ABSTRACT
Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.
Subject(s)
Gene Expression Regulation, Fungal , Histones/metabolism , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Silencing , Histones/genetics , Humans , Molecular Sequence Data , Multiprotein Complexes , Nuclear Proteins/genetics , Open Reading Frames , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , TATA-Box Binding Protein/genetics , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
The virus/host interactions during the acute phase of human immunodeficiency virus (HIV) infection help determine the course of disease. During this time period, virus enters the brain. Here, we report clusters of genes whose transcripts are significantly upregulated in the frontal lobe of the brain during acute simian immunodeficiency virus (SIV) infection of rhesus monkeys. Many of these genes are involved in interferon (IFN) and/or interleukin (IL)-6 pathways. Although neither IFNalpha nor IFNgamma are elevated in the brain, IL6 is increased. Both IFNalpha and IL6 are elevated in plasma during this acute phase. The upregulation of STAT1, verified by immunohistochemical staining, can be due to both central nervous system (CNS) (SIV and IL6) and peripheral (IFNalpha and IL6) causes, and can itself drive the expression of many of these genes. Examination of the levels of expression of the upregulated genes in the post-acute and long-term phases of infection, as well as in SIV encephalitis, reveals increased expression throughout SIV infection, which may serve to protect the brain, but can have untoward long-term consequences.
Subject(s)
Brain/virology , Interferons/metabolism , Interleukin-6/metabolism , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Immunodeficiency Virus/physiology , Analysis of Variance , Animals , Brain/metabolism , DNA-Binding Proteins/metabolism , Disease Progression , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunohistochemistry/methods , Interferons/genetics , Interleukin-6/genetics , Macaca mulatta , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , STAT1 Transcription Factor , Simian Acquired Immunodeficiency Syndrome/genetics , Simian Acquired Immunodeficiency Syndrome/virology , Time Factors , Trans-Activators/metabolism , Up-Regulation , Viral Load/methodsABSTRACT
Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II. Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process. Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation. First, we identified Chd1 in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation. Secondly, we show through co-immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3. Thirdly, we demonstrate that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II. Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin. Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation.
