Biomedical subjects
Publications and source records attributed to Noora Kotaja.
The chromatoid body, a unique cloud-like structure of male germ cells, moves dynamically in the cytoplasm of haploid spermatids, but its function has remained elusive for decades. Recent findings indicate that microRNA and RNA-decay pathways converge to the chromatoid body. This highly specialized structure might function as an intracellular focal domain that organizes and controls RNA processing in male germ cells.
The Aurora kinases are cell cycle-regulatory serine-threonine kinases that have been implicated in the function of the centrosomes, kinetechores, chromosome dynamics, and cytokinesis. In comparison with other tissues, there are high levels of expression of Aurora-B and -C in testis. What their respective roles in mammalian spermatogenesis are is an open question. Here we describe the expression and distribution patterns of the three kinases in mouse testis using in situ hybridization and immunohistochemistry. Importantly, the localization of Aurora-B is tightly regulated during spermatogenesis, whereas Aurora-C expression appears to be testis specific. To address the function of Aurora-B in spermatogenesis, we have generated transgenic mice using a pachytene-stage-specific promoter driving the expression of either wild-type Aurora-B or an inactive form of the kinase. Expression of the inactive Aurora-B results in abnormal spermatocytes, increased apoptosis, spermatogenic arrest, and subfertility defects. The function of Aurora-C may also be targeted in the Aurora-B transgenic mutants. To address the function of Aurora-C in testis, we generated Aurora-C knockout mice by homologous recombination. Remarkably, Aurora-C null mice were viable, yet the males had compromised fertility. Aurora-C mutant sperm display abnormalities that included heterogenous chromatin condensation, loose acrosomes, and blunted heads. These findings indicate that Aurora-B and Aurora-C serve specialized functions in mammalian spermatogenesis.
Chromatoid bodies are thought to act as male-germ-cell-specific platforms for the storing and processing of haploid transcripts. The molecular mechanisms governing the formation and function of these germ-cell-specific structures have remained elusive. In this study, we show that the kinesin motor protein KIF17b, which is involved in the nucleocytoplasmic transport of RNA and of a transcriptional coactivator, localizes in chromatoid bodies. The chromatoid body moves actively and non-randomly in the cytoplasm of round spermatids, making frequent contacts with the nuclear envelope. The localization of KIF17b thereby offers a potential mechanism for microtubule-dependent mobility of chromatoid bodies, as well as for the transport of the specific components in and out of the chromatoid body. Interestingly, we demonstrate that KIF17b physically interacts with a testis-specific member of the PIWI/Argonaute family, MIWI, a component of chromatoid bodies implicated in RNA metabolism. A functional interplay between KIF17b and MIWI might be needed for the loading of haploid RNAs in the chromatoid body. Importantly, chromatoid bodies from round spermatids of miwi-null mice are not fully compacted and remain as a diffuse chromatoid material, revealing the essential role played by MIWI in the formation of chromatoid bodies. These results shed new light on the function of chromatoid bodies in the post-transcriptional regulation of gene expression in haploid germ cells.
The chromatoid body is a perinuclear, cytoplasmic cloud-like structure in male germ cells whose function has remained elusive. Here we show that the chromatoid body is related to the RNA-processing body of somatic cells. Dicer and components of microRNP complexes (including Ago proteins and microRNAs) are highly concentrated in chromatoid bodies. Furthermore, we show that Dicer interacts with a germ cell-specific chromatoid body component, the RNA helicase MVH (mouse VASA homolog). Thus, chromatoid bodies seem to operate as intracellular nerve centers of the microRNA pathway. Our findings underscore the importance of posttranscriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.
FLI-1 is a transcription factor of the ETS family that is involved in several developmental processes and that becomes oncogenic when overexpressed or mutated. As the functional regulators of FLI-1 are largely unknown, we performed a yeast two-hybrid screen with FLI-1 and identified the SUMO E3 ligase PIASxalpha/ARIP3 as a novel in vitro and in vivo binding partner of FLI-1. This interaction involved the ETS domain of FLI-1 and required the integrity of the SAP domain of PIASxalpha/ARIP3. SUMO-1 and Ubc9, the ubiquitin carrier protein component in the sumoylation pathway, were also identified as interactors of FLI-1. Both PIASxalpha/ARIP3 and the closely related PIASxbeta isoform specifically enhanced sumoylation of FLI-1 at Lys(67), located in its N-terminal activation domain. PIASxalpha/ARIP3 relocalized the normally nuclear but diffusely distributed FLI-1 protein to PIASxalpha nuclear bodies and repressed FLI-1 transcriptional activation as assessed using different ETS-binding site-dependent promoters and different cell systems. PIASxalpha repressive activity was independent of sumoylation and did not result from inhibition of FLI-1 DNA-binding activity. Analysis of the properties of a series of ARIP3 mutants showed that the repressive properties of PIASxalpha/ARIP3 require its physical interaction with FLI-1, identifying PIASxalpha as a novel corepressor of FLI-1.
Kinesins are motor proteins that transport their cargos along microtubules in an ATP-dependent manner. The testis-specific kinesin KIF17b was shown to directly regulate cAMP-response element modulator (CREM)-dependent transcription by determining the subcellular localization of the activator of CREM in testis (ACT), the testis-specific coactivator of CREM in postmeiotic male germ cells. CREM is a crucial transcriptional regulator of many important genes required for spermatid maturation, as demonstrated by the complete block of sperm development at the first steps of spermiogenesis in crem-null mice. To better understand the complex regulation of postmeiotic germ cell differentiation, we further characterized the ACT-KIF17b interaction, the function of KIF17b, and the signaling pathways governing its action. In this study, we demonstrated that the abilities of KIF17b to shuttle between the nuclear and the cytoplasmic compartments and to transport ACT are neither dependent on its motor domain nor on microtubules, thus revealing a novel microtubule-independent function for kinesins. We also showed that the cyclic AMP-dependent protein kinase A mediates the phosphorylation of KIF17b, and this modification is important for its subcellular localization. These results indicate that cyclic AMP signaling controls CREM-mediated transcription in male germ cells through modification of KIF17b function.
Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell involving replacement of the somatic histones by protamines packing the DNA into the condensed spermatid nucleus and elimination of the cytoplasm during the elongation phase. We describe H1T2, an histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis. In round and elongating spermatids, H1T2 specifically localizes to a chromatin domain at the apical pole, revealing a polarity in the spermatid nucleus. Inactivation by homologous recombination shows that H1T2 is critical for spermiogenesis as male H1t2(-/-) mice have greatly reduced fertility. Analysis of spermiogenesis in H1t2 mutant mice shows delayed nuclear condensation and aberrant elongation. As a result, mutant spermatids are characterized by the presence of residual cytoplasm, acrosome detachment, and fragmented DNA. Hence, H1T2 is a protein required for proper cell restructuring and DNA condensation during the elongation phase of spermiogenesis.
ACT [activator of cAMP-responsive element modulator (CREM) in testis] is a LIM-only protein that interacts with transcription factor CREM in postmeiotic male germ cells and enhances CREM-dependent transcription. CREM regulates many crucial genes required for spermatid maturation, and targeted mutation of the Crem gene in the mouse germ-line blocks spermatogenesis. Here we report the phenotype of mice in which targeted disruption of the act gene was obtained by homologous recombination. Whereas the seminiferous tubules of the act(-/-) mice contain all of the developmental stages of germ cells and the mice are fertile, the amount of mature sperm in the epididymis is drastically reduced. The residual sperm display severe abnormalities, including fully folded tails and aberrant head shapes. These results indicate that numerous postmeiotic genes under CREM control require the coactivator function of ACT. Thus, the fine-tuning of sperm development is achieved by the coordinated action of two transcriptional regulators.
The differentiation of male germ cell requires spermatogenic stage and cell-specific gene expression that is achieved by unique chromatin remodelling, transcriptional control, and the expression of testis-specific genes or isoforms. Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding (TBP) protein family and its associated co-factors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator CREM represents an established paradigm. In somatic cells, activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT, which confers a powerful, phosphorylation-independent activation capacity. The function of ACT is regulated by a testis-specific kinesin, KIF17b. This study discusses some aspects of the testis-specific transcription machinery, the function of which is essential for the process of spermatogenesis.
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Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.
Male germ-cell differentiation requires spermatogenic stage- and cell-specific gene expression that is achieved by unique chromatin remodeling, transcriptional control and the expression of testis-specific genes or isoforms. Recent findings have shown that the testis has specialized transcription complexes that coordinate the differentiation program of spermatogenesis. There are male germ cell-specific differences in the components of the general transcription machinery. These include upregulated expression of the TATA-binding protein (TBP) family and its associated cofactors. Importantly, a member of the TBP family, TBP-like factor (TLF), has a distribution pattern that is dependent on the spermatogenic cycle and is essential for spermatogenesis. Interestingly TBP-associated factor (TAF7), a factor of the transcription factor (TF)IID complex, is exchanged at a critical stage in germ cell development for the testis-specific paralogue TAF7L. A compelling amount of data has established that cAMP-response-element modulator (CREM), a transcription factor responsive to the cAMP signal transduction pathway, drives expression of key testis-specific genes. In this review we summarize recent advances in the transcription machinery that is testis-specific, gene-selective and necessary for the process of spermatogenesis.
Signal transducer and activator of transcription 1 (STAT1) is a critical mediator of interferon-gamma (IFN-gamma)-induced transcription that is regulated through posttranslational modifications and through transacting proteins such as protein inhibitor of activated STAT1 (PIAS1). PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases, and sumoylation has been identified as a modulatory mechanism for several transcription factors. Here we show that STAT1 is subject to SUMO-1 modification, and sumoylation occurs in vivo and in vitro at a single, evolutionary conserved amino acid residue Lys703. Members of the PIAS family of proteins were found to strongly stimulate sumoylation of STAT1. Furthermore, activation of STAT1 by IFN-gamma or pervanadate induced SUMO-1 conjugation. Mutation of Lys703 in STAT1 resulted in increased IFN-gamma-mediated transactivation, suggesting a negative regulatory function for sumoylation. These results indicate that STAT1 is covalently modified by SUMO-1 in cytokine signaling and that PIAS proteins promote SUMO-1 conjugation to STAT1.
The steroid receptor coactivator (SRC) proteins comprise a well-characterized family of nuclear receptor (NR) coactivators that increase transcriptional activation by NRs via covalent modification of chromatin proteins and recruitment of other coactivators. We have recently shown that the SRC family member GRIP1 interacts with a class of SUMO-1 (small ubiquitin-like modifier 1) E3 ligases, the PIAS proteins, and that the coactivator is subjected to SUMO-1 modifications (sumoylation). In this work, we demonstrate that lysine residues 239, 731, and 788 of GRIP1 serve as principal attachment sites for SUMO-1. Lys-731 and Lys-788 are located in the NR interaction domain (NID), and their substitution by arginines impairs the ability of GRIP1 to colocalize with androgen receptor (AR) in nuclei. Likewise, Lys-731 and Lys-788 mutants of GRIP1 have attenuated ability to enhance AR-dependent transcription and fail to synergize with PIASx beta-mediated activation of AR function, indicating that sumoylation modifies the ability of GRIP1 to function as a steroid receptor coactivator. The Lys-731 sumoylation site is conserved in SRC-3 and SRC-1, and the NIDs of the latter coactivators harbor one or two additional sites matching with the consensus sites for SUMO-1 attachment, respectively, suggesting a more general role for the modification in the regulation of SRC protein activity.
Androgen receptor (AR)-interacting protein 3 (ARIP3/PIASxalpha) is a coregulator capable of modulating transcriptional activity of various steroid receptors. We have characterized functional regions of ARIP3 and studied its interaction with the glucocorticoid receptor (GR)-interacting protein 1 (GRIP1). We find that the potential zinc-binding domain is critical for ARIP3 to function as a coactivator; the deletion of amino acids 347-418 or the mutation of the conserved cysteines 385 and 388 to serines converts ARIP3 to a transcriptional repressor from AR-dependent minimal promoters and abolishes its ability to activate GR. By contrast, mutations in the two LXXLL motifs of ARIP3 have relatively minor effects on its ability to regulate AR or GR function. ARIP3 is able to interact with different regions of GRIP1, but the strongest interaction is detected with the C-terminal region (amino acids 1122-1462) of GRIP1. The interaction of ARIP3 with the latter GRIP1 domain or full-length GRIP1 and the ability of ARIP3 to cooperate with GRIP1 in the regulation of AR- or GR-dependent transcription are dependent on the ARIP3 zinc-binding region. We also find a strong synergism between GRIP1 and two other PIAS family members, Miz1 and PIAS1. Taken together, our results suggest that PIAS proteins and GRIP1 interact functionally in transcriptional regulation.
PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins xalpha, xbeta, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASxalpha enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASxalpha on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.
DAX-1 (NROB1) is an atypical member of the nuclear receptor family that is predominantly expressed in mammalian reproductive tissues. While a receptor function of DAX-1 remains enigmatic, previous work has indicated that DAX-1 inhibits the activity of the orphan receptor steroidogenic factor 1 and the estrogen receptors (ERs), presumably via direct occupation of the coactivator-binding surface and subsequent recruitment of additional corepressors. In vivo evidence points at a particular role of DAX-1 for the development and maintenance of male reproductive functions. In this study, we have identified the androgen receptor (AR) NR3C4 as a novel target for DAX-1. We show that DAX-1 potently inhibits ligand-dependent transcriptional activation as well as the interaction between the N- and C-terminal activation domains of AR. We provide evidence for direct interactions of the two receptors that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Moreover, DAX-1, known to shuttle between the cytoplasm and the nucleus, is capable of relocalizing AR in both cellular compartments, suggesting that intracellular tethering is associated with DAX-1 inhibition. These results implicate novel inhibitory mechanisms of DAX-1 action with particular relevance for the modulation of androgen-dependent gene transcription in the male reproductive system.