PBAF chromatin remodeler complexes that mediate meiotic transitions in mouse.

Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.

Mouse Chd4-NURD is required for neonatal spermatogonia survival and normal gonad development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

SHOC1 is a ERCC4-(HhH)2-like protein, integral to the formation of crossover recombination intermediates during mammalian meiosis.

Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.

The chromatin-remodeling subunit Baf200 promotes homology-directed DNA repair and regulates distinct chromatin-remodeling complexes.

The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.

Nef neutralizes the ability of exosomes from CD4+ T cells to act as decoys during HIV-1 infection.

Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4- T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface.

Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.

Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants.

The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.

Tyrosines in the carboxyl terminus regulate Syk kinase activity and function.

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcepsilonRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcepsilonRI-induced degranulation, nuclear factor for T cell activation and NFkappaB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.