• DNA methylation
• Klf4
• SP1
• human dental pulp cells
• odontoblastic differentiation

• Animals
• Apoptosis
• Cell Differentiation
• Cell Proliferation
• Cells, Cultured
• CpG Islands
• DNA (Cytosine-5-)-Methyltransferase 1/genetics
• DNA (Cytosine-5-)-Methyltransferase 1/metabolism
• DNA Methylation
• Dental Pulp/cytology
• Dental Pulp/metabolism
• Gene Expression Regulation
• Humans
• Kruppel-Like Factor 4
• Kruppel-Like Transcription Factors/genetics
• Kruppel-Like Transcription Factors/metabolism
• Mice
• Odontoblasts/cytology
• Odontoblasts/metabolism
• Promoter Regions, Genetic
• Sp1 Transcription Factor/genetics
• Sp1 Transcription Factor/metabolism

• R01 DE019802 NIDCR NIH HHS

• Alleles
• Animals
• Animals, Genetically Modified
• Biological Assay
• Chromatin/chemistry
• Chromosomes, Human, Pair 1/chemistry
• Cleft Lip/diagnosis
• Cleft Lip/genetics
• Cleft Lip/pathology
• Cleft Palate/diagnosis
• Cleft Palate/genetics
• Cleft Palate/pathology
• Enhancer Elements, Genetic
• GTPase-Activating Proteins/genetics
• GTPase-Activating Proteins/metabolism
• Gene Expression
• Genes, Reporter
• Genetic Loci
• Genetic Predisposition to Disease
• Genome-Wide Association Study
• Humans
• Linkage Disequilibrium
• Luciferases/genetics
• Luciferases/metabolism
• Polymorphism, Single Nucleotide
• Promoter Regions, Genetic
• Risk Factors
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Zebrafish

• R37 DE008559 NIDCR NIH HHS
• U01 DE018993 NIDCR NIH HHS
• R56 DE023575 NIDCR NIH HHS
• K99 DE025060 NIDCR NIH HHS
• U01 HG005925 NHGRI NIH HHS
• R01 DE023575 NIDCR NIH HHS
• R01 DE014581 NIDCR NIH HHS
• R01 DE016148 NIDCR NIH HHS
• R00 DE025060 NIDCR NIH HHS
• R01 DE008559 NIDCR NIH HHS

ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo “library preparation” to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing.

In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a “gold standard” reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling.

We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

The online version of this article (doi:10.1186/s12864-016-3135-y) contains supplementary material, which is available to authorized users.

• Chromatin immunoprecipitation
• HTS
• Low-input
• Micro-ChIP
• NGS

• Cell Culture Techniques/methods
• Cell Lineage
• Cells, Cultured
• Gene Expression Profiling
• Human Embryonic Stem Cells/cytology
• Humans
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Myocytes, Cardiac/cytology
• Neurons/cytology
• Stem Cell Transplantation

• K01AG024496 NIA NIH HHS
• R21HD056530 NICHD NIH HHS

Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.

• Embryonic stem cells
• Cancer stem cells
• Pluripotency
• Self-renewal
• Tumorigenicity

Chromatin is a dynamic but highly regulated structure. DNA-binding proteins such as transcription factors, epigenetic and chromatin modifiers are responsible for regulating specific gene expression pattern and may result in different phenotypes. To reveal the identity of the proteins associated with the specific region on DNA, chromatin immunoprecipitation (ChIP) is the most widely used technique. ChIP assay followed by next generation sequencing (ChIP-seq) or microarray (ChIP-chip) is often used to study patterns of protein-binding profiles in different cell types and in cancer samples on a genome-wide scale. However, only a limited number of bioinformatics tools are available for ChIP datasets analysis.

We present ChIPseek, a web-based tool for ChIP data analysis providing summary statistics in graphs and offering several commonly demanded analyses. ChIPseek can provide statistical summary of the dataset including histogram of peak length distribution, histogram of distances to the nearest transcription start site (TSS), and pie chart (or bar chart) of genomic locations for users to have a comprehensive view on the dataset for further analysis. For examining the potential functions of peaks, ChIPseek provides peak annotation, visualization of peak genomic location, motif identification, sequence extraction, and comparison between datasets. Beyond that, ChIPseek also offers users the flexibility to filter peaks and re-analyze the filtered subset of peaks. ChIPseek supports 20 different genome assemblies for 12 model organisms including human, mouse, rat, worm, fly, frog, zebrafish, chicken, yeast, fission yeast, Arabidopsis, and rice. We use demo datasets to demonstrate the usage and intuitive user interface of ChIPseek.

ChIPseek provides a user-friendly interface for biologists to analyze large-scale ChIP data without requiring any programing skills. All the results and figures produced by ChIPseek can be downloaded for further analysis. The analysis tools built into ChIPseek, especially the ones for selecting and examine a subset of peaks from ChIP data, provides invaluable helps for exploring the high through-put data from either ChIP-seq or ChIP-chip. ChIPseek is freely available at http://chipseek.cgu.edu.tw.

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.

• Cell Cycle/physiology
• Gene Expression Regulation
• Genetic Association Studies/methods
• Genotype
• Humans
• Internet
• Neoplasms/genetics
• Neoplasms/metabolism
• Phenotype
• Software
• Substance-Related Disorders/genetics
• Substance-Related Disorders/metabolism
• Time Factors
• User-Computer Interface

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.

• Human embryonic stem cell
• cell therapy
• heart disease
• neurological disease
• pluripotent
• regenerative medicine
• stem cell
• tissue engineering

• Human embryonic stem cell
• cell therapy
• heart disease
• neurological disease
• pluripotent
• regenerative medicine
• stem cell
• tissue engineering

Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers.

We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.

The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.