Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells.

Krüppel-like factors (Klfs) have a pivotal role in maintaining self-renewal of mouse embryonic stem cells (mESCs). The functions of three Klf family members (Klf2, Klf4 and Klf5) have been identified, and are suggested to largely overlap. For further dissection of their functions, we applied an inducible knockout system for these Klf family members and assessed the effects of combinatorial loss of function. As a result, we confirmed that any one of Klf2, Klf4 and Klf5 was sufficient to support self-renewal, whereas the removal of all three compromised it. The activity of any single transcription factor, except for a Klf family member, was not sufficient to restore self-renewal of triple-knockout mESCs. However, some particular combinations of transcription factors were capable of the restoration. The triple-knockout mESCs were successfully captured at primed state. These data indicate that the pivotal function of a Klf family member is transduced into the activation of multiple transcription factors in a naïve-state-specific manner.

MEAF6 is essential for cell proliferation and plays a role in the assembly of KAT7 complexes.

Myst family genes encode lysine acetyltransferases that mainly mediate histone acetylation to control transcription, DNA replication and DNA damage response. They form tetrameric complexes with PHD-finger proteins (Brpfs or Jades) and small non-catalytic subunits Ing4/5 and Meaf6. Although all the components of the complex are well-conserved from yeast to mammals, the function of Meaf6 and its homologs has not been elucidated in any species. Here we revealed the role of Meaf6 utilizing inducible Meaf6 KO ES cells. By elimination of Meaf6, proliferation ceased although histone acetylations were largely unaffected. In the absence of Meaf6, one of the Myst family members Myst2/Kat7 increased the ability to interact with PHD-finger proteins. This study is the first indication of the function of Meaf6, which shows it is not essential for HAT activity but modulates the assembly of the Kat7 complex.

Distinct transcriptional programs of SOX2 in different types of small cell lung cancers.

SOX2 is recognized as an oncogene in human small cell lung cancer (SCLC), which is an aggressive neuroendocrine (NE) tumor. However, the role of SOX2 in SCLC is not completely understood, and strategies to selectively target SOX2 in SCLC cells remain elusive. Here, we show, using next-generation sequencing, that SOX2 expressed in the ASCL1-high SCLC (SCLC-A) subtype cell line is dependent on ASCL1, which is a lineage-specific transcriptional factor, and is involved in NE differentiation and tumorigenesis. ASCL1 recruits SOX2, which promotes INSM1 and WNT11 expression. Immunohistochemical studies revealed that SCLC tissue samples expressed SOX2, ASCL1, and INSM1 in 18 out of the 30 cases (60%). Contrary to the ASCL1-SOX2 signaling axis controlling SCLC biology in the SCLC-A subtype, SOX2 targets distinct genes such as those related to the Hippo pathway in the ASCL1-negative, YAP1-high SCLC (SCLC-Y) subtype. Although SOX2 knockdown experiments suppressed NE differentiation and cell proliferation in the SCLC-A subtype, they did not sufficiently impair the growth of the SCLC-Y subtype cell lines in vitro and ex vivo. The present results support the importance of the ASCL1-SOX2 axis as a main subtype of SCLC, and suggest the therapeutic potential of targeting the ASCL1-SOX2 axis.

Invasion of histiocytic sarcoma into the spinal cord of HTLV-1 tax transgenic mice with HTLV-1-associated myelopathy/tropical spastic paraparesis-like disease.

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Transgenic (Tg) mice expressing HTLV-1 Tax also develop T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. We found that 8 of 297 Tax-Tg mice developed HAM/TSP-like disease with symmetrical paraparesis of the hind limbs, but these symptoms were absent in non-Tg littermates and in other mice strains at our animal facilities. We could perform detailed evaluations for five of these mice. These evaluations showed that the disease was not inflammatory, unlike that in HAM/TSP patients, but instead involved the invasion of histiocytic sarcoma cells into the lumbar spinal cord from the bone marrow where they had undergone extensive proliferation.

Meiosis-specific ZFP541 repressor complex promotes developmental progression of meiotic prophase towards completion during mouse spermatogenesis.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.

VMAT2 Safeguards ß-Cells Against Dopamine Cytotoxicity Under High-Fat Diet-Induced Stress.

Vesicular monoamine transporter 2 (VMAT2) uptakes cytoplasmic monoamines into vesicles for storage. VMAT2 plays a role in modulating insulin release by regulating dopamine levels in the pancreas, although the exact mechanism remains elusive. We found that VMAT2 expression in ß-cells specifically increases under high blood glucose conditions. The islets isolated from ß-cell-specific Vmat2 knockout (ßVmat2KO) mice show elevated insulin secretion levels in response to glucose stimulation. Under prolonged high-fat diet feedings, the ßVmat2KO mice exhibit impaired glucose and insulin tolerance and progressive ß-cell dysfunction. Here we demonstrate VMAT2 uptake of dopamine to protect dopamine from degradation by monoamine oxidase, thereby safeguarding ß-cells from excess reactive oxygen species (ROS) exposure. In the context of high demand for insulin secretion, the absence of VMAT2 leads to elevated ROS in ß-cells, which accelerates ß-cell dedifferentiation and ß-cell loss. Therefore, VMAT2 controls the amount of dopamine in ß-cells, thereby protecting pancreatic ß-cells from excessive oxidative stress.

MEIOSIN Directs the Switch from Mitosis to Meiosis in Mammalian Germ Cells.

The mechanisms regulating meiotic initiation in mammals are enigmatic. It is known that retinoic acid (RA) signaling plays a pivotal role during meiotic initiation. STRA8, which is expressed in response to RA, is thought to be a key factor promoting meiotic initiation. However, the specific role of STRA8 in meiotic initiation has remained elusive. Here, we identified MEIOSIN as a germ-cell-specific factor that associates with STRA8. MEIOSIN, like STRA8, is expressed in response to RA and plays an essential role in meiotic initiation in both males and females. Functional analyses revealed that MEIOSIN acts as a transcription factor together with STRA8, and that both factors are critical for driving meiotic gene activation. Furthermore, temporally restricted expression of MEIOSIN leads to meiotic entry decision during spermatogenesis. The present study demonstrates that MEIOSIN, in collaboration with STRA8, plays a central role in regulating the mitosis to meiosis germ cell fate decision in mammals.

Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly.

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.

Natural infection of murine norovirus in conventional and specific pathogen-free laboratory mice.

Noroviruses cause most cases of acute viral gastroenteritis worldwide. The lack of a cell culture infection model for human norovirus necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict norovirus inactivation. Murine norovirus (MNV) may be used to construct a small animal model for studying the biology and pathogenesis of noroviruses because MNV is the only norovirus that replicates in cell culture and a small animal model. However, recent studies have shown that natural MNV infection is widespread in laboratory mouse colonies. We investigated MNV infection in both conventional and specific pathogen-free (SPF) genetically modified mice from Japan and the US, and commercial mice from several animal breeders in Japan, using serological and molecular techniques. MNV antibodies were detected in 67.3% of conventional mice and 39.1% of SPF mice from Japan and 62.5% of conventional mice from the US. MNV antibodies were also found in 20% of commercial SPF C57BL/6 mice from one of three breeders. Partial gene amplification of fecal isolates from infected animals showed that the isolates were homologous to reported MNV sequences. These results suggest that both conventional and SPF laboratory mice, including commercial mice, are widely infected with MNV, which might require considerable attention as an animal model of human disease.

Conserved origin of the ventral pancreas in chicken.

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.

Kinesin-II is not essential for mitosis and cell growth in Chlamydomonas.

The FLA10 gene product (Fla10p) in Chlamydomonas, a heterotrimeric kinesin-II, plays a crucial role in flagellar assembly as a motor protein driving intraflagellar transport. This protein has also been suggested to play a role in mitosis based on its localization to mitotic spindle. A role for Fla10p in mitosis has been difficult to test because to date only conditional (temperature-sensitive) mutant alleles were available, and it is not known whether these retain residual function for mitosis at the non-permissive temperature. In this report, we describe a null allele of fla10 produced by insertional mutagenesis. This mutant does not assemble flagella, but proliferates at a rate identical to that of wild type cells. Observation of microtubule organization in the cell body revealed that normal mitotic spindles are formed in dividing mutant cells. Thus, we conclude that FLA10 kinesin plays no significant roles in mitosis.