The Gregory Lab
Dr. Richard Gregory is a Principal Investigator at Children's Hospital Boston and an Assistant Professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School.
Dr. Gregory received a Ph.D. from Cambridge University, UK in 2001 studying genomic imprinting at the Babraham Institute. His postdoctoral work was performed at the Fox Chase Cancer Center and the Wistar Institute, Philadelphia. Dr. Gregory’s postdoctoral research focused on mechanisms of miRNA biogenesis and function, and was supported by a Jane Coffin Childs Research Fellowship. He joined Children’s Hospital and Harvard Medical School as an Assistant Professor in 2006. He was named a 2008 Pew Scholar.
Gregory Lab Research Summary:
The laboratory research focus is to understand how small regulatory RNAs are generated, how they exert their gene regulatory function, how they play a role in the self-renewal and pluripotency of embryonic stem cells (ES cells), and how they are relevant to human disease. RNA interference describes the recently identified phenomenon whereby small non-coding RNAs can silence gene expression. It is emerging that cells possess a wide repertoire of tiny regulatory RNAs that are critical for a variety of biological pathways and can repress genes via numerous mechanisms. For posttranscriptional gene silencing, microRNAs, and small inhibitory RNAs, function as guide molecules inducing messenger RNA (mRNA) degradation or translational repression. More than 700 human microRNAs have been identified. Each of these is thought to be capable of regulating the expression of hundreds or even thousands of target mRNAs. Therefore microRNA-mediated repression is considered to be an important and pervasive level of gene regulation and is essential for normal development. Indeed, recent predictions indicate that most human genes may be targeted by microRNAs. Moreover, altered microRNA expression is associated with human diseases including cancer. Mature ~22 microRNAs are generated by a two-step processing pathway that begins in the cell nucleus where long primary microRNA transcripts are specifically cleaved by the Microprocessor complex to yield ~60-70nucleotide precursor microRNAs. The Microprocessor is a ~600 kDa complex comprising Drosha and its essential cofactor, the double-stranded RNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8). Genetic studies in mice have demonstrated that DGCR8 is essential for normal development and for the rapid proliferation and differentiation of ES cells. Moreover, in vitro reconstitution experiments have established that DGCR8 is essential for recruiting Drosha catalytic activity for primiry microRNA processing. The precursor microRNAs are exported into the cytoplasm where they are processed by Dicer to ~22nucleotide microRNAs. One strand of the mature microRNA is incorporated into the RNA-induced silencing complex that recognizes target mRNAs based on sequence complementarity between the guide microRNA and the mRNA transcript and results in either Ago2-mediated endonucleolytic mRNA cleavage or translational repression.
Control of microRNA Processing and Function in Stem Cells and Cancer:
Perturbation of the microRNA biogenesis pathway impacts the differentiation potential of mouse embryonic stem cells. Moreover, a selective block in microRNA processing has been observed in ES cells, embryonal carcinoma cells (EC cells), and primary tumors. These data support the notion that microRNAs are important for cell differentiation and that unidentified mechanisms exist in stem cells and certain cancers to promote rapid cell proliferation and to prevent microRNA-mediated cell differentiation. We identified the developmentally regulated RNA-binding protein Lin28 as the first negative regulator of microRNAprocessing. It had been reported let-7 biogenesis in ES cells and cancer cells is regulated post-transcriptionally, the mechanism for which had remained elusive. Using pre-let-7 RNA affinity purification we identified the developmentally regulated RNA-binding protein Lin28, and demonstrated that Lin28 is directly responsible for the blockade of let-7 microRNA processing in ES cells and cancer cells. Lin28 selectively regulates the let-7 family of microRNAs, known to be key players in cancer, and was also recently shown to help reprogram human fibroblast cells to induced pluripotent stem cells (iPS cells). More recently, we identified a novel enzyme, the 3′ terminal uridylyl transferase (TUTase), Zcchc11, responsible for Lin28-mediated pre-let-7 uridylation and blockade of let-7 processing. We demonstrated that the activity of Zcchc11 is UTP-dependent, selective for the let-7 family, and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 microRNAs and repression of let-7 target reporter genes in embryonic cells. Our results establish a novel role for Zcchc11-catalyzed pre-let-7 uridylation in the control of microRNA biogenesis, and identify Zcchc11 as a possible therapeutic target in cancers in which Lin28 or Lin28B restrict the expression of the tumor suppressor let-7 microRNAs.
Since microRNAs regulate cancer cell differentiation, proliferation, survival, and metastasis, manipulating microRNA expression could provide a powerful therapeutic strategy to interfere with cancer progression. Significantly, many different primary human tumors express a globally lower level of microRNAs than the corresponding normal tissue, likely the result of a defect in microRNA processing. Other studies have observed altered expression of DGCR8 and/or Drosha, components of the Microprocessor complex, in numerous different human cancers, and microRNA deficiency through experimental depletion of Drosha or DGCR8 promotes tumorigenesis in mice. The Microprocessor complex should therefore be considered an important future target for cancer therapy. We aim to develop a novel cell-based reporter system suitable for the high-throughput screening and identify small molecules that specifically target the Microprocessor to control microRNA biogenesis. The development of this assay will enable a chemical genetics approach to discover how the Microprocessor is regulated, and may lead to the development of novel chemotherapeutic strategies to restore normal microRNA levels in tumors.
Members of the Trim-NHL protein family were found to positively regulate microRNA function in C.elegans and mice. Certain Trim-NHL genes are essential for normal developmental timing in C.elegans, regulating the proliferation of Drosophila ovarian stem cells, as well as controlling neural progenitor cell fate in both Drosophila and mice. We have recently identified an uncharacterized, stem cell-specific, Trim-NHL family member–Trim71–as a novel Ago2-interacting protein that promotes microRNA-mediated gene repression in ES cells. Moreover, our preliminary data indicate that Trim71 controls the cell-cycle to promote rapid ES cell proliferation. Our ongoing work aims to understand the molecular mechanism by which Trim71 functions in ES cells.
Piwi-interacting RNAs: A New Class of Small Non-Coding RNA Molecules:
Members of the Argonaute family of proteins play a central role in RNA silencing pathways that regulate transcription, heterochromatin, genome integrity, and mRNA stability. Argonaute proteins are divided into two subfamilies: Ago and Piwi. Ago members are ubiquitously expressed and are required for post-transcriptional gene silencing. MicroRNAs and short-interfering RNAs of 22 nucleotides in length interact directly with Ago proteins and guide these complexes to target mRNAs of complementary sequence. The expression of the members of the Piwi sub-family is restricted to stem cells and germ cells. Recently, a novel class of abundant small RNAs known as Piwi-interacting RNAs (piRNAs) that range in size from 26-31 nucleotides was identified in mammalian cells. This exciting discovery raises several fundamental questions of significant biological and biomedical relevance: What is the function of piRNAs? How are piRNAs generated? What is the identity of Piwi-interacting proteins? Although piRNA biogenesis and function is not yet well understood, Piwi proteins are highly expressed during mouse spermatogenesis and are important for gamete formation. We hypothesize that Piwi proteins and associated small RNAs have additional stem cell functions in mammals. We therefore aim to biochemically define the composition of Piwi-containing ribonucleoprotein complexes in human and mouse stem cells, towards our goal of understanding the mechanism of piRNA biogenesis, the function of the Piwi-complexes, and their requirement for stem cell biology.
GREGORY LABORATORY STAFF
Hao-Ming Chang, Postdoctoral Fellow
John Hagan, Postdoctoral Fellow
Robinson Triboulet, Postdoctoral Fellow
Natalia Martinez, Postdoctoral Fellow
Elena Piskounova, Graduate student
James Thornton, Graduate Student
Robert LaPierre, Research Assistant
Hagan J.P.*, Piskounova E*, Gregory R.I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat Struct Mol Biol. 2009 16, 1021-5. (*equal contribution).
Triboulet R., Chang H., LaPierre R. J., and Gregory R. I., Posttranscriptional control of DGCR8 expression by the Microprocessor. RNA 2009 15, 1005-11.
Piskounova E., Viswanathan S. R., Janas, M., Lapierre R. J., Daley G. Q., Sliz P., and Gregory R. I. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. Journal of Biological Chemistry 2008; 283: 21310-4.
Viswanathan S. R., Daley G. Q., and Gregory R. I. Selective Blockade of MicroRNA Processing by Lin28. Science 2008; 320, 97-100.
Li Q., and Gregory R. I. microRNA Regulation of Stem Cell Fate. Cell Stem Cell 2008; 2, 195-6.
Gregory R. I., and Shiekhattar, R. MicroRNA biogenesis: Isolation and characterization of the Microprocessor complex. “MicroRNA Protocols”, Methods in Molecular Biology 2006; 342, 33-47.
Gregory R. I., Chendrimada T., Cooch N., and Shiekhattar R. Human RISC couples microRNA maturation and posttranscriptional gene silencing. Cell 2005;123:631-40.
Gregory R. I., and Shiekhattar, R. MicroRNA biogenesis and cancer. Cancer Research 2005; 65:3509-12.
Chendrimada T.*, Gregory R. I.*, Kumaraswamy E.*, Norman J., Cooch N., Nishikura K., and Shiekhattar R. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005;436:740-4. (*equal contribution).
Gregory R. I.*, Yan K.*, Amuthan G., Chendrimada T., Doratotaj B., Cooch N. and Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs. Nature 2004;432:235-240. (*equal contribution).