Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation.

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling.

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation.

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Ligand-induced architecture of the leptin receptor signaling complex.

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT-GTP and ß1γ1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and ß1γ1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ß2-adrenergic receptor-GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.

Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human.

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies.

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ß(2)-adrenoceptor (ß(2)AR) in complex with the heterotrimeric G protein Gs (Gαsßγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

A hypomorphic Artemis human disease allele causes aberrant chromosomal rearrangements and tumorigenesis.

The Artemis gene encodes a DNA nuclease that plays important roles in non-homologous end-joining (NHEJ), a major double-strand break (DSB) repair pathway in mammalian cells. NHEJ factors repair general DSBs as well as programmed breaks generated during the lymphoid-specific DNA rearrangement, V(D)J recombination, which is required for lymphocyte development. Mutations that inactivate Artemis cause a human severe combined immunodeficiency syndrome associated with cellular radiosensitivity. In contrast, hypomorphic Artemis mutations result in combined immunodeficiency syndromes of varying severity, but, in addition, are hypothesized to predispose to lymphoid malignancy. To elucidate the distinct molecular defects caused by hypomorphic compared with inactivating Artemis mutations, we examined tumor predisposition in a mouse model harboring a targeted partial loss-of-function disease allele. We find that, in contrast to Artemis nullizygosity, the hypomorphic mutation leads to increased aberrant intra- and interchromosomal V(D)J joining events. We also observe that dysfunctional Artemis activity combined with p53 inactivation predominantly predisposes to thymic lymphomas harboring clonal translocations distinct from those observed in Artemis nullizygosity. Thus, the Artemis hypomorphic allele results in unique molecular defects, tumor spectrum and oncogenic chromosomal rearrangements. Our findings have significant implications for disease outcomes and treatment of patients with different Artemis mutations.

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation.

The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

The Effect of Longer-Acting vs Shorter-Acting Testosterone Therapy on Follicle Stimulating Hormone and Luteinizing Hormone.

INTRODUCTION: Testosterone (T) replacement therapy causes suppression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that can lead to decrease in semen parameters and possible infertility. Different T formulations may have varying suppression on FSH and LH. OBJECTIVE: To study whether shorter-acting T (multiple daily dosing) has less suppression on FSH and LH serum levels compared with longer-acting T (transdermal gel, injectable). METHODS: A systematic literature search was conducted by following the protocol based on Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocols. We comprehensively reviewed the literature by systematically searching manuscripts indexed in PubMed from 1995 to March 13, 2019 to identify studies reporting changes in FSH and LH in hypogonadal men treated with exogenous T which evaluated the effect of exogenous T on FSH and LH. RESULTS: A total of 8 studies reported the effect of T on FSH and LH in 793 hypogonadal men: 2 used long-acting injectables (enanthate or undecanoate) in a total of 16 men, 5 used intermediate-acting daily topical gels or patches in a total of 471 men, and 1 used short-acting intranasal T (125 µL/nostril, twice a day or three times a day) in 306 men. Long-acting injectables decreased FSH by 86.3%, intermediate-acting daily gels/patches decreased FSH by 60.2%, and short-acting intranasal gel decreased FSH by 37.8%. Long-acting injectables decreased LH by 71.8%, intermediate-acting daily gels/patches decreased LH by 59.2%, and short-acting intranasal gel decreased LH by 47.3%. CONCLUSIONS: Our findings suggest that short-acting T preparations do not decrease serum FSH or LH to the same extent as longer-acting transdermal gels and injectables. However, further clinical trial data are necessary to determine whether the effect of short-acting TRT on gonadotropins translates into similar changes in semen parameters and fertility. Masterson TA, Turner D, Vo D, et al. The Effect of Longer-Acting vs Shorter-Acting Testosterone Therapy on Follicle Stimulating Hormone and Luteinizing Hormone. Sex Med Rev 2021;9:143-148.

Impact of a hypomorphic Artemis disease allele on lymphocyte development, DNA end processing, and genome stability.

Artemis was initially discovered as the gene inactivated in human radiosensitive T(-)B(-) severe combined immunodeficiency, a syndrome characterized by the absence of B and T lymphocytes and cellular hypersensitivity to ionizing radiation. Hypomorphic Artemis alleles have also been identified in patients and are associated with combined immunodeficiencies of varying severity. We examine the molecular mechanisms underlying a syndrome of partial immunodeficiency caused by a hypomorphic Artemis allele using the mouse as a model system. This mutation, P70, leads to premature translation termination that deletes a large portion of a nonconserved C terminus. We find that homozygous Artemis-P70 mice exhibit reduced numbers of B and T lymphocytes, thereby recapitulating the patient phenotypes. The hypomorphic mutation results in impaired end processing during the lymphoid-specific DNA rearrangement known as V(D)J recombination, defective double-strand break repair, and increased chromosomal instability. Biochemical analyses reveal that the Artemis-P70 mutant protein interacts with the DNA-dependent protein kinase catalytic subunit and retains significant, albeit reduced, exo- and endonuclease activities but does not undergo phosphorylation. Together, our findings indicate that the Artemis C terminus has critical in vivo functions in ensuring efficient V(D)J rearrangements and maintaining genome integrity.

Switch recombination and somatic hypermutation are controlled by the heavy chain 3' enhancer region.

Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.