RESEARCH

DNA modifications in stem cells and neuroepigenetics 

DNA cytosine methylation (5-methylcytosine) is an evolutionarily conserved epigenetic mark and has a profound impact on transcription, development and genome stability. Historically, 5-methylcytosine (mC) is considered as a highly stable chemical modification that is mainly required for long-term epigenetic memory. The recent discovery that ten-eleven translocation (TET) proteins can iteratively oxidize mC in the mammalian genome represents a paradigm shift in our understanding of how mC may be enzymatically reversed. It also raises the possibility that three oxidized mC bases (hmC: hydroxymethylcytosine, fC: formylcytosine and caC: carboxymethylcytosine) generated by TET may act as a new class of epigenetic modifications.

 

As the most abundant oxidized forms of mC, hmC is of particular interest as it is preferentially enriched in central nervous systems, reaching levels as high as 40% of all modified cytosines in adult neurons. Neuronal hmC builds up postnatally, coinciding with the peak of synaptogenesis and synaptic pruning. The unique profile and dynamics of neuronal hmC suggest that hmC may play unique gene regulatory roles in brain maturation and aging.

We employ high-throughput sequencing technologies, bioinformatics, mammalian genetic models, as well as epigenome editing tools to investigate the epigenetic gene regulatory mechanisms by which proteins that write, read and erase DNA modifications in mammalian pluripotent stem cells as well as brain development and maturation.

Our research and review papers on this topic:

1. Schutsky et al., Nature Biotechnology (2018). PMID: 30295673

2. Wu H*, Wu X*, Zhang Y†, Nature Protocol (2016). PMID: 27172168

3. Wu H, Zhang Y†, Nature Structural & Molecular Biology (2015). PMID: 26333715

4. Wu H*,†, Wu X*, et al., Nature Biotechnology (2014). PMID: 25362244

5. Wu H, Zhang Y†, Cell (2014). PMID: 24439369

6. Shen L*, Wu H*,†, et al., Cell (2013). PMID: 23602152

7. Wu H, Zhang Y†, Genes & Development (2011). PMID: 22156206

8. Wu H†, Zhang Y†, Cell Cycle (2011). PMID: 21750410

9. Wu H*, D'Alessio AC*, et al., Nature (2011). PMID: 21451524

10. Wu H et al., Genes & Development (2011). PMID: 21460036

11. Wu H*,†, et al., Science (2010). PMID: 20651149

Massively parallel and time-resolved RNA sequencing in single cells 

Dynamic changes in RNA levels are regulated by the interplay of RNA transcription, processing, and degradation. Understanding the mechanisms by which the transcriptome is regulated in functionally diverse cell-types within multi-cellular organisms thus requires cell-type-specific measurements of the kinetics of RNA biogenesis and decay. Recent advances in single-cell RNA sequencing (scRNA-Seq) technologies are leading to a more complete understanding of heterogeneity in cell types and states. However, standard scRNA-Seq methods capture a mixture of newly-transcribed (“new”) and pre-existing (“old”) RNAs without being able to temporally resolve RNA dynamics.

To overcome these constraints, we developed single-cell metabolically labeled new RNA tagging sequencing (scNT-Seq), a high-throughput and UMI-based scRNA-Seq method that simultaneously measures both new and old transcriptomes from the same cell. In scNT-Seq, integration of metabolic RNA labeling, droplet microfluidics, and chemically induced recoding of 4sU to cytosine analog permits highly scalable and time-resolved single-cell analysis of cellular RNA dynamics. We demonstrate that the method is easy to set up and substantially improves the time and cost. We show scNT-Seq enables temporally resolved analysis of gene regulatory networks and RNA trajectories. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

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Epigenetic regulation of cardiac maturation, 

aging, and repair

The life of all animals, from the beginning of their life and throughout adulthood, depends on the normal function of life. As the mammalian hearts have very limited regenerative potential, the loss or pathological remodeling of specific cardiac cell types, such as ventricular cardiomyocytes, during injury and diseases often leads to heart failure and sudden death. However, limited understanding of the precise composition and functional heterogeneity of resident cell-types in developing and adult human hearts represents a major barrier to the development of effective therapies to treat heart diseases. Standard single-cell RNA sequencing approaches require the preparation of intact, single-cell suspensions from fresh tissues. This requirement poses a particular challenge for mammalian hearts, which is difficult to be dissociated without altering the cell-type composition. 

 

To overcome these challenges, We and others have recently developed highly scalable single-nucleus RNA-seq (snRNA-seq) approaches. Using nuclei allows the decoupling of biospecimen collection and molecular assays because intact nuclei can be isolated from cryopreserved tissues. Moreover, use of nuclei can avoid potential alteration of transcriptome due to tissue dissociation and minimize biased representation of cells of certain sizes. With our snRNA-seq method (termed sNucDrop-seq), we dissected cell-type composition and activity-dependent transcriptional program in adult brains (Hu* and Fabyanic* et al., 2017). Next, we demonstrated that unbiased profiling ~20,000 nuclear transcriptomes not only accurately determines subtype composition, maturation state and proliferation index of cardiomyocytes, but also identifies changes in transcription induced by inactivation of key transcriptional factors that drive cardiac maturation in postnatal mouse hearts (Hu et al., 2018). More recently, we applied sNucDrop-seq to analyze human pluripotent stem cell-derived cardiomyocytes, frozen-archived human adult hearts to systematically investigate the molecular basis of human cardiac cell-type maturation and aging.

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