Multi-organ Site Metastatic Reactivation Mediated by Non-canonical Discoidin Domain Receptor 1 Signaling.

Genetic screening identifies the atypical tetraspanin TM4SF1 as a strong mediator of metastatic reactivation of breast cancer. Intriguingly, TM4SF1 couples the collagen receptor tyrosine kinase DDR1 to the cortical adaptor syntenin 2 and, hence, to PKCα. The latter kinase phosphorylates and activates JAK2, leading to the activation of STAT3. This non-canonical mechanism of signaling induces the expression of SOX2 and NANOG; sustains the manifestation of cancer stem cell traits; and drives metastatic reactivation in the lung, bone, and brain. Bioinformatic analyses and pathological studies corroborate the clinical relevance of these findings. We conclude that non-canonical DDR1 signaling enables breast cancer cells to exploit the ubiquitous interstitial matrix component collagen I to undergo metastatic reactivation in multiple target organs.

Immune surveillance of brain metastatic cancer cells is mediated by IFITM1.

Brain metastasis, most commonly originating from lung cancer, increases cancer morbidity and mortality. Although metastatic colonization is the rate-limiting and most complex step of the metastatic cascade, the underlying mechanisms are poorly understood. Here, in vivo genome-wide CRISPR-Cas9 screening revealed that loss of interferon-induced transmembrane protein 1 (IFITM1) promotes brain colonization of human lung cancer cells. Incipient brain metastatic cancer cells with high expression of IFITM1 secrete microglia-activating complement component 3 and enhance the cytolytic activity of CD8+ T cells by increasing the expression and membrane localization of major histocompatibility complex class I. After activation, microglia (of the innate immune system) and cytotoxic CD8+ T lymphocytes (of the adaptive immune system) were found to jointly eliminate cancer cells by releasing interferon-gamma and inducing phagocytosis and T-cell-mediated killing. In human cancer clinical trials, immune checkpoint blockade therapy response was significantly correlated with IFITM1 expression, and IFITM1 enhanced the brain metastasis suppression efficacy of PD-1 blockade in mice. Our results exemplify a novel mechanism through which metastatic cancer cells overcome the innate and adaptive immune responses to colonize the brain, and suggest that a combination therapy increasing IFITM1 expression in metastatic cells with PD-1 blockade may be a promising strategy to reduce metastasis.

Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

A cascade of transcriptional repression determines sexual commitment and development in Plasmodium falciparum.

Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new ApiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions.

Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks.

Aberrant DNA methylation is a distinguishing feature of cancer. Yet, how methylation affects immune surveillance and tumor metastasis remains ambiguous. We introduce a novel method, Guide Positioning Sequencing (GPS), for precisely detecting whole-genome DNA methylation with cytosine coverage as high as 96% and unbiased coverage of GC-rich and repetitive regions. Systematic comparisons of GPS with whole-genome bisulfite sequencing (WGBS) found that methylation difference between gene body and promoter is an effective predictor of gene expression with a correlation coefficient of 0.67 (GPS) versus 0.33 (WGBS). Moreover, Methylation Boundary Shift (MBS) in promoters or enhancers is capable of modulating expression of genes associated with immunity and tumor metabolism. Furthermore, aberrant DNA methylation results in tissue-specific enhancer switching, which is responsible for altering cell identity during liver cancer development. Altogether, we demonstrate that GPS is a powerful tool with improved accuracy and efficiency over WGBS in simultaneously detecting genome-wide DNA methylation and genomic variation. Using GPS, we show that aberrant DNA methylation is associated with altering cell identity and immune surveillance networks, which may contribute to tumorigenesis and metastasis.

A PARP1-BRG1-SIRT1 axis promotes HR repair by reducing nucleosome density at DNA damage sites.

Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.

Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability.

Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.

Differential analysis of chromatin accessibility and histone modifications for predicting mouse developmental enhancers.

Enhancers are distal cis-regulatory elements that modulate gene expression. They are depleted of nucleosomes and enriched in specific histone modifications; thus, calling DNase-seq and histone mark ChIP-seq peaks can predict enhancers. We evaluated nine peak-calling algorithms for predicting enhancers validated by transgenic mouse assays. DNase and H3K27ac peaks were consistently more predictive than H3K4me1/2/3 and H3K9ac peaks. DFilter and Hotspot2 were the best DNase peak callers, while HOMER, MUSIC, MACS2, DFilter and F-seq were the best H3K27ac peak callers. We observed that the differential DNase or H3K27ac signals between two distant tissues increased the area under the precision-recall curve (PR-AUC) of DNase peaks by 17.5-166.7% and that of H3K27ac peaks by 7.1-22.2%. We further improved this differential signal method using multiple contrast tissues. Evaluated using a blind test, the differential H3K27ac signal method substantially improved PR-AUC from 0.48 to 0.75 for predicting heart enhancers. We further validated our approach using postnatal retina and cerebral cortex enhancers identified by massively parallel reporter assays, and observed improvements for both tissues. In summary, we compared nine peak callers and devised a superior method for predicting tissue-specific mouse developmental enhancers by reranking the called peaks.

Sin3a-Tet1 interaction activates gene transcription and is required for embryonic stem cell pluripotency.

Sin3a is a core component of histone-deacetylation-activity-associated transcriptional repressor complex, playing important roles in early embryo development. Here, we reported that down-regulation of Sin3a led to the loss of embryonic stem cell (ESC) self-renewal and skewed differentiation into mesendoderm lineage. We found that Sin3a functioned as a transcriptional coactivator of the critical Nodal antagonist Lefty1 through interacting with Tet1 to de-methylate the Lefty1 promoter. Further studies showed that two amino acid residues (Phe147, Phe182) in the PAH1 domain of Sin3a are essential for Sin3a-Tet1 interaction and its activity in regulating pluripotency. Furthermore, genome-wide analyses of Sin3a, Tet1 and Pol II ChIP-seq and of 5mC MeDIP-seq revealed that Sin3a acted with Tet1 to facilitate the transcription of a set of their co-target genes. These results link Sin3a to epigenetic DNA modifications in transcriptional activation and have implications for understanding mechanisms underlying versatile functions of Sin3a in mouse ESCs.

Chemical cocktails enable hepatic reprogramming of human urine-derived cells with a single transcription factor.

Human liver or hepatocyte transplantation is limited by a severe shortage of donor organs. Direct reprogramming of other adult cells into hepatic cells may offer a solution to this problem. In a previous study, we have generated hepatocyte-like cells from mouse fibroblasts using only one transcription factor (TF) plus a chemical cocktail. Here, we show that human urine-derived epithelial-like cells (hUCs) can also be transdifferentiated into human hepatocyte-like cells (hiHeps) using one TF (Foxa3, Hnf1α, or Hnf4α) plus the same chemical cocktail CRVPTD (C, CHIR99021; R, RepSox; V, VPA; P, Parnate; T, TTNPB; and D, Dznep). These hiHeps express multiple hepatocyte-specific genes and display functions characteristic of mature hepatocytes. With the introduction of the large T antigen, these hiHeps can be expanded in vitro and can restore liver function in mice with concanavalin-A-induced acute liver failure. Our study provides a strategy to generate functional hepatocyte-like cells from hUCs by using a single TF plus a chemical cocktail.

A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces.

Hundreds of different proteins regulate and implement transcription in Saccharomyces. Yet their interrelationships have not been investigated on a comprehensive scale. Here we determined the genome-wide binding locations of 200 transcription-related proteins, under normal and acute heat-shock conditions. This study distinguishes binding between distal versus proximal promoter regions as well as the 3' ends of genes for nearly all mRNA and tRNA genes. This study reveals (1) a greater diversity and specialization of regulation associated with the SAGA transcription pathway compared to the TFIID pathway, (2) new regulators enriched at tRNA genes, (3) a global co-occupancy network of >20,000 unique regulator combinations that show a high degree of regulatory interconnections among lowly expressed genes, (4) regulators of the SAGA pathway located largely distal to the core promoter and regulators of the TFIID pathway located proximally, and (5) distinct mobilization of SAGA- versus TFIID-linked regulators during acute heat shock.

Improved recombinant protein production by regulation of transcription and protein transport in Chinese hamster ovary cells.

OBJECTIVES: To identify genes that affected protein expression in Chinese hamster ovary (CHO) cells was significant, and we identified the changes in the transcriptome and the functional gene sets that would contribute to increase expression of recombinant protein. RESULTS: Here two sub-clones from a methotrexate-treated parental recombinant CHO cell line were selected. The two sub-clones, with different expression levels (qp were 42.8 pg/cell/day and 14.0 pg/cell/day), were analyzed through RNA-seq. More than 600 genes were identified as differently expressed, and we found that the differentially expressed genes were involved in processes such as RNA processing, transcription, protein catabolism, and protein transport. Among these, we cloned genes encoding proteins that were involved in transcription and protein transport to investigate their effect on protein production. CONCLUSIONS: We found that some genes involved in transcription and protein transport would improve recombinant protein production in CHO cells.

Opposing Roles of Wnt Inhibitors IGFBP-4 and Dkk1 in Cardiac Ischemia by Differential Targeting of LRP5/6 and ß-catenin.

BACKGROUND: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/ß-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/ß-catenin pathway activation. However, their roles in the adult heart remain unexplored. METHODS: To understand the role of LRP5/6 and ß-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and ß-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and ß-catenin. RESULTS: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of ß-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/ß-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting ß-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. CONCLUSIONS: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and ß-catenin signaling within the Wnt/ß-catenin pathway.

Genetic analysis of heterogeneous sub-clones in recombinant Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells have been widely used for production of recombinant proteins and therapeutic antibodies. However, owing to the instability and heterogeneity of CHO cells, the development of stable and high-expression recombinant CHO cell lines is often time-consuming. To investigate the mechanisms associated with heterogeneity in protein productivity, we performed transcriptome analysis on the subclones derived from a stable parental CHO clone. Two high-expression subclones and one low-expression subclone were selected based on their similar genomic background and subjected to RNA-seq analysis. Over 100 differentially expressed genes were identified between the subclones with high and low productivity. The molecular functions of the differentially expressed genes were enriched for translational elongation, sterol biosynthetic process, and regulation of secretion. In addition, analyses of the two subclones with high protein expression levels identified over 300 differentially expressed genes involved in DNA metabolic processes, cellular macromolecule catabolic processes, cell cycle, protein catabolic processes, and RNA processing and transcription. A subset of the differentially expressed genes was overexpressed in CHO cells to identify their effects on protein production. Together, these results indicate that transcriptome variation can cause significant inter-cellular heterogeneity in CHO cells and a better understanding of the molecular mechanism underlying heterogeneity might help to improve the production of recombinant proteins by CHO cells.

Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome.

A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here, we examine on a genomic scale several protein-nucleosome interactions, including those that (1) bind histones (Bdf1/SWR1 and Srm1), (2) bind specific DNA sequences (Rap1 and Reb1), and (3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a dinucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of nucleosomal DNA. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions and that some gene regulators have position-specific nucleosomal interactions.

Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling.

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self-renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self-renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs.

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

Unusual combinatorial involvement of poly-A/T tracts in organizing genes and chromatin in Dictyostelium.

Dictyostelium discoideum is an amoebozoa that exists in both a free-living unicellular and a multicellular form. It is situated in a deep branch in the evolutionary tree and is particularly noteworthy in having a very A/T-rich genome. Dictyostelium provides an ideal system to examine the extreme to which nucleotide bias may be employed in organizing promoters, genes, and nucleosomes across a genome. We find that Dictyostelium genes are demarcated precisely at their 5' ends by poly-T tracts and precisely at their 3' ends by poly-A tracts. These tracts are also associated with nucleosome-free regions and are embedded with precisely positioned TATA boxes. Homo- and heteropolymeric tracts of A and T demarcate nucleosome border regions. Together, these findings reveal the presence of a variety of functionally distinct polymeric A/T elements. Strikingly, Dictyostelium chromatin may be organized in di-nucleosome units but is otherwise organized as in animals. This includes a +1 nucleosome in a position that predicts the presence of a paused RNA polymerase II. Indeed, we find a strong phylogenetic relationship between the presence of the NELF pausing factor and positioning of the +1 nucleosome. Pausing and +1 nucleosome positioning may have coevolved in animals.

Nucleosome positioning and gene regulation: advances through genomics.

Knowing the precise locations of nucleosomes in a genome is key to understanding how genes are regulated. Recent 'next generation' ChIP-chip and ChIP-Seq technologies have accelerated our understanding of the basic principles of chromatin organization. Here we discuss what high-resolution genome-wide maps of nucleosome positions have taught us about how nucleosome positioning demarcates promoter regions and transcriptional start sites, and how the composition and structure of promoter nucleosomes facilitate or inhibit transcription. A detailed picture is starting to emerge of how diverse factors, including underlying DNA sequences and chromatin remodelling complexes, influence nucleosome positioning.