Xiaobai Zhang

Coordination of PAD4 and HDAC2 in the regulation of p53 target gene expression

Histone Arg methylation and Lys acetylation have been found to cooperatively regulate the expression of p53-target genes. Peptidylarginine deiminase 4 (PAD4) is an enzyme that citrullinates histone arginine and monomethyl-arginine residues thereby regulating histone Arg methylation. We have recently found that PAD4 serves as a p53 corepressor to regulate histone Arg methylation at the p53-target gene p21/WAF1/CIP1 promoter. However, it has not been tested whether histone Arg citrullination coordinates with other histone modifications to repress transcription. Here, we show that histone deacetylase (HDAC2) and PAD4 interact with p53 through distinct domains and simultaneously associate with the p21 promoter to regulate gene expression. After DNA damage, PAD4 and HDAC2 dissociate from several p53-target gene promoters (for example, p21, GADD45, and PUMA) with a concomitant increase in histone Lys acetylation and Arg methylation at these promoters. Furthermore, PAD4 promoter association and histone Arg modifications are regulated by p53 and HDAC activity. In contrast, HDAC2 promoter association and histone Lys acetylation are affected by p53 and PAD4 activity at minor degrees. Importantly, PAD4 inhibitor Cl-amidine and HDAC inhibitor suberoylanilide hydroxamic acid show additive effects in inducing p21, GADD45, and PUMA expression and inhibiting cancer cell growth in a p53-dependent manner. Our results unveil an important crosstalk between histone deacetylation and citrullination, suggesting that a combination of PAD4 and HDAC2 inhibitors as a potential strategy for cancer treatment.

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self‐renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β‐thalassemia into transgene‐free naïve iPSCs with molecular signatures of ground‐state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross‐species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene‐correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient‐specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy.

Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

Phylogenetic affinity of tree shrews to Glires is attributed to fast evolution rate

Previous phylogenetic analyses have led to incongruent evolutionary relationships between tree shrews and other suborders of Euarchontoglires. What caused the incongruence remains elusive. In this study, we identified 6845 orthologous genes between seventeen placental mammals. Tree shrews and Primates were monophyletic in the phylogenetic trees derived from the first or/and second codon positions whereas tree shrews and Glires formed a monophyly in the trees derived from the third or all codon positions. The same topology was obtained in the phylogeny inference using the slowly and fast evolving genes, respectively. This incongruence was likely attributed to the fast substitution rate in tree shrews and Glires. Notably, sequence GC content only was not informative to resolve the controversial phylogenetic relationships between tree shrews, Glires, and Primates. Finally, estimation in the confidence of the tree selection strongly supported the phylogenetic affiliation of tree shrews to Primates as a monophyly.

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5′ ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

Gene expression profiling analysis of bisphenol A-induced perturbation in biological processes in ER-negative HEK293 cells.

Bisphenol A (BPA) is an environmental endocrine disruptor which has been detected in human bodies. Many studies have implied that BPA exposure is harmful to human health. Previous studies mainly focused on BPA effects on estrogen receptor (ER)-positive cells. Genome-wide impacts of BPA on gene expression in ER-negative cells is unclear. In this study, we performed RNA-seq to characterize BPA-induced cellular and molecular impacts on ER-negative HEK293 cells. The microscopic observation showed that low-dose BPA exposure did not affect cell viability and morphology. Gene expression profiling analysis identified a list of differentially expressed genes in response to BPA exposure in HEK293 cells. These genes were involved in variable important biological processes including ion transport, cysteine metabolic process, apoptosis, DNA damage repair, etc. Notably, BPA up-regulated the expression of ERCC5 encoding a DNA endonuclease for nucleotide-excision repair. Further electrochemical experiment showed that BPA induced significant DNA damage in ER-positive MCF-7 cells but not in ER-negative HEK293 cells. Collectively, our study revealed that ER-negative HEK293 cells employed mechanisms in response to BPA exposure different from ER-positive cells.

Toxic effects of decabromodiphenyl ether (BDE-209) on human embryonic kidney cells.

Polybrominated diphenyl ethers (PBDEs) are widely used as flame-retardant additives in consumer and household products and can escape into the environment over time. PBDEs have become a global environmental organic pollutant due to the properties of persistence, toxicity, and bioaccumulation. The well-studied toxic effects of PBDEs mainly include thyroid hormone disruption and neurotoxicity. There is no consistent conclusions on the carcinogenic potential of PBDEs to date. Here, we explored the toxic effects of BDE-209 on human embryonic kidney cells (HEK293T). The comparison of the gene expression profiles of HEK293T cells with BDE-209 treatment and the negative control found that BDE-209 exposure may alter nucleosome organization through significantly changing the expression of histone gene clusters. The remodeled chromatin structure could further disturb systemic lupus erythematosus as one of the toxic effects of BDE-209. Additionally, gene sets of different cancer modules are positively correlated with BDE-209 exposure. This suggests that BDE-209 has carcinogenic potential for a variety of tumors. Collectively, BDE-209 has a broader toxicity not limited to disruption of thyroid hormone-related biological processes. Notably, the toxic effects of BDE-209 dissolved in dimethyl sulfoxide (DMSO) is not the simply additive effects of BDE-209 and DMSO alone.

Dynamically reorganized chromatin is the key for the reprogramming of somatic cells to pluripotent cells

Nucleosome positioning and histone modification play a critical role in gene regulation, but their role during reprogramming has not been fully elucidated. Here, we determined the genome-wide nucleosome coverage and histone methylation occupancy in mouse embryonic fibroblasts (MEFs), induced pluripotent stem cells (iPSCs) and pre-iPSCs. We found that nucleosome occupancy increases in promoter regions and decreases in intergenic regions in pre-iPSCs, then recovers to an intermediate level in iPSCs. We also found that nucleosomes in pre-iPSCs are much more phased than those in MEFs and iPSCs. During reprogramming, nucleosome reorganization and histone methylation around transcription start sites (TSSs) are highly coordinated with distinctively transcriptional activities. Bivalent promoters gradually increase, while repressive promoters gradually decrease. High CpG (HCG) promoters of active genes are characterized by nucleosome depletion at TSSs, while low CpG (LCG) promoters exhibit the opposite characteristics. In addition, we show that vitamin C (VC) promotes reorganizations of canonical, H3K4me3- and H3K27me3-modified nucleosomes on specific genes during transition from pre-iPSCs to iPSCs. These data demonstrate that pre-iPSCs have a more open and phased chromatin architecture than that of MEFs and iPSCs. Finally, this study reveals the dynamics and critical roles of nucleosome positioning and chromatin organization in gene regulation during reprogramming.

Pwp1 Is Required for the Differentiation Potential of Mouse Embryonic Stem Cells Through Regulating Stat3 Signaling

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self‐renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD‐40 repeat‐containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self‐renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation‐seq results revealed that the PWP1‐occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs. Stem Cells 2015;33:661–673

Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos.

Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification.