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The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The discoid shape of resting platelets is maintained by a peripheral, circular bundle of microtubules called marginal band. Marginal band microtubules are acetylated on lysine 40 of the alpha-tubulin subunits. We have previously shown that the deacetylase HDAC6 is responsible for tubulin deacetylation in platelets and that the hyperacetylated state of the microtubules in HDAC6KO platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification. So far, the question about the reverse enzyme, responsible for tubulin acetylation in platelets, has remained unanswered. Several enzymes have been described as having tubulin acetylation activity. Here we identify αTAT1 as the enzyme responsible for the acetylation of marginal band microtubules. We show that αTAT1 deficiency has only minor consequences for platelet production and function. A residual tubulin acetylation level in αTAT1 deficient platelet lysates suggests the presence of an additional tubulin-acetylating enzyme that is unable to acetylate marginal band microtubules.

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin., Competing Interests: The authors declare no conflict of interest.

Background: Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown., Results: Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription-splicing territories and changes to pre-mRNA splicing are observed., Conclusion: We have produced the first H2A.B knockout mouse using the TALEN approach.

Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dramatic and unique genome reorganizations accompany the differentiation of haploid male germ cells, characterized by a gradual loss of the vast majority of histones leading to a final tight compaction of the genome by protamines. Despite being essential for procreation and the life cycle, the mechanisms driving the transformation of nucleosomes into nucleoprotamines remain poorly understood. To address this issue, our laboratory has developed a number of specific approaches, ranging from the purification of spermatogenic cells at specific stages, the analysis of chromatin transitional states, the functional characterization of histone variants, histone-replacing proteins and their chaperones. This chapter will detail all related relevant techniques with a particular emphasis on methods allowing the functional studies of histone variants and the genome organizational states associated with the studied histones in spermatogenic cells undergoing histone-to-protamine exchange.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β ( ck2β -/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2 β -/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β -/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca 2+ ) release. Presumably due to a blunted increase in the concentration of cytosolic Ca 2+ , activation-dependent increases of α and dense-granule secretion and integrin α IIb β 3 activation, and aggregation were abrogated in ck2β -/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2β -/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2β -/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2β fl/fl mice. The present observations reveal CK2β as a novel powerful regulator of thrombopoiesis, Ca 2+ -dependent platelet activation, and arterial thrombosis in vivo., (© 2017 by The American Society of Hematology.)

Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Isolation of pools of spermatogenic cells at specific developmental stages is essential for the investigations of molecular events controlling critical transitions during spermatogenesis. Large-scale cell purification techniques allow for combined proteomics, genomics, and transcriptomics studies. Herein, we describe a procedure for the purification of meiotic and post-meiotic male germ cells from adult mouse testes. We also describe how the fractionated cell populations could be used for further studies. In our laboratory, these protocols are routinely used to specifically investigate the molecular basis of histone acetylation/acylation-driven epigenetic programming.

Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Ectopic activation of tissue-specific genes accompanies malignant transformation in many cancers. Prolactin (PRL) aberrant activation in lung cancer was investigated here to highlight its value as a biomarker., Results: PRL is ectopically activated in a subset of very aggressive lung tumors, associated with a rapid fatal outcome, in our cohort of 293 lung tumor patients and in an external independent series of patients. Surprisingly PRL receptor expression was not detected in the vast majority of PRL-expressing lung tumors. Additionally, the analysis of the PRL transcripts in lung tumors and cell lines revealed systematic truncations of their 5' regions, including the signal peptide-encoding portions. PRL expression was found to sustain cancer-specific gene expression circuits encompassing genes that are normally responsive to hypoxia. Interestingly, this analysis also indicated that histone deacetylase (HDAC) inhibitors could counteract the PRL-associated transcriptional activity., Innovation and Conclusion: Altogether, this work not only unravels a yet unknown oncogenic mechanism but also indicates that the specific category of PRL-expressing aggressive lung cancers could be particularly responsive to an HDAC inhibitor-based treatment.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

We report the identification of a new type of histone mark, lysine 2-hydroxyisobutyrylation (Khib), and identify the mark at 63 human and mouse histone Khib sites, including 27 unique lysine sites that are not known to be modified by lysine acetylation (Kac) and lysine crotonylation (Kcr). This histone mark was initially identified by MS and then validated by chemical and biochemical methods. Histone Khib shows distinct genomic distributions from histone Kac or histone Kcr during male germ cell differentiation. Using chromatin immunoprecipitation sequencing, gene expression analysis and immunodetection, we show that in male germ cells, H4K8hib is associated with active gene transcription in meiotic and post-meiotic cells. In addition, H4K8ac-associated genes are included in and constitute only a subfraction of H4K8hib-labeled genes. The histone Khib mark is conserved and widely distributed, has high stoichiometry and induces a large structural change. These findings suggest its critical role on the regulation of chromatin functions.

S100A1 and S100B are induced by the SOX trio transcription factors (SOX9, SOX5, and SOX6) in chondrocytes, and inhibit their hypertrophic differentiation in culture. However, functional roles of S100A1 and S100B during in vivo skeletal development are yet to be determined. Here we show that mice deficient of both the S100a1 and S100b genes displayed normal skeletal growth from embryonic stage to adulthood. Although no compensatory upregulation of other S100 family members was observed in S100a1/S100b double mutants, the related S100a2, S100a4, S100a10, and S100a11 were expressed at similarly high levels to S100a1 and S100b in mouse primary chondrocytes. Furthermore, overexpression of these other S100 members suppressed the hypertrophic differentiation of chondrocytes in vitro as efficiently as S100A1 and S100B. Taken together, the present study demonstrates that S100A1 and S100B are dispensable for endochondral ossification during skeletal development, most likely because their deficiency may be masked by other S100 proteins which have similar functions to those of S100A1 and S100B.

The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle, yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified, we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B reassembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant that plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg.

HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a 'progenitor cells' gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2β) revealed a viability defect. However, analysis of CK2β functions along the neural lineage established CK2β as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2β involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis.

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Genetic programs that govern neural stem/progenitor cell (NSC) proliferation and differentiation are dependent on extracellular cues and a network of transcription factors, which can be regulated posttranslationally by phosphorylation. However, little is known about the kinase-dependent pathways regulating NSC maintenance and oligodendrocyte development. We used a conditional knockout approach to target the murine regulatory subunit (beta) of protein kinase casein kinase 2 (CK2beta) in embryonic neural progenitors. Loss of CK2beta leads to defects in proliferation and differentiation of embryonic NSCs. We establish CK2beta as a key positive regulator for the development of oligodendrocyte precursor cells (OPCs), both in vivo and in vitro. We show that CK2beta directly interacts with the basic helix-loop-helix (bHLH) transcription factor Olig2, a critical modulator of OPC development, and activates the CK2-dependent phosphorylation of its serine-threonine-rich (STR) domain. Finally, we reveal that the CK2-targeted STR domain is required for the oligodendroglial function of Olig2. These findings suggest that CK2 may control oligodendrogenesis, in part, by regulating the activity of the lineage-specific transcription factor Olig2. Thus, CK2beta appears to play an essential and uncompensated role in central nervous system development.

The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.

Knocking out the regulatory beta subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2beta (CK2beta+/-) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2B+/- inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2beta+/- ES (Embryonic Stem) cells express a considerably lower level of CK2beta than wild-type ES cells, whereas the level of CK2beta in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2beta levels during development in the majority of, but not in all, cases (Mol Cell Biol 23: 908-915, 2003). In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2beta+/- ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2beta+/- embryos. No correlation between CK2beta levels in normal looking and retarded CK2beta+/- embryos were found. However, a different post-translational form of CK2beta protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2beta+/- cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2beta.

Protein kinase CK2 (formerly known as casein kinase 2) was among the first protein kinases to be identified and characterized. Surprisingly, in spite of intense efforts, the regulation and cellular functions of CK2 remain obscure. However, recent data on its molecular structure, its signal-mediated intracellular dynamic localization and its unexpected function in cell survival have raised new interest in this enzyme. These studies reveal unique features of CK2 and highlight its importance in the transduction of survival signals.

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE-cadherin is able to mediate a homotypic type of cellular interaction in a Ca2+-dependent manner. In the mouse embryo, VE-cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE-cadherin expression is required for the assembly of endothelial cells into vascular structures, we generated VE-cadherin-negative mouse embryonic stem cells (VE-cadherin-/- ES cells) by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, we observed that endothelial cells remained dispersed and failed to organize a vessel-like pattern in VE-cadherin-/- ES-derived EBs. However, dispersed VE-cadherin-/- ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the VE-cadherin locus did not interfere with the hematopoietic differentiation potential of ES cells. These in vitro experiments are consistent with a pivotal role of VE-cadherin in vascular structure assembly.

A monoclonal antibody, AC7, directed against the RGD (Arg-Gly-Asp) binding site on the GpIIIa subunit of the platelet fibrinogen receptor, interacts only with activated platelet. In order to identify the regions of AC7 that interact with the receptor, cDNA sequences of AC7 immunoglobulin heavy and light chain variable regions were determined. Among the six complementarity-determining regions (CDRs) of AC7, the CDR3 heavy chain (H3) contains homology to the RGDF sequence within fibrinogen. A synthetic peptide encompassing the H3 region (H3, RQMIRGYFDV) inhibited platelet aggregation and fibrinogen binding to platelet (IC50 = 700 microM). The inhibitory potencies of modified H3 peptides suggest that the RGYF sequence within the H3 peptide mimic the receptor recognition sequence in fibrinogen.

Adenovirus-(Ad)- E1A proteins carry two conserved domains (CR1 and CR2) required for transformation of primary rodent cells and essential for association with cellular proteins, including p105RB, p58cyclin A and p33cdk2. We show that in normal rat kidney 49F (NRK) cell lines expressing various mutant Ad5-E1A genes, CR2-, but not CR-1-, deletion mutants induce a typical transformed phenotype as characterized by morphology, absence of density arrest and loss of serum requirement. This indicates that induction of these transformed properties is a function of CR1. The fact that E1A proteins with deletions in CR2 show a greatly reduced association with RB, cyclin A and p33cdk2 suggests that these associations are dispensable for E1A-mediated transformation of NRK cells. Induction of the transformed properties is accompanied by a CR1-dependent increase in Proliferating Cell Nuclear Antigen and cyclin A gene expression. Elevated mRNA and protein levels of cyclin A were also found in Ad12-E1-transformed NRK cells but not in ras-transformed NRK cells. On the other hand, cyclin D expression is decreased in a CR1-dependent manner. Although Ad5-E1A proteins are sufficient to transform NRK cells, further deregulation of growth is obtained when Ad5-E1B proteins are co-expressed. One of the Ad5-E1B effects is the sequestration of the p53 protein into a cytoplasmic body containing the p53/Ad5-E1B-55 kD complex. Interestingly, in NRK cell lines expressing Ad5-E1B-55 kD, cyclin A could be detected not only in the nucleus but also in the cytoplasmic bodies. These results indicate that the deregulation of cell cycle control by the Adenovirus-E1 region may be due to a CR1-dependent alteration of the expression of cyclins A and D.

Quiescent benzo[alpha]pyrene-transformed BALB/c 3T3 fibroblasts (line BP-A31), continue to express 'competence' genes (such as c-myc) and do not require platelet-derived growth factor ('competence' factor) in order to resume the cell division cycle. Insulin-like growth factor I (IGF-I), as well as insulin (at high concentrations, where it interacts with IGF-I-receptors) are potent mitogens in these cells. In contrast with the original non-transformed A31 cell line, we show that insulin/IGF-I (even in the absence of de novo protein synthesis) induce actin transcription in BP-A31 cells. We have verified that 'CArG' boxes, major actin promoter elements, can act as insulin-inducible elements in BP-A31 cells. Insulin-induced actin transcription is also observed in quiescent A31 cells stably transfected with a myc expression vector, suggesting a correlation between constitutive myc expression and insulin-induced actin transcription.

The chick oviduct cytosol progesterone receptor, when complexed with ligand, can be exposed to urea concentrations as high as 3 M (at 0 degrees C) without loss of steroid binding capacity. The ligand dissociation rate is increased greater than or equal to 10 fold under these conditions. The "native" 8 S form of the receptor is progressively converted to a 4 S species by urea (greater than 2 M) as seen in ultracentrifugation analysis. This conversion is inhibited by Na2MoO4 (5-50 mM) suggesting that molybdate stabilizes the 8 S molecule by direct interaction. At urea concentrations above 2 M, the ligand-free receptor looses progressively its binding capacity. The "transformed", 4 S receptor was less stable than the 8 S species, and could not be protected by molybdate.

Mitogenic effects of agents activating either the protein kinase C (PDGF; phorbol esters) or the insulin-like growth factor 1 (IGF1)-receptor pathway were studied in quiescent chemically transformed mouse fibroblasts (BP-A31), by evaluating the rate of [3H]thymidine incorporation. Each of these pathways alone was found to be sufficient to sustain progression through the entire cell division cycle. The mitogenic activity of phorbol 12-myristate 13-acetate (PMA) but not that of insulin was blocked by staurosporine (an inhibitor of protein kinase C), in support of the notion that protein kinase C activation was required for the PMA-induced cell cycle progression. The mitogenic effects of PMA were potentiated by cycloheximide pretreatment, and they were abolished by 3-isobutyl-1-methyl xanthine (IBMX; a cyclic nucleotide phosphodiesterase inhibitor). PDGF (known to activate the phospholipase C-protein kinase C pathway) also displayed mitogenic activity in the cycloheximide-pretreated BP-A31 cells, and its effects were prevented by IBMX. In contrast, the mitogenic effects of insulin (at concentrations where it activates the IGF1 receptor) or of IGF1 neither were notably influenced by cycloheximide pretreatment nor were inhibited by IBMX (in the presence of IBMX, the onset of S-phase was delayed by several hours). The expression of the c-fos gene was absent at quiescence; its induction by growth factors was not proportional to their mitogenic potency. Thus, c-fos expression was strongly induced by PMA but only weakly by insulin. IBMX was a powerful inducer of c-fos gene expression but caused a decrease in the level of c-myc mRNA.

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [gamma-32P]-ATP in the presence of Ca2+ but not of Mg2+ ions, while the 110K component was phosphorylated in the presence of Mg2+, but not of Ca2+. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg2+. These data suggest that components of the chick oviduct PR display protein kinase activity.

Steroid hormones produce a response in target cells by binding to hormone-specific soluble receptors, which undergo a transformational change, leading to their interaction with chromatin and to modified gene expression. In a previous paper, we described a monoclonal antibody, BF4, that specifically recognizes and binds the non-transformed '8S' form of chicken oviduct progesterone receptor (8S-PR). We now show that BF4 does not form an immune complex with the 4S transformed form of 3H-progestin-labelled progesterone receptor, but does interact with the 8S non-transformed forms of the oestrogen, androgen and glucocorticosteroid receptors. Our results suggest that the antigenic determinant recognized by BF4 is present on a non-hormone binding unit, which we identify as a polypeptide of molecular weight (MW) 90,000 in the case of the progesterone receptor, and that this unit is common to other 8S non-transformed chicken steroid receptors.

The mitogenic effect of serum pulses, observed previously in quiescent BP-A31 cells, is an artifact due to adsorption of (unknown) serum mitogens to the culture dish; the continuous presence of growth factors is necessary for these cells to traverse the G1 phase. When pretreated with cycloheximide (CH) during the last 8-24 h of quiescence, the BP-A31 cells are particularly sensitive to the adsorbed serum mitogens, as well as to low concentrations of fetal calf serum (less than 0.25%) and to basic fibroblast growth factor. In contrast, the mitogenic activity of insulin was not influenced by CH pretreatment. The expression of the "competence genes" c-myc and JE was only transiently elevated in quiescent BP-A31 cells during CH exposure and did not correlate with the enhancement of the cells' sensitivity to mitogens.

A rabbit was immunized with the highly purified B-subunit (110kDa) (20 to 50 micrograms per injection) of the chick oviduct progesterone receptor (PR). Specific antibodies (IgG-RB) were observed 2 weeks after the first booster injection and high antibody titers in the serum were found after the second and third booster injections (with Kdeq of interaction integral of 2 nM). IgG-RB were tested by immunoprecipitation, immunoblotting, density gradient ultracentrifugation and protein A-sepharose assay methods. They recognized not only the B-subunit but also the A-subunit (79K), the nuclear PR, the mero-receptor (proteolytic cleavage product) and the "non-activated" molybdate-stabilized "8S" PR. However, IgG-RB did not interact with the 90K non hormone-binding component of this 8S-PR. IgG-RB did not affect the binding of the hormone to PR, whether incubated with the receptor before or after labelling with tritiated progesterone. They did not cross-react with glucocorticosteroid receptor of the chick oviduct. Weak interaction was observed with estrogen receptor of the chick oviduct and with KC1 activated "4S" forms of the rabbit and human uterus PR.

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)

Quiescent, chemically transformed (benzo-alpha-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (less than 4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable 'memory' that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this 'memory' probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

A protein kinase activity was copurified with the chick oviduct progesterone receptor. The enzyme is magnesium dependent and can use the B subunit of progesterone receptor or histones as substrates. The physiochemical parameters of the kinase were determined [pI approximately 5.3; Stokes radius approximately 7.2 nm; sedimentation coefficient (S 20,w) approximately 5.6] and compared to those of the purified B subunit. The results were consistent with the presence of an unique enzyme distinct from the receptor itself. The physiological significance of receptor phosphorylation was investigated in oviduct cells grown in primary culture. Cells were labeled with [32P]orthophosphate in presence or absence of progesterone and the receptor components were immunoprecipitated with a specific polyclonal antibody. Although progesterone treatment lead to the attachment of most of the receptor (approximately 80%) to nuclear structures, the 32P-labeled B subunit was only recovered in the cytosol fraction. Different procedures to extract the nuclear receptor did not allow detection of any 32P-labeled form in the nuclear-soluble fractions, suggesting that the B subunit was not further phosphorylated upon the exposure of cells to progesterone.