Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide.

Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-ß and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.

Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage.

Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence-dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.

Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing.

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

RATIONALE: Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. OBJECTIVE: We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. METHODS AND RESULTS: We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. CONCLUSIONS: Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.

Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest.

cis-regulatory changes play a central role in morphological divergence, yet the regulatory principles underlying emergence of human traits remain poorly understood. Here, we use epigenomic profiling from human and chimpanzee cranial neural crest cells to systematically and quantitatively annotate divergence of craniofacial cis-regulatory landscapes. Epigenomic divergence is often attributable to genetic variation within TF motifs at orthologous enhancers, with a novel motif being most predictive of activity biases. We explore properties of this cis-regulatory change, revealing the role of particular retroelements, uncovering broad clusters of species-biased enhancers near genes associated with human facial variation, and demonstrating that cis-regulatory divergence is linked to quantitative expression differences of crucial neural crest regulators. Our work provides a wealth of candidates for future evolutionary studies and demonstrates the value of "cellular anthropology," a strategy of using in-vitro-derived embryonic cell types to elucidate both fundamental and evolving mechanisms underlying morphological variation in higher primates.

Regulation of Peripheral Nerve Myelin Maintenance by Gene Repression through Polycomb Repressive Complex 2.

Myelination of peripheral nerves by Schwann cells requires coordinate regulation of gene repression as well as gene activation. Several chromatin remodeling pathways critical for peripheral nerve myelination have been identified, but the functions of histone methylation in the peripheral nerve have not been elucidated. To determine the role of histone H3 Lys27 methylation, we have generated mice with a Schwann cell-specific knock-out of Eed, which is an essential subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3 Lys27. Analysis of this mutant revealed no significant effects on early postnatal development of myelin. However, its loss eventually causes progressive hypermyelination of small-diameter axons and apparent fragmentation of Remak bundles. These data identify the PRC2 complex as an epigenomic modulator of mature myelin thickness, which is associated with changes in Akt phosphorylation. Interestingly, we found that Eed inactivation causes derepression of several genes, e.g., Sonic hedgehog (Shh) and Insulin-like growth factor-binding protein 2 (Igfbp2), that become activated after nerve injury, but without activation of a primary regulator of the injury program, c-Jun. Analysis of the activated genes in cultured Schwann cells showed that Igfbp2 regulates Akt activation. Our results identify an epigenomic pathway required for establishing thickness of mature myelin and repressing genes that respond to nerve injury.

Differential Sox10 genomic occupancy in myelinating glia.

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. GLIA 2015;63:1897-1914.

In silico pooling of ChIP-seq control experiments.

As next generation sequencing technologies are becoming more economical, large-scale ChIP-seq studies are enabling the investigation of the roles of transcription factor binding and epigenome on phenotypic variation. Studying such variation requires individual level ChIP-seq experiments. Standard designs for ChIP-seq experiments employ a paired control per ChIP-seq sample. Genomic coverage for control experiments is often sacrificed to increase the resources for ChIP samples. However, the quality of ChIP-enriched regions identifiable from a ChIP-seq experiment depends on the quality and the coverage of the control experiments. Insufficient coverage leads to loss of power in detecting enrichment. We investigate the effect of in silico pooling of control samples within multiple biological replicates, multiple treatment conditions, and multiple cell lines and tissues across multiple datasets with varying levels of genomic coverage. Our computational studies suggest guidelines for performing in silico pooling of control experiments. Using vast amounts of ENCODE data, we show that pairwise correlations between control samples originating from multiple biological replicates, treatments, and cell lines/tissues can be grouped into two classes representing whether or not in silico pooling leads to power gain in detecting enrichment between the ChIP and the control samples. Our findings have important implications for multiplexing samples.

Genome editing-enabled HTS assays expand drug target pathways for Charcot-Marie-tooth disease.

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.

Reorganization of enhancer patterns in transition from naive to primed pluripotency.

Naive and primed pluripotency is characterized by distinct signaling requirements, transcriptomes, and developmental properties, but both cellular states share key transcriptional regulators: Oct4, Sox2, and Nanog. Here, we demonstrate that transition between these two pluripotent states is associated with widespread Oct4 relocalization, mirrored by global rearrangement of enhancer chromatin landscapes. Our genomic and biochemical analyses identified candidate mediators of primed state-specific Oct4 binding, including Otx2 and Zic2/3. Even when differentiation cues are blocked, premature Otx2 overexpression is sufficient to exit the naive state, induce transcription of a substantial subset of primed pluripotency-associated genes, and redirect Oct4 to previously inaccessible enhancer sites. However, the ability of Otx2 to engage new enhancer regions is determined by its levels, cis-encoded properties of the sites, and the signaling environment. Our results illuminate regulatory mechanisms underlying pluripotency and suggest that the capacity of transcription factors such as Otx2 and Oct4 to pioneer new enhancer sites is highly context dependent.

MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf), as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

Multivalent recognition of histone tails by the PHD fingers of CHD5.

The chromodomain, helicase, DNA-binding protein 5 (CHD5) is a chromatin remodeling enzyme which is implicated in tumor suppression. In this study, we demonstrate the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD(1) and PHD(2) alone and the tandem PHD(1-2) construct. Both domains displayed similar binding preferences for histone H3, where modification (e.g., methylation, acetylation, and phosphorylation) at H3R2, H3K4, H3T3, H3T6, and H3S10 disrupts high-affinity binding, and the three most N-terminal amino acids (ART) are crucial for binding. The tandem CHD5-PHD(1-2) displayed similar preferences to those displayed by each PHD finger alone. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5-PHD(1-2) simultaneously engages two H3 N-termini and results in a 4-11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin-binding modules function to interpret the combinatorial PTM capacity written in chromatin.

Genome-wide analysis of EGR2/SOX10 binding in myelinating peripheral nerve.

Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell development. Egr2/Krox20 expression is activated by axonal signals in myelinating Schwann cells, and is required for cell cycle arrest and myelin formation. To elucidate the integrated function of these two transcription factors during peripheral nerve myelination, we performed in vivo ChIP-Seq analysis of myelinating peripheral nerve. Integration of these binding data with loss-of-function array data identified a range of genes regulated by these factors. In addition, although SOX10 itself regulates Egr2/Krox20 expression, leading to coordinate activation of several major myelin genes by the two factors, there is a large subset of genes that are activated independent of EGR2. Finally, the results identify a set of SOX10-dependent genes that are expressed in early Schwann cell development, but become subsequently repressed by EGR2/KROX20.

Distal enhancers upstream of the Charcot-Marie-Tooth type 1A disease gene PMP22.

Myelin insulates axons in the peripheral nervous system to allow rapid propagation of action potentials, and proper myelination requires the precise regulation of genes encoding myelin proteins, including PMP22. The correct gene dosage of PMP22 is critical; a duplication of PMP22 is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth Disease (CMT) (classified as type 1A), while a deletion of PMP22 leads to another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies. Recently, duplications upstream of PMP22, but not containing the gene itself, were reported in patients with CMT1A like symptoms, suggesting that this region contains regulators of PMP22. Using chromatin immunoprecipitation analysis of two transcription factors known to upregulate PMP22-EGR2 and SOX10-we found several enhancers in this upstream region that contain open chromatin and direct reporter gene expression in tissue culture and in vivo in zebrafish. These studies provide a novel means to identify critical regulatory elements in genes that are required for myelination, and elucidate the functional significance of non-coding genomic rearrangements.

SOX10 regulates expression of the SH3-domain kinase binding protein 1 (Sh3kbp1) locus in Schwann cells via an alternative promoter.

The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells-specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells.

Developmental regulation of microRNA expression in Schwann cells.

Schwann cell differentiation and subsequent myelination of the peripheral nervous system require the action of several transcription factors, including Sox10, which is vital at multiple stages of development. The transition from immature to myelinating Schwann cell is also regulated posttranscriptionally and depends upon Dicer-mediated processing of microRNAs (miRNAs). Although specific miRNA targets have begun to be identified, the mechanisms establishing the dynamic regulation of miRNA expression have not been elucidated. We performed expression profiling studies and identified 225 miRNAs differentially expressed during peripheral myelination. A subset of 9 miRNAs is positively regulated by Sox10, including miR-338 which has been implicated in oligodendrocyte maturation. In vivo chromatin immunoprecipitation (ChIP) of sciatic nerve cells revealed a Sox10 binding site upstream of an alternate promoter within the Aatk gene, which hosts miR-338. Sox10 occupied this site in spinal cord ChIP experiments, suggesting a similar regulatory mechanism in oligodendrocytes. Cancer profiling studies have identified clusters of miRNAs that regulate proliferation, termed "oncomirs." In Schwann cells, the expression of many of these proproliferative miRNAs was reduced in the absence of Sox10. Finally, Schwann cells with reduced Sox10 and oncomir expression have an increase in the CDK inhibitor p21 and a concomitant reduction in cell proliferation.

Regulation of the PMP22 gene through an intronic enhancer.

Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as peripheral myelin protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in three copies of PMP22, is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth disease. The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we found a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites not only for Egr2 but also for Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression.

Locus-wide identification of Egr2/Krox20 regulatory targets in myelin genes.

Myelination of peripheral nerves by Schwann cells depends upon a gene regulatory network controlled by early growth response Egr2/Krox20, which is specifically required for Schwann cells to initiate and maintain myelination. To elucidate the mechanism by which Egr2 regulates gene expression during myelination, we have performed chromatin immunoprecipitation analysis on myelinating rat sciatic nerve in vivo. The resulting samples were applied to a tiled microarray consisting of a broad spectrum of genes that are activated or repressed in Egr2-deficient mice. The results show extensive binding within myelin-associated genes, as well as some genes that become repressed in myelinating Schwann cells. Many of the Egr2 peaks coincide with regions of open chromatin, which is a marker of enhancer regions. In addition, further analysis showed that there is substantial colocalization of Egr2 binding with Sox10, a transcription factor required for Schwann cell specification and other stages of Schwann cell development. Finally, we have found that Egr2 binds to promoters of several lipid biosynthetic genes, which is consistent with their dramatic up-regulation during the formation of lipid-rich myelin. Overall, this analysis provides a locus-wide profile of Egr2 binding patterns in major myelin-associated genes using myelinating peripheral nerve.

Regulation of memory CD8 T-cell differentiation by cyclin-dependent kinase inhibitor p27Kip1.

Induction of potent T-cell memory is the goal of vaccinations, but the molecular mechanisms that regulate the formation of memory CD8 T cells are not well understood. Despite the recognition that controls of cellular proliferation and apoptosis govern the number of memory T cells, the cell cycle regulatory mechanisms that control these key cellular processes in CD8 T cells during an immune response are poorly defined. Here, we have identified the cyclin-dependent kinase inhibitor p27(Kip1) as a critical regulator of the CD8 T-cell homeostasis at all phases of the T-cell response to an acute viral infection in mice. By acting as a timer for cell cycle exit, p27(Kip1) curtailed the programmed expansion of interleukin-2-producing memory precursors and markedly limited the magnitude and quality of CD8 T-cell memory. In the absence of p27(Kip1), CD8 T cells showed superior recall responses shortly after vaccination with recombinant Listeria monocytogenes. Additionally, we show that p27(Kip1) constrains proliferative renewal of memory CD8 T cells, especially of the effector memory subset. These findings provide critical insights into the cell cycle regulation of CD8 T-cell homeostasis and suggest that modulation of p27(Kip1) could bolster vaccine-induced T-cell memory and protective immunity.