Kyoichi Isono

Background: Because of the high frequency of chronic edema formation in the current “aged” society, analyses and detailed observation of post-surgical edema are getting more required. Post-surgical examination of the dynamic vasculature including L.V. (Lymphatic Vasculature) to monitor edema formation has not been efficiently performed. Hence, procedures for investigating such vasculature are essential. By inserting transparent sheet into the cutaneous layer of mouse tails as a novel surgery model (theTailEdema bySilicone sheet mediatedTransparency protocol; TEST), the novel procedures are introduced and analyzed by series of histological analyses including video-based L.V. observation and 3D histological reconstruction of vasculatures in mouse tails. Results: The dynamic generation of post-surgical main and fine (neo) L.V. connective structure during the edematous recovery process was visualized by series of studies with a novel surgery model. Snapshot images taken from live binocular image recording for TEST samples suggested the presence of main and elongating fine (neo) L.V. structure. After the ligation of L.V., the enlargement of main L.V. was confirmed. In the case of light sheet fluorescence microscopy (LSFM) observation, such L.V. connections were also suggested by using transparent 3D samples. Finally, the generation of neo blood vessels particularly in the region adjacent to the silicone sheet and the operated boundary region was suggested in 3D reconstruction images. However, direct detection of elongating fine (neo) L.V. was not suitable for analysis by such LSFM and 3D reconstruction procedures. Thus, such methods utilizing fixed tissues are appropriate for general observation for the operated region including of L.V. Conclusions: The current surgical procedures and analysis on the post-surgical status are the first case to observe vasculatures in vivo with a transparent sheet. Systematic analyses including the FITC-dextran mediated snap shot images observation suggest the elongation of fine (neo) lymphatic vasculature. Post-surgical analyses including LSFM and 3D histological structural reconstruction, are suitable to reveal the fixed structures of blood and lymphatic vessels formation.

Significance: Androgen plays essential roles for sex differentiation of external genitalia through regulating male-biased genes. Even females can differentiate into male-type external genitalia depending on the degree of androgenic exposure. However, the mechanisms underlying androgen responsiveness in females are poorly defined. Here, we demonstrate Sp1 regulates the enrichment of H3K27ac on androgen receptor-mediated regulatory elements in both males and females, indicating transcriptional competency for male-biased genes is conserved by Sp1 in both sexes. Compound-MafBmutant mice lacking a tissue-specific enhancer in theMafB3′ UTR show incomplete male-type external differentiation similar to female-type differentiation. These findings underscore the idea that transcriptional competency for male-biased genes is preserved in both sexes, thus helping define the potential sexual fate of external genitalia.

The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation ofPhc2in mice causes a severe defect in HSPC mobilization through the derepression ofVcam1in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 inPhc2-deficient mice reverses the symptoms. We further determine that Phc2-dependentVcam1repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5. These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.

The CCR4–NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre–B cell transition. CNOT3 regulated generation of germline transcripts in the V_H region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4–NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA.

The presence of somatic mutations in splicing factor 3b subunit 1 (SF3B1) in patients with Myelodysplastic syndromes with ring sideroblasts (MDS-RS) highlights the importance of the RNA-splicing machinery in MDS. We previously reported the presence of bone marrow (BM) RS in Sf3b1 heterozygous (Sf3b1+/−) mice which are rarely found in mouse models of MDS. Sf3b1+/− mice were originally engineered to study the interaction between polycomb genes and other proteins. We used routine blood tests and histopathologic analysis of BM, spleen, and liver to evaluate the hematologic and morphologic characteristics of Sf3b1+/− mice in the context of MDS by comparing the long term follow-up (15 months) of Sf3b1+/− and Sf3b1+/+ mice. We then performed a comprehensive RNA-sequencing analysis to evaluate the transcriptome of BM cells from Sf3b1+/− and Sf3b1+/+ mice. Sf3b1+/− exhibited macrocytic anemia (MCV: 49.5 ± 1.6 vs 47.2 ± 1.4; Hgb: 5.5 ± 1.7 vs 7.2 ± 1.0) and thrombocytosis (PLTs: 911.4 ± 212.1 vs 878.4 ± 240.9) compared to Sf3b1+/+ mice. BM analysis showed dyserythropoiesis and occasional RS in Sf3b1+/− mice. The splenic architecture showed increased megakaryocytes with hyperchromatic nuclei, and evidence of extramedullary hematopoiesis. RNA-sequencing showed higher expression of a gene set containing Jak2 in Sf3b1+/− compared to Sf3b1+/+. Our study indicates that Sf3b1+/− mice manifest features of low risk MDS-RS and may be relevant for preclinical therapeutic studies.

In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.