Megan F. Cole

Core transcriptional regulatory circuitry in human embryonic stem cells.

The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.

Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

Genome-wide Map of Nucleosome Acetylation and Methylation in Yeast

Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.

Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells.

Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.

Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas—an organ that critically requires cyclin D1 function—cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term ‘genetic–proteomic’, can be used to study the in vivo function of essentially any protein.

Mapping Key Features of Transcriptional Regulatory Circuitry in Embryonic Stem Cells

The process by which a single fertilized egg develops into a human being with more than 200 cell types--each with a distinct gene expression pattern controlling its cellular state--is poorly understood. Knowledge of the transcriptional regulatory circuitry that establishes and maintains gene expression programs in mammalian cells is fundamental to understanding development and should provide the foundation for improved diagnosis and treatment of disease. Although it is not yet feasible to map the entirety of this circuitry in vertebrate cells, recent work in embryonic stem (ES) cells has demonstrated that core features of the circuitry can be discovered through studies involving selected regulators. Here, we highlight the fundamental insights that have emerged from studies that examined the role of transcription factors, chromatin regulators, signaling pathways, and noncoding RNAs in the regulatory circuitry of ES cells. Maps of regulatory circuitry and the insights that have emerged from these studies have improved our understanding of global gene expression and are facilitating efforts to reprogram cells for disease therapeutics and regenerative medicine.

Exploiting Models of Molecular Evolution to Efficiently Direct Protein Engineering

Directed evolution and protein engineering approaches used to generate novel or enhanced biomolecular function often use the evolutionary sequence diversity of protein homologs to rationally guide library design. To fully capture this sequence diversity, however, libraries containing millions of variants are often necessary. Screening libraries of this size is often undesirable due to inaccuracies of high-throughput assays, costs, and time constraints. The ability to effectively cull sequence diversity while still generating the functional diversity within a library thus holds considerable value. This is particularly relevant when high-throughput assays are not amenable to select/screen for certain biomolecular properties. Here, we summarize our recent attempts to develop an evolution-guided approach, Reconstructing Evolutionary Adaptive Paths (REAP), for directed evolution and protein engineering that exploits phylogenetic and sequence analyses to identify amino acid substitutions that are likely to alter or enhance function of a protein. To demonstrate the utility of this technique, we highlight our previous work with DNA polymerases in which a REAP-designed small library was used to identify a DNA polymerase capable of accepting non-standard nucleosides. We anticipate that the REAP approach will be used in the future to facilitate the engineering of biopolymers with expanded functions and will thus have a significant impact on the developing field of ‘evolutionary synthetic biology’.

Reconstructing evolutionary adaptive paths for protein engineering.

Reconstructing Evolutionary Adaptive Paths (REAP) is one of several methods to improve enzyme -functionality. This approach incorporates computational and theoretical aspects of protein engineering to create a focused library of protein variety with a high degree of functionality. In contrast to other -techniques like DNA shuffling, REAP allows a library to have diverse functionality among relatively few variants. REAP is a low-throughput method which takes advantage of natural selection and uses ancestral protein sequences to direct gene mutations, thereby creating a library with a high density of viable proteins. These proteins must then be assayed to characterize their functionality to identify which variants have the desired traits such as acid stability or thermostability.

Interchanging Functionality Among Homologous Elongation Factors Using Signatures of Heterotachy

Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.