Zi Peng Fan

Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.

Summary Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.

Lineage Regulators Direct BMP and Wnt Pathways to Cell-Specific Programs during Differentiation and Regeneration

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation

Visualizing the beginnings of melanoma In cancer biology, a tumor begins from a single cell within a group of precancerous cells that share genetic mutations. Kaufman et al. used a zebrafish melanoma model to visualize cancer initiation (see the Perspective by Boumahdi and Blanpain). They used a fluorescent reporter that specifically lit up neural crest progenitors that are only present during embryogenesis or during adult melanoma tumor formation. The appearance of this tumor correlated with a set of gene regulatory elements, called super-enhancers, whose identification and manipulation may prove beneficial in detecting and preventing melanoma initiation. Science, this issue p. 10.1126/science.aad2197; see also p. 453 Melanocytes with oncogenic or tumor suppressor mutations revert to expressing the crestin gene early in melanoma formation. [Also see Perspective by Boumahdi and Blanpain] INTRODUCTION The “cancerized field” concept posits that cells in a given tissue sharing an oncogenic mutation are cancer-prone, yet only discreet clones within the field initiate tumors. Studying the process of cancer initiation has remained challenging because of (i) the rarity of these events, (ii) the difficulty of visiualizing initiating clones in living organisms, and (iii) the transient nature of a newly transformed clone emerging before it expands to form an early tumor. A more complete understanding of the molecular processes that regulate cancer initiation could provide important prognostic information about which precancerous lesions are most prone to becoming cancer and also implicate druggable molecular pathways that, when inhibited, may prevent the cancer from ever starting. RATIONALE The majority of benign nevi carry oncogenic BRAFV600E mutations and can be considered a cancerized field of melanocytes, but they only rarely convert to melanoma. In an effort to define events that initiate cancer, we used a melanoma model in the zebrafish in which the human BRAFV600E oncogene is driven by the melanocyte-specific mitfa promoter. When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward. RESULTS We developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors. We show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. Early crestin+ patches of cells expand and are transplantable in a manner consistent with their possessing tumorigenic activity, and they exhibit a gene expression pattern consistent with the NCP identity readout by the crestin reporter. The crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, whereas CRISPR/Cas9 targeting of sox10 delayed melanoma onset. We show activation of super-enhancers at NCP genes in both zebrafish and human melanomas, identifying an epigenetic mechanism for control of this NCP signature leading to melanoma. CONCLUSION This work using our zebrafish melanoma model and in vivo reporter of NCP identity allows us to see cancer from its birth as a single cell and shows the importance of NCP-state reemergence as a key event in melanoma initiation from a field of cancer-prone melanocytes. Thus, in addition to the typical fixed genetic alterations in oncogenes and tumor supressors that are required for cancer development, the reemergence of progenitor identity may be an additional rate-limiting step in the formation of melanoma. Preventing NCP reemergence in a field of cancer-prone melanocytes may thus prove therapeutically useful, and the association of NCP genes with super-enhancer regulatory elements implicates the associated druggable epigenetic machinery in this process. Neural crest reporter expression in melanoma. The crestin:EGFP transgene is specifically expressed in melanoma in BRAFV600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expressing crestin:EGFP expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation (bracket). (Bottom) A fully formed melanoma specifically expresses crestin:EGFP, whereas the rest of the fish remains EGFP-negative. The “cancerized field” concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAFV600E mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity

Summary Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.

Direct Lineage Conversion of Adult Mouse Liver Cells and B Lymphocytes to Neural Stem Cells

Summary Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency (Cell Stem Cell (2014) 15 (471-487))

Thorold W. Theunissen, Benjamin E. Powell, Haoyi Wang, Maya Mitalipova, Dina A. Faddah, Jessica Reddy, Zi Peng Fan, Dorothea Maetzel, Kibibi Ganz, Linyu Shi, Tenzin Lungjangwa, Sumeth Imsoonthornruksa, Yonatan Stelzer, Sudharshan Rangarajan, Ana D’Alessio, Jianming Zhang, Qing Gao, Meelad M. Dawlaty, Richard A. Young, Nathanael S. Gray, and Rudolf Jaenisch* *Correspondence: jaenisch@wi.mit.edu http://dx.doi.org/10.1016/j.stem.2014.08.002

The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs

Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis.

Abstract PR06: The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes

Within a group of cancer-prone cells that harbor a shared oncogenic mutation, only rare clones transition to a malignant state. These rare and transient events occurring during cancer initiation remain incompletely understood, and we thus sought to visualize and molecularly characterize this crucial early step in oncogenesis of melanoma. We previously found that the zebrafish crestin gene, which specifically marks embryonic neural crest and normally turns off at three days of life, is specifically re-expressed in BRAFV600E/p53 mutant melanoma tumors in adult zebrafish. We cloned the crestin promoter/enhancer and developed an EGFP reporter that recapitulates the embryonic expression pattern of crestin mRNA and, crucially, marks de novo melanomas in living BRAFV600E/p53 mutant zebrafish. Remarkably, we also found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. These crestin:EGFP positive patches are transplantable, and these precursor lesions are enriched for expression of neural crest progenitor (NCP) genes including the transcription factors sox10 and dlx2a, among other melanoma- and NCP-associated genes. In order to favor the readoption of an NCP-state, we forced misexpression of sox10 in melanocytes of BRAFV600E/p53 melanoma-prone zebrafish, and this led to enhanced melanoma formation. We then analyzed the chromatin landscape of both human and zebrafish melanoma cells using ChIP-Seq and ATAC-Seq and identified super-enhancers at crestin (in zebrafish), sox10, dlx2a, and other NCP/melanoma loci, which together described an overall chromatin signature consistent with features of the NCP state. These data support a model in which 1) an NCP program stochastically reactivates, as read out by crestin expression, in rare BRAFV600E/p53 mutated melanocytes at the initiation of melanoma formation and 2) reemergence of this NCP state is an important and potentially rate-limiting event in melanoma initiation. We anticipate that progenitor identity reemergence will prove to be a general feature of cancer initiation. This abstract is also presented as Poster B19. Citation Format: Charles K. Kaufman, Christian Mosimann, Andrew Thomas, Zi Peng Fan, Song Yang, Justin Tan, Rachel D. Fogley, Ellen van Rooijen, Elliott Hagedorn, Christie Ciarlo, Richard White, Dominick Matos, Ann-Christin Puller, Cristina Santoriello, Eric Liao, Richard A. Young, Leonard I. Zon. The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR06.

Abstract 3230: Genome-wide localization of anti-cancer drugs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide new and important insights into the mechanisms by which such small molecules interfere with tumor cell functions. We have developed a method that couples affinity capture of chemical entities and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small molecules throughout the human genome. Using Chem-seq, we have uncovered the full repertoire of the genomic sites bound by a BET bromodomain inhibitor, a cyclin-dependent kinase (CDK) inhibitor and a DNA intercalating drug. Moreover, by combining Chem-seq with ChIP-seq, we have characterized the interactions of drugs with their targets throughout the genome of tumor cells. These methods provide a powerful approach to enhance understanding of therapeutic action and characterize the specificity of drugs that interact with DNA or genome-associated proteins. Citation Format: Lars Anders, Matthew G. Guenther, Jun Qi, Zi Peng Fan, Jason J. Marineau, Peter B. Rahl, Jakob Loven, Alla A. Sigova, William B. Smith, Tong Ihn Lee, James E. Bradner, Richard A. Young. Genome-wide localization of anti-cancer drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2014-3230