Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1.

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway - a developmental pathway with many implications in health and disease - yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.

Neural stem cell metabolism revisited: a critical role for mitochondria.

Metabolism has emerged as a key regulator of stem cell behavior. Mitochondria are crucial metabolic organelles that are important for differentiated cells, yet considered less so for stem cells. However, recent studies have shown that mitochondria influence stem cell maintenance and fate decisions, inviting a revised look at this topic. In this review, we cover the current literature addressing the role of mitochondrial metabolism in mouse and human neural stem cells (NSCs) in the embryonic and adult brain. We summarize how mitochondria are implicated in fate regulation and how substrate oxidation affects NSC quiescence. We further explore single-cell RNA sequencing (scRNA-seq) data for metabolic signatures of adult NSCs, highlight emerging technologies reporting on metabolic signatures, and discuss mitochondrial metabolism in other stem cells.

mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites.

Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.

BAF subunit switching regulates chromatin accessibility to control cell cycle exit in the developing mammalian cortex.

mSWI/SNF or BAF chromatin regulatory complexes are dosage-sensitive regulators of human neural development frequently mutated in autism spectrum disorders and intellectual disability. Cell cycle exit and differentiation of neural stem/progenitor cells is accompanied by BAF subunit switching to generate neuron-specific nBAF complexes. We manipulated the timing of BAF subunit exchange in vivo and found that early loss of the npBAF subunit BAF53a stalls the cell cycle to disrupt neurogenesis. Loss of BAF53a results in decreased chromatin accessibility at specific neural transcription factor binding sites, including the pioneer factors SOX2 and ASCL1, due to Polycomb accumulation. This results in repression of cell cycle genes, thereby blocking cell cycle progression and differentiation. Cell cycle block upon Baf53a deletion could be rescued by premature expression of the nBAF subunit BAF53b but not by other major drivers of proliferation or differentiation. WNT, EGF, bFGF, SOX2, c-MYC, or PAX6 all fail to maintain proliferation in the absence of BAF53a, highlighting a novel mechanism underlying neural progenitor cell cycle exit in the continued presence of extrinsic proliferative cues.

LSH mediates gene repression through macroH2A deposition.

The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.

Rapid and reversible epigenome editing by endogenous chromatin regulators.

Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin.

Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.

Dynamics of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic states.

The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.

Programming Hippocampal Neural Stem/Progenitor Cells into Oligodendrocytes Enhances Remyelination in the Adult Brain after Injury.

Demyelinating diseases are characterized by a loss of oligodendrocytes leading to axonal degeneration and impaired brain function. Current strategies used for the treatment of demyelinating disease such as multiple sclerosis largely rely on modulation of the immune system. Only limited treatment options are available for treating the later stages of the disease, and these treatments require regenerative therapies to ameliorate the consequences of oligodendrocyte loss and axonal impairment. Directed differentiation of adult hippocampal neural stem/progenitor cells (NSPCs) into oligodendrocytes may represent an endogenous source of glial cells for cell-replacement strategies aiming to treat demyelinating disease. Here, we show that Ascl1-mediated conversion of hippocampal NSPCs into mature oligodendrocytes enhances remyelination in a diphtheria-toxin (DT)-inducible, genetic model for demyelination. These findings highlight the potential of targeting hippocampal NSPCs for the treatment of demyelinated lesions in the adult brain.

SPOT14-positive neural stem/progenitor cells in the hippocampus respond dynamically to neurogenic regulators.

Proliferation of neural stem/progenitor cells (NSPCs) in the adult brain is tightly controlled to prevent exhaustion and to ensure proper neurogenesis. Several extrinsic stimuli affect NSPC regulation. However, the lack of unique markers led to controversial results regarding the in vivo behavior of NSPCs to different stimuli. We recently identified SPOT14, which controls NSPC proliferation through regulation of de novo lipogenesis, selectively in low-proliferating NSPCs. Whether SPOT14-expressing (SPOT14+) NSPCs react in vivo to neurogenic regulators is not known. We show that aging is accompanied by a marked disappearance of SPOT14+ NSPCs, whereas running, a positive neurogenic stimulus, increases proliferation of SPOT14+ NSPCs. Furthermore, transient depletion of highly proliferative cells recruits SPOT14+ NSPCs into the proliferative pool. Additionally, we have established endogenous SPOT14 protein staining, reflecting transgenic SPOT14-GFP expression. Thus, our data identify SPOT14 as a potent marker for adult NSPCs that react dynamically to positive and negative neurogenic regulators.

Adult neurogenesis: mechanisms and functional significance.

New neurons are generated throughout life in distinct regions of the mammalian brain. This process, called adult neurogenesis, has been implicated in physiological brain function, and failing or altered neurogenesis has been associated with a number of neuropsychiatric diseases. Here, we provide an overview of the mechanisms governing the neurogenic process in the adult brain and describe how new neurons may contribute to brain function in health and disease.

Temporal control of retroviral transgene expression in newborn cells in the adult brain.

Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in distinct areas of the adult mammalian brain. Besides classical transgenesis-based approaches, retrovirus-mediated genetic manipulation is frequently used to study mechanisms that regulate neurogenesis in the nervous system. Here, we show that fusion of a tamoxifen-regulatable estrogen receptor (ER(T2)) motif to transcription factors (i.e., ASCL1 and NEUROD1) enables temporal control of transgene expression in adult mouse NSPCs in vitro and in vivo. Thus, the approach described here represents a versatile strategy for regulating gene expression to study gene function in dividing cells and their progeny.

Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.

Mechanisms controlling the proliferative activity of neural stem and progenitor cells (NSPCs) have a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (Fasn), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of Fasn in mouse NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene previously implicated in lipid metabolism, that we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for Fasn to fuel lipogenesis. Thus, we identify here a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation.

Previews. Crossing boundaries: direct programming of fibroblasts into neurons.

In a recent paper in Nature, Vierbuchen et al. (2010) show that fibroblasts can be directly converted into functional neurons by defined factors. This finding sheds new light on the biology underlying cell-fate restrictions and might offer a new avenue for studying neurological diseases.