The first bromodomain of the testis-specific double bromodomain protein Brdt is required for chromocenter organization that is modulated by genetic background

Mice homozygous for a mutation (Brdt^?BD1/?BD1) lacking the first bromodomain of Brdt, a testis-specific member of the BET family of double-bromodomain containing proteins, are sterile and exhibit profound defects in chromatin remodeling during spermiogenesis. We have now observed that a prominent feature of the aberrant spermatid nuclei is a fragmented chromocenter, a structure comprised of peri-centromeric heterochromatin. There was a concomitant increase in the levels of heterochromatin protein 1 alpha (Hp1α), suggesting that the presence of multiple chromocenters was correlated with a spread of heterochromatin beyond the normal centromeric region. Brdt protein was normally present throughout the nucleus but was excluded from the chromocenter. A more densely staining region of Brdt protein appeared to separate sirtuin 1 (Sirt1) protein from contact with the chromocenter. Although still nuclear, this unique localization of Brdt protein was lost in Brdt^?BD1/?BD1 mutant spermatids and Brdt and Sirt1 overlapped around the chromocenters. There was also ectopic localization of the H1 histone family, member N, testis-specific (H1fnt) protein in Brdt^?BD1/?BD1 round spermatids, which may be linked to the previously reported loss of polarized localization of peri-nuclear heterochromatin foci. The extent of chromocenter fragmentation was more severe and penetrant in mutant testes on a pure 129Sv/Ev as compared to a pure C57Bl/6 background. Indeed, all aspects of the mutant phenotype were more severe on the 129 Sv/Ev background. Contrary to previous studies in genetic models where fragmented chromocenters were observed in spermatids, the Brdt^?BD1/?BD1 mutant spermatids do not undergo apoptosis (on either background). These observations suggest that the first bromodomain of Brdt is critical in the formation and/or maintenance of an intact chromocenter and implicate this structure in proper remodeling of the chromatin architecture of the sperm head.     

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.     

Stk33 is required for spermatid differentiation and male fertility in mice

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.     

The full-length isoform of the mouse pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth

PHIP was isolated as an insulin receptor substrate 1 (IRS-1) interacting protein. To date, the physiological roles of PHIP remain unknown. Here we show that mice lacking PHIP1, the full-length isoform of PHIP, are born at normal size but suffer a 40% growth deficit by weaning. PHIP1 mutant mice develop hypoglycemia and have an average lifespan of 4-5 weeks. PHIP1-deficient mouse embryonic fibroblasts (MEFs) grow markedly slower than wild-type MEFs, but exhibit normal AKT phosphorylation and an increased cell proliferation in response to IGF-1 treatment. Together these results suggest that PHIP1 regulates postnatal growth in an IGF-1/AKT pathway-independent manner.     

The prion or the related Shadoo protein is required for early mouse embryogenesis

The prion protein PrP has a key role in transmissible spongiform encephalopathies but its biological function remains largely unknown. Recently, a related protein, Shadoo, was discovered. Its biological properties and brain distribution partially overlap that of PrP. We report that the Shadoo-encoding gene knockdown in PrP-knockout mouse embryos results in a lethal phenotype, occurring between E8 and E11, not observed on the wild-type genetic background. It reveals that these two proteins play a shared, crucial role in mammalian embryogenesis, explaining the lack of severe phenotype in PrP-knockout mammals, an appreciable step towards deciphering the biological role of this protein family.     

Brg1 is required for murine neural stem cell maintenance and gliogenesis

Epigenetic alterations in cell-type-specific gene expression control the transition of neural stem cells (NSCs) from predominantly neurogenic to predominantly gliogenic phases of differentiation, but how this switch occurs is unclear. Here, we show that brahma-related gene 1 (Brg1), an ATP-dependent chromatin remodeling factor, is required for the repression of neuronal commitment and the maintenance of NSCs in a state that permits them to respond to gliogenic signals. Loss of Brg1 in NSCs in conditional brg1 mutant mice results in precocious neuronal differentiation, such that cells in the ventricular zone differentiate into post-mitotic neurons before the onset of gliogenesis. As a result, there is a dramatic failure of astrocyte and oligodendrocyte differentiation in these animals. The ablation of brg1 in gliogenic progenitors in vitro also prevents growth-factor-induced astrocyte differentiation. Furthermore, proteins implicated in the maintenance of stem cells, including Sox1, Pax6 and Musashi-1, are dramatically reduced in the ventricular zones of brg1 mutant mice. We conclude that Brg1 is required to repress neuronal differentiation in NSCs as a means of permitting glial cell differentiation in response to gliogenic signals, suggesting that Brg1 regulates the switch from neurogenesis to gliogenesis.     

The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis

Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation.     

Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis

Mta1, a representative of the MTA gene family, is believed to be involved in the metastasis of malignant tumors. However, a systematic study of its physiological function has not been performed. It has been found in normal mouse organs at relatively low levels, except for in testis, suggesting a potential function in the male reproductive system. In order to explore the role of Mta1 protein during spermatogenesis, its expression in adult mouse testis was compared with that in developing mouse testis and in testis from adult mice treated with methoxyacetic acid, which selectively depletes primary spermatocytes. Quantitative analysis revealed that Mta1 protein gradually increased in the testis from 14 days postnatally. Immunolocalization analysis demonstrated strong signals in the seminiferous tubules, and Mta1 was predominantly present in the nucleus of primary spermatocytes and spermatogonia from 14 days postnatally. The most intensive staining was located in the nucleus of pachytene spermatocytes in mature testes. The expression pattern of Mta1 during spermatogenesis was also shown to be stage-specific by immunohistochemistry analysis. Finally, dramatic loss of Mta1 expression from pachytene spermatocytes was observed in the spermatogenic-arrested adult mouse testis. These results collectively demonstrate that Mta1 appears during postnatal testis development and suggest that this expression may be crucial for spermatogenesis. This study was supported by the Natural Science Foundation of China (2006: 30570982; 2003: 30370750; 2003: 30371584).     

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).     

A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 degrees C. To identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human SMU1 gene. Comparison of sequences of the Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39 degrees C. Furthermore, cdc2 kinase was not activated during G2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.     

Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes

During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid–liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.     

The transmembrane protein TMEM16A is required for normal development of the murine trachea

Pathological collapsibility of the upper airways, caused by many different genetic and environmental insults, is known as tracheomalacia in humans. We determined that Tmem16a, a member of an evolutionarily conserved family of predicted transmembrane proteins, is expressed in the developing trachea. We report that all mice homozygous for a null allele of Tmem16a died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea. In addition, the development of the trachealis muscle that spans the dorsal aspect of the trachea was abnormal in Tmem16a mutants. Since the chondrogenic mesenchyme does not express Tmem16a at any time, we propose that the cartilage ring defect observed in Tmem16a mutants is secondary to an expansion of the embryonic trachea that might result from improper stratification of the embryonic tracheal epithelium or the abnormal trachealis muscle. Our data identify Tmem16a as a novel regulator of epithelial and smooth muscle cell organization in murine development. This mutant, the first knockout of a vertebrate TMEM16 family member, provides a mouse model of tracheomalacia.     

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest.     

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.     

The tumor suppressor Chd5 is induced during neuronal differentiation in the developing mouse brain

Epigenetic regulation of gene expression orchestrates dynamic cellular processes that become perturbed in human disease. An understanding of how subversion of chromatin-mediated events leads to pathologies such as cancer and neurodevelopmental syndromes may offer better treatment options for these pathological conditions. Chromodomain Helicase DNA-binding protein 5 (CHD5) is a dosage-sensitive tumor suppressor that is inactivated in human cancers, including neural-associated malignancies such as neuroblastoma and glioma. Here we report a detailed analysis of the temporal and cell type-specific expression pattern of Chd5 in the mammalian brain. By analyzing endogenous Chd5 protein expression during mouse embryogenesis, in the neonate, and in the adult, we found that Chd5 is expressed broadly in multiple brain regions, that Chd5 sub-cellular localization undergoes a switch from the cytoplasm to the nucleus during mid-gestation, and that Chd5 expression is retained at high levels in differentiated neurons of the adult. These findings may have important implications for defining the role of CHD5-mediated chromatin dynamics in the brain and for elucidating how perturbation of these epigenetic processes leads to neuronal malignancies, neurodegenerative diseases, and neurodevelopmental syndromes.     

The INO80 complex is required for damage-induced recombination

The budding yeast INO80 complex has a role in remodeling chromatin structure, and the SWR1 complex replaces a H2A/H2B dimer with a variant dimer, H2A.Z (Htz1)/H2B. It has been reported that these chromatin remodeling complexes contain Arp4 (actin-related protein) and actin in common and are recruited to HO endonuclease-induced DNA double-strand break (DSB) site. Reportedly, Ino80 can evict nucleosomes surrounding a HO-induced DSB; however, it has no apparent role to play in the repair of HO-induced DSB. Here we show that an essential factor for INO80 chromatin remodeling activity, Arp8, is involved in damage-induced sister chromatid recombination and interchromosomal recombination between heteroalleles. In contrast, arp6 mutants are proficient for recombination, indicating that the SWR1 complex does not promote recombination. Our data suggest that the remodeling of chromatin by the INO80 complex facilitates efficient homologous recombination upon DNA damages.     

The tumor suppressor Chd5 is induced during neuronal differentiation in the developing mouse brain

Epigenetic regulation of gene expression orchestrates dynamic cellular processes that become perturbed in human disease. An understanding of how subversion of chromatin-mediated events leads to pathologies such as cancer and neurodevelopmental syndromes may offer better treatment options for these pathological conditions. Chromodomain Helicase DNA-binding protein 5 (CHD5) is a dosage-sensitive tumor suppressor that is inactivated in human cancers, including neural-associated malignancies such as neuroblastoma and glioma. Here we report a detailed analysis of the temporal and cell type-specific expression pattern of Chd5 in the mammalian brain. By analyzing endogenous Chd5 protein expression during mouse embryogenesis, in the neonate, and in the adult, we found that Chd5 is expressed broadly in multiple brain regions, that Chd5 sub-cellular localization undergoes a switch from the cytoplasm to the nucleus during mid-gestation, and that Chd5 expression is retained at high levels in differentiated neurons of the adult. These findings may have important implications for defining the role of CHD5-mediated chromatin dynamics in the brain and for elucidating how perturbation of these epigenetic processes leads to neuronal malignancies, neurodegenerative diseases, and neurodevelopmental syndromes.