Shira Rockowitz

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility. Here we show that super-enhancers underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters (‘epicentres’) of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

ETS factors reprogram the androgen receptor cistrome and prime prostate tumorigenesis in response to PTEN loss

Studies of ETS-mediated prostate oncogenesis have been hampered by a lack of suitable experimental systems. Here we describe a new conditional mouse model that shows robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, the mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG markedly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included the restoration of AR transcriptional output and upregulation of genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETS variant 1 (ETV1) positively regulated the AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, expression of ERG and ETV1 correlated with higher AR transcriptional output in PTEN-deficient prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.

An H3K36 Methylation-Engaging Tudor Motif of Polycomb-like Proteins Mediates PRC2 Complex Targeting

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.

Development of Patient-Specific Neurons in Schizophrenia Using Induced Pluripotent Stem Cells

Abstract: Induced pluripotent stem cell (iPSC) technology has the potential to transform regenerative medicine. It also offers a powerful tool for establishing in vitro models of disease, in particular, for neuropsychiatric disorders where live human neurons are essentially impossible to procure. Using iPSCs derived from three schizophrenia (SZ) patients, one of whom has 22q11.2del (velocardiofacial syndrome; VCFS), the authors developed a culture system to study SZ on a molecular and cellular level. SZ iPSCs were differentiated into functional, primarily glutamatergic neurons that were able to fire action potentials after ∼8 weeks in culture. Early differentiating neurons expressed a number of transcription factors/chromatin remodeling proteins and synaptic proteins relevant to SZ pathogenesis, including ZNF804A, RELN, CNTNAP2, CTNNA2, SMARCA2, and NRXN1. Although a small number of lines were developed in this preliminary study, the SZ line containing 22q11.2del showed a significant delay in the reduction of endogenous OCT4 and NANOG expression that normally occurs during differentiation. Constitutive expression of OCT4 has been observed in Dgcr8-deficient mouse embryonic stem cells (mESCs); DGCR8 maps to the 22q11.2-deleted region. These findings demonstrate that the method of inducing neural differentiation employed is useful for disease modeling in SZ and that the transition of iPSCs with 22q11.2 deletions towards a differentiated state may be marked by subtle changes in expression of pluripotency-associated genes.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999s on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using forebrain and lens chromatin from mice. A total of 3514 (forebrain) and 3723 (lens) Pax6-containing peaks were identified, with ∼70% of them found in both tissues and thereafter called ‘common’ peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, Pax6-PD/HD and Pax6-HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit via components of FGF (Fgfr2, Prox1 and Ccnd1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis.

Characterization of human pseudogene-derived non-coding RNAs for functional potential.

Thousands of pseudogenes exist in the human genome and many are transcribed, but their functional potential remains elusive and understudied. To explore these issues systematically, we first developed a computational pipeline to identify transcribed pseudogenes from RNA-Seq data. Applying the pipeline to datasets from 16 distinct normal human tissues identified ∼3,000 pseudogenes that could produce non-coding RNAs in a manner of low abundance but high tissue specificity under normal physiological conditions. Cross-tissue comparison revealed that the transcriptional profiles of pseudogenes and their parent genes showed mostly positive correlations, suggesting that pseudogene transcription could have a positive effect on the expression of their parent genes, perhaps by functioning as competing endogenous RNAs (ceRNAs), as previously suggested and demonstrated with the PTEN pseudogene, PTENP1. Our analysis of the ENCODE project data also found many transcriptionally active pseudogenes in the GM12878 and K562 cell lines; moreover, it showed that many human pseudogenes produced small RNAs (sRNAs) and some pseudogene-derived sRNAs, especially those from antisense strands, exhibited evidence of interfering with gene expression. Further integrated analysis of transcriptomics and epigenomics data, however, demonstrated that trimethylation of histone 3 at lysine 9 (H3K9me3), a posttranslational modification typically associated with gene repression and heterochromatin, was enriched at many transcribed pseudogenes in a transcription-level dependent manner in the two cell lines. The H3K9me3 enrichment was more prominent in pseudogenes that produced sRNAs at pseudogene loci and their adjacent regions, an observation further supported by the co-enrichment of SETDB1 (a H3K9 methyltransferase), suggesting that pseudogene sRNAs may have a role in regional chromatin repression. Taken together, our comprehensive and systematic characterization of pseudogene transcription uncovers a complex picture of how pseudogene ncRNAs could influence gene and pseudogene expression, at both epigenetic and post-transcriptional levels.

Comparison of REST Cistromes across Human Cell Types Reveals Common and Context-Specific Functions

Recent studies have shown that the transcriptional functions of REST are much broader than repressing neuronal genes in non-neuronal systems. Whether REST occupies similar chromatin regions in different cell types and how it interacts with other transcriptional regulators to execute its functions in a context-dependent manner has not been adequately investigated. We have applied ChIP-seq analysis to identify the REST cistrome in human CD4+ T cells and compared it with published data from 15 other cell types. We found that REST cistromes were distinct among cell types, with REST binding to several tumor suppressors specifically in cancer cells, whereas 7% of the REST peaks in non-neuronal cells were ubiquitously called and <25% were identified for ≥5 cell types. Nevertheless, using a quantitative metric directly comparing raw ChIP-seq signals, we found the majority (∼80%) was shared by ≥2 cell types. Integration with RNA-seq data showed that REST binding was generally correlated with low gene expression. Close examination revealed that multiple contexts were correlated with reduced expression of REST targets, e.g., the presence of a cognate RE1 motif and cellular specificity of REST binding. These contexts were shown to play a role in differential corepressor recruitment. Furthermore, transcriptional outcome was highly influenced by REST cofactors, e.g., SIN3 and EZH2 co-occupancy marked higher and lower expression of REST targets, respectively. Unexpectedly, the REST cistrome in differentiated neurons exhibited unique features not observed in non-neuronal cells, e.g., the lack of RE1 motifs and an association with active gene expression. Finally, our analysis demonstrated how REST could differentially regulate a transcription network constituted of miRNAs, REST complex and neuronal factors. Overall, our findings of contexts playing critical roles in REST occupancy and regulatory outcome provide insights into the molecular interactions underlying RESTs diverse functions, and point to novel roles of REST in differentiated neurons.

Significant expansion of the REST/NRSF cistrome in human versus mouse embryonic stem cells: potential implications for neural development

Recent studies have employed cross-species comparisons of transcription factor binding, reporting significant regulatory network ‘rewiring’ between species. Here, we address how a transcriptional repressor targets and regulates neural genes differentially between human and mouse embryonic stem cells (ESCs). We find that the transcription factor, Repressor Element 1 Silencing Transcription factor (REST; also called neuron restrictive silencer factor) binds to a core group of ∼1200 syntenic genomic regions in both species, with these conserved sites highly enriched with co-factors, selective histone modifications and DNA hypomethylation. Genes with conserved REST binding are enriched with neural functions and more likely to be upregulated upon REST depletion. Interestingly, we identified twice as many REST peaks in human ESCs compared to mouse ESCs. Human REST cistrome expansion involves additional peaks in genes targeted by REST in both species and human-specific gene targets. Genes with expanded REST occupancy in humans are enriched for learning or memory functions. Analysis of neurological disorder associated genes reveals that Amyotrophic Lateral Sclerosis and oxidative stress genes are particularly enriched with human-specific REST binding. Overall, our results demonstrate that there is substantial rewiring of human and mouse REST cistromes, and that REST may have human-specific roles in brain development and functions.