The Role of Celf2 in the Signal Induced Alternative Splicing of Lef1 Exon 6 in T Cells

Alternative splicing is the process by which an exon is preferentially included or excluded from an mRNA transcript. Recent global sequencing studies have shown that \u3e95% of the transcriptome undergoes some form of alternative splicing. Such regulation often alters protein isoform expression, as is especially apparent in T cells of the immune system that change their expression of RNA and protein according to signaling cues. The focus of this thesis is on one alternative exon in the pre-mRNA of transcription factor LEF1 and its regulation by the splicing factor CELF2. LEF1 is crucial for T cell function as it upregulates the expression of TCRα. Upon signal induction in T-cells, CELF2 promotes the inclusion of exon 6 in LEF1 (LEF1-E6) in the final mRNA transcript. This increase in LEF-E6 inclusion generates an isoform of LEF1 that is preferentially active in promoting transcription of TCRα. CELF2 regulates LEF1-E6 inclusion upon stimulation by increasing its binding to two conserved elements (USE60 and DSE120) in the upstream and downstream introns flanking exon 6. My goal is to understand how the increase of binding of CELF2 to the USE60 and DSE120 upon stimulation results in an increase in LEF1-E6 inclusion. Using a combination of in vivo minigene assays, in vitro splicing assays and UV-crosslinking assays I correlate the binding of CELF2 to the function of the USE60 and DSE120. I show that the USE60 and DSE120 do not work synergistically to enhance inclusion but function antagonistic to each other. The USE60 is a repressor of splicing while the DSE120 is an enhancer. In order to achieve an increase in exon 6 inclusion only upon stimulation, CELF2 binding is highly regulated between the USE60 and DSE120. In unstimulated T cells, binding is biased towards the repressive USE60 and upon stimulation the increase in CELF2 binding happens purely on the activating DSE120. This bolus of CELF2 binding on the DSE120 upon stimulation leads to an increase in exon 6 inclusion. These studies reveal a model where binding of CELF2 to the DSE120 is inhibited in unstimulated cells and this inhibition is relieved upon stimulation

Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination

Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors

Aurora B activity is inhibited when centromeric repeat sequences are absent, although kinetochores can still assemble

Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts

Broad-Spectrum Anti-Human Immunodeficiency Virus (HIV) Potential of a Peptide HIV Type 1 Entry Inhibitor

The AIDS epidemic continues to spread at an alarming rate worldwide, especially in developing countries. One approach to solving this problem is the generation of anti-human immunodeficiency virus (HIV) compounds with inhibition spectra broad enough to include globally prevailing forms of the virus. We have examined the HIV type 1 (HIV-1) envelope specificity of a recently identified entry inhibitor candidate, HNG-105, using surface plasmon resonance spectroscopy and pseudovirus inhibition assays. The combined results suggest that the HNG-105 molecule may be effective across the HIV-1 subtypes, and they highlight its potential as a lead for developing therapeutic and microbicidal agents to help combat the spread of AIDS

Introducing metallocene into a triazole peptide conjugate reduces its off‐rate and enhances its affinity and antiviral potency for HIV‐1 gp120

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar K(D) in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1(YU-2) gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC(50) for inhibiting cell infection by HIV-1(BaL) whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments

Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.</p

Alternative splicing networks regulated by signaling in human T cells

The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway–specific regulation of alternative splicing during T-cell stimulation.</jats:p

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)*

Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD