R. Nicholas Laribee

Assistant Professor

Office: 264 Cancer Research Building
Tel: (901) 448-2609
rlaribee@uthsc.edu

Education

  • Ph.D., Indiana University School of Medicine, Bloomington, IN, Microbiology and Immunology
  • B.S., Indiana University-Bloomington, Bloomington, IN, Biology

Research Interest/Specialty

 Epigenetics, gene regulation and signal transduction

Research Keywords

 Epigenetics; Chromatin; Gene transcription; nutrient signaling; mTORC1

Research Description

 While DNA is the purveyor of genetic information, the human genome is not composed solely of DNA.  Instead, it is packaged with histone proteins to form individual nucleosomes which is the basic unit of chromatin found in all eukaryotic cells.  Individual nucleosome particles, and their internucleosomal interactions, form compacted three-dimensional structures that allow genomes to be packaged into nuclei.  This genomic compaction into chromatin is essential for compressing and storing the genetic material in nuclei; however, the physical state of chromatin is resistant to biochemical reactions requiring access to the underlying DNA template.  Therefore, eukaryotic cells ranging from yeast to man have evolved highly conserved biochemical mechanisms that control chromatin structure and function to regulate these DNA-templated processes.  These mechanisms include histone post-translational modifications, DNA methylation, ATP-dependent chromatin remodeling, histone chaperone-dependent nucleosome regulation and the incorporation of histone variants.

 The modulation of chromatin structure is typically regarded as “epigenetic” regulation due to the potential for these altered chromatin states to be propagated through multiple cell divisions and, in some cases, through multiple organismal generations.  Epigenetic regulation can be significantly influenced by environmental factors, including such things as diet and stress.  Similarly, changes in cellular metabolism also contribute to the regulation of chromatin as many of the cofactors needed in enzymatic reactions necessary for DNA and histone modifications are generated as a consequence of cellular metabolism.   As such, the dysregulation of epigenetic processes through many different mechanisms can either contribute to, or cause, a number of human diseases including cancer, cardiovascular disease, and many neurodevelopmental disorders.  Furthermore, the process of aging alters epigenetic pathways that impair the maintenance of the chromatin fiber.  Aging-dependent chromatin alterations ultimately lead to the physical degradation of chromatin and can be a contributing factor in the enhanced susceptibility to diseases such as cancer that occurs in older individuals.  Therefore, determining how epigenetic processes function is essential for understanding how the environment affects human health and disease.  Our laboratory uses budding yeast as a genetic and biochemical model to understand the molecular mechanisms by which the environment affects chromatin regulation.  Yeast provide unparalleled genetic and biochemical approaches for teasing apart the complex nature of these processes yet the information gained from these studies is highly applicable to the corresponding pathways in human cells as these processes are highly conserved.  For example, human replication-independent histone H3, one of the core histone proteins contained in the nucleosome, differs from yeast histone H3 by only three amino acids!   Our laboratory has several on-going projects to dissect these mechanisms which are outlined briefly below.

Role of the Ccr4-Not complex in cell growth and proliferation.  The Ccr4-Not complex is a nine subunit protein complex conserved from yeast to man.  This complex functions in every step of the gene expression process, including the regulation of chromatin structure, to control cell growth, proliferation and development.  In mammals, this complex is required for the maintenance of embryonic stem cells and is essential for embryogenesis.  Our laboratory is currently defining how nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway utilizes this multiprotein complex to regulate gene transcription necessary for cell growth and proliferation.  We have several ongoing projects related to the function of this complex in eukaryotic cells as described below.

 1.  Ccr4-Not is a novel transcriptional co-regulatory for RNA polymerase I (Pol I).  Ccr4-Not is a known RNA polymerase II (Pol II) transcriptional co-regulator that can both positively and negatively regulate Pol II transcription initiation in poorly understood ways.  Recent studies have also defined a positive role for this complex in promoting Pol II transcription elongation through mechanisms that are largely not defined.  Our laboratory has recently identified a novel role for Ccr4-Not as a Pol I transcriptional co-regulator (manuscript submitted, Fall 2014).  Specifically, we have found that loss of the Ccr4-Not specific mRNA deadenylase subunit Ccr4 or the ubiquitin ligase Not4 subunit increases Pol I binding to ribosomal DNA (rDNA) which leads to increased Pol I transcription and increased ribosomal RNA (rRNA) synthesis.  Surprisingly, the increased Pol I transcription only occurs under nutrient rich conditions and is a consequence of elevated Pol I interactions with the essential transcription factor, Rrn3, which result in the transcription initiation competent form of the Pol I holoenzyme.  Surprisingly, the Rrn3-Pol I complex is resistant to disruption by decreased mTORC1 signaling thus implicating the Ccr4-Not complex as essential for bridging mTORC1 signaling with Pol I transcriptional regulation.  Ccr4-Not additionally functions to promote Pol I transcription elongation and/or termination.  Non-essential subunits of the Pol I holoenzyme modulate Ccr4-Not association with Pol I and Pol I elongation mutants combined with Ccr4-Not mutants result in synergistic Pol I transcriptional and rRNA synthesis defects.  Surprisingly, while individual Ccr4-Not mutants are hypersensitive to pharmacological mTORC1 inhibitors, a Ccr4-Not mutant paired with a Pol I elongation mutant results in cells that are considerably less sensitive to mTORC1 inhibition.  These novel data suggest that the majority of Ccr4-Not specific sensitivity to mTORC1 inhibition is due to deregulation of Pol I transcription.  Our laboratory is continuing to delineate the mechanisms by which Ccr4-Not functions in Pol I transcriptional regulation and we will ultimately expand these studies into human cell line models to determine how conserved these pathways and whether they are disrupted in diseases such as cancer.  Since mTORC1 inhibitors such as rapamycin and rapamycin-based molecules (rapalogs) are in clinical testing as anti-cancer agents, these results suggest the possibility that targeting Ccr4-Not function could be a beneficial means of enhancing the anti-cancer activity of these mTORC1 inhibitors.

 2.  The biological functions of the Not4 ubiquitin ligase.  Ccr4-Not contains the Not4 ubiquitin ligase which is rather unique within the budding yeast proteome as it is the only RING domain containing ubiquitin ligase that also has a putative RNA recognition motif (RRM) domain.  Whether this RRM domain binds RNA and, if so, how RNA binding might modulate the ubiquitin ligase function of this protein remains unknown.  Furthermore, this ubiquitin ligase only has a few identified substrates and none of these substrates can individually explain the severe phenotypes that occur upon loss of Not4.  Our laboratory is currently exploring both the role of this domain in Not4 function as well as attempting to identify novel substrates for this ligase in order to understand its critical role in cell growth and proliferation.

 3.  Role of Ccr4-Not in control of chromatin structure.  Ccr4-Not is known to interact with multiple chromatin regulators, including a histone demethylase and a histone acetyltransferase.  Our laboratory has unpublished results demonstrating that Ccr4-Not also interacts with an ATP-dependent chromatin remodeling complex that is necessary for nucleosome remodeling and the regulation of multiple DNA-templated processes.  We are currently exploring the role of Ccr4-Not in these aspects of chromatin regulation and how the contribute to gene transcription mediated by both Pol II and Pol I complexes.

  Mechanisms of environmentally-induced epigenetic regulation.  The environment has a profound effect on human development and disease susceptibility.  While genome alterations due to DNA mutation are well-defined mechanisms by which the environment affects human health, a number of complex diseases cannot be explained through this simple mechanism.  Instead, many diseases, including a large number of cancers, cardiovascular disease and neurodevelopmental disorders are thought to be caused by epigenetic dysfunction.  Because epigenetic dysregulation affects organismal phenotype without altering the genetic material, these processes are potentially reversible which makes them ideal targets for drug development.  Our laboratory is currently delineating how mTORC1, which is regulated by environmental nutrients, transmits this information to the chromatin machinery necessary for controlling cell growth, proliferation and development.  mTORC1 dysregulation occurs in almost all human cancers and dysfunctional control of this pathway within the developing nervous system is implicated in several neurodevelopmental disorders as well.  Therefore, defining how mTORC1 regulates epigenetic processes will define how the environment can affect phenotype as well as identify novel areas for pharmacological intervention to treat multiple human diseases.

1.  Role of mTORC1-regulated histone post-translational modifications in gene transcription.  Our laboratory recently published a genetic screen of a library of histone H3 and histone H4 mutants to identify those individual histone residues exhibiting genetic interactions with the mTORC1 signaling pathway (Chen et al, Epigenetics & Chromatin, 2013).  We identified several sites on both histone H3 and H4 that are functionally linked to mTORC1, including the identification of an absolutely essential and novel role for histone H3 lysine 37 (H3K37) in mTORC1-dependent cell growth control.  Mutation of H3K37 to alanine (H3K37A) causes cell death by necrosis when mTORC1 signaling is decreased which is a surprising result since pharmacological mTORC1 inhibition typically results in cytostatic, but not cytoxic, effects.  We have determined that one role for H3K37 in the mTORC1 signaling pathway may be to anchor high mobility group (HMG) proteins to chromatin and that specific disruption of HMG chromatin binding induces cell death.  Our laboratory is exploring the concept that mTORC1 signaling actively regulates multiple histone post-translational modifications that promote HMG chromatin binding and suppression of necrosis as well as those modifications that function in mTORC1-dependent transcriptional regulation.  Our long term goals with this project are to both understand how mTORC1 regulates histone modifications to affect chromatin structure as well as identify chromatin pathways to target pharmacologically to enhance the anti-cancer activities of mTORC1 inhibitors.

 2.  mTORC1-dependent chromatin regulation via histone chaperones.  Our laboratory recently published that the histone H3 lysine 56 acetylation (H3K56ac) pathway, regulated by the histone chaperone Asf1 and acetyltransferase Rtt109, are critical for normal mTORC1-dependent cell growth and proliferation.  We determined that this pathway functions in the mTORC1 signaling pathway in part by regulating Pol I-dependent rRNA biogenesis.  Specifically, H3K56ac is required to create a chromatin architecture on the rDNA necessary for binding of key Pol I transcriptional regulators, specifically the HMG factor Hmo1 and the small subunit (SSU) processome complex.  Perturbing the H3K56ac pathway results in profound sensitivity to mTORC1 inhibitors, may involve sirtuin histone deacetylases that target H3K56ac, and results in co-transcriptional rRNA processing defects due to decreased rDNA binding by the SSU processome.  We are currently exploring in greater detail the role of the H3K56ac pathway in rDNA transcription and rRNA biogenesis while also expanding how the Asf1 histone chaperone contributes to the structure and function of nucleoli.  Our long term goals of this project will be to expand these studies into human cell lines since one of the two human Asf1 orthologs, Asf1a, localizes to nucleoli yet the functions of this histone chaperone in nucleolar regulation is unknown.  Because nucleoli are exquisitely sensitive to environmental stress, these studies in human cells could elucidate how cells balance cell growth and proliferation with environmental stress responses through the regulation of nucleolar-specific chromatin states.

 Long-term vision statement.  Our laboratory currently utilizes budding yeast to address mechanistic questions regarding the control of chromatin structure and function via environmentally-regulated signaling pathways such as mTORC1.  The utilization of a simple genetic and biochemical model system is essential to probe these scientific questions as they are both complex in nature and quite difficult to experimentally address.  However, our long term goals are to expand these studies into human cell lines to understand how these processes function in normal gene transcription as well as to understand how dysregulation of these processes promote development of diseases such as cancer.  Each pathway and/or process we study in yeast is highly conserved in human cells so the information generated by our yeast model will ultimately inform our experimental studies in human cells.                                                                                                                              

Publications

  1. Workman, JJ, Chen, H, Laribee, RN. Environmental signaling through the mechanistic target of rapamycin complex 1: mTORC1 goes nuclear. Cell Cycle, 13 (5), 714-25, 2014.
  2. Chen, H, Workman, JJ, Tenga, A, Laribee, RN. Target of rapamycin signaling regulates high mobility group protein association to chromatin, which functions to suppress necrotic cell death. Epigenetics Chromatin, 6 (1), 29, 2013.
  3. Chen, H, Fan, M, Pfeffer, LM, Laribee, RN. The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis. Nucleic Acids Res, 40 (14), 6534-46, 2012.
  4. Kerr, SC, Azzouz, N, Fuchs, SM, Collart, MA, Strahl, BD, Corbett, AH, Laribee, RN. The Ccr4-Not complex interacts with the mRNA export machinery. PLoS One, 6 (3), e18302, 2010.
  5. Fuchs, SM, Laribee, RN, Strahl, BD. Protein modifications in transcription elongation. Biochim Biophys Acta, 1789 (1), 26-36, 2009.
  6. Laribee, RN, Fuchs, SM, Strahl, BD. H2B ubiquitylation in transcriptional control: a FACT-finding mission. Genes Dev, 21 (7), 737-43, 2007.
  7. Laribee, RN, Shibata, Y, Mersman, DP, Collins, SR, Kemmeren, P, Roguev, A, Weissman, JS, Briggs, SD, Krogan, NJ, Strahl, BD. CCR4/NOT complex associates with the proteasome and regulates histone methylation. Proc Natl Acad Sci U S A, 104 (14), 5836-41, 2007.
  8. Xiao, T, Shibata, Y, Rao, B, Laribee, RN, O'Rourke, R, Buck, MJ, Greenblatt, JF, Krogan, NJ, Lieb, JD, Strahl, BD. The RNA polymerase II kinase Ctk1 regulates positioning of a 5' histone methylation boundary along genes. Mol Cell Biol, 27 (2), 721-31, 2007.
  9. Laribee, RN, Klemsz, MJ. Histone H4 HDAC activity is necessary for expression of the PU.1 gene. Biochim Biophys Acta, 1730 (3), 226-34, 2005.
  10. Laribee, RN, Krogan, NJ, Xiao, T, Shibata, Y, Hughes, TR, Greenblatt, JF, Strahl, BD. BUR kinase selectively regulates H3 K4 trimethylation and H2B ubiquitylation through recruitment of the PAF elongation complex. Curr Biol, 15 (16), 1487-93, 2005.
  11. Chang, HC, Zhang, S, Thieu, VT, Slee, RB, Bruns, HA, Laribee, RN, Klemsz, MJ, Kaplan, MH. PU.1 expression delineates heterogeneity in primary Th2 cells. Immunity, 22 (6), 693-703, 2005.
  12. Zhang, B, Laribee, RN, Klemsz, MJ, Roman, A. Human papillomavirus type 16 E7 protein increases acetylation of histone H3 in human foreskin keratinocytes. Virology, 329 (1), 189-98, 2004.
  13. Laribee, RN, Klemsz, MJ. Loss of PU.1 expression following inhibition of histone deacetylases. J Immunol, 167 (9), 5160-6, 2001.
  14. Liu, Y, Jones, M, Hingtgen, CM, Bu, G, Laribee, N, Tanzi, RE, Moir, RD, Nath, A, He, JJ. Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med, 6 (12), 1380-7, 2000.