Image: Royalactin binding of mouse embryonic fibroblasts
Image: Royalactin binding of mouse embryonic fibroblasts
Anna Luan, Michael S. Hu, Tripp Leavitt, Elizabeth A. Brett, Kevin C. Wang, Michael T. Longaker, and Derrick C. Wan. Noncoding RNAs in Wound Healing: A New and Vast Frontier.
Significance: Wound healing requires a highly orchestrated coordination of processes that are not yet fully understood. Therefore, available clinical therapies are thus far limited in their efficacy in preventing and treating both chronic wounds and scars. Current gene-based therapeutics is largely based on our under-standing of the protein-coding genome and proteins involved in
known wound healing pathways.
Recent Advances: Noncoding RNAs such as microRNAs and long noncoding RNAs have recently been found to be significant modulators of gene expression in diverse cellular pathways. Research has now implicated noncoding RNAs in nearly every stage of the wound healing process, suggesting that they may
serve as clinical therapeutic targets. Noncoding RNAs are critical regulators in processes such as angiogenesis and cutaneous cell migration and proliferation, including classically described biological pathways previously attributed to mostly protein constituents.
Critical Issues: The complexity and diversity of the interactions of noncoding RNAs with their targets and other binding partners require thorough characterization and understanding of their functions before they may be altered to modulate human wound healing pathways.
Future Directions: Research in the area of noncoding RNAs continues to rapidly expand our understanding of their potential roles in physiological and pathological wound healing. Coupled with improving technologies to enhance or suppress target non- coding RNA in vivo, these advances hold great promise in the development of new therapies for wound healing.
Wang, K.C., Chang, H.Y. (2017). Epigenomics: Technologies and Applications. 27 Apr 2018 Circulation Research. 2018; 122:1191-1199.
The advent of high-throughput epigenome mapping technologies has ushered in a new era of multiomics where powerful tools can now delineate and record different layers of genomic output. Integrating various components of the epigenome from these multiomics measurements allows the interrogation of cellular heterogeneity in addition to the discovery of molecular connectivity maps between the genome and its functional output. Mapping of chromatin accessibility dynamics and higher-order chromatin structure has enabled new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease. We provide a perspective on the progress of the epigenomics field and applications and anticipate an even greater revolution in our understanding of the human epigenome for years to come.
Wang, K.C., Chang, H.Y. (2017). Transcription coactivator and IncRNA duet evoke Hox genes. PLoS Genet 13(6): e1006797. 2017 Jun 29;13(6):e1006797. doi: 10.1371/journal.pgen.1006797.
Mammalian genomes are pervasively transcribed [1, 2] to produce thousands of long noncoding RNAs (lncRNAs) [3±5], transcripts that are more than 200 nucleotides in length that do not code for proteins. Although only a handful of functional lncRNAs have been well characterized to date, recent work suggests that some lncRNAs have crucial roles in the control of gene expression during both normal development and disease, through multiple mechanisms
[6, 7]. As new lncRNAs are being discovered at a rapid pace, their molecular mechanisms are continuing to be enriched and diversified. For example, a few lncRNAs have been shown to
affect expression of nearby genes through recruitment of protein regulatory complexes [8±10], while it is suggested that others function akin to enhancers and local regulators in cis [11, 12].
In fact, such local gene-regulatory mechanisms have been invoked to explain the observation that lncRNA expression is often correlated with the expression of nearby genesÐthe so-called
"guilt by association".
Morgan, Stefanie L, Mariano, Natasha C., Bermudez, Abel, Arruda, Nicole L., Wu, Fangting, Luo, Uynhai, Shankar, Gautam, Jia, Lin, Chen, Huiling, Hu, Ji-Fan, Hoffman, Andrew R., Huang, Chiao-Chain, Pitteri, Sharon J., & Wang, K.C., (2017). Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nature Communications; 8:15993. doi: 10.1038.
Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.
Luo, Y, Morgan, Stefanie L, & Wang, KC. PICSAR: Long Noncoding RNA in Cutaneous Squamous Cell Carcinoma. Journal of Investigative Dermatology; 2016 Aug;136(8):1541-2.
It is increasingly evident that long noncoding RNAs may play the roles of both oncogenes and tumor suppressors during cancer development. A new study from Piipponen et al. provides evidence that a long noncoding RNA, PICSAR, promotes cutaneous squamous cell carcinoma development through activation of extracellular signal-regulated kinase signaling. Because specific inhibition of PICSAR suppresses tumor growth, this long noncoding RNA may serve as a useful diagnostic marker and therapeutic target for cutaneous squamous cell carcinoma.
Morgan, Stefanie L, Seggio, JA, Nascimento, NF, Huh, DD, Hicks, JA, Sharp, KA, Axelrod, JD, & Wang, KC, (2016). The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific. PLoS One; 2016 Aug 3;11(8):e0159456. doi: 10.1371/journal.pone.0159456. eCollection 2016.
The role for royal jelly (RJ) in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species.
Luan, A, Paik, KJ, Li, J, Zielins, ER, Atashroo, DA, Spencley, A, Momeni, A, Longaker, MT, Wang, KC, & Wan, DC. RNA Sequencing for Identification of Differentially Expressed Noncoding Transcripts during Adipogenic Differentiation of Adipose-Derived Stromal Cells. Plastic and Reconstructive Surgery, 2015 Oct; 136(4):752-763.
Adipose-derived stromal cells represent a relatively abundant source of multipotent cells, with many potential applications in regenerative medicine. The present study sought to demonstrate the use of RNA sequencing in identifying differentially expressed transcripts, particularly long noncoding RNAs, associated with adipogenic differentiation to gain a clearer picture of the mechanisms responsible for directing adipose-derived stromal cell fate toward the adipogenic lineage.
Wan, DC, Wang, KC. Long noncoding RNA: significance and potential in skin biology. Cold Springs Harbor Perspectives in Medicine, 2014 May 1;4(5). pii: a015404.
Over the past few years, advances in genome analyses have identified an emerging class of noncoding RNAs that play critical roles in the regulation of gene expression and epigenetic reprogramming. Given their transcriptional pervasiveness, the potential for these intriguing macromolecules to integrate a myriad of external cellular cues with nuclear responses has become increasingly apparent. Recent studies have implicated noncoding RNAs in epidermal development and keratinocyte differentiation, but the complexity of multilevel regulation of transcriptional programs involved in these processes remains ill defined. In this review, we discuss the relevance of noncoding RNA in normal skin development, their involvement in cutaneous malignancies, and their role in the regulation of adult stem-cell maintenance in stratified epithelial tissues. Furthermore, we provide additional examples highlighting the ubiquity of noncoding RNAs in diverse human diseases.
Yang, YW, Flynn, RA, Chen, Y, Qu, K, Wan, B, Wang, KC, Lei, M, Chang, HY. Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife, 2014 Feb 12;3:e02046.
The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates.
Wang, K. C., & Chang, H. Y. (2011). Molecular Mechanisms
of Long Noncoding RNAs. Molecular Cell, 43(6),
Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute—as signals, decoys, guides, and scaffolds.
For each archetype, examples from several disparate biological contexts illustrate the commonality of the molecular mechanisms,and these mechanistic views provide useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular mechanisms of lncRNAs are likely to be enriched and diversified.
Chen, Y., Wan, B., Wang, K.C., Cao, F., Yang, Y., Protacio, A., Dou, Y., Chang, H.Y., Lei, M. (2011). Crystal structure of the N-terminal region of human Ash2L shows a winged-
helix motif involved in DNA binding. EMBO reports, 12(8), 797-803.
Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2LWH (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.
Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., ... Chang, H.H. (2011). A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature, 472 (7341), 120-126.
The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.
Image: Early embryonic development of Gallus gallus domesticus
Image: Early embryonic development of Gallus gallus domesticus
Wang, K.C., Kim, J.A., Sivasankaran, R., Segal, R., He, Z. (2002). P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature, 420(6911), 74-8.
In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66), oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG), exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75-NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR-p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.
Wang K.C., Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z. (2002).Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 417(6892):941-4.
The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS). In addition to myelin-associated glycoprotein (MAG) and Nogo-A, available evidence suggests the existence of additional inhibitors in CNS myelin. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR) as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.