Our Research
Direction 1
The regulatory R-loops in pluripotency, embryo development, and B-cell development and lymphomas
Direction 2
The RNA binding protein and mRNA epigenetics in pluripotent stem cells (PSCs) maintenance and differentiation
Direction 3
The crosstalk between transcription factors and ATP-dependent chromatin remodelers in human pluripotency and diseases
1. The regulatory R-loops in pluripotency, embryo development, and B-cell development and lymphomas.
Our preliminary work strongly suggests a functional association between Zfp281 and R-loops in the transcriptional control of mouse and human PSCs. R-loops are three-stranded nucleic acid structures composed of a DNA hybrid and a free single-stranded DNA. These structures can lead to DNA damage, posing a risk to genome integrity. However, growing evidence suggests that R-loops also regulate gene transcription, mitosis, and homologous recombination, contributing to fundamental biological processes. The dual nature of R-loops necessitates tight regulation of their formation and resolution, as improper accumulation may contribute to abnormal human development and disease. Our preliminary data indicated that Zfp281 recruits Tet1 for DNA demethylation at promoters and transcriptionally activates Dnmt3a/3b for DNA methylation at gene bodies in mouse PSCs. Additionally, it has been reported that R-loops are associated with DNA damage and that Zfp281 prevents R-loop accumulation during DNA replication. Therefore, we will explore the functions of regulatory R-loops in pluripotency and early embryo development through the following pathways: 1) Zfp281/Tet1-mediated R-loop formation for gene activation; 2) Zfp281- and Dnmt3a/3b-mediated R-loop restriction for gene repression; and 3) Zfp281/Brca2-mediated R-loop resolution for genome stability. Furthermore, we will develop a research program to explore R-loop regulatory functions in disease settings. A recent study reported that R-loops accumulate aberrantly in Tet-deficient B-cells, disrupting B-cell homeostasis and accelerating the growth of diffuse large B-cell lymphoma (DLBCL)-like tumors in mice. Using a Zfp281-floxed (Zfp281fl/fl) mouse line we recently created, we will investigate the detrimental effects of aberrantly accumulated R-loops induced by Zfp281 loss-of-function on B-cell homeostasis. This will involve both in vitro studies using human B-cell (lymphoma) lines and in vivo studies using a B-cell-specific Zfp281-deficient animal model.
2. The RNA binding protein and mRNA epigenetics in pluripotent stem cells (PSCs) maintenance and differentiation.
Building on our research into RNA binding proteins (RBPs), noncoding RNAs (ncRNAs), and transposable elements (TEs), including TE-transcribed ncRNAs, in pluripotent stem cells (PSCs), we developed a research project focusing on short interspersed nuclear elements (SINEs)-mediated mRNA m5C modification in pluripotency control. TEs, such as LINEs, SINEs, and ERVs, are the most abundant DNA elements in the mouse and human genome. Historically considered “junk” DNA, it is now widely accepted that they play significant roles in various cellular processes. Unlike other TEs, SINEs are often embedded in noncoding regions (e.g., introns and UTRs) within genes, affecting host mRNA expression. From an RNA bisulfite sequencing (BS-seq) dataset in mouse ESCs, we identified that pluripotency-related mRNAs (e.g., Nanog) with embedded SINE elements are enriched with the m5C modification. Additionally, the RBP Alyref, within the transcription-export complex, was identified as an m5C reader, suggesting that m5C may facilitate the nuclear export of mRNAs. We hypothesize that the self-renewal and differentiation of ESCs are controlled by m5C-mediated nuclear export and retention of pluripotency mRNAs bearing SINE elements, respectively. To investigate this, we will perform the following experiments: 1) examine the regulation of m5C-modified pluripotency mRNA (e.g., Nanog) for nuclear export in ESC self-renewal; 2) Determine if the active nuclear export of pluripotency mRNAs is mediated by the m5C reader Alyref; 3) Profile the Alyref RNA targets by eCLIP-seq and test if Alyref mediates nuclear export of m5C-modified mRNA by outcompeting another RBP, Pspc1, for nuclear retention. Additionally, I will use an immunoprecipitation−mass spectrometry (IP-MS) approach to explore if additional cofactors of Alyref are required to recognize and export mRNAs with a SINE-like structure and m5C modification.
3. The crosstalk between transcription factors and ATP-dependent chromatin remodelers in human pluripotency and diseases.
We aim to further study the functional importance of chromatin remodelers in human development and diseases, following our research on OCT4 and chromatin remodelers in human ESCs (hESCs). Mammalian chromatin remodelers, including BAF (SWI/SNF) and ISWI complexes, utilize BRG1/BRM and SNF2H/SNF2L as core ATPases, respectively. Our published work on OCT4 interactomes revealed that BRM is a naive-specific ATPase in the BAF complex, while SNF2L is a primed-specific ATPase in the ISWI complex. BRG1 and SNF2H are shared ATPases in both the BAF and ISWI complexes in naive and primed states. In our unpublished work, we performed ChIP-seq analysis of these factors and identified a cluster of primed-specific enhancers with OCT4/BRG1/SNF2L co-binding. Motif analysis indicated that these enhancers are related to neural lineage development, involving other key TFs such as SOX2. As heterozygous mutations in chromatin remodelers (e.g., BAF) drive many neural disorders, including autism spectrum disorders (ASD) and Coffin-Siris syndrome (CSS), we will focus on neural differentiation using hESC models. We will focus on the roles of these chromatin remodelers, alongside TFs and epigenetic regulators, in human naive/primed pluripotent states and (neural) lineage differentiation. Using CRISPR/KO of specific ATPase in hESCs, we aim to understand: 1) the importance of BRG1 and BRM in maintaining naive pluripotency; 2) The functional interactions among OCT4, SOX2, and chromatin remodelers BRG1 (BAF) and SNF2L (ISWI) in primed pluripotency; 3) The regulatory roles of OCT4 in recruiting BRG1 and SNF2L to the SOX2 locus during hESC-to-neural progenitor cells (NPCs) differentiation.
Moreover, multiple pan-cancer studies have documented BAF mutations in human tumors, with various aberrations observed. For instance, BRG1 is described as a tumor suppressor in cancers such as brain, lung, ovarian cancers, and lymphoma, while it is described as an oncogene in other cancers like breast and prostate cancers. The functional consequences of specific BAF mutations in different cancer types are poorly understood and likely context-dependent. One of our long-term research goals is to integrate the mutagenesis of chromatin remodelers with human PSCs/iPSCs to create robust and versatile cancer models. These models will provide valuable insights into cancer biology and therapeutic development. We are particularly interested in the B-cell lymphomas and have established collaborations with colleagues and clinicians to advance this research.