Search

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.

A shot noise suppression process of the emission-limited or the “soft” reset in an image sensor is investigated by analytically solving a master (Kolmogorov–Bateman) equation for a continuous-time Markov chain with correlated carrier emission rates. An explicit form of the probability distribution function, the hypoexponential type, is derived. An analytical form of the soft-reset (SR) noise is derived as the second-order moment, that is, variance, of the distribution function. It describes all the main characteristics of the SR noise previously reported; time-dependent shot noise suppression approaching an asymptotic limit of the half amount of noise charges as those of the fast or “hard” reset. The noise suppression mechanism is attributed to cascaded transitions of the storage node potential on a potential ladder giving the variance composed of a uniformly decreasing convergent series. The noise reduction factor is shown to be determined by the ladder spacing of a single-carrier charging potential. From the first-order moment analysis, an average time or lifetime dependence identical to that of the deterministic diode-decay equation is derived. Thus, the SR noise can be characterized by the governing probability law and its moments.

We present modeling and analysis of carrier dynamics in a single-photon avalanche diode (SPAD) operated with a capacitive quenching (CQ) method. The CQ method is regarded as a conventional resistive quenching (RQ) with its quenching resistance infinite or an open circuit. The SPAD is modelled as a lumped circuit consisting of a voltage dependent charge generator representing an avalanching depletion region and a capacitance of the depletion region and parasitic components. The carrier dynamics inside the device is described by time-dependent bipolar continuity equations (BCE) derived from the carrier continuity equations. We solve the BCE numerically with a 0.1 ps time resolution and investigate numbers of carriers in each circuit element as functions of time and of excess bias voltage (|𝑉ex|). We find two important characteristics of the CQ method; (1) a single-photon triggered Geiger-mode pulse is guaranteed to be quenched in a stable state (2) a voltage drop of the internal bias of SPAD due to the charges stored on the capacitance is proportional to |𝑉ex| with the proportionality factor of two. The results, in turn, enables one to design a SPAD free from after-pulse and from overflow. Such a SPAD pixel is shown to be compatible with a conventional complementary metal-oxide semiconductor (CMOS) image sensor (CIS) with a four transistors configuration pixel circuit. Finally, effectiveness of the present methodology is demonstrated by the subrange synthesis (SRS) time-of-flight (ToF) ranging experiments using a 6 μm size 400 × 400 pixels SPAD-based CIS.

N 6-methyladenosine (m6A), the most abundant modification in eukaryotic mRNAs, plays an important role in mRNA metabolism and functions. When adenosine is transcribed as the first cap-adjacent nucleotide, it is methylated at the ribose 2′-O and N6 positions, thus generating N6, 2′-O-dimethyladenosine (m6Am). Phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1) is a novel cap-specific adenine N6-methyltransferase responsible for m6Am formation. As PCIF1 specifically interacts with the Ser5-phosphorylated CTD of RNA polymerase II (Pol II), which is a marker for the early phase of transcription, PCIF1 is speculated to be recruited to the early elongating Pol II. In this study, subcellular fractionation and immunofluorescence microscopy demonstrated that PCIF1 is mainly localized to the transcriptionally active chromatin regions in HeLa cells. Chromatin immunoprecipitation (ChIP) revealed that PCIF1 was predominantly localized to the promoter of a broad range of Pol II-transcribed genes, including several protein-coding genes and non-coding RNA genes. Moreover, PCIF1 accumulation on these promoters depended entirely on transcriptional activity and Ser5 phosphorylation of the CTD. These results suggest that PCIF1 dynamically localizes to the Pol II early in transcription and may efficiently catalyze N6-methylation of the first adenosine residue of nascent mRNAs cotranscriptionally.

Long-range (~250m) measurement systems with 3D resolution are highly anticipated for various applications such as automotive, surveillance and robotics systems. Direct time-of-flight (ToF) systems based on CMOS image sensors (CIS) with avalanche photodiodes with the Geiger-mode or single-photon avalanche diodes (SPAD) have been developed for these purposes. One difficulty is to meet practical requirements of long-range (~250m) measurement capability together with both lateral and depth resolution. Although indirect-ToF systems based on modulation and phase sensitive detection have fine depth resolution of the order of sub-cm, their full range is limited only to ~4m with standard Si-photodiodes [1] and ~20m with the gain assist of APD [2]. Direct-ToF systems based on SPAD pixels with time-to-digital converters (TDC) have long range measurement capability but have not reached megapixel resolution [3], [4]. Another direct-ToF of sub-range syntheses (SRS) system, which detects single photons returned from an object located in each sub-range by synchronous gating, was demonstrated to have a long range (250m) measurement capability with high lateral resolution of 10cm. However, it has a depth resolution limited by the width of optical pulse source [5]. In order to circumvent this issue, in this work, we develop an SRS-ToF system based on a 6µm pitch, 1200×900pixels, Geiger-mode operated vertical avalanche photodiodes (VAPD) CIS that enables one to configure depth resolution down to 10cm for short-distance (

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

In eukaryotes, the Mediator complex has important roles in regulation of transcription by RNA polymerase II. Mediator is a large complex with more than 20 subunits that form head, middle, tail and CDK/cyclin modules. Among them, CDK8 and/or CDK19 (CDK8/19), and their counterpart cyclin C, form the CDK/cyclin module together with Mediator subunits MED12 and MED13. Despite evidences of both activation and repression, the precise functional roles of CDK8/19 in transcription are still elusive. Our previous results indicate that CDK8/19 recruits epigenetic regulators to repress immunoresponse genes. Here, this study focused on Toll-like receptors (TLRs), which exert innate immune responses through recognition of pathogen-associated molecular patterns and examined the functional roles of CDK8/19. As a result, CDK8/19 regulated transcription of inflammatory genes on stimulation of TLR9 in myeloma-derived RPMI8226 cells, which led to expression of inflammation-associated genes such as IL8, IL10, PTX3 and CCL2. Mediator subunits CDK8/19 and MED1, inflammation-related transcriptional activator NF-κB and C/EBPβ, and general transcription factors TFIIE and TFIIB colocalized at the promoter regions of these genes under this condition. Our results show that CDK8/19 positively regulates inflammatory gene transcription in cooperation with NF-κB and C/EBPβ on stimulation of TLR9.

The intensive development of Single-photon avalanche photodiode (SPAD) based CMOS image sensors (CIS) continues, with rapid progress [1–6]. Yet, due to unestablished quenching operation [5,6], realization of SPADs onto a CIS alongside conventional pixel circuitry has been a fundamental challenge.

INTRODUCTION N6-methyladenosine (m6A), an abundant modification in eukaryotic mRNAs and long-noncoding RNAs, has been recognized as a major epitranscriptome mark that plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start site of capped mRNAs in vertebrates. Previous studies reported that an eraser protein, FTO, demethylates N6-methyl group of m6Am and destabilizes a subset of mRNAs, suggesting a possible function of m6Am in stabilizing A-starting capped mRNAs. However, the biogenesis and functional role of m6Am remain elusive. RATIONALE To reveal the functional and physiological roles of m6Am, it is necessary to identify a writer protein for N6-methylation of m6Am. We first established a highly sensitive method to analyze 5′-terminal chemical structures of the capped mRNAs using mass spectrometry (RNA-MS), and then measured m6Am methylation status accurately. We employed RNA-MS to identify the writer gene by a reverse genetic approach. We chose several candidates of uncharacterized methyltransferases (MTases) that are conserved in vertebrates, but not in yeast, which does not have m6Am. Each of the candidates was knocked out in human cells. If the target gene is disrupted, RNA-MS can detect the absence of m6Am in mRNAs prepared from the knockout cells. RESULTS RNA-MS analysis revealed that m6Am modification in human mRNAs is more abundant (92%) than previously estimated. We identified human PCIF1 as cap-specific adenosine-N6-MTase (CAPAM) responsible for N6-methylation of m6Am. Indeed, m6Am disappeared completely and converted to Am modification in mRNAs prepared from the CAPAM knockout (KO) cells. The CAPAM KO cells were viable, but sensitive to oxidative stress, implying the physiological importance of m6Am. We showed that CAPAM catalyzes N6-methylation of m6Am in the capped mRNAs in an S-adenosylmethionine (SAM)–dependent manner. A series of biochemical studies revealed that CAPAM specifically recognizes the 7-methylguanosine (m7G) cap structure and preferentially N6-methylates m7GpppAm rather than m7GpppA, indicating the importance of the 2′-O-methyl group of the target site for efficient m6Am formation. CAPAM has a N-terminal WW domain that specifically interacts with the Ser5-phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), suggesting that the CAPAM is recruited to the early elongation complex of RNAPII and introduces m6Am in a nascent mRNA chain cotranscriptionally. We also solved the crystal structure of CAPAM complexed with the cap analog and SAM analog. The core region of CAPAM is composed of MTase and helical domains. The m7G cap is bound to a pocket formed by these two domains. The SAM analog is recognized by an active site with the characteristic NPPF motif in the MTase domain. This structure reveals the molecular basis of cap-specific m6A formation. RNA-sequencing analysis of the CAPAM KO cells revealed that loss of m6Am does not affect transcriptome alteration. This result does not support the proposed function of m6Am in stabilizing A-starting capped mRNAs. Instead, ribosome profiling of the CAPAM KO cells showed that N6-methylation of m6Am promotes the translation of capped mRNAs. CONCLUSION We identified PCIF1/CAPAM as a cap-specific m6A writer for vertebrate mRNAs. Structural analysis revealed the molecular basis of cap-specific m6A formation by CAPAM. The ribosome profiling experiment revealed that CAPAM-mediated m6Am formation promotes translation of A-starting mRNAs, rather than stabilization of mRNAs.

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit contains tandem repeats of the heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is subject to dynamic phosphorylation during transcription, mainly at serine residues (Ser2, Ser5 and Ser7). Regulation of CTD phosphorylation by specific kinases and phosphatases is crucial for coordinating transcription with RNA processing and histone modification. Human small CTD phosphatase 4 (SCP4), also called CTDSPL2 or HSPC129, is a putative CTD phosphatase belonging to the FCP/SCP family and implicated in control of ε- and γ-globin gene expression. Here, we report the biochemical and functional characterization of SCP4. SCP4 exhibited Ser5-preferential CTD phosphatase activity in vitro, while small interfering RNA-mediated SCP4 knockdown in HeLa cells increased phosphorylation levels of Pol II at Ser5 and Ser7, but not at Ser2. Furthermore, cell fractionation, chromatin immunoprecipitation and immunofluorescence assays revealed an exclusive localization for SCP4 in the chromatin, particularly at transcriptionally silenced chromosomal regions. Interestingly, SCP4 was gradually released from the chromatin fraction during hemin-induced erythroid differentiation of K562 cells, with concomitant cytoplasmic accumulation. Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation.

We developed a stacked CMOS image sensor overlaid with a thin-film gallium oxide (Ga2O3)/crystalline selenium (c-Se) heterojunction photodiode. Uniform c-Se films laminated on the CMOS circuits were fabricated through a low-temperature (200 °C) tellurium-diffused crystallization process. This stacking structure has several advantages, such as a high aperture ratio, low crosstalk, and high sensitivity. We successfully controlled the size of the polycrystalline particles to much smaller than the pixel size of the image sensors by adjusting the thickness of the c-Se films. Therefore, we herein present the first high-resolution images obtained with such a device. Furthermore, the dark current, which is induced by the carrier injection from an external electrode, was significantly reduced by employing a wide bandgap Ga2O3 film as a high hole-injection barrier.

We present an analysis of carrier dynamics of the single-photon detection process, i.e., from Geiger mode pulse generation to its quenching, in a single-photon avalanche diode (SPAD). The device is modeled by a parallel circuit of a SPAD and a capacitance representing both space charge accumulation inside the SPAD and parasitic components. The carrier dynamics inside the SPAD is described by time-dependent bipolar-coupled continuity equations (BCE). Numerical solutions of BCE show that the entire process completes within a few hundreds of picoseconds. More importantly, we find that the total amount of charges stored on the series capacitance gives rise to a voltage swing of the internal bias of SPAD twice of the excess bias voltage with respect to the breakdown voltage. This, in turn, gives a design methodology to control precisely generated charges and enables one to use SPADs as conventional photodiodes (PDs) in a four transistor pixel of a complementary metal-oxide-semiconductor (CMOS) image sensor (CIS) with short exposure time and without carrier overflow. Such operation is demonstrated by experiments with a 6 &micro, m size 400 &times, 400 pixels SPAD-based CIS designed with this methodology.

We have developed a direct time-of-flight (TOF) 250 m ranging Complementary Metal Oxide Semiconductor (CMOS) image sensor (CIS) based on a 688 &times, 384 pixels array of vertical avalanche photodiodes (VAPD). Each pixel of the CIS comprises VAPD with a standard four transistor pixel circuit equipped with an analogue capacitor to accumulate or count avalanche pulses. High power near infrared (NIR) short (&lt, 50 ns) and repetitive (6 kHz) laser pulses are illuminated through a diffusing optics. By globally gating the VAPD, each pulse is counted in the in-pixel counter enabling extraction of sub-photon level signal. Depth map imaging with a 10 cm lateral resolution is realized from 1 m to 250 m range by synthesizing subranges images of photon counts. Advantages and limitation of an in-pixel circuit are described. The developed CIS is expected to supersede insufficient resolution of the conventional light detection and ranging (LiDAR) systems and the short range of indirect CIS TOF.

We have developed a direct time-of-flight (TOF) 220 m ranging CMOS image sensor (CIS) based on a 688 × 384 pixel array of vertical avalanche photodiodes. An area scene at 220 m ahead can be ranged and imaged by one high power light pulse illuminated through diffusing (without scanning) optics and sub-photon level signals are extracted by in-pixel pulse count circuits. Depth map imaging with a 10 cm lateral resolution and with a 30 cm depth precision is realized from 1 m to 220 m range. The developed direct-TOF CIS with SPSE is expected to supersede the insufficient resolution of the conventional light detection and ranging (LiDAR) systems and the short range of indirect CIS TOF.

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation.

In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.

We present a sensitivity-boosting technique by incorporating an avalanche photodiode into a normal photo-conversion region. Under a dark scene, an avalanche photodiode operation is selected, where the photo-electrons are multiplied up to 105 electrons. Under a bright scene, a photodiode operation is selected, where photo-electrons in proportional to light intensity are generated in a similar way to conventional CMOS image sensors. Alternating the two operations enables wide operational range extending to dark conditions.

Continuous improvements in sensitivity have opened up applications for image sensors such as camcorders, digital still cameras, mobile phones, and surveillance cameras. Even though leading-edge image sensors have reached the noise floor of a few electrons [1,2], a thrust towards darker levels still continues down to an illumination level equivalent to being under a crescent moon (i.e., 10−2 down to 10−4 lux). This requires single-photon detection with typical digital cameras pixel size, i.e., 1.5 to 5µm. Although huge-size pixel [3] or single-photon avalanche photodiode (SPAD) based image sensors [4] have been presented for such a purpose, in general, both have to pay area and dark current penalties. Thus, an image sensor capable of both single-photon detection and normal imaging providing us with a high dynamic range is a huge technological challenge.

The Mediator complex consists of more than 20 subunits. This is composed of four modules: head, middle, tail and CDK/Cyclin. Importantly, Mediator complex is known to play pivotal roles in transcriptional regulation, but its molecular mechanisms are still elusive. Many studies, including our own, have revealed that CDK8, a kinase subunit of the CDK/Cyclin module, is one of the key subunits involved in these roles. Additionally, we previously demonstrated that a novel CDK component, CDK19, played similar roles. It is assumed that various factors that directly affect transcriptional regulation target these two CDKs; thus, we conducted yeast two-hybrid screenings to isolate the CDK19-interacting proteins. From a screening of 40 million colonies, we obtained 287 clones that provided positive results encoded mRNAs, and it turned out that 59 clones of them encoded nuclear proteins. We checked the reading frames of the candidate clones and obtained three positive clones, all of which encoded the transcriptional cofactors, Brahma-related gene 1, B-cell CLL/lymphoma 6 and suppressor of zeste 12 homolog. Intriguingly, these three cofactors are also related to chromatin regulation. Further studies demonstrated that those could bind not only to CDK19 but also to CDK8. These results help elucidate the functional mechanism for the mutual regulations between transcription and chromatin.

We demonstrate a stacked complementary metal-oxide semiconductor (CMOS) image sensor overlaid with a chlorine (Cl)-doped crystalline selenium (c-Se) photoconversion layer. The size of the polycrystalline particles (grains) of c-Se, which is strongly related to dark current pattern noise, is controlled by Cl doping to c-Se; hence, the resulting device provides clear images. Furthermore, avalanche multiplication was successfully observed in a Cl-doped c-Se film fabricated on a glass substrate at a relatively low applied voltage.

Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate the in vivo transcriptional activities of the complexes, we used a luciferase assay in combined with siRNA-mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16-dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context-specific transcriptional regulator.

NF 3 was decomposed over various ortho, pyrophosphates and some fluorides at 450–600 K. Mn 2 P 2 O 7 and CePO 4 were the most active. The catalytic activity of Co 2 P 2 O 7 , Co 3 (PO 4 ) 2 , CrPO 4 , Cu 2 P 2 O 7 , and Cr 4 (P 2 O 7 ) 3 followed in this order. Zn 3 (PO 4 ) 2 and AlPO 4 were less active. Activity of pyrophosphate was comparable to that of ortho-phosphate. Products were NO 2 and a very small amount of N 2 O except for HF. An essential reaction was 2NF 3 + 3H 2 O → 6HF + NO + NO 2 . Catalytic activity of phosphates was proportional to not the amount but the concentration of surface hydroxyls, suggesting that the reaction proceeds via a bidentate surface intermediate.

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit undergoes reversible phosphorylation during transcription cycle. The phosphorylated CTD plays critical roles in coordinating transcription with chromatin modification and RNA processing by serving as a scaffold to recruit various proteins. Recently, we identified a novel human WW domain-containing protein PCIF1 as a phosphorylated CTD-interacting factor and demonstrated that PCIF1 negatively modulates Pol II activity in vivo. In the present study, to explore cellular functions of PCIF1, we generated PCIF1-deficient chicken DT40 cell lines. We observed significant up-regulation of WW domain-containing prolyl isomerase Pin1 in two independently established PCIF1-deficient mutant clones. As reconstitution of PCIF1 in the mutants did not reduce Pin1 expression, PCIF1 may not be a negative regulator of Pin1 expression. We assume that Pin1 over-expression might suppress defects caused by PCIF1 deficiency in DT40 cells. We furthermore compared PCIF1 and Pin1 for their functional properties and found that these two proteins exhibit most similar target specificity among other CTD-binding WW proteins, overlapping subcellular localization and comparative inhibitory effects on transcriptional activation by Pol II in human cultured cells. These results suggest that Pin1 may have overlapping cellular function with PCIF1 in vertebrate cells.

Phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) regulates transcription cycle and coordinates recruitment of RNA processing factors and chromatin regulators. Recently, we reported the identification of human PCIF1 as a novel protein that directly binds to the phosphorylated CTD via its WW domain, which is highly homologous to the WW domain of human peptidylprolyl isomerase Pin1. Although PCIF1 has been shown to associate with phosphorylated Pol II, functional consequence of the interaction remains unclear. Here we further characterized the cytological, structural, and functional properties of human PCIF1. Immunofluorescence microscopy revealed that endogenous PCIF1 was colocalized with the phosphorylated Pol II and the transcription elongation factor DSIF in the cell nucleus. We also found that PCIF1 WW domain inhibits the CTD phosphatase activity of SCP1 in vitro. By examining the effect of either PCIF1 overexpression or knockdown on the transactivation of reporter gene expression by various transcriptional activation domains, we found that PCIF1 significantly repressed the transactivation depend on its CTD binding ability. These data suggest that PCIF1 modulates phosphorylation status of the CTD and negatively regulates gene expression by Pol II.

RNA polymerase II (Pol II) is the only polymerase to possess heptapeptide repeats in the C-terminal domain (CTD) of its large subunit. During transcription, CTD phopshorylation occurs and is maintained from initiation to termination. To date, among the three known CTD kinases possessing CDK-cyclin pairs, TFIIH is the only one that forms a preinitiation complex. The Mediator complex plays essential roles in transcription initiation and during the transition from initiation to elongation by transmitting signals from transcriptional activators to Pol II. P-TEFb specifically plays a role in transcription elongation. TFIIH and mediator phosphorylate serine 5 (Ser5) of the CTD heptapeptide repeat sequence, whereas P-TEFb phosphorylates serine 2 (Ser2). Recently, it has become clear that CTD phosphorylation is not only essential for transcription, but also as a platform for RNA processing and chromatin regulation. In this review, we discuss the central role of Pol II phosphorylation in these nuclear events.

To examine a possible mechanism of endogenous estrogen-induced carcinogenesis, we studied the effect of the catechol-O-methyltransferase (COMT) inhibitor Ro 41-0960 on cell transforming and clastogenic activities of 2 catechol estrogens 2- and 4-hydroxyestrone (2- or 4-OHE1) using Syrian hamster embryo (SHE) cells. COMT activity was assayed by determining the methylation of 2- or 4-OHE1 using gas chromatography. The production of 2-methoxyestrone in cultures treated with 2-OHE1 was approximately 2-fold that of 4-methoxyestrone in cultures treated with 4-OHE1. 4-OHE1 induced morphological transformation at a higher frequency than 2-OHE1 did and the frequencies of cell transformation and chromosome aberrations were not significantly changed in cells treated with 4-OHE1 in the presence of Ro 41-0960. In contrast, the frequencies of cell transformation and chromosome aberrations were markedly increased in cells treated with 2-OHE1 along with Ro 41-0960 when compared to cells treated with 2-OHE1 alone. In addition, both catechol estrogens induced P53 protein expression and apoptosis. The frequencies of apoptotic cells induced by the catechol estrogens were modified by the COMT inhibition in a manner similar to those observed with the chromosome aberrations assay and the cell transformation assay, indicating that each effect by the catechol estrogens at the three measured endpoints might be caused by a mechanism similar to the others. Our findings indicate that COMT activity has an influence on cell transforming activity and its related genetic effects of catechol estrogens in SHE cells, which implies that an individual activity of COMT may be one of the etiological factors in endogenous estrogen-induced carcinogenesis.

The human thyroid hormone receptor-associated proteins (TRAP)/Mediator and related complexes mediate transcription through regulatory factors. To further understand the structural and functional diversity of these complexes we established three HeLa cell lines each expressing one of three epitope-tagged human TRAP/Mediator subunits, MED6, MED7, and CDK8 and isolated the complexes in which these subunits were contained by affinity and HPLC-gel filtration chromatography. The largest complexes from each cell line had a molecular mass of 1.5 MDa and possessed almost identical subunit compositions; we designated these complexes TRAP/Mediator-like complex 1 (TMLC1). Two potential subcomplexes were additionally observed: a 1-MDa complex from the CDK8-cell line (TMLC2) and a 600-kDa complex from the MED6-cell line (TMLC3). All three complexes regulated transcription in vitro; TMLC1 and TMLC3 augmented transcriptional activation, whereas TMLC2 repressed it. TMLC1 and TMLC2 phosphorylated RNA polymerase II (Pol II), but TMLC3 did not. Furthermore, TMLC1 predominantly interacted with the general transcription factors TFIIE, TFIIF, and TFIIH, which function during transcription initiation and the transition to elongation. In a final experiment, knockdown of CDK8 using RNA interference prevented transcriptional activation by Gal4-VP16 in a luciferase-assay. This, together with the effect of TMLC1 on transcription in vitro, suggests that CDK8 play positive roles in transcriptional activation.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD of Pol II undergoes reversible phosphorylation during the transcription cycle, mainly at Ser2, Ser5, and Ser7. Dynamic changes in the phosphorylation patterns of the CTD are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Human RNA polymerase II-associated protein 2 (RPAP2) was originally identified as a Pol II-associated protein and was subsequently shown to function as a novel Ser5-specific CTD phosphatase. Although a recent study suggested that RPAP2 is required for the efficient expression of small nuclear RNA genes, the role of RPAP2 in controlling the expression of protein-coding genes is unknown. Here, we demonstrate that the C-terminal region of RPAP2 interacts directly with the Pol II subunit Rpb6. Chromatin immunoprecipitation analyses of the MYC and GAPDH protein-coding genes revealed that RPAP2 occupied the coding and 3apos; regions. Notably, siRNA-mediated knockdown of RPAP2 caused defects in 3apos;-end formation of the MYC and GAPDH pre-mRNAs. These results suggest that RPAP2 controls Pol II activity through a direct interaction with Rpb6 and participates in pre-mRNA 3apos;-end formation.

We present results of some novel AlGaN/GaN heterojunction field-effect transistors (HFETs) specifically developed for RF front-end and power applications. To reduce the parasitic resistance, two unique techniques: selective Si doping into contact area and a superlattice (SL) cap structure, are developed. With the selective Si doping method, a transistor with an on-state resistance as low as 1.86 Ω·mm and a Tx/Rx switch IC with very low insertion loss (0.26 dB) and very high power handling capability (P1dB over 40 dBm) were obtained. With the SL cap HFETs, an ultra low source resistance of 0.4 Ω·mm was achieved and excellent DC and RF performances were demonstrated. The typical characteristics of these HFETs are: maximum transconductance of over 400 mS/mm, maximum drain current of 1.2 A/mm, cut-off frequency of 60 GHz, maximum oscillation frequency of 140 GHz, and a very low noise figure of 0.7 dB with 15 dB gain at 12 GHz. For power applications, in order to significantly reduce fabrication cost, we fabricated the AlGaN/GaN HFET on a conductive Si substrate with a source-via grounding (SVG) structure. The device has a very low on-state sheet resistance of 1.9 mΩ·cm2, a high off-state breakdown voltage of 350 V, and a current handling capability of 150 A. In addition, a sub-nano second switching response with t r of 98 ps and t f of 96 ps with a current density as high as 2.0 kA/cm2 is demonstrated for the first time.

L-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked L-proline dehydrogenase (L-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and p...