Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch.

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function.

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modification. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases.

Histologic findings of anterior cruciate ligament reconstruction with Achilles allograft.

The purpose of the current study was to evaluate the remodeling process of Achilles allograft for anterior cruciate ligament reconstruction under light microscopic and electron microscopic evaluations. In 12 patients, histologic examinations were done preoperatively and during arthroscopy at 6, 12, and 24 months. Gross observations were made with respect to width, vascularity, elasticity, and stability of the Achilles allograft. Biopsy specimens were obtained at the followup arthroscopic study. Light microscopic examinations done at 6 months showed many fibroblasts with spindle-shaped nuclei and hypercellularity. At 12 months, the small folds pattern of collagen bundles and decreased cellularity were observed. At 24 months, gross and findings of the light microscopic evaluations of the allograft tendons were similar to those of the normal anterior cruciate ligaments. On electron microscopic examinations done at 24 months, the allograft had fibroblasts with much cytoplasm and densely packed parallel-laid collagen fibrils, which showed the characteristic cross striations, but the Achilles allograft ligaments did not show similar findings compared with biopsy samples from normal anterior cruciate ligaments.

Extension block secondary to partial anterior cruciate ligament tear on the femoral attachment of the posterolateral bundle.

PURPOSE: The purpose of this investigation was to prove that a partial tear of the anterior cruciate ligament (ACL) at the femoral attachment of the posterolateral bundle can result in mechanical knee locking and trigger the injury mechanism of an isolated ACL injury. TYPE OF STUDY: Case series. METHODS: From February 1993 through June 1999, 19 cases of knee locking with observation of a torn ACL, confirmed under arthroscopy, were investigated. Patients' ages ranged from 22 to 54 years. The causes of injury were sporting activities in 14 patients (8 skiing, 4 soccer, and 2 badminton); slipping and falling in 2 cases; a pedestrian car accident in 2 cases; and an unknown low-velocity injury in 1 case. RESULTS: On physical examination, each patient indicated that the chief complaint was knee pain; all had a locked knee at 5 degree to 20 degree of full extension and 4 cases showed a locked knee at full flexion. Of the 19 cases, Lachman testing was positive in 3. With respect to intra-articular injuries, 3 patients had a tear in the medial meniscus and 1 had a tear in the lateral meniscus. The average time span between arthroscopy examinations was 3 months with a range of 1 to 10 months. Arthroscopic diagnosis and treatment were performed and meniscal tear was ruled out as the cause of locking in all patients. CONCLUSIONS: The mechanisms of injury for isolated ACL were knee hyperextension and internal rotation of lower extremity brought on especially by a low-velocity injury. The partial ACL tears were found on the femoral attachment site of the posterolateral bundle. The torn segment of the ACL was interposed between the lateral femoral condyle and the lateral tibial condyle and acted as a mechanical obstruction, giving rise to the locking symptom. In all 19 cases, the torn portion of the ACL was excised under arthroscopy and the locked knees were treated properly.