IU Indianapolis ScholarWorks :: Browsing by Subject "transcription"

Browsing by Subject "transcription"

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Analysis of Histone Lysine Methylation Using Mass Spectrometry
(2012-12-11) True, Jason Donald; Goebl, Mark G.; Mosley, Amber L.; Witzmann, F. A. (Frank A.)
Histones are highly basic proteins which when digested by trypsin are hard to analyze using mass spectrometry. Because histones are basic nuclear proteins, a nuclei prep followed by acid extraction is the best purification strategy to increase overall abundance of purified histones. Blocking the lysine residues and cleaving with trypsin is a useful technique to increase detection of histone peptides using MudPIT. In particular, carbamylation and propionylation are the best two methods to block lysine residues. Using both propionylation and carbamylation along with no treatment has been shown to increase the identification of unmodified and modified histone peptides when coupled with MudPIT analysis.
Changes in the Rpb3 Interactome Caused by the Deletion of RPB9 in Saccharomyces cerevisiae
(2016-08-02) Talbert, Eric A.; Mosley, Amber L.; Goebl, Mark G.; Hudmon, Andy
RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions.
The Chimeric Fusion Protein SETMAR Functions as a Chromatin Organizing Factor
(2020-08) Bates, Alison Melissa; Georgiadis, Millie M.; Mosley, Amber L.; Quilliam, Lawrence A.; Fehrenbacher, Jill C.
About 50 million years ago, an Hsmar1 transposon invaded an early primate genome and inserted itself downstream of a SET methyltransferase gene, leading to the birth of a new chimeric protein now called SETMAR. While all other Hsmar1 sequences in the human genome have suffered inactivating mutational damage, the transposase domain of SETMAR has remained remarkably intact, suggesting that it has gained a novel, evolutionarily advantageous function. While SETMAR can no longer transpose itself throughout the genome, it has retained its ancestral sequence-specific DNA binding activity, the importance of which is currently unknown. To investigate this, we performed ChIP-seq to examine SETMAR binding in the human genome. We also utilized RNA-sequencing to assess SETMAR overexpression as well as SETMAR deletion on the human transcriptome. Additionally, we explored SETMAR’s transposase-derived chromatin-looping ability using chromosome-conformation-capture-on-ChIP (4C) in the presence of SETMAR overexpression and performed genome-wide Hi-C to assess the impact of complete SETMAR silencing on global chromatin interactions. ChIP-seq revealed that SETMAR amassed 7,332 unique binding sites, 69% of which included a TIR motif. RNA-sequencing in cells overexpressing SETMAR indicated 177 differentially regulated transcripts, including repression of 17 histone transcripts, suggesting a possible role in chromatin dynamics. RNA-sequencing of parental and SETMAR knockout clones highlighted an average of 5,000 altered transcripts in each cell line, with 343 transcripts significantly differentially expressed in all three knockout clones, many of which participate in embryonic development pathways. 4C analysis in the presence of SETMAR overexpression discovered multiple intrachromosomal looping interactions, and Hi-C analysis of SETMAR knockout cell lines uncovered genome-wide loss of chromatin interactions and disruption of TAD boundaries. The prevalence of SETMAR binding in the human genome combined with its chromatin looping capability and its dramatic effects on the transcriptome suggest a previously undiscovered role for SETMAR as a novel chromatin organizing factor.
A distinct transcriptional profile in response to endothelial monocyte activating polypeptide II is partially mediated by JAK-STAT3 in murine macrophages
(American Physiological Society, 2019-09-01) Lee, Daniel D.; Hochstetler, Alexandra; Murphy, Christina; Lowe, Chinn-Woan; Schwarz, Margaret A.; Pediatrics, School of Medicine
Macrophages are important responders to environmental changes such as secreted factors. Among the secreted factors in injured tissues, the highly conserved endothelial monocyte activating polypeptide II (EMAP II) has been characterized to limit vessel formation, to be locally expressed near sites of injury labeling it a “find-me” signal, and to recruit macrophages and neutrophils. The molecular mechanisms mediated by EMAP II within macrophages once they are recruited are unknown. In this study, using a model of partially activated, recruited thioglycollate-elicited peritoneal macrophages, a transient, transcription profile of key functional genes in macrophages exposed to EMAP II was characterized. We found that EMAP II-mediated changes were elicited mainly through signal transducer and activator of transcription 3 (STAT3) as evidenced by increased Y705 phosphorylation and changes in activity and upstream of it, Janus associated kinase (JAK)1/2 upstream. Both inhibition of JAK1/2 and knockdown of Stat3 abrogated a subset of genes that are upregulated by EMAP II. Our results identify a rapid EMAP II-mediated STAT3 activation that coincides with altered pro- and anti-inflammatory gene expression in macrophages.
The Eukaryotic SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus
(2020-10) Gibson, Ryan Taylor; Androphy, Elliot; Guo, Haitao; Yu, Andy; Mayo, Lindsey
Human papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. High-risk HPV (HR-HPV) infection causes nearly all cervical cancers and an increasing number of head and neck cancers. While prophylactic vaccinations have reduced the incidence of HPV infection and attributable cancers, currently there is no cure for pre-existing HPV infection. As such, HPV remains a global health threat and a better understanding of HPV biology remains of significant medical importance for identification of novel therapeutic targets. The multi-subunit structural maintenance of chromosomes 5/6 complex (SMC5/6) is comprised of SMC5, SMC6 and NSE1-4. SMC5/6 is essential for homologous recombination DNA repair and reportedly functions as an antiviral factor during hepatitis B and herpes simplex-1 viral infections. Intriguingly, SMC5/6 has been found to associate with HR-HPV E2 proteins, which are multifunctional transcription factors essential to regulation of viral replication and transcription. The function of SMC5/6 associations with E2, as well as its role during HR-HPV infection remain unclear and we explored this question in the context of HR-HPV- 31. SMC6 interacted with HPV-31 E2 and co-immunoprecipitation of SMC6/E2 complexes required the E2 transactivation domain, inferring SMC6 association is limited to the full-length E2 isoform. Depletion of SMC6 and NSE3 increased HPV replication and transcription in keratinocytes stably maintaining episomal HPV-31, suggesting that the SMC5/6 complex represses these processes. Neither SMC6 nor NSE3 co-IP the viral E1 DNA helicase alone or E1/E2 complexes but the association of SMC6 with E2 was reduced in the presence of E1, indicating that SMC6 competes with E1 for E2 binding. This infers that SMC6 repression of the viral replicative program may involve inhibiting initiation of viral replication by disrupting E2 interactions with E1. Chromatin immunoprecipitation determined that SMC6 is present on episomal HPV-31 genomes, alluding to a possible role for SMC5/6 in modifying the chromatin state of viral DNA. Taken together, these findings describe a novel function for SMC5/6 as a repressor of the HPV-31 replicative program.
Intrinsic Disorder in Transcription Factors
(2005-08) Liu, Jiangang (Al); Perumal, Narayanan B.
Reported evidence suggested that high abundance of intrinsic disorder in eukaryotic genomes in comparison to bacteria and archaea may reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. The major advantage of intrinsically disordered proteins or disordered regions is their inherent plasticity for molecular recognition, and this advantage promotes disordered proteins or disordered regions in binding their targets with high specificity and low affinity and with numerous partners. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported and the biological functions associated with their corresponding structural properties have been examined, so far no specific systematic analysis of intrinsically disordered proteins has been reported. To test for a generalized prevalence of intrinsic disorder in transcriptional regulation, we first used the Predictor Of Natural Disorder Regions (PONDR VL-XT) to systematically analyze the intrinsic disorder in three Transcription Factor (TF) datasets (TFSPTRENR25, TFSPNR25, TFNR25) and two control sets (PDBs25 and RandomACNR25). PONDR VL-XT predicts regions of ≥30 consecutive disordered residues for 94.13%, 85.19%, 82.63%, 54.51%, and 18.64% of the proteins from TFNR25, TFSPNR25, TFSPTRENR25, RandomACNR25, and PDBs25, respectively, indicating significant abundance of intrinsic disorder in TFs as compared to the two control sets. We then used Cumulative Distribution Function (CDF) and charge-hydropathy plots to further confirm this propensity for intrinsic disorder in TFs. The amino acid compositions results showed that the three TF datasets differed significantly 5 from the two control sets. All three TF datasets were substantially depleted in order-promoting residues such as W, F, I, Y, and V, and significantly enriched in disorder-promoting residues such as Q, S, and P. H and C were highly over-represented in TF datasets because nearly a half of TFs contain several zinc-fingers and the most popular type of zinc-finger is C2H2. High occurrence of proline and glutamine in these TF datasets suggests that these residues might contribute to conformational flexibility needed during the process of binding by co-activators or repressors during transcriptional activation or repression. The data for disorder predictions on TF domains showed that the AT-hooks and basic regions of DNA Binding Domains (DBDs) were highly disordered (the overall disorder scores are 99% and 96% respectively). The C2H2 zinc-fingers were predicted to be highly ordered; however, the longer the zinc finger linkers, the higher the predicted magnitude of disorder. Overall, the degree of disorder in TF activation regions was much higher than that in DBDs. Our studies also confirmed that the degree of disorder was significantly higher in eukaryotic TFs than in prokaryotic TFs, and the results reflected the fact that the eukaryotes have well-developed elaborated gene transcription mechanism, and such a system is in great need of TF flexibility. Taken together, our data suggests that intrinsically disordered TFs or partially unstructured regions in TFs play key roles in transcriptional regulation, where folding coupled to binding is a common mechanism.
Lifespan profiles of Alzheimer's disease–associated genes and their products in monkeys and mice.
(IOS, 2009) Dosunmu, Remi; Wu, Jinfang; Adwan, Lina; Maloney, Bryan; Basha, Md Riyaz; McPherson, Christopher A.; Harry, G. Jean; Rice, Deborah C.; Zawia, Nasser H.; Lahiri, Debomoy K.; Department of Psychiatry, IU School of Medicine
Alzheimer's disease (AD) is characterized by plaques of amyloid–beta (Aβ) peptide, cleaved from amyloid–β precursor protein (AβPP). Our hypothesis is that lifespan profiles of AD-associated mRNA and protein levels in monkeys would differ from mice, and that differential lifespan expression profiles would be useful to understand human AD pathogenesis. We compared profiles of AβPP mRNA, AβPP protein, and Aβ levels in rodents and primates. We also tracked a transcriptional regulator of the AβPP gene, specificity protein 1 (SP1), and the β amyloid precursor cleaving enzyme (BACE1). In mice, AβPP and Sp1 mRNA and their protein products were elevated late in life; Aβ levels declined in old age. In monkeys, Sp1, AβPP, and BACE1 mRNA declined in old age, while protein products and Aβ levels rose. Proteolytic processing in both species did not match production of Aβ. In primates, AβPP and Sp1 mRNA levels coordinate, but an inverse relationship exists with corresponding protein products, as well as Aβ levels. Comparison of human DNA and mRNA sequences to monkey and mouse counterparts revealed structural features that may explain differences in transcriptional and translational processing. These findings are important for selecting appropriate models for AD and other age–related diseases.
Nkx2.5 regulates Endothelin Converting Enzyme-1 during pharyngeal arch patterning
(Wiley, 2017-03) Iklé, Jennifer M.; Tavares, Andre L. P.; King, Marisol; Ding, Ding; Colombo, Sophie; Firulli, Beth A.; Firulli, Firulli; Targoff, Kimara L.; Yelon, Deborah; Clouthier, David E.; Anatomy and Cell Biology, School of Medicine
In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediates this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.
A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin*.
(ASBMB, 2015-10-23) Simon, Jeremy M.; Parker, Joel S.; Liu, Feng; Rothbart, Scott B.; Ait-Si-Ali, Slimane; Strahl, Brian D.; Jin, Jian; Davis, Ian J.; Mosley, Amber L.; Pattenden, Samantha G.; Department of Biochemistry and Molecular Biology, IU School of Medicine
Background: G9a-GLP lysine methyltransferases mono- and di-methylate histone H3 lysine 9 (H3K9me2).Results: Widely interspaced zinc finger (WIZ) regulates H3K9me2 levels through a mechanism that involves retention of G9a on chromatin.Conclusion: The G9a-GLP-WIZ complex has unique functions when bound to chromatin that are independent of the H3K9me2 mark.Significance: Combining pharmacologic and genetic manipulations is essential to any translational hypotheses related to G9a function.
The SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus Type 31
(MDPI, 2020-09) Gibson, Ryan T.; Androphy, Elliot J.; Microbiology and Immunology, School of Medicine
The multi-subunit structural maintenance of chromosomes (SMC) 5/6 complex includes SMC6 and non-SMC element (NSE)3. SMC5/6 is essential for homologous recombination DNA repair and functions as an antiviral factor during hepatitis B (HBV) and herpes simplex-1 (HSV-1) viral infections. Intriguingly, SMC5/6 has been found to associate with high-risk human papillomavirus (HPV) E2 regulatory proteins, but the functions of this interaction and its role during HPV infection remain unclear. Here, we further characterize SMC5/6 interactions with HPV-31 E2 and its role in the HPV life cycle. Co-immunoprecipitation (co-IP) revealed that SMC6 interactions with HPV-31 E2 require the E2 transactivation domain, implying that SMC5/6 interacts with full-length E2. Using chromatin immunoprecipitation, we found that SMC6 is present on HPV-31 episomes at E2 binding sites. The depletion of SMC6 and NSE3 increased viral replication and transcription in keratinocytes maintaining episomal HPV-31, indicating that SMC5/6 restricts the viral replicative program. SMC6 interactions with E2 were reduced in the presence of HPV-31 E1, suggesting that SMC6 and E1 compete for E2 binding. Our findings demonstrate SMC5/6 functions as a repressor of the viral replicative program and this may involve inhibiting the initiation of viral replication.

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