Proteomic analysis of FOXP proteins reveals interactions between cortical transcription factors associated with neurodevelopmental disorders

FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical as

Alternative Splicing and Gene Duplication in the Evolution of the FoxP Gene Subfamily

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1–P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system

Forkhead Transcription Factors Foxp1 and Foxp4 Regulate T Cell Development and Function

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the FoxP family of Forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for regulatory T cell generation and function, and Foxp1 is necessary for the development of naïve T cells. FoxP family member Foxp4 is highly homologous to Foxp1 and has been shown to dimerize with other FoxP proteins. In this thesis, we report the first studies of Foxp4 in T lymphocytes. Using a CD4Cre-mediated conditional knockout approach we evaluated the roles for Foxp4 regulation in the T lineage. T cell development and homeostasis are normal in the absence of Foxp4. Despite effective control of infection with Toxoplasma gondii or acute Lymphocytic choriomeningitis virus in vivo, cytokine production during antigen-specific rechallenge is reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal memory T cell recall responses to antigen in acutely or chronically infected mice. Next we determined whether FoxP family members compensate for one another in Foxp1- or Foxp4-knockout models. We utilized a similar CD4Cre approach to delete both Foxp1 and Foxp4 in T cells. Foxp1/4-deficient T cells exhibit abnormal thymic development and T cell receptor signaling. Loss of Foxp1/4 results in significantly reduced T cell numbers, and altered T cell effector function, reminiscent of Foxp1-deficient T cells. Lastly, we examined the functions of Foxp1/4 in Foxp3+ regulatory T cells (Tregs). Tregs are critical for prevention of autoimmunity and controlling immune responses during infection. While conditional deletion of either Foxp1 or Foxp4 in T cells has little effect on Tregs, combined deletion results in abnormal Treg generation. Foxp1/4-deficient Tregs exhibited significant defects in both development and homeostasis. Under competitive conditions, double-deficient Tregs are at a significant developmental disadvantage relative to wild-type competitors. Furthermore, Foxp1/4-deficient Tregs exhibit impaired cytokine-induced STAT5 phosphorylation and reduced expression of Foxp3, suggesting Foxp1/4 is required for normal Treg generation. Together, these findings demonstrate that the FoxP family regulates multiple facets of T cell development and function, and actively contributes to the maintenance of immunological tolerance

Novel markers in pediatric-type follicular lymphoma

The aim of this study was to review the histopathological, phenotypic, and molecular characteristics of pediatric-type follicular lymphoma (PTFL) and to assess the diagnostic value of novel immunohistochemical markers in distinguishing PTFL from follicular hyperplasia (FH). A total of 13 nodal PTFLs were investigated using immunohistochemistry, fluorescence in situ hybridization (FISH), and PCR and were compared with a further 20 reactive lymph nodes showing FH. Morphologically, PTFL cases exhibited a follicular growth pattern with irregular lymphoid follicles in which the germinal centers were composed of numerous blastoid cells showing a starry-sky appearance. Immunohistochemistry highlighted preserved CD10 (13/13) and BCL6 (13/13) staining, CD20 (13/13) positivity, a K light chain predominance (7/13), and partial BCL2 expression in 6/13 cases (using antibodies 124, E17, and SP66). The germinal center (GC)–associated markers stathmin and LLT-1 were positive in most of the cases (12/13 and 12/13, respectively). Interestingly, FOXP-1 was uniformly positive in PTFL (12/13 cases) in contrast to reactive GCs in FH, where only a few isolated positive cells were observed. FISH revealed no evidence of BCL2, BCL6, or MYC rearrangements in the examined cases. By PCR, clonal immunoglobulin gene rearrangements were detected in 100% of the tested PTFL cases. Our study confirmed the unique morphological and immunophenotypic features of PTFL and suggests that FOXP-1 can represent a novel useful diagnostic marker in the differential diagnosis between PTFL and FH

T Regulatory Cell Responses to Immunization with a Soluble Egg Antigen in Schistosoma mansoni-Infected Mice

The aim of the study is to characterize the phenotypes of CD4+ CD25+ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naïve C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4+ CD25+) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-γ, IL-4, and TNF-α, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology

Energetics and dynamics of the FOXP2 forkhead domain-DNA interaction

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 15 May 2018.The members of the forkhead box (FOX) family of transcription factors are key regulators in the development and metabolism of a wide variety of tissues in humans. The FOX transcription factors are classified by the presence of a canonical forkhead winged-helix DNA binding domain and are further divided into subfamilies based on sequence divergence of the forkhead domain. To date, only the FOXP subfamily is known to require dimerisation for transcriptional activity. Dimerisation of the FOXP subfamily members occurs at two distinct interfaces, a conserved leucine zipper domain, and through domain-swapping of a C-terminal forkhead domain. The role of the domain-swapped forkhead domain is unclear, although several attempts have been made to clarify this. Due to the orientation of the recognition motifs in the domain-swapped dimer, it has been speculated that it is capable of binding and congregating two distal promoter response elements, suggesting a role in cross-chromosomal gene co-regulation. The unique capability of the FOXP forkhead domain to dimerise is attributed to an evolutionary mutation (proline to alanine) that occurs in the hinge loop connecting the second and third α-helices. Further to this, the hinge loop has also been implicated in altering the specificity of the forkhead domain. Here, we aim to elucidate how the evolutionary proline to alanine mutation facilitates dimerisation and whether it has any role in defining the DNA binding specificity of the FOXP2 forkhead domain. To do this all experiments were conducted on an obligate monomeric mutant (A539P) and an engineered obligate dimeric mutant (F541C) FOXP2 forkhead domain in addition to the wild-type. High and low-resolution DNA binding studies involving electrophoretic mobility shift assays (EMSA), fluorescence polarisation (FP) studies and isothermal titration calorimetry (ITC) revealed that the FOXP2 forkhead domain preferentially binds to the FOXP2 consensus site as a monomer, despite having the capacity to form dimers in the absence of DNA. During these studies, a significant difference in the thermodynamic signatures of DNA binding was observed between the wild-type and A539P mutant FOXP2 forkhead domain. Further dissection of the thermodynamic results revealed that the hinge loop mutation significantly alters the mechanism of DNA binding. The wild-type FOXP2 forkhead domain undergoes significant conformational changes upon DNA binding, shown by hydrogen-deuterium exchange mass spectrometry, in addition to making two additional contacts with the sugar-phosphate backbone of the consensus site. The large conformational changes incurred by DNA binding, stabilises the monomeric form of the FOXP2 forkhead domain and is indicative of a searchrecognition conformational switch that is unique to the FOXP subfamily. Furthermore, in vivo studies, involving dual-luciferase reporter assays, show that dimerisation of the FOXP2 forkhead domain acts as a regulatory mechanism controlling the transcriptional activity of FOXP2. Together the work presented here proposes that the DNA binding by FOXP2, and by extension FOXP1 and 4, follows a monomeric pathway whereby FOXP2 translocate to the site of action as a monomer and in a context-dependent manner either dimerises or remains monomeric to fine-tune the regulation of target genes. This work provides the first detailed assessment of the energetics and dynamics that occur during DNA binding for not only the FOXP2 forkhead domain but any of the FOX forkhead domains. Furthermore, presented here is the first proposed mechanism of transcriptional regulation through the oligomeric state of the FOXP2 forkhead domain.LG201

Identification and analysis of evolutionary selection pressures acting at the molecular level in five forkhead subfamilies

<p>Abstract</p> <p>Background</p> <p>Members of the forkhead gene family act as transcription regulators in biological processes including development and metabolism. The evolution of forkhead genes has not been widely examined and selection pressures at the molecular level influencing subfamily evolution and differentiation have not been explored. Here, <it>in silico </it>methods were used to examine selection pressures acting on the coding sequence of five multi-species FOX protein subfamily clusters; FoxA, FoxD, FoxI, FoxO and FoxP.</p> <p>Results</p> <p>Application of site models, which estimate overall selection pressures on individual codons throughout the phylogeny, showed that the amino acid changes observed were either neutral or under negative selection. Branch-site models, which allow estimated selection pressures along specified lineages to vary as compared to the remaining phylogeny, identified positive selection along branches leading to the FoxA3 and Protostomia clades in the FoxA cluster and the branch leading to the FoxO3 clade in the FoxO cluster. Residues that may differentiate paralogs were identified in the FoxA and FoxO clusters and residues that differentiate orthologs were identified in the FoxA cluster. Neutral amino acid changes were identified in the forkhead domain of the FoxA, FoxD and FoxP clusters while positive selection was identified in the forkhead domain of the Protostomia lineage of the FoxA cluster. A series of residues under strong negative selection adjacent to the N- and C-termini of the forkhead domain were identified in all clusters analyzed suggesting a new method for refinement of domain boundaries. Extrapolation of domains among cluster members in conjunction with selection pressure information allowed prediction of residue function in the FoxA, FoxO and FoxP clusters and exclusion of known domain function in residues of the FoxA and FoxI clusters.</p> <p>Conclusion</p> <p>Consideration of selection pressures observed in conjunction with known functional information allowed prediction of residue function and refinement of domain boundaries. Identification of residues that differentiate orthologs and paralogs provided insight into the development and functional consequences of paralogs and forkhead subfamily composition differences among species. Overall we found that after gene duplication of forkhead family members, rapid differentiation and subsequent fixation of amino acid changes through negative selection has occurred.</p