The architecture of mammalian ribosomal protein promoters

BACKGROUND: Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp) promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. RESULTS: A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y)(2)C(+1)TY(T)(2)(Y)(3). At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. CONCLUSIONS: This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes

Studies on mammalian ribosomal protein S7

http://www.ester.ee/record=b1163838~S1*es

Mammalian ribosomal protein S3a genes and intron-encoded small nucleolar RNAs U73 and U82

http://www.ester.ee/record=b4330778~S58*es

Intron Retention Of JARID2 Regulates Gene Expression During Temperature-Dependent Sex Determination

Temperature-dependent sex determination (TSD) is the developmental process in which cell fate decisions in the bipotential gonads are influenced by temperature and drive the gonad toward one of two distinct organs, an ovary or testis. In species with TSD, temperature establishes the gene expression patterns required for cell fate decisions and gonad differentiation. However, the precise mechanism that temperature regulates gene expression is unknown. Here we have shown that Jarid2, which encodes a Polycomb Repressive Complex 2 (PRC2) accessory protein, exhibits temperature-dependent intron retention that results in three distinct transcripts encoding distinct proteins with functional similarities and differences in TSD. We found in a long-read sequencing assay that the transcripts encoding these proteins exhibited different expression patterns in response to a temperature shift from a male- to female-producing temperature. We also found that overexpression of these proteins in a bipotential gonad cell culture model yielded both similar and differing effects on gene expression, suggesting that they may play unique roles in regulating gene expression in TSD. Our results demonstrate that JARID2 is alternately spliced and the resulting isoforms are likely involved in establishing the transcriptional profiles required for cell fate decisions in TSD. We anticipate that this study will provide a foundation from which to further probe the function of JARID2 in the thermal response during TSD

Molecular Mechanisms of Nuclear Hormone Receptor Transcriptional Synergy and Autoinduction.

Thyroid hormone (TH) and glucocorticoids (GC) play critical roles in the development and function of the central nervous system (CNS) by binding to their cognate nuclear hormone receptors (NRs), which function as ligand-activated transcription factors. In my dissertation, I studied the TH and GC-mediated regulation of Krüppel like factor 9 (Klf9/Basic Transcription Element Binding Protein 1; Bteb1), a member of the Sp1/KLF family of zinc finger transcription factors that bind GC rich genomic sequences, and plays an important role in neuronal development and plasticity. In prior work Klf9 was found to be a direct TH receptor (TR) target gene. I showed that Klf9 is also directly targeted by the GC receptor (GR), and that TH and GCs cause synergistic induction of Klf9. This synergistic regulation is phylogenetically ancient, and was likely present in the earliest tetrapods and has been evolutionarily conserved from frogs to mammals. I identified a genomic region in the 5’ flanking region of the Klf9 gene (the ‘Klf9 synergy module’) that contains TR/GR binding sites and confers synergistic gene regulation by TH and GC. The synergistic effect of TH and GC on Klf9 can be explained by a TR-dependent increase in the recruitment of the GR and enhanced association of stalled RNA polymerase II at the Klf9 synergy module, and an interaction between the synergy module and the Klf9 promoter by chromosomal looping. I also conducted a genome-wide microarray analysis, the first study to identify transcriptional targets that are coordinately regulated by TH and GC in the brain. Lastly, I demonstrated a role for KLF9 as an accessory transcription factor to support TH-dependent expression of TRβ (autoinduction), a process that is necessary for the progression of amphibian metamorphosis, and normal mammalian brain development. Given the synergistic regulation of the Klf9 gene by TR and GR, and its role in TRβ autoinduction, my findings support that KLF9 is an important intermediate that functions to integrate TH and GC by enhancing the cell sensitivity to hormonal signals. Taken together, my thesis broadens our understanding of the molecular mechanisms of NR cooperativity and autoinduction, with important implications for animal development.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93977/1/piab_1.pd

RNA polymerase II CTD Evolutionary Diversity and Associated Protein Identification in Green and Red Algae

In model eukaryotes, the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II is composed of tandemly repeated heptads with the consensus sequence YSPTSPS. Both the core motif and tandem structure generally are highly conserved across many model taxa, including animals, yeasts and higher plants. Broader investigations quickly revealed that the CTDs of many organisms deviate substantially from this canonical structure; however, limited sampling made it difficult to determine whether disordered sequences represent the CTD's ancestral state, or reflect degeneration from an originally repetitive structure. Therefore, I undertook the broadest investigation to date of the evolution of the RNAP II CTD across eukaryotic diversity. The results indicate that a tandem heptad CTD-structure existed in the ancestors of each major taxon, and that degeneration and reinvention of this ordered structure are common features of CTD evolution. Lineage specific modifications of heptads that were amplified initially appear to be associated with greater developmental complexity in multicellular taxa. The pattern has been taken to an extreme in both fungi and red algae. Overall, loss and reinvention of varied repeats have punctuated CTD evolution, occurring independently and sometimes repeatedly in various groups.    Although present in simple, ancestral red algae, CTD tandem repeats have undergone extensive modifications and degeneration during the evolutionary transition to developmentally complex rhodophytes. In contrast, CTD repeats are conserved in both green algae and their more complex land plant relatives. Understanding the mechanistic differences that underlie these variant patterns of CTD evolution requires knowledge of CTD-associated proteins in these two lineages. To provide an initial baseline comparison, potential phospho-CTD associated proteins (PCAPs) were bound to artificially synthesized and phosphorylated CTD repeats from the unicellular green alga Chlamydomonas reinhardtii and red alga Cyanidioschyzon merolae. My results indicate that red and green algae share a number of PCAPs, including kinases and proteins involved in mRNA export. There also are important taxon-specific differences, including mRNA splicing-related PCAPs recovered from Chlamydomonas but not Cyanidioschyzon, consistent with the relative intron densities in green and red algae. This work also offers the first experimental indication that different proteins bind the two types of repeats in Cyanidioschyzon, suggesting a division of function between the proximal and distal CTD, similar to patterns identified in more developmentally complex model organisms.  Ph.D

Differentially expressed genes in aortic cells from atherosclerosis-resistant and atherosclerosis-susceptible pigeons

Representational Difference Analysis (RDA) was used to identify genes that were differentially expressed between White Carneau (WC) and Show Racer (SR) pigeon aortic smooth muscle cells. The gene(s) responsible for atherosclerotic resistance in cultured SR smooth muscle cells (SMC) were hypothesized to be silent or down regulated in the WC. In the reciprocal experiments, it was hypothesized that the gene(s) contributing to the spontaneous atherosclerotic phenotype in cultured WC SMC would not be expressed in the SR. Total RNA was extracted from primary cultured cells of each breed, converted to cDNA, and compared in four reciprocal RDA experiments. Seventy-four transcripts were identified exclusively in the WC cells, and 63 were unique to the SR. Genes representing several biochemical pathways were distinctly different between aortic cells from susceptible (WC) and resistant (SR) pigeons. The most striking genetic differences were observed in energy metabolism and smooth muscle contractility. The WC cells derived their energy from glycolysis, while the SR cells utilized oxidative phosphorylation to produce energy. Myosin light chain kinase and alpha actin were exclusively expressed by the SR SMC, whereas beta actin and collagen were dominant in the WC. Because of the compressed in vitro time frame compared with in vivo development, it was not obvious whether insufficient ATP synthesis is preventing the WC aortic cells from performing their contractile function or if the lack of functional contractile elements in the WC causes the mitochondrial ATP synthesis to down regulate. Either way, energy production was successfully coupled to muscle contraction in the SR, but not in the WC. This difference was observed prior to lipid accumulation in the WC cells, and appears to be a major contributing factor in pigeon atherogenesis. One hundred forty five pigeon transcripts were homologous to the chicken. However, the mitochondrial genes expressed in the pigeon were more closely related to non-domestic birds such as the turkey vulture and oriental stork. Despite this categorical exception, the recently published chicken genome was an ideal resource for identifying differentially expressed genes in the pigeon. The results were interpreted in the context of current hypotheses of human atherogenesis. The pigeon transcripts can also be used in comparative studies of avian genomics