The HMG-box transcription factor SOX2 has critical roles in the function of multiple stem cell types including pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs). How this master regulator can control diverse transcriptional programs has remained an important and unresolved question in the field. While SOX2 occupied many promoters in both cell types, the major class of genomic elements occupied by SOX2 in ESCs and NPCs were distal enhancers ( and ). While our data displayed high concordance among replicates and with published data sets in ESCs, SOX2 binding in NPCs was less correlated with prior data 
). This is likely due to the different protocols used to derive and culture NPCs. NPCs with similar developmental potential but distinct molecular profiles exist throughout development 
, and these populations respond differently to external signaling cues present in culture media 
. Thus, it is perhaps not surprising that SOX2 binding is more variable in NPCs relative to ESCs.
We derived NPCs directly from genetically identical ESCs allowing us to directly analyze SOX2 binding as these cells transition between states. Several criteria support the high quality of our data. First, we identified many known SOX2 binding sites including promoters and enhancers in both ESCs and NPCs. Second, while many binding sites were distinct, we identified a canonical SOX2 motif as highly enriched in both cell types. Third, SOX2 overlapped significantly with POU partner factors in ESCs and NPCs consistent with the expectation that these transcription factor families function together to regulate developmental progression. In addition, we identified a SOX-OCT composite motif as enriched in these co-bound sites.
SOX2 occupied largely exclusive sites in ESCs and NPCs, despite using the same DNA motif to recognize these genomic targets. Moreover, SOX2 occupied distinct regions in the same promoter and distinct enhancers associated with the same gene. These data indicated that additional factors dictated SOX2 binding site specificity. While SOX2 co-occupied many binding sites with OCT4 in ESCs, partner factors in NPCs have not been well defined. We found the recognition motif for the Class III POU factor BRN2 was enriched in SOX2 bound regions in NPCs. The evolutionary conservation of the SOX-POU interaction, the co-expression of Sox2
in neurogenic regions of the brain, and the neurodevelopmental defects associated with Brn2
loss-of-function suggested that SOX2 and BRN2 together regulate a subset of genes important for neural fate. Consistent with this, we defined a large group of enhancer elements co-bound by SOX2 and BRN2 in NPCs. We identified known functional enhancers bound by SOX2 and BRN2 in NPCs, such as the Nes30
enhancer of the Nestin
and the 3′ enhancer of the Sox2
, and extended this list to include hundreds of additional neural-specific enhancers.
Consistent with a positive role in regulating neural cell state, forced expression of Brn2
led to up-regulation of neural markers and to differentiation toward the neural lineage. Our work is in agreement with several studies that have implicated Brn2
as an early marker of neural commitment 
. Notably, ectopic BRN2 was sufficient to recruit SOX2 to hundreds of novel sites in differentiating ESCs that corresponded to a subset of enhancers also bound in NPCs. The recruitment of SOX2 by BRN2 to specific loci was sufficient to induce expression of nearby genes and to alter chromatin state in some cases. These data are in agreement with the notion that SOX proteins require partner factors to tightly bind to genomic targets and modulate transcriptional outcomes 
. Interestingly, ectopic expression of OCT4 alone in NPCs was sufficient to reprogram cells into induced pluripotent stem cells, presumably by partnering with endogenous SOX2 
, consistent with the idea that POU factors can recruit SOX2 to specific targets. Furthermore, ectopic expression of Sox2
, and the forkhead factor Foxg1
can transdifferentiate fibroblasts to NPC-like cells 
. Taken together, these data may facilitate efforts to define the minimal set of genes needed to promote the transition from undifferentiated cells to the neural lineage. Thus, our results implicate BRN2 as a SOX2 partner factor and suggest that together these factors are important for neural specification and NPC function.
While the motifs occupied by these factors were highly similar, the arrangement of SOX and OCT motifs in SOX2-POU target sites displayed differences in ESCs and NPCs. Regulation of SOX-POU target genes appears to depend not only on the presence of a SOX and an OCT motif in close proximity to each other, but also on other DNA sequence determinants, including the spacing and orientation of these motifs with respect to each other 
. However, these observations related to only a few genes and had not been extended genome-wide. While we found that SOX2-OCT4 and SOX2-BRN2 preferred similar composite motifs when they were bound in close proximity to each other, examination of co-bound regions found that peaks of SOX2 and BRN2 in NPCs were often spaced farther apart than peaks of SOX2 and OCT4 in ESCs. Thus, allosteric interactions between transactivation domains of SOX and POU factors may be key in stabilizing ternary complexes and in setting the stage for additional interactions that determine binding specificity and transcriptional output at target genes 
Combinatorial interactions among transcription factors allow cells to respond to environmental and developmental cues in a tissue-specific manner. A classical example involves the regulation of interferon-β expression through cooperative binding of transcription factors and chromatin proteins to an enhancer, collectively known as the interferon-β enhancesome 
. In ESCs, SOX2 and OCT4 are known to physically interact with other transcription factors at many loci, including enhancers 
, suggesting that SOX2-POU factors may also nucleate specific enhancesomes. We identified a set of candidate factors that may interact with SOX2-BRN2 that included RFX and NF-I family members. NF-I factors are expressed in NPCs in vivo
and their loss in development leads to defects in central nervous system formation and specifically NPC dysfunction 
. RFX family members also play essential roles in proper brain development 
. For example, RFX4 regulates Sonic Hedgehog (SHH) signaling in the developing nervous system and loss of function resulted in pleiotropic brain defects linked to SHH signaling 
. Defects associated with conditional ablation of Sox2
in the brain were also shown to be partially mediated by aberrant SHH signaling 
. Additional studies revealed that SOX2 co-localized with the ATP-dependent histone remodeler CHD7 in NPCs 
. Thus, interactions with chromatin modifiers or other epigenetic regulators may also be critical for binding site selection and establishment of NPC-specific gene expression programs in response to particular signals.
Recent data showed that SOX2 functions as a pioneer factor in ESCs by marking a subset of genes for activation by other SOX family members, namely SOX4 in the B-cell lineage, SOX3 in NPCs and SOX11 in immature neurons 
. Interestingly, the POU factor Brn5
, like Sox11
, is expressed in differentiated cell types in the CNS and thought to play a role in regulating cell state 
, thus elucidation of BRN5 targets in these cells may reveal another layer of SOX-POU regulation of neurogenesis. Taken together, these data suggest that transitions in SOX-POU partners regulate the earliest stages of development through terminal differentiation. Ultimately, characterization of combinatorial interactions among transcription factors and chromatin regulators at distal enhancers will be central to understanding the complex mechanisms that control cell state throughout development.