ho et al., 2009a
). Perhaps this requirement for CREST in motor neurons is why mutant CREST causes ALS, a fatal motor neuron disease characterized by loss of coordination and atrophy of limbs. Interestingly, CREST-null mice also have coordination defects and smaller cerebellums (Aizawa et al., 2004
). In contrast to the roles of BAF complexes in mental retardation and microcephaly, ALS is thought to result from deterioration in neural function and connectivity. Therefore, BAF complexes appear to function during normal human neural development and are also required to maintain circuitry and/or viability over a lifetime. However, additional studies will be required to confirm these possibilities.
SS18 and CREST subunits are 62% identical, yet SS18 cannot substitute for CREST in postmitotic neurons. SS18 has 65 proline residues and CREST has 27. Proline residues make proteins more rigid, so the secondary structure of SS18 will be more rigid than that of CREST and thus could give rise to different functions. In addition, the paralogs may have different posttranslational modifications, because SS18 has 31 serine and 8 threonine residues compared with 53 serines and 22 threonines in CREST. There are two splice variants of SS18, the short isoform (lacking exon 8) being the more abundant of the two (B). Both SS18 splice variants functioned as a dominant-negative and blocked activity-dependent dendritic outgrowth ( and data not shown). This indicates that exon 8, which CREST contains, does not confer CREST-like function to SS18. Therefore, the unique functionality of SS18 and CREST must come from other biochemical differences.
Recent exome sequencing studies have revealed frequent mutations in BAF complex subunits in CSS, NBS, and sporadic mental retardation. In addition, mutations have been reported in Brg and Brm in schizophrenia and in BAF155, BAF170, BAF180, and REST in human autism (Neale et al., 2012
; O'Roak et al., 2012
). These mutations were dominant and considered to be dominant-negatives. In the NBS study, the authors suggested the dominant mutations in BRM
(one of two ATPases that in neurons are components of neural-specific nBAF complexes) to be dominant-negatives and also affect Brg-based complexes. Genetic dominance can have many different mechanistic underpinnings, including the production of dominant-negative proteins that can block WT protein function, abnormal polymerization processes in which the mutated protein blocks polymer extension, and haploinsufficiency due to a nonsense mutation in a protein involved in a rate-limiting process. To clarify this issue, we reexamined our proteomic analysis done with purified nBAF complexes. We found that peptides from Brg exceeded peptides recovered from Brm by more than threefold. Because both are large proteins, these differences are unlikely to be due to peptide recovery differences and likely reflect the endogenous levels of the proteins. Many of these BRM
dominant mutations are surprisingly conservative amino acid changes and cluster to the ATP binding and Helicase domains (Ronan et al., 2013
), suggesting that rather than being strong dominant-negatives, the mutations affect a rate-limiting catalytic process. ATP-dependent chromatin remodeling is well suited for a rate-limiting role in biological processes, because the rate of chromatin alteration may be limited by the intrinsic rate of energy utilization by these complexes rather than by ATP levels, which are generally not rate limiting. The dominant role of BAF subunits in human diseases fits nicely with the observation that, in mice, mutations in Brg, BAF155, and BAF53a all have dominant phenotypes and are apparently haploinsufficient. Rate-limiting functions are often instructive, so these considerations are consistent with the apparent instructive role of the microRNA/chromatin switch in converting fibroblasts to neurons (Yoo et al., 2011
). Interestingly, the ALS mutations we have described also appear to be dominant (Chesi et al., 2013
Our kinetic analysis of conversion of ES cells to neurons reveals the nature of the negative cascade network motif underlying the triple-negative genetic circuit we described as contributing to neural fate determination (Yoo et al., 2009
; Yoo et al., 2011
). The timing of repression of REST, activation of mir-9/9* and miR-124, subsequent repression of BAF53a, and stabilization of BAF53b protein nicely fit the negative cascade network motif model in which downstream genes are sequentially repressed and activated when their regulators reach the relevant threshold (Shoval and Alon, 2010
). Our kinetic analysis also indicates that more than a single pathway controls the microRNA/chromatin switch. This follows from the observation that BAF45c is induced without RA and before miR-9/9* and miR-124. The requirement for two pathways to bring about this epigenetic switch indicates that the npBAF to nBAF switch could be a hub or integration site for neurogenesis, which is supported by recent genome sequencing efforts in human neurologic diseases.
The discovery that CREST is a dedicated, nonexchangeable subunit of nBAF complexes indicates that these complexes have three neuronal-specific “letters” endowing them with the ability to provide specific patterns of gene expression in neurons. These complexes in neurons are additionally polymorphic by virtue of alternative assembly of other subunits such as Brg versus Brm, BAF250a versus BAF250b versus BAF200, BAF60a versus BAF60b versus BAF60c, and BAF45b versus BAF45c. Therefore, they should be referred to as npBAF complexes and nBAF complexes, each with a complexity of 36 possible assemblies based on gene numbers (A). An even larger number of combinatorial possibilities might result from the extensive alternative splicing shown for BAF250b and SS18. The functional target(s) of the neuron-specific surfaces of the nBAF complexes is not known, but is likely to reflect the emergent features of complexes (i.e., those that emerge not from the subunit itself, but rather from the composite surface produced by each subunit and its adjacent subunits). Our findings indicate that the widely different neurologic diseases causally or possibly related to mutations in BAF subunits (mental retardation, schizophrenia, autism, ALS, and microcephaly) must be interpreted in the context of these different BAF assemblies rather than a monomorphic mSWI/SNF complex.