Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells.

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our chromatin immunoprecipitation followed by sequencing (ChIP-seq) results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II (RNA Pol II Ser2P), and Ser5-phosphorylated RNA Pol II (RNA Pol II Ser5P). Furthermore, depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P.

Histone H3 lysine 36 methyltransferase Whsc1 promotes the association of Runx2 and p300 in the activation of bone-related genes.

The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1-/- embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression.

Urothelium-dependent and urothelium-independent detrusor contractility mediated by nitric oxide synthase and cyclooxygenase inhibition.

AIMS: The urothelium has been implicated in regulating detrusor smooth muscle contractility but the identity of the putative urothelium-derived inhibitory factor remains unconfirmed. There was inconclusive evidence on the role of nitric oxide synthase (NOS) and cyclooxygenase (COX) in mediating detrusor contractions. This study examined varying regulation by NOS and COX in transverse and longitudinal carbachol (CCh)-induced and unstimulated phasic contractions. METHODS: Rat detrusor strips with the urothelium-intact (+UE) and urothelium-denuded (-UE) were isolated in both transverse and longitudinal directions. Isometric tension of the detrusor strips was recorded both during stimulation with CCh and at the unstimulated state. In the unstimulated state, phasic contractile activity was measured. Tension recordings were made with and without the NOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) and COX inhibitor indomethacin (Indo). RESULTS: Only transverse +UE strips responded convincingly to L-NAME and Indo treatment, generating larger CCh-induced contractions. In unstimulated tissues, L-NAME treatment increased phasic amplitude in -UE strips only. Indo treatment failed to elicit any change in the amplitude but suppressed frequency of the phasic activity in transverse +UE strips. There was no significant Indo-mediated change in other strips. CONCLUSIONS: The data suggested heterogeneity in the regulation of directional detrusor contractility via NOS- and COX-associated mechanisms.

ß-Adrenoceptor-mediated differences in transverse and longitudinal strips from the rat detrusor.

Contractions and relaxations of the urinary bladder occur in all directions to facilitate urine release and storage. Transverse and longitudinal contractility of detrusor smooth muscle have been studied before using various pharmacologic stimuli but not ß agonists. Given the importance of ß-adrenoceptors in mediating bladder relaxation, the effects of isoprenaline (IPNA) in transverse and longitudinal contractility were examined. Pretreatment with a low concentration of IPNA (0.1 or 1 µM) suppressed carbachol (CCh)-induced contractions, more in the transverse than longitudinal direction. Increasing the IPNA concentration to 10 or 100 µM resulted in greater inhibition of longitudinal contractions. Also in the longitudinal direction, IPNA-induced relaxation was greater than in the transverse direction. When precontracted with a submaximal concentration of CCh (1 µM), IPNA increased the phasic activity in the longitudinal direction only. In summary, ß-adrenoceptor-mediated differences between transverse and longitudinal contractility were revealed. In testing the relaxant properties of selective ß-agonists, the findings here should be considered such that other than the conventional longitudinal contractions, measurements are also made in other directions.