2The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing growth regimens. Before they can transcribe from specific DNA promoter sequences, bacterial core RNA polymerases (with subunit composition ␣ 2 ␤␤Ј) must combine with a dissociable sigma subunit () to form RNA polymerase holoenzyme (␣ 2 ␤␤Ј). Since the discovery of factors (6), it has become clear that these proteins are central to the function of the RNA polymerase holoenzyme. The reversible binding of alternative factors allows formation of different holoenzymes able to distinguish groups of promoters required for different cellular functions. In addition to double-strand DNA promoter recognition and binding, proteins are closely involved in promoter melting (e.g., references 31,36,49,51,74,76,128), inhibit nonspecific initiation, are targets for activators, and control early transcription through promoter clearance and release from RNA polymerase (48,49,53). Here we describe the functioning of the bacterial 54 -RNA polymerase that is the target for sophisticated signal transduction pathways (103) involving activation via remote enhancer elements (5, 95).Based on structural and functional criteria, the different factors identified in bacteria can be grouped in two classes, one of which has a single member, 54 . Many factors belong to the 70 class, the major factor which is involved in expression of most genes during exponential growth (72). 54 (also called N ) differs both in amino acid sequence and in transcription mechanism from the 70 class (80). Despite the lack of any significant sequence similarity, both types of bind the same core RNA polymerase. Nonetheless, they produce holoenzymes with different properties.With the recognition that the 54 protein represented an entirely new class of factor, what had once been regarded as an aspect of transcription restricted to higher organisms became a well-established feature of certain bacterial regulatory systems, particularly those associated with nitrogen metabolism. Activation of 54 -RNA polymerase employs specialized bacterial enhancer-binding proteins whose activating function requires nucleotide hydrolysis (94,96,122) (Fig. 1). In this system, initiation rates are controlled via regulation of the DNA melting step that is necessary for establishing the open promoter complex (85,94,97). Bacterial enhancer-dependent transcription can be studied with just two purified proteins (an activator and the 54 -RNA polymerase holoenzyme) and the appropriate DNA template, facilitating progress in understanding mechanistic aspects of 54 functioning. Below we review the biology and biochemistry of the 54 -RNA polymerase.

Upon fertilization, drastic chromatin reorganization occurs during preimplantation development . However, the global chromatin landscape and its molecular dynamics in this period remain largely unexplored in humans. Here we investigate chromatin states in human preimplantation development using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) . We find widespread accessible chromatin regions in early human embryos that overlap extensively with putative cis-regulatory sequences and transposable elements. Integrative analyses show both conservation and divergence in regulatory circuitry between human and mouse early development, and between human pluripotency in vivo and human embryonic stem cells. In addition, we find widespread open chromatin regions before zygotic genome activation (ZGA). The accessible chromatin loci are readily found at CpG-rich promoters. Unexpectedly, many others reside in distal regions that overlap with DNA hypomethylated domains in human oocytes and are enriched for transcription factor-binding sites. A large portion of these regions then become inaccessible after ZGA in a transcription-dependent manner. Notably, such extensive chromatin reorganization during ZGA is conserved in mice and correlates with the reprogramming of the non-canonical histone mark H3K4me3, which is uniquely linked to genome silencing. Taken together, these data not only reveal a conserved principle that underlies the chromatin transition during mammalian ZGA, but also help to advance our understanding of epigenetic reprogramming during human early development and in vitro fertilization.

Polycystic ovary syndrome (PCOS) is the most frequent endocrinopathy in women of reproductive age. It is difficult to treat PCOS because of its complex etiology and pathogenesis. Here, we characterized the roles of gut microbiota on the pathogenesis and treatments in letrozole (a nonsteroidal aromatase inhibitor) induced PCOS rat model. Changes in estrous cycles, hormonal levels, ovarian morphology and gut microbiota by PCR-DGGE and real-time PCR were determined. The results showed that PCOS rats displayed abnormal estrous cycles with increasing androgen biosynthesis and exhibited multiple large cysts with diminished granulosa layers in ovarian tissues. Meanwhile, the composition of gut microbiota in letrozole-treated rats was different from that in the controls. Lactobacillus, Ruminococcus and Clostridium were lower while Prevotella was higher in PCOS rats when compared with control rats. After treating PCOS rats with Lactobacillus and fecal microbiota transplantation (FMT) from healthy rats, it was found that the estrous cycles were improved in all 8 rats in FMT group, and in 6 of the 8 rats in Lactobacillus transplantation group with decreasing androgen biosynthesis. Their ovarian morphologies normalized. The composition of gut microbiota restored in both FMT and Lactobacillus treated groups with increasing of Lactobacillus and Clostridium, and decreasing of Prevotella. These results indicated that dysbiosis of gut microbiota was associated with the pathogenesis of PCOS. Microbiota interventions through FMT and Lactobacillus transplantation were beneficial for the treatments of PCOS rats.

The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded͞ single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.

Scope: The accumulation of misfolded α-synuclein in dopaminergic neurons is the leading cause of Parkinson’s disease (PD). Resveratrol (RV), a polyphenolic compound derived from grapes and red wine, exerts a wide range of beneficial effects via activation of sirtuin 1 (SIRT1) and induction of vitagenes. Here, we assessed the role of RV in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model of PD and explored its potential mechanisms. Methods and results: RV and EX527, a specific inhibitor of SIRT1, were administered before and after MPTP treatment. RV protected against MPTP-induced loss of dopaminergic neurons, and decreases in tyrosine hydroxylase and dopamine levels, as well as behavioral impairments. Meanwhile, RV administration activated SIRT1. Microtubule-associated protein 1 light chain 3 (LC3) was then deacetylated and redistributed from the nucleus to the cytoplasm, which provoked the autophagic degradation of α-synuclein in dopaminergic neurons. Furthermore, EX527 antagonized the neuroprotective effects of RV by reducing LC3 deacetylation and subsequent autophagic degradation of α-synuclein. Conclusion: We showed that RV ameliorated both motor deficits and pathological changes in MPTP-treated mice via activation of SIRT1 and subsequent LC3 deacetylation-mediated autophagic degradation of α-synuclein. Our observations suggest that RV may be a potential prophylactic and/or therapeutic agent for PD.

Results of binding assays using DNA fork junction probes indicate that sigma 54 contains multiple determinants that regulate melting to allow RNA polymerase to remain in closed promoter complexes in order to respond to enhancers. Gel mobility shift studies indicate that the -12 promoter element and parts of sigma 54 act together to form a molecular switch that controls melting. The DNA sequences and the sigma 54 N-terminus help direct polymerase to the location within the -12 promoter element where melting will initiate. However, the fork junction that would lead to melting does not form, due to the action of an inhibitory DNA element. Such unregulated melting is inhibited further by the lack of availability of the single-strand binding elements, which are needed to spread opening from the junction to the transcription start site. Thus, in the absence of looping enhancer protein, proper regulation is maintained as the sigma 54 polymerase remains bound in an inactive state. These complex protein-DNA interactions allow the controls over protein recruitment and DNA melting to be separated, enhancing the diversity of accessible mechanisms of transcription regulation.

Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both 54 and 70 holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that 54 imposes tighter regulation by creating a complex molecular switch at −12/−11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes 54 to alter its cross-linking pattern near −11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent 70 promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.

Background: The sperm DNA fragmentation index (DFI) is widely regarded as a key measure for assessing male fertility, but the predictive value of the DFI for outcomes of assisted reproductive technology (ART) remains under debate. In this study, we used a large sample to analyze the effect of sperm DFI on pregnancy outcomes following ART and its relationship with oocyte fertilization and embryo development in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). We also explore the value of sperm DNA fragmentation (SDF) and its associated factors in assessing male fertility. Methods:The relationship between the DFI measured with the sperm chromatin structure assay (SCSA) and pregnancy outcomes following ART were retrospectively analyzed in 2,622 ART treatment cycles, of which 1,185 were intrauterine insemination (IUI) cycles, 1,221 were IVF cycles and 216 were ICSI cycles.Rates of pregnancy, early abortion, oocyte fertilization and good quality embryos from IVF and ICSI cycles were compared between the groups of low DFI (DFI ≤15%), medium DFI (15%< DFI <30%) and high DFI (DFI ≥30%). Additionally, the relationships between sperm DFI and male lifestyle variables such as age, body mass index, smoking, and alcohol consumption, as well as routine semen parameters, were analyzed.Results: Clinical pregnancy rates following IUI among high, medium, and low sperm DFI groups were 12.5% (11/88), 14.3% (48/336), and 13.4% (102/761), respectively, with no statistical difference between the groups (P=0.88); however, early abortion rates among these groups were 27.3% (3/11), 14.6% (7/48), and 4.9% (5/102), respectively, and the difference was statistically significant (P=0.02). No significant differences in the rates of clinical pregnancy, early abortion, oocyte fertilization, or good quality embryos in IVF or ICSI cycles were detected among different DFI groups (P<0.05). Sperm DFI was negatively associated with sperm density, vitality and normal morphology; It was positively correlated with age, abstinence time and unhealthy lifestyles.Conclusions: As an increasingly common technique for reproductive testing, sperm DFI has proven to be very valuable in male fertility evaluation, but its significance as a predictor of pregnancy outcomes following ART requires further investigation.