Bifidobacterium breve UCC2003. These TFs appear to not only control transcription of genes involved in the bifid shunt and each other, but also seem to commonly and directly affect transcription of other TF-encoding genes, as well as genes related to uptake and metabolism of various carbohydrates. This complex and interactive network of AraQ/MalR1-mediated gene regulation provides previously unknown insights into the governance of carbon metabolism in bifidobacteria.Bifidobacteria resident in the gastrointestinal tract (GIT) are subject to constantly changing environmental conditions, which require rapid adjustments in gene expression. Here, we show that two predicted LacI-type transcription factors (TFs), designated AraQ and MalR1, are involved in regulating the central, carbohydrate-associated metabolic pathway  of the gut commensal  Of particular interest is the genus Bifidobacterium, a member of the Actinobacteria phylum, that represent Gram-positive, non-motile, non-spore forming, saccharoclastic anaerobic bacteria that commonly colonize the gut of a wide variety of animals, including birds, insects and mammals3. Several bifidobacterial strains/species are designated as probiotic, which means that when such bacteria are administered in adequate and viable numbers, they confer a health benefit to their (human) host4. These benefits include, among others, modulation of the host immune response5, mitigation of lactose intolerance, the ability to lower serum cholesterol levels in humans8, pathogen inhibition/exclusion9 and prophylaxis of tumour growth in certain cancers12. Bifidobacteria are among the first and predominant colonisers of the infant GIT, and persist throughout adulthood though at lower relative abundance (compared to the infant gut), with a further reduction in the elderly gut15.The microbial communities within the human gastrointestinal tract (GIT), commonly referred to as the gut microbiota, have enjoyed intense scientific scrutiny and are predominantly made up of members of the bacterial phyla Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria17. These estimates are consistent with the well-documented ability of bifidobacteria to utilise a diverse array of carbohydrates18. This remarkable genetic dedication to a particular metabolic behaviour probably reflects the importance of carbohydrate utilisation for bifidobacterial gut colonization and persistence. Just as important is their ability to control expression of the genes involved in the associated metabolic pathways in order to prevent wasting energy and resources on something which is unnecessary or inefficient. This is a widespread phenomenon, exemplified by the lac operon of Escherichia coli, which is only switched on if the substrate lactose is present and if a better (i.e. more energy generating) substrate such as glucose is absent19.Approximately 8–15% of the bifidobacterial coding capacity is dedicated to carbohydrate uptake and metabolism, depending on species and functional gene assignment20, while metabolic intermediates of carbon metabolism (such as pyruvate) are used as precursors for a range of biosynthetic processes. Carbohydrate metabolism of bifidobacteria is performed by a rather unique pathway, the so-called phosphoketolase pathway or bifid shunt, which allows this taxonomic group to produce more energy in the form of ATP from carbohydrates than fermentative pathways employed by other bacteria21. From 1 mol of glucose the bifid shunt theoretically yields 2.5 mol ATP, along with 1.5 mol of acetate and 1 mol of lactate22, whereas for example homofermentative lactic acid bacteria (LAB) produce 2 mol of ATP and 2 mol of lactic acid per mol of glucose23. This superior ATP production ability is due to the presence of a phosphoketolase called X5P/F6P phosphoketolase  which is unique in exhibiting comparable affinities for both xylulose 5-P (X5P) and fructose 6-P (F6P)24. The bifid shunt has been investigated in terms of its enzymatic functions in animal-derived species, such as Bifidobacterium globosum26, and it has been established that the bifid shunt is indeed the sole carbohydrate metabolism pathway to satisfy all energy demands in Bifidobacterium animalis subsp. lactis Bb-12 when grown on either glucose or lactose24. All members of the Bifidobacteriaceae family are said to possess xfpK homologs, and thus employ the bifid shunt27. Phylogenetic and protein modelling studies have recently suggested that the bifidobacterial XFPK enzyme is related to that found in members of the Coriobacteriales order and that xfpK was horizontally transferred between (an ancestor of) these two groups29. Another interesting difference between bifidobacteria  and other bacteria is that they lack a number of enzymes, such as phosphofructokinase, which are crucial to control central carbohydrate metabolism30.Additionally, carbon flux control enables bifidobacteria to balance energy and biosynthetic demands, thereby allowing optimal growth and redox balance management, as based on environmental conditions. This control is of crucial importance to bifidobacteria, as carbohydrate metabolism is their sole mechanism to produce ATP by substrate phosphorylation39. Several carbohydrate metabolism-related LacI-type regulators are known in Bifidobacterium breve UCC200337 and are typically involved in local (i.e. the gene encoding the LacI-type regulator is located in the vicinity of the genes it regulates) transcriptional regulation of genes that are responsible for the metabolism of a particular carbohydrate. These LacI-type transcription factors (TFs) typically limit , yet may sometimes enhance (then designated as activators), access or progression of the RNA polymerase to the promoter region of the regulated gene. This ability to act as a repressor or activator depends on a number of factors such as the location of the Transcription Factor Binding Site (TFBs) of the TF with respect to the -10/-35 sequences of the promoter region and the presence or absence of effector molecules, which may bind to the TF to alter its conformation and consequently its DNA-binding ability  are involved in the metabolism of starch-like carbohydrates, while AraQ was envisaged to be responsible for transcriptional control of genes encoding a number of bifid shunt enzymes40. We show here that two of the previously identified LacI-type TFs appear to be directly involved in transcriptionally regulating genetic components of the bifid shunt, as well as genes involved in a range of specific carbohydrate metabolic pathways, thereby revealing a previously undiscovered global carbohydrate control network, which is predicted to operate in many bifidobacterial species.It had previously been predicted that the B. breve UCC200340. In the course of our experiments we found that MalR1 was in fact functionally more similar to AraQ and for this reason our experimental scope was narrowed to specifically characterise AraQ and MalR1. The encoded products of araQ and malR1 represent proteins of 371 and 338 amino acids, respectively, which exhibit 26% overall identity to each other (91% query coverage). Their respective DNA binding domains  exhibit a higher level of identity at 49% (93% query coverage) (BlastP). This may be a reflection of the conserved nature of the DNA-binding helix-turn-helix regions of LacI-type proteins in general41, coupled to the finding that, as will be outlined below, AraQ and MalR1 recognize (near) identical operator sequences.This study was initially aimed at the functional characterization of six previously described LacI-type transcription factors (TFs), namely AraQ, MalR1, MalR2, MalR3, MalR5 and MalR6 along with their proposed regulation of central metabolic pathways and malto-oligosaccharide utilisation pathways in 40, and to a number of  genes that were identified in microarray analyses involving insertion mutants of araQ and malR1 (discussed below). Obtaining purified and active AraQ or MalR1 protein from B. breve initially proved to be troublesome, in line with similar issues observed for other bifidobacterial LacI-type proteins42. We adapted our original protocol so as to allow purification of active (i.e. DNA-binding competent) AraQ and MalR1 , which were tested for their ability to bind to DNA sequences encompassing the operator sequence or transcription factor binding site (TFBS) previously predicted for AraQ40. The ability of AraQ and MalR1 to bind DNA fragments was graded utilising a non-linear, arbitrary scale . This EMSA analysis revealed that AraQ and MalR1 bind with varying degrees of affinity to the promoter regions of 23 and 20 genes, respectively, out of a total of 33 DNA regions tested. Figure araQ/malR1 mutants (see below), indicating that such differential transcription reflects indirect regulatory effects.In order to assess AraQ and MalR1 interaction with their possible operator sequences, electrophoretic mobility shift assays (EMSAs) were carried out using DNA fragments encompassing upstream regions of genes corresponding to the previously predicted AraQ and MalR1 regulons43 . The ability of MalR1 to transcriptionally control genes that are required for the metabolism of starch-like/-derived carbohydrates is consistent with in silico predictions40. However, MalR1’s presumed ability to control genes involved in central carbon metabolism was an unexpected finding, as these genes were not predicted to be members of MalR1’s predicted regulon40.Six of the genes/gene clusters whose promoter regions were clearly shown to be bound by AraQ and MalR1 encode enzymes that are part of the bifid shunt as illustrated in the schematic representation of central metabolism in Fig. Based on their binding behaviour AraQ and MalR1 appear to have a similar affinity for the six promoter regions of the central metabolism genes Table . Furtherin-vitro affinity for various promoter regions, including those that correspond to several central metabolic genes. In order to identify the TFBSs that are recognized by either AraQ or MalR1, fragmentation analysis was carried out  and pyk (encoding pyruvate kinase and corresponding to locus tag Bbr_0757). These genes were selected as they which hold key roles in central carbon metabolism , while the 48-bp DNA segment located upstream of the pyk gene contains a very similar 20 bp palindrome CAGTGTGAGC><GCTCACAACA.As shown above, EMSA analyses employing AraQ and MalR1 clearly demonstrate their ism Fig. . DNA fraB. breve. For this task two PWM’s were used , resulting in the identification of several additional genes/gene clusters that appear to contain this TFBS in their predicted promoter region . The full list of genes assigned to the AraQ/MalR1 regulon is present in Supplemental Table Bifidobacteriaceae45. Some B. breve-specific genes of the AraQ/MalR1 regulon include Bbr_0037/0038 , Bbr_0221-Bbr_0222 (iron-uptake system BfeU-BfeO)47, Bbr_0603 (predicted cholate transporter Ctr), Bbr_1316 and Bbr_1685   approach was employed to identify additional genes which may be regulated by AraQ and MalR1 in eno and pyk), which form the core of the regulon. Furthermore, this analysis found that AraQ and MalR1 bind with the highest affinity to promoter regions of genes involved in central metabolism. This observed high affinity may be explained by the conserved G, C, and A in positions 9, 12 and 15, respectively, of the AraQ/MalR1 binding motif  away from the transcriptional start sites (TSS). Finally, analysis of the deduced TSS with respect to the location of the TFBSs of AraQ/MalR1 upstream of carD, glgP and cldE revealed that the TFBS is overlapping with predicted −35/−10 regions, indicating that AraQ/MalR1 in these cases act as repressors, being more typical of LacI-type TFs48.Interestingly, the deduced TFBSs of AraQ and MalR1 are located upstream of recently assigned −10/−35 promoter sites of six bifid shunt-associated genes/gene clusters Table 24. This araQ DNA promoter region, in the presence of each of a variety of possible (available/relevant) effectors , or perhaps that the molecule is the product of another metabolic pathway outside of central carbon metabolism.Bioinformatic and EMSA analyses allowed the deduction of the AraQ/MalR1 regulons and associated operator sequences. We next wanted to gain further insight into the nature of the environmental/metabolic signals that impact on the recognition ability and/or binding affinity of AraQ and MalR1 to their target DNA sequences. For this purpose, EMSA analyses were performed whereby AraQ or MalR1 was incubated with one of its binding targets, in this case the araQ and malR1 in controlling central carbon metabolism, insertional mutants were created in each of these genes in B. breve UCC2003, resulting in strains B. breve UCC2003-araQ and B. breve UCC2003-malR1 . These two mutants were then employed to assess transcriptional changes due to either of these mutations using microarray analysis.In order to further investigate the role of B. breve UCC2003-araQ, when compared to that of B. breve UCC2003 50 is significantly up-regulated in B. breve UCC2003-araQ . In silico analysis via a PWM-based approach identified AraQ/MalR1 TFBSs upstream of the above-mentioned gene cluster, the location of these TFBSs indicates that AraQ/MalR1 act as repressors of the Bbr_0118-Bbr_0121 gene cluster. Similarly, malR3  and apuB  were up regulated 3.7 and 4.3-fold, respectively (p < 0.001) in B. breve UCC2003-araQ as compared with the wild type. rbsD (Bbr_1416), encoding a D-ribose pyranase, which is located in a ribose uptake cluster was also upregulated 2-fold (p < 0.001), PWM approach also identified the AraQ/MalR1 TFBS upstream of this cluster.The transcriptome of B. breve UCC2003-araQ or B. breve UCC2003-malR1  and vice versa (for strain B. breve UCC2003-araQ). Attempts to create a double mutant of both araQ and malR1 were unsuccessful, indicating that these two regulators form an essential transcriptional circuit of the bifid shunt genes.Most bifid shunt-associated genes whose promoter regions contain binding sites for AraQ/MalR1 were not significantly altered in the transcriptional analysis employing B. breve UCC2003-malR1 revealed, when compared to that of the B. breve UCC2003 WT , to be transcriptionally up-regulated above a fold change of 2.0, while no genes were found to be significantly (fold change of >2.0) down-regulated in the transcriptomic analysis of the B. breve UCC2003-malR1 mutant. In silico analysis via a PWM-based approach identified a MalR1 TFBS upstream of malE1 (Bbr_0118), where the location of this TFBS indicates that MalR1 acts as a repressor of this gene. Two genes, apuB and malG1, that form part of the predicted malR1-governed regulon40, were up-regulated to a lower, yet significant degree . Notably, some of the central metabolic genes were found to be up-regulated at a rather modest, yet statistically significant level .The transcriptome of araQ and malR1 genes in a set of 70 Bifidobacteriaceae genomes. This was carried out by aligning the 70 Bifidobacteriaceae genomes using a PATtyfam approach and the BLAST tool, followed by alignment with MUSCLE . The two regulator-encoding genes showed somewhat different patterns of distribution; araQ was fully conserved among all currently known species of the genus Bifidobacterium, while missing in genomes of closely related genera such as Scardovia and Pseudoscardovia , whereas malR1 was conserved in 50 genomes   is a distant LacI family regulator from B. breve UCC2003, this TF was employed as an out group as its function is predicted to be different from those of MalR1 and AraQ  are confined to genera within the Bifidobacteriaceae family , along with experimental validation through EMSA analysis. Thus, the possibility that MalR1 operates differently in other bifidobacteria or closely related genera cannot be entirely excluded. We were also unable to identify conserved joint AraQ/MalR1 binding sites in Gardnerella vaginalis ATCC 14019 using a PWM approach, the only analysed strain that lacks araQ yet possesses malR1. This observation suggests that in this bacterium MalR1 recognizes a different binding motif, and presumably works as a local TF regulating maltose/maltodextrin utilization.AraQ has been identified in distant lineages of Actinobacteria Bifidobacterium species56. For this reason, B. breve UCC2003 WT, B. breve UCC2003-araQ and B. breve UCC2003-malR1 mutants were analysed for their ability to grow in the absence (control) as compared to the presence of 1% porcine bile with OD600nm readings taken at 24-hour post inoculation. Figure 600nm following 24-hour growth in the presence of 1% porcine bile as compared to the control. A significant fold reduction in OD600nm readings was observed for all three strains when grown in the presence of porcine bile, as compared to OD600nm readings without porcine bile. However, the fold reduction in growth of B. breve UCC2003-araQ and B. breve UCC2003-malR1 mutants was more pronounced compared to the wild type strain B. breve UCC2003: in the presence of 1% porcine bile, the B. breve UCC2003-araQ and B. breve UCC2003-malR1 mutants demonstrated a fold reduction of 11.1  and 6.4  in OD600nm readings, respectively, whereas the fold reduction for the control strain B. breve UCC2003 was 3.5.Several publications have reported that the (in)ability to survive bile stress is linked with shifts in the expression of genes involved in central metabolism and with the concentration of its resulting end products in various The distal gut does not supply bacteria with a reliable carbon source; available carbohydrates are dependent on many factors such as host diet and other (competing) bacteria present in the GIT. Therefore, it is necessary for bifidobacteria to be capable of utilising a multitude of carbon sources and to do so efficiently in order to be competitive. Gene regulation is one way in which bifidobacteria ensure that their energy generation is in line with available carbohydrate substrates, and that energy and biosynthetic demands are kept in balance.45. In the current study, we experimentally validated these predictions. Additionally, it appears that AraQ and MalR1 together impose global shifts in gene transcription in B. breve UCC2003 through their apparent ability to regulate a number of TFs  and consequently the regulons of these TFs. AraQ and MalR1 were also found to control the transcription of key genes of the bifid shunt in B. breve UCC2003  gene duplication event. Additionally, it is also apparent from the phylogenetic analysis that AraQ and MalR1 form two non-orthologous groups  limitations of the EMSA approach, namely, non-specific binding of AraQ/MalR1 to sites of other TFs from the LacI family present in promoter regions of these genes; (ii) limitations of the PWM approach, specifically insufficient sensitivity of designed matrices.Analysis of 48, and which binds 14 bp upstream of the −35 site when functioning as an activator. These authors suggested that CcpA binds to the same side of the helix face as the position of the −35 site. The AraQ/MalR1 TFBS was found (in some cases) to be located at a similar distance upstream of the −35 site implying that AraQ/MalR1 may also bind on the same side of the helix face as the position of the −35 site. One important feature of LacI-type TFs is that they can alter the local DNA structure48, which in this case may enhance the affinity of the RNA polymerase holoenzyme to its cognate −35/−10 sites.The binding sites of AraQ/MalR1, where AraQ/MalR1 are believed to function as activators, were found to bind at a distance of a multiple of one turn plus one half, away from the TSS. This is consistent with findings for another LacI-type family member, CcpA, which may act as a repressor or activatorB. breve UCC2003-araQ vs the B. breve UCC2003 WT strain demonstrated that transcription of a number of genes is repressed by AraQ, whereas genes where AraQ is predicted to act as a transcriptional activator (based on the location of the TFBS in relation to the −35 site) showed no significant change in expression. This result is in line with our predictions as insertional mutagenesis should reduce the amount of active AraQ protein in the cell, therefore the expression of those genes which AraQ activates should remain unchanged.Transcriptional analysis for Bifidobacterium species56. For this reason, growth assays were carried out on B. breve UCC2003-araQ and B. breve UCC2003-malR1, along with the wild type control strain B. breve UCC2003, when exposed to porcine bile  and an iron uptake system (Bbr_0121-0122)47, indicating that these TFs control a number of genes involved in bile and other cell stress responses. AraQ and MalR1 ability to control central metabolism may also aid survival under bile stress conditions by increasing ATP production from the bifid shunt, while fine tuning the expression on essential central metabolic genes, a role which would benefit from the presence of two distinct TF proteins.The biological significance of a bacterium producing two distinct proteins which appear to have overlapping functions is currently unknown. We hypothesize that there may be a link between the presence of these two proteins and the stress response of the bacterium. Several publications have reported that the (in)ability to survive bile stress is linked with shifts in the expression of genes involved in central metabolism and with the concentration of its resulting end products in various ile Fig. . This anin vitro assays, EMSA and transcriptomic analyses have their limitations and it is important to consider these factors on interpretation of the obtained results. Nonetheless, our findings indicate that AraQ and MalR1 play an important role in regulating the metabolic flux of central metabolism in a large proportion of the Bifidobacteriaceae, presumably contributing to metabolic agility and associated ability to adapt to stressful conditions. In conclusion, this work revealed a novel and apparently unique regulatory mechanism employed by most human-derived bifidobacteria. This regulatory system appears to be crucial to provide adaptability and competitiveness in the constantly changing environment of the gastrointestinal tract.As with all B. breve strains were routinely grown at 37 °C in either de Man Rogosa and Sharpe medium , modified de Man Rogosa and Sharpe (mMRS) medium made from first principles60, or reinforced clostridial medium  supplemented with 0.05% cysteine-HCl. Bifidobacteria were incubated anaerobically in a modular, atmosphere-controlled system . Escherichia coli strains were routinely grown in Luria Bertani (LB) broth at 37 °C with liquid cultures agitated at 120 rpm. Where appropriate growth medium was supplemented with tetracycline , chloramphenicol , ampicillin , erythromycin  or kanamycin  for plasmid/strain selection and/or maintenance.Bacterial strains and plasmids are listed in Table 61 genome annotations of the B. breve UCC2003 genome sequence62. Database searches were carried out using non-redundant sequences accessible at the National Centre for Biotechnology Information internet site (http://www.ncbi.mlm.nih.gov) utilising the basic local alignment search tool (BLAST). Sequence analysis was performed employing the Seqbuilder and Seqman programs of the DNASTAR software package . Protein functions were assigned with the use of BLASTP, and homology detection and structure prediction by HMM-HMM comparison and HHpred64.Sequence data were obtained from the Artemis-mediatedBifidobacteriaceae genomes was created. The search for AraQ and MalR1 orthologs in these genomes was carried out using the PATtyfam approach and the BLAST tool integrated into the SEED genomic platform65. Sequences of orthologs were collected into one file and then aligned via MUSCLE66. The alignment was subsequently manually filtered to reduce the number of gaps. A phylogenetic tree was built using IQ-TREE52 with the following parameters: automatic substitution model selection, ultrafast bootstrap with 1000 replicates, and employing BglR (Bbr_1659), a distant LacI family regulator from B. breve UCC2003 with predicted different function, as an outgroup.For comparative studies, a non-redundant set of 70 67. Restriction enzymes and T4 DNA ligase were obtained from Roche Diagnostics , and were used according to the manufacturer’s instructions. PCRs were performed using either Q5® High-Fidelity DNA polymerase  or Extensor Long Range PCR Enzyme master mix . Synthetic oligonucleotides were synthesized by Eurofins  and are listed in supplemental Table E. coli and B. breve by electroporation, and large-scale preparation of chromosomal DNA from B. breve was performed as described previously68. Plasmid DNA was obtained from B. breve and E. coli using the Roche High Pure plasmid isolation kit . An initial lysis step was performed using 30 mg ml−1 of lysozyme for 30 min at 37 °C as part of the plasmid purification protocol for B. breve.DNA manipulations were carried out based on previously published protocolsaraQ; fragment used was 402 bp in length, representing codons 74 through to 208 of the 371 codons of this gene) and Bbr_1846  were amplified by PCR using B. breve UCC2003 chromosomal DNA as a template . Insertional mutants and the WT strain were cultivated in mMRS medium supplemented with 0.5% ribose until an OD600nm of ∼0.6 was achieved. We employed ribose for such transcriptome analyses as this sugar has been utilized previously as a reference carbohydrate for various metabolic studies72. Furthermore, transcriptome analysis for B. breve UCC2003-araQ and B. breve UCC2003 WT was carried out when these strains were grown on lactose as a sole carbon source, which generated similar results as compared to ribose-grown cells except for the expected sugar-specific differences. Cells were harvested by centrifugation at 10,000 rpm for 2 min at room temperature and immediately frozen at −80 °C prior to RNA isolation. DNA microarrays containing oligonucleotide primers representing each of the annotated genes on the genome of B. breve UCC2003 were designed by and obtained from Agilent Technologies . Cell disruption, RNA isolation, RNA quality control, and cDNA synthesis and labelling were performed as described previously73. The labelled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-ColorMicroarrayBased Gene Expression Analysis v4.0 manual (G4140-90050).The transcriptome of 75. Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test76. A gene was considered to exhibit a significantly different transcription level relative to the control when p < 0.001 and an expression ratio of >2 or <0.25. The microarray data sets obtained in this study have been deposited in NCBI’s Gene Expression Omnibus database and are accessible through GEO series accession number GSE108949.Following hybridization, the microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). The generated scans were converted to data files with Agilent’s Feature Extraction software (v9.5). The DNA microarray data sets were processed as previously describedaraQ and pQE60 + malR1, DNA fragments encompassing araQ (corresponding to locus tag Bbr_0411) and malR1 (corresponding to locus tag Bbr_1846) were generated by PCR amplification employing chromosomal DNA of B. breve UCC2003 as a template, Q5 high-fidelity DNA polymerase, and primers araQ_F and araQ_R, and malR1_F and malR1_R, respectively . The plasmid contents of a number of AmpR transformants were screened by restriction analysis, and the integrity of positively identified clones was verified by sequencing. One verified clone of plasmid pQE60 + araQ and pQE60 + malR1  . Bacterial cells were disrupted by bead beating in a mini-bead beater  using glass beads. Cellular debris was removed by centrifugation to produce a crude cell extract. Recombinant AraQ and MalR1 proteins, each tagged with an incorporated C-terminal His10 sequence, were purified from a crude cell extract using a nickel-nitrilotriacetic acid column  according to the manufacturer’s instructions . Lysis, wash and elution buffers were supplemented with 10% glycerol as this considerably improved the (binding) activity of AraQ and MalR1. Elution fractions were analysed by SDS-polyacrylamide gel electrophoresis, as described previously77, on a 12.5% polyacrylamide gel. Following electrophoresis, gels were fixed and stained with Coomassie brilliant blue to identify fractions containing the purified protein. Colour Prestained Protein Standard, Broad Range (11–245 kDa)  was used to estimate the molecular weights of the purified proteins. Protein was concentrated using Amicon® Ultra Filters from Merck Millipore and dialysed into EMSA binding buffer . Concentration of the purified AraQ/MalR1 protein and subsequent dialysis into EMSA binding buffer also improved the binding ability of these two LacI-type proteins. Protein concentrations were determined using the Qubit® fluorometer as per manufacturer’s instructions . Purified protein was aliquoted and stored at -80 °C for subsequent use in EMSAs.78. All binding reactions were carried out with poly(dI-dC) (0.05 µg µl−1), DNA probe (0.5 nmol), BSA (0.2 µg µl−1), binding buffer  and water to a final volume of 20 μl. Binding reactions were typically carried out with 150 nM purified AraQ or MalR1 and 0.5 nM Ird-700-labelled DNA, and incubated for 20 min at 37 °C prior to loading onto a 6% non-denaturing polyacrylamide (PAA) gel prepared in TAE buffer  and bound/unbound DNA fragments were then separated by electrophoresis on a 0.5X to 2.0X gradient of TAE at 100 V for 90 min in an Atto Mini PAGE system .Promoter regions of genes of interest were amplified by PCR utilising individual primer pairs, of which one or both were 5’ Ird-700-labelled  as listed in supplemental Table Signals and percentage binding inferred from the Integrated Intensity (II) were detected/calculated using an Odyssey infrared imaging system , and images were electronically captured with the use of Odyssey software v3.0. The ability of AraQ and MalR1 to bind DNA fragments was assessed employing an arbitrary, non-linear scale which spanned from no affinity, low, medium to high affinity . This non-linear scale was correlated to the percentage of DNA which was bound as compared to unbound target DNA within a given reaction with – representing no DNA bound, + represents up to 15% DNA bound, ++ represents 15% – 50% DNA bound and +++ represents 50–100% DNA bound. In some cases, when a DNA fragment was shown to exhibit binding affinity, the binding site was more precisely determined by generating sub-fragments by PCR, followed by EMSAs as described above. To identify possible effectors for AraQ or MalR1, 10 mM of a particular compound  a PWM used for the AraQ regulon reconstruction in bifidobacteria based on a previous study40; (ii) a new AraQ/MalR1 PWM which was built using the EMSA data as a training set  or high affinity (+++). A graphical representation of the identified motifs was obtained using WebLOGO software79.Motif searches were carried out using Position Weight Matrix (PWM) approach as described previouslyB. breve UCC2003 and its isogenic derivatives B. breve UCC2003-araQ and B. breve UCC2003-malR1 were inoculated at 1% (v/v) from stock into mMRS medium supplemented with 1% maltose and 0.5% (v/v) L-cysteine HCl and were cultured overnight under anaerobic conditions at 37 °C. The above strains were then inoculated at 1% (v/v) into mMRS medium containing 1% maltose, 0.5% (v/v) L-cysteine HCl along with increasing concentration of porcine bile at 0, 1 and 2% (v/v). mMRS without the addition of a carbohydrate source served as a negative control. Bacteria were evaluated for their ability to grow in the presence of bile with Optical density (OD600nm) readings taken at 10 and 24 hr post-inoculation. Samples were assessed as biologically independent triplicates. Statistical analysis was carried out using the Microsoft Excel Data Analysis ToolPak. The statistical significance was determined for the two mutants, by comparing the difference in their OD600nm readings as compared with the WT control when exposed to bile.In order to assess the effects of bile on growth, Supplementary Information


Rac1 regulation in diabetes.Oxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 promoter. The effect of inhibition of Dnmts/Tets  on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreally Dnmt1-siRNA.Using human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnmt and Tets were quantified at the Rac1 promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Dnmts/Tets prevented increase in 5hmC and NF-kB binding, and attenuated Rac1 activation. Similarly, in mouse retinal microvessels, Dnmt1-siRNA ameliorated diabetes-induced increase in Rac1 transcripts and activity, and decreased ROS levels.Despite high glucose-induced increased binding of Dnmt1, 5mC levels remained subnormal at Rac1. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.Thus, despite Dnmts activation, concomitant increase in Tets rapidly hydroxymethylates 5mC, allowing NF-κB to bind and activate  Experimental models have shown that the activation of Rac1-Nox2-ROS precedes mitochondrial damage, and the development of diabetic retinopathy.5Diabetic retinopathy, a slow progressing complication, is the leading cause of blindness in working age adults. The pathology of the disease is complex and involves many interrelated metabolic abnormalities initiated by persistent high circulating glucose.5 Hyperglycemic milieu also increases Rac1 transcription, and our recent work has shown that hyperglycemia promotes this transcriptional activation by increasing the binding of nuclear transcriptional factor-κB  at the Rac1 promoter.6 However, the putative mechanism underlying increased transcription factor binding remains to be explored.Functional activation of Rac1 requires specific guanine exchange factors, including Tiam1 and Sos1, and in diabetes, Tiam1-mediated activation of Rac1 is implicated in retinal Nox2-ROS production.7 Many epigenetic modifications, including DNA methylation and histone acetylation, are shown to play a role in the development of diabetic complications, including retinopathy.812 Addition of a methyl group to the cytosine by DNA methyl transferases (Dnmts) forms 5-methylcytosine (5mC), and methylated cytosine impedes transcriptional factor binding, resulting in gene repression. However, 5mC can be rapidly oxidized by Ten-Eleven Translocation dioxygenases (Tets), and the resulting formation of 5-hydroxymethylcytosine (5hmC) stimulates gene expression.1316 In diabetes, both Dnmts and Tets are activated in the retina and its vasculature, and among these two families of enzymes, Dnmt1 and Tet2 are the only respective isoforms that are upregulated.9 How these opposing enzymes regulate DNA methylation status of Rac1 is not clear.Epigenetic modifications, the modifications that can alter gene expression without affecting the DNA sequence, play an important role in regulating the binding of the transcription factors in many disease conditions.Rac1. Using human retinal endothelial cells (HRECs), we investigated the effect of high glucose on DNA methylation status of the Rac1 promoter. The role of DNA methylation in Rac1 transcriptional activation was further established using pharmacological inhibitors and specific small interfering RNAs (siRNAs) of Dnmt1 and Tet2. Key parameters were confirmed in an in vivo model using retinal microvessels from streptozotocin-induced diabetic mice, receiving intravitreal administration of Dnmt1-siRNA or negative control siRNA.The aim of this study was to investigate the role of epigenetic modifications in the transcriptional regulation of 2 and 5% CO2, as described previously.17 Cells from the sixth to eighth passage were incubated in 5-mM or 20-mM D-glucose (glucose) for 6 to 96 hours in the presence or absence of inhibitors of Dnmts  or Tets .18 The role of p65 in Rac1 transcriptional activation was confirmed in the cells incubated with a cell-permeable inhibitor of NF-κ B activation, SN 50 .19 A group of cells was transfected with a single siRNA pool of Dnmt1 or Tet2  using siRNA transfection reagent , and the transfected cells were incubated in 5-mM or 20-mM glucose for 96 hours.9 The transfection efficiency was evaluated by quantifying mRNA levels of the respective genes (Dnmt1 or Tet2) by SYBR green–based real-time quantitative reverse transcription PCR (qRT-PCR) and by protein expression (western blotting), and it was ∼40% for Dnmt1-siRNA and >50% for Tet2-siRNA. Untransfected cells and cells transfected with Dnmt1-siRNA or Tet2-siRNA had similar Dnmt3a or Tet1 expression, confirming the specificity of the Dnmt1- and Tet2- siRNA. , were cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 containing 10% heat-inactivated fetal bovine serum, endothelial cell growth supplement (15 μg/mL), insulin transferrin selenium (1%), Glutamax (1%) and antibiotic/antimycotic (1%) in an environment of 95% O- siRNA. . Paralle20 Soon after establishment of diabetes, a group of diabetic mice received intravitreal administration of 2-μg Dnmt1-siRNA ; this stealth Dnmt1 siRNA is a combination of sequences targeting three positions at exon 4. Dnmt1-siRNA was suspended in 2-μL nuclease-free water and mixed with Invivofectamine . The right eye received Dnmt1-siRNA and the left eye received a medium GC content negative control siRNA . Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg ketanest and 12 mg/kg xylazine), and using a 32-gauge needle attached to a 5-μL glass syringe (Hamilton), intravitreal injections were performed under a dissecting microscope. Care was taken to position the needle 1 mm posterior to the limbus, and siRNA mixture was slowly injected into the vitreous chamber.21 Four weeks after administration of siRNA, the mice were killed, and the retina was quickly isolated. Age-matched normal mice and diabetic mice, without receiving any siRNA, served as controls. The treatment of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the Wayne State University's Institutional Animal Care and Use Committee.Seven- to 8-week-old C57BL/6J mice (either sex) were made diabetic by streptozotocin injection (55 mg/kg body weight for 4 consecutive days). Mice with blood glucose more than 250 mg/dL 2 days after the last injection were considered diabetic.6Freshly isolated retina was used to prepare microvessels by incubating it in 5-mL de-ionized water for 60 minutes in a shaking water bath at 37°C. The nonvascular tissue was gently removed under the microscope.Rac1 promoter was analyzed by qRT-PCR using specific primers.9Genomic DNA was isolated using Qiagen DNA isolation kit , and was immunoprecipitated with 5mC or 5hmC antibodies. The levels of 5mC and 5hmC were quantified using methylated or hydroxymethyldated DNA Immunoprecipitation (MeDIP)/(hMeDIP) kits, respectively . The enrichment of 5mC or 5hmC at the Rac1 promoter was determined by ChIP assay using cross-linked samples sonicated in ChIP lysis buffer. Protein-DNA complex (100 μg) was immunoprecipitated with Dnmt1, Tet2, or p65 antibody . Each experiment included antibody control, where the samples were immunoprecipitated with IgG . The immunoprecipitated complex was captured using Protein A Agarose/Salmon Sperm DNA , washed and de-crosslinked at 65°C for 6 hours, followed by DNA isolation with phenol:choloroform:isoamylalcohol, using the methods reported previously.8 Relative binding of Dnmt1/Tet2/p65 at the promoter was quantified by qRT-PCR using Rac1 promoter specific primer , at the c primer . The tarβ-actin (human) or 18S (mice), and the relative fold change was calculated using delta delta Ct method.8Gene expression was quantified using gene/species-specific primers by qRT-PCR , and the2 Fold change was calculated considering the values obtained from cells in normal glucose as one.Active Rac1 was measured by G-LISA colorimetric assay kit  in 20- to 25-μg protein, as described previously.22Total ROS levels were quantified using 2′,7′-dichlorofluorescein diacetate . Briefly, 5-μg protein was incubated with 4-μM DCFH-DA for 20 minutes, and the fluorescence was measured at 485-nm excitation wavelength and 535-nm emission wavelengths. Fold change was calculated as percent change with respect to values obtained from cells in normal glucose.t-test; P < 0.05 was considered significant.Data are presented as mean ± SD. Comparison between groups was made using 1-way ANOVA followed by Dunn's 22; to investigate the role of DNA methylation in Rac1 transcriptional regulation, 5mC levels at the Rac1 promoter were quantified in the cells exposed to high glucose for 96 hours, a duration when cell apoptosis is detected.2324 and hyperglycemic milieu activates both Dnmts and Tets,9 the effect of high glucose on hydroxymethylation of 5mC was investigated; compared with cells in normal glucose, cells incubated in high glucose had 2-fold higher 5hmC levels at Rac1 promoter  or molecular (Dnmt1-siRNA) inhibitors was analyzed; both 5-Aza and Dnmt1-siRNA prevented glucose-induced increase in Dnmt1 binding at Rac1 promoter  of exposure of high glucose, at 6 hours Dnmt1 expression was significantly decreased  of high glucose incubation, 5hmC levels, Tet2 binding, and its gene transcripts were significantly elevated, and they remained elevated at 48 to 96 hours of high glucose exposure , due to inhibition of Dnmts, the promoter is hypomethylated. However, at 24 hours, although the binding of Dnmt1 is increased, concomitant activation of Tets hydroxymethylates 5mC to 5hmC, resulting in Rac1 activation. Role of active DNA methylation-hydroxymethylation in regulation of Rac1 transcription is further confirmed by our results showing increased 5hmC levels at the Rac1 promoter in the retinal microvessels from mice diabetic for 2 weeks, and amelioration of diabetes-induced activation of Rac1 (transcription and activity) by Dnmt1-siRNA. These results have significant implications for diabetic retinopathy because, although the appearance of retinal histopathology, characteristic of diabetic retinopathy, in rodent models is visible only after 6 to 8 months of diabetes, Rac1-Nox2-ROS signaling is an early event, which can be seen within 2 weeks after induction of diabetes.Diabetic retinopathy has a complex underlying pathophysiology, and in spite of cutting edge research in the field, molecular events contributing to its development remain unclear. Among many abnormalities implicated in the development of diabetic retinopathy, experimental models have documented oxidative stress to be at a central place.26 DNA methylation is mainly associated with repression of gene expression by changing the chromatin structure and restricting access of the transcription factors to the gene promoter.24 However, errors in DNA methylation are observed with many genes in chronic diseases including cancer and diabetes.2729 Human Rac1 promoter has four CpG islands within −1200 bases upstream of transcription start site, and the binding site for the transcription factors lies in the CpG island close to the transcription start site between −317 to 147 bp.30 Our previous work has shown that in diabetes, the binding of transcriptional factor NF-κB at Rac1 promoter is increased in the retinal vasculature,6 and here we show that at a duration of high glucose insult when mitochondrial damage and apoptosis are also observed (96 hours), 5mC levels are also decreased. As mentioned above, Dnmts catalyze the transfer of a methylation residue from S-adenosyl-L-methionine to cytosine, and among these, Dnmt3a and Dnmt3b are the de novo methyltransferases, Dnmt1 is considered as the maintenance methyltransferase and recognizes hemimethylated sites restoring symmetric methylation during replication.31 In diabetes, Dnmts are activated, and among this family of enzymes, only Dnmt1 is upregulated in the retina and its vasculature.9 At 96 hours, despite decrease in 5mC levels, binding of Dnmt1 at Rac1 promoter is increased, suggesting that the 5mC formed by Dnmt1 could be demethylated. The results from the in vivo model further support the role of active DNA methylation in Rac1 activation, as administration of Dnmt1-siRNA ameliorates both Rac1 transcription and activation, and decreases ROS levels, seen in the retinal microvasculature of diabetic rodents.Addition of a methyl group to carbon-5 of a cytosine base, forming 5mC, is considered as a fundamental epigenetic mediator, and is an important component in many cellular processes, including genomic imprinting and chromosome stability.13 Demethylation of cytosine is considered necessary for epigenetic reprogramming of genes, and is directly involved in many diseases including tumor progression.14 Demethylation of DNA can either be passive or active, or a combination of both, and the enzyme-based oxidation of 5mC to 5hmC promotes DNA demethylation by binding to CpG-rich regions to prevent unwanted Dnmt activity.13 Retinal Tets are activated in diabetes, and among this family of enzymes, Tet2 is the only member with upregulated gene transcripts.9 The results presented here clearly show that, although Dnmt1 binding at Rac1 promoter is increased in diabetes, due to increased Tet2 binding, 5hmC levels are elevated, suggesting a rapid hydroxymethylation of 5mC formed by Dnmt1. This increase in 5hmC allows binding of the transcription factor, increasing Rac1 expression. Further, we show that inhibition of Tets, in addition to regulating DNA methylation status of Rac1 promoter, also inhibits NF-κB binding and Rac1 transcription, further confirming the role of active DNA methylation in Rac1 activation. In support, diabetes modulates transcription of matrix metalloproteinase MMP-9, an enzyme implicated in mitochondrial damage, via dynamic DNA methylation-hydroxymethylation of its promoter.9 Moreover, hyperglycemia has a direct impact on changes in the epigenome, and high DNA methylation and hydroxymethylation levels are observed in poorly controlled diabetic patients compared with healthy individuals.32 We recognize that Tets can further oxidize 5hmC to 5-formylcytosine, and 5-formylcytosine to 5-carboxylcytosine33; their role in regulation of Rac1 transcription cannot be ruled out. Although methylated CpGs are considered to influence transcription factor binding at their recognition site, and DNA methylation at promoter generally locks genes in a stable silent state, many transcription factor recognition sites do not possess CpGs in their recognition sequence,10 and their role in maintaining Rac1 gene transcription remains to be examined. However, amelioration of diabetes-induced increased NF-κB binding at the Rac1 promoter by both Dnmt and Tet inhibitors, observed in the present study, clearly suggests that dynamic CpG methylation is playing a critical role in the regulation of binding of this transcription factor.DNA is a dynamic structure, and the methylated cytosine can be rapidly demethylated.2 DNA methylation varies in a global and local context, and active changes in DNA methylation are observed during aging.3437 Site-specific hypermethylation are observed predominantly in promoter regions,39 and methylation marks are also considered to predict the age of the organism.40 Our data show that in the early stages of diabetic retinopathy (6 hours of high glucose exposure), although Tet2 is not affected, Dnmt1 is repressed, and promoter DNA is hypomethylated, resulting in Rac1 transcriptional activation. However, as the disease progresses, the two enzymes with opposing functions, Dnmts and Tets, are both activated. Although there could be increase in 5mC at the promoter, activated Tets overpower Dnmts activation, and hydroxymethylate 5mC to 5hmC, transcriptionally activating Rac1.Diabetic retinopathy is a progressive disease, and activation of Rac1-Nox2 is one of the early events in its pathogenesis. Rac1-Nox2–mediated cytosolic ROS play a major role in damaging the mitochondria, leading to the development of histopathology characteristic of diabetic retinopathy.There are some limitations of our study, including the following:4143 The possibility of a similar mechanism(s) operating in retinal pericytes, and in other nonvascular cells, cannot be ruled out.Retinal endothelial cells are not the only cells that are lost in the early stages of diabetic retinopathy; instead, pericytes of the vasculature also undergo accelerated apoptosis before any pathology, characteristic of diabetic retinopathy, can be observed, and in diabetes, photoreceptors are also a major source of ROS.45 raising a possibility that the in vitro model might not fully reproduce the in vivo model. However, most of the mechanism-based data are comparable between the in vitro and in vivo models.17Diabetic retinopathy is a slow progressing disease, mitochondrial damage and capillary cell apoptosis precedes the development of microvascular histopathology, and the histopathology not visible until 6 to 8 months of diabetes in rodents,Dnmt1-siRNA for only 4 weeks. In support of a possible link between early changes in DNA methylation and the onset of diabetic retinopathy, our previous work has shown that inhibition of the enzymes responsible for maintaining DNA methylation ameliorates mitochondrial damage and capillary cell apoptosis, the events that precede retinal microvascular pathology in the in vivo models of diabetic retinopathy.46As mentioned above, high glucose-induced increase in Rac1-ROS can be seen within 6 hours in retinal endothelial cells and within 15 days of diabetes in rodents, and increase in mitochondrial damage and capillary cell apoptosis within 96 hours in cells and approximately 6 months of diabetes in mice. However, our retinal microvessel Rac1-ROS data, presented here, are from diabetic mice receiving Rac1 promoter DNA methylation in high glucose and normal glucose conditions using HRECs from the same passage (and the same batch). Moreover, experiments in in vitro models for many chronic diseases. including diabetic retinopathy, using appropriate controls, have provided consistent results as seen in the in vivo disease models.48Because epigenetic modifications are influenced by external factors, and in vitro culture of endothelial cells may introduce additional epigenetic modifications, this may interfere with the true recapitulation of the disease state. To alleviate the effect of cell passaging, the present study compared Rac1 in the development of diabetic retinopathy. Although DNA methylation is an epigenetic repressive mark, hydroxymethylation of 5mC is also now considered as an independent epigenetic marker; imbalance in genomic 5mC/5hmC levels are associated with aberrant gene activation and oncogenic transformation.50 Thus, despite increased 5mC formed at the Rac1 promoter due to activation of Dnmt1, concomitant increase in Tet2 hydroxymethylate it to 5hmC. This allows the transcription factor to bind, and activate Rac1 transcription. Rac1, via Nox2, elevates cytosolic ROS levels, and ROS damage mitochondria and increase capillary cell apoptosis, which, ultimately, results in diabetic retinopathy (In summary, despite the limitations stated above, our convincing data clearly demonstrate a role of active DNA methylation-demethylation in the regulation of inopathy . These d


In eukaryotes, the general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. However, the mechanism by which these transcription factors incorporate the preinitiation complex and coordinate their action during RNA polymerase II transcription remains elusive. Here we show that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module (CAK) within the preinitiation complex. In addition, we show that while RNA polymerase II phosphorylation and DNA opening occur, CAK and TFIIEα are released from the promoter. This dissociation is impeded by either ATP-γS or CDK7 inhibitor THZ1, but still occurs when XPB activity is abrogated. Finally, we show that the Core-TFIIH and TFIIEβ are subsequently removed, while elongation factors such as DSIF are recruited. Remarkably, these early transcriptional events are affected by TFIIE and TFIIH mutations associated with the developmental disorder, trichothiodystrophy. The general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. Here the authors provide evidence that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module within the preinitiation complex before their release during transcription. Among them, the general transcription factors (GTFs), including TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH assemble at the transcription start site with RNA Polymerase II (Pol II) to form the preinitiation complex (PIC). While the different GTFs have been characterized almost 30 years ago, little is known about the chronological order of the enzymatic activities that occurr during transcription initiation. Nonetheless, a conventional assembly order for a Pol II-PIC emerged, in which TBP (TATA Binding Protein), as part of TFIID, initially binds to the promoter and is further stabilized by TFIIB and TFIIA. Pol II and TFIIF then align to make a stable TFIID–TFIIA–TFIIB–Pol II–TFIIF promoter complex, which is finally completed by the addition of TFIIE and TFIIH4. TFIIE interacts with different factors within the PIC, including Pol II6 as well as with DNA immediately upstream of the transcription bubble region8. Furthermore, TFIIE seems to influence TFIIH activity9, although it is not clear how this molecular process can occur.The heterodimer TFIIE (composed of the TFIIEα and TFIIEβ subunits) seems to play a pivotal role in transcription by directly influencing the transition from initiation to elongation11. The role of TFIIH during transcription initiation mainly relies on its XPB and CDK7 enzymatic activities, XPD having essentially a structural function12. Observations revealed that XPB is not a conventional helicase14, but an ATPase/translocase that pumps downstream DNA toward the Pol II to generate torsional and mechanical strain, leading to the formation of the DNA bubble18. For its part, CDK7 phosphorylates the serine 5 (Ser5-P) of the C-terminal domain (CTD) of the largest Pol II subunit (RPB1), thus contributing to promoter escape21. The CDK7 action is not restricted to Pol II, since other transcription factors are targeted by this kinase, including nuclear receptors (NRs), such as the retinoic acid receptor alpha (RARα)22.TFIIH, which also intervenes during the nucleotide excision repair (NER) pathway, contains 10 subunits that can be resolved into two sub-complexes: the Core  and the CAK . Both sub-complexes interact with XPD subunit, whose ATPase/helicase activity promotes DNA opening during NER24. Remarkably, mutations within the TFIIEβ subunit recently have recently been associated with trichothiodystrophy (TTD), an autosomal recessive developmental disorder mainly related to TFIIH mutations (mostly in ERCC2/XPD gene and few cases in ERCC3/XPB or GFT2H5/p8/TTD-A genes) and characterized by brittle hairs with alternating dark and light (“Tiger tail”) banding with polarized microscopy and low content of sulfur-containing amino acids26. TFIIE/TTD patients, like TFIIH/TTD patients also have dry, ichthyotic skin, short stature, microcephaly, cerebellar dysfunction, developmental delay, happy engaging personality, attention deficit hyperactivity disorder, and myopia27.A better understanding of the transcription mechanism arose from the study of diseases related to mutations in genes encoding transcription machinery components XPD or the TFIIEβ-coding gene (GTF2E2) similarly disrupt these processes, which could explain why alterations of TFIIE or TFIIH lead to the same clinical syndrome.Our work reveals an unexpected dynamic process during which TFIIEα and TFIIEβ act as key factors to recruit the CAK of TFIIH within the PIC. Furthermore, we show that Pol II phosphorylation is accompanied by the release of the CAK and TFIIEα from the promoter, a process that takes place before DNA opening. Once the CAK and TFIIEα are released, RNA synthesis is initiated, a process during which the Core-TFIIH and TFIIEβ are also removed while elongation factors including DRB sensitivity-inducing factor (DSIF) are recruited. Strikingly, TTD-related mutations in either 29, we first examined the levels of components of the transcription machinery in fibroblasts isolated from TTD patients bearing either TFIIEβ/A150P or /D187Y mutations. Western Blot analysis showed that both α and β subunits of TFIIE were reduced in TTD/TFIIE-mutated cells when compared to wild-type fibroblasts  inducible lls Fig. . Intereslls Fig. , revealit = 0 on the RARβ2 promoter, progressively decreased in normal fibroblasts  assays were then performed to determine whether deficiencies occurred during the PIC assembly. In wild-type fibroblasts, the recruitment of both Pol II , TFIIH (CDK7), and TFIIB (p33) was reduced in KI-IIEβ/A150P cells when compared with that observed in IIEβ/WT cells Fig. , which clls Fig. ; its reclls Fig. . Intereslls Fig. ; howeverRARβ2 gene expression30 and the very distal position of exon 4 from the transcription start site (~140 kb). Strikingly, Pol II phosphorylation was strongly disrupted in KI-IIEβ/A150P cells  was clearly impaired in mutated cells  in IIEβ/WT cells after 6 h t-RA treatment Fig. . Persistlls Fig. . It is wlls Fig. ; only balls Fig. . Finallylls Fig. , suggest32, did not influence the removal of the damaged oligonucleotides  containing rIIEα/WT subunit with either rIIEβ/WT, /A150P, or /D187Y were produced and purified Fig. . We firs12. We observed that 5, 10, and 20 min after addition of nucleoside triphosphates , rIIEαβ/A150P and /D187Y led to a lower RNA synthesis than rIIEαβ/WT , TFIIA, TFIIB, TFIID (TBP), TFIIF, and TFIIH as well as Pol II/WT Fig. . The def34. Upon addition of radiolabelled CTP and increasing amounts of either rIIEαβ/A150P or /D187Y, trinucleotide (CpApC) synthesis was impaired (lanes 5–7 and 8–10). The defect was greater with rIIEαβ/A150P than /D187Y, which paralleled what was observed during in vitro run-off assays , Pol II, and either rIIEαβ/WT, rIIEαβ/A150P, or rIIEαβ/D187Y Fig. . After s35. Coimmunoprecipitation between TFIIE and TFIIH components were slightly reduced in the presence of rIIEαβ/A150P or rIIαβ/D187Y variants  and the Core (XPB) sub-complexes  led to accumulation of Ser5-P of Pol II , a chemical compound known to inhibit the ATPase activity of XPB and to block transcription  in the presence of NTPs Fig. . While RTaken together, these results strongly suggested that TFIIEα and the CAK, followed by the Core-TFIIH and TFIIEβ, are sequentially released from the promoter while elongation factors take place to pursue transcription.We next investigated how TTD mutations might perturb the transcription initiation process. Immobilized AdMLP was first incubated with Pol II, the GTFs  and either rIIEαβ/WT, rIIEαβ/A150P, or rIIEαβ/D187Y in the presence of ATP Fig. . After wIn vitro transcription assays were also performed in the presence of the basal transcription machinery with the different rIIEs and rIIHs variants Fig. . ReactioTaken together, these results suggested that TFIIEβ and XPD mutations cause in common a perturbation of Pol II phosphorylation by the CAK.The present study aimed to dissect the interplay between TFIIE and TFIIH during transcription, from their integration within the PIC until their release before the upcoming recruitment of elongation factors. This work also revealed how TTD mutations within TFIIE and TFIIH disrupt such processes, resulting in a decrease in transcription.2, the whole forming a structure ready to incorporate TFIIE and TFIIH  sites(s) and to determine the function(s) of such modification(s) during transcription.The ATP-dependent release of TFIIEα and the CAK raises the question of the fate of the Core-TFIIH and TFIIEβ within the remaining complex. The Core-TFIIH is required for next transcriptional events; in particular, the translocase activity of XPB allows DNA pumping into the active site cleft of Pol IIken Fig. . Concernken Fig. , which mer Figs. ; its reler Figs. , 5e, f, TP Figs. , 5 and 626, which is so far mainly related to TFIIH mutations. The results presented here show that TTD-related mutations within TFIIE and TFIIH have in common (i) decreasing the cellular concentration of these complexes  afxes Fig. , (iii) hxes Fig.  and therxes Fig.  and ex vxes Fig. . ConsequThe key reagents and resources  are given in Supplementary Table Cells were grown in DMEM/Ham-F10 (1:1) supplemented with 10% FCS and 40 µg/ml gentamicin. Treatments with t-RA (10 µM) were performed in the presence of red phenol-free medium containing 5% charcoal-treated FCS and 40 µg/ml gentamicin.GTF2E2 with the missense mutation c.448G>C [p.Ala150Pro]. The GFP-positive cells were sorted 24 h post-transfection, and were diluted 48 h later as 0.5 cells/well in p96 plates. Cells were grown in DMEM (1 g/l glucose) supplemented with 10% FCS and 40 µg/ml gentamicin. Clones were screened by PCR and enzymatic digestion followed by further sequencing to confirm the correct knock-in in the desired locus.Human osteosarcoma epithelial U2OS cells were co-transfected with the px2-Cas9WT(GFP)-Puro plasmid  and a single-stranded oligodeoxynucleotide (ssODN) corresponding to an exon 5 region of RARβ2 mRNA expression represents the ratio between values obtained from treated and untreated cells normalized to 18S RNA. The primer sequences are given in Supplementary Table Total RNAs (2 µg) were reverse transcribed with Moloney murine leukemia virus RT (Invitrogen) using random hexanucleotides. Real-time qPCR  was done using the “FastStart DNA Master SYBR Green” kit and the LightCycler apparatus (Roche Diagnostics). The 59. Non-specific controls were performed with samples incubated with magnetic beads in the absence of antibodies. Primers were designed to amplify regions located either at the proximal promoter or at exon 4 of RARB2 (see 30. The primer sequences are given in Supplementary Table Subconfluent cells were treated with t-RA 10 µM) and ChIP experiments were next performed with Protein G-magnetic beads ARB2 see 30. The p0 µM and E. coli. Baculoviruses overexpressing Flag-XPD/WT, /R112H, /R722W, as well as XPB, p62, p52, p44, p34, cdk7, cyclin H, MAT1, and p8 were previously generated61. To purify entire rIIHs or XPD alone, Sf21 insect cells were infected with the corresponding baculoviruses. The whole cell extracts (WCE) were then incubated with agarose beads bound to anti-M2-Flag antibody. Recombinant TFIIH and the different forms of XPD were eluted with an epitope peptide.Flag-TFIIEα as well as His-TFIIEβ/WT, /A150P and /D187Y were produced in 62. Dual incision assay was carried out in the presence of XPG, XPF/ERCC1, XPC/hHR23B, RPA, XPA, the different rIIHs and rIIEs (when indicated)61.The single lesion (Pt-GTG) plasmid was prepared as described elsewhere63. RNA synthesis was initiated by the addition of NTPs (200 μM), including radiolabelled CTP (0.15 μM). In order to study Pol II phosphorylation during transcription, classical run-off transcription assays were carried out in presence of cold NTPs; after SDS–PAGE, specific monoclonal antibody was used to reveal Ser5-P of Pol II.The AdMLP template was preincubated for 15 min at 25 °C with Pol II, TFIIA, TBP, TFIIB, TFIIF, and the different rIIEs and rIIHs2 (6.5 mM), dATP (4 μM), and radiolabelled CTP (1 μM). After 30 min of CpApC synthesis, the reactions were stopped in the presence of proteinase K (0.5 mg/ml).The AdMLP template was preincubated with the GTFs  for 15 min at 25 °C. Phosphodiester bond synthesis was then initiated by the addition of priming dinucleotides CpA (0.5 mM), MgClBiotinylated AdMLP bound to streptavidin magnetic beads was incubated 20 min at 25 °C with Pol II, TFIIA, IIB, IIF, TBP, and the different rIIEs and rIIHs. After several washings at 50 mM NaCl, the bound fractions were resolved by SDS–PAGE followed by immunoblottings. The abundance of each protein was assessed by immunoblot densitometry analysis (using ImageJ software). Each signal was quantified three times (mean ± s.d.) and plotted in arbitrary units (a.u.).Depending on the coimmunoprecipitation assay, purified Pol II, rIIEs, and rIIHs were coincubated together with specific antibody  bound to protein G magnetic beads. After extensive washings (at either 150 or 500 mM KCl), Western blots were performed using antibodies raised against the proteins of interest.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Co-transcriptional methylations of histone H3 at lysines 4 and 36, highly conserved methyl marks from yeast to humans, have profound roles in regulation of histone acetylation. These modifications function to recruit and/or activate distinct histone acetyltransferases (HATs) or histone deacetylases (HDACs). Whereas H3K4me3 increases acetylation at promoters via multiple HATs, H3K4me2 targets Set3 HDAC to deacetylate histones in 5′ transcribed regions. In 3′ regions of genes, H3K36me2/3 facilitates deacetylation by Rpd3S HDAC and slows elongation. Despite their important functions in deacetylation, no strong effects on global gene expression have been seen under optimized or laboratory growth conditions. Instead, H3K4me2-Set3 HDAC and Set2-Rpd3S pathways primarily delay the kinetics of messenger RNA (mRNA) and long noncoding RNA (lncRNA) induction upon environmental changes. A majority of mRNA genes regulated by these pathways have an overlapping lncRNA transcription either from an upstream or an antisense promoter. Surprisingly, the distance between mRNA and lncRNA promoters seems to specify the repressive effects of the two pathways. Given that co-transcriptional methylations and acetylation have been linked to many cancers, studying their functions in a dynamic condition or during cancer progression will be much more important and help identify novel genes associated with cancers. This mark also acts as a binding site for bromodomain proteins associated with factors that regulate chromatin structure and transcription.1 Histone acetylation is dynamically regulated by the antagonistic functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). How HATs and HDACs control histone acetylation at specific regions of a genome remains to be completely understood, but recent studies propose that site-specific histone methylations may affect local histone acetylation by targeting HATs or HDACs or stimulating their activities.5 Histone ubiquitination also regulates histone acetylation at a specific genomic position via H3K4 and H3K79 methylation.7Post-translational modifications on histone tails including acetylation, methylation, phosphorylation and ubiquitination play important roles in eukaryotic RNA polII transcription.8 Instead, single gene studies revealed that many chromatin regulators primarily modulated the kinetics of gene induction without changes in basal or final levels of expression.11 Furthermore, loss of chromatin regulators resulted in greater effects on global gene expression during activation or repression upon environmental changes.12 Therefore, unlike gene-specific transcriptional activators or repressors turning on or off their target genes, chromatin regulators can function as a transcriptional modulator that controls the kinetics of gene induction or repression. In addition, long noncoding RNA (lncRNA) transcription and lncRNA itself also play critical roles in gene regulation.13 Interestingly, ~12% of yeast genes are overlapped with lncRNA transcription.14 A majority of these genes tend to be inducible suggesting the importance of overlapping lncRNA transcription in regulation of gene expression dynamics.Although chromatin factors have been thought to be essential for global gene regulation, a recent study revealed a highly specific effect. Many chromatin regulators had little or no detectable effect on global gene expression in a steady state growth condition.In natural growth conditions, cells must reprogram their gene expression patterns to adapt to rapidly changing environmental conditions. Fine tuning of gene expression dynamics by chromatin regulators can coordinate cellular processes to cell adaptation and fitness upon environmental changes. Here we review recent findings on modulation of gene expression dynamics by co-transcriptional methylations, their downstream effectors and overlapping lncRNA transcription.1 Factors involved in transcription repression contribute to recruitment of repressive histone methyltransferases (HMTs) for H3K9 or H3K27 methylation. In contrast, methylation of histone H3 at lysines 4 or 36 correlates positively with RNA polII transcription rate, and HMTs for these methyl marks interact with elongating RNA polII to localize histone methylations in transcribed regions.17Histone methylations can be targeted by multiple distinct mechanisms.18 Whereas phosphorylation of the CTD repeat heptad sequence on serine 5 by Kin28, a subunit of TFIIH, peaks in transcription start site and 5′ transcribed regions, Ctk1 phosphorylates serine 2 of CTD in 3′ regions.18 CTD phosphorylated at serine 5 plays important roles in initiation and early elongation of transcription. This modification mediates a direct interaction between messenger RNA (mRNA) capping enzyme and RNA polII.18 Furthermore, transcription termination of snRNAs and short mRNA genes by Nrd1-exosome is stimulated via the interaction between Nrd1 and CTD phosphorylated at serine 5  of Rpb1, the RNA polII largest subunit, is differentially phosphorylated by at least two kinases .18 Whereserine 5 . Phosphomination .185 Serine 5 phosphorylation is known to stimulate H3K4 methylation by Set1-COMPASS in 5′ transcribed regions.19 Although a direct interaction between phosphorylated CTD at serine 5 and Set1-COMPASS has not been reported yet, inactivation of Kin28 reduced Set1 crosslinking.19 In budding yeast, Set1 is the sole methyltransferase for all states of H3K4 methylation. Whereas H3K4me3 and Set1 crosslinking are observed at transcription initiation sites, H3K4me2 and H3K4me1 peak in 5′ and 3′ transcribed regions, respectively.20 The factors that affect the gradients of H3K4 methylation need to be determined. It is possible that Set1 activity/stability or composition of COMPASS may be altered during transcription elongation.21 Alternatively, Rad6-Bre1 may affect the distribution of H3K4 methylation. H2B ubiquitination on K123 by Rad6-Bre1 is required for H3K4me3 and H3K4me2  may maintain optimal levels of histone H3 methylation at lysines 4 or 36  is the first enzyme discovered that demethylates H3K4me2 and H3K4me1, but not H3K4me3. 4 or 36 .37, 384 These effector proteins are often found in large protein complexes including HATs, HDACs or chromatin remodeling complexes.6 The interaction between a site-specific methylation and an effector protein can regulate local chromatin structure and RNA polII transcription.Unlike acetylation, histone methylation is not known to directly affect chromatin structure. Instead, site-specific methylations act as binding sites for various downstream effector proteins containing chromodomains, tudor domains, PHD fingers and PWWP domains.20 Many proteins that stimulate RNA polII transcription can bind H3K4me3. In budding yeast, Yng1, Yng2 and Sgf29 are associated with distinct HATs, and these were shown to strongly bind H3K4me3.40 Yng1 and Yng2 are PHD proteins and the yeast homologs of the ING (INhibitor of Growth) gene family that may function as tumor suppressors.41 Yng1 is a component of NuA3 HAT, which preferentially acetylates histone H3 at K14.44 Yng2 is a subunit of NuA4, a major HAT for histone H4.45 The Sgf29 tudor domain protein promotes chromatin binding of SAGA HAT and histone H3 acetylation  HDAC, directly bind to H3K4me3 via their PHD fingers.51 Mammalian ING2, a mSin3a-HDAC1 complex subunit also preferentially binds to H3K4me3 to promote gene repression  HDAC and NuA4 HAT.58 Both Not1 and Pdp3 are components of NuA3 HAT.56 These findings suggest both a positive and negative role of H3K36me3 in histone acetylation. The interaction between Ioc4 PWWP domain and H3K36me3 helps maintain chromatin integrity during RNA polII elongation  and nuclear exosome (rrp6Δ), but only the short sense transcripts were seen upon deletion of RCO1 alone.75 These suggest that antisense transcripts from STE11 and CTT1 are rapidly degraded by nuclear exosome.A previous study reported that although 1685 genes showed increased acetylation within open reading frames in 71 We identified 118 internal cryptic promoters generating a short sense transcript and 639 producing an antisense transcript in SET2 deletion cells during carbon source shifts.74 Surprisingly, 416 of them were induced by specific carbon sources (PCA1 gene that are repressed by Set2 differently responded to nutritional shifts.74 One was constitutively active but the other two were induced or repressed during carbon source shifts. These results suggest that in addition to mRNA genes, the Set2-Rpd3S pathway can delay the induction of lncRNAs upon environmental changes.Another interesting possibility is that cryptic promoters within genes may be inducible and therefore active only under certain conditions. Previous studies revealed that some cryptic transcripts were induced by nutritional shifts. sources . Further74 However, the genes regulated by both pathways have a common feature. A majority of them are overlapped by lncRNA transcription either from a distal or an antisense promoter. The overlapping lncRNA transcription, rather than the lncRNA itself, may target H3K4me2 or H3K36me3 to promoter regions of distinct genes and delay the kinetics of gene induction.Two recent studies from our group have revealed that H3K4me2-Set3 HDAC and the Set2-Rpd3S pathway modulate the induction of mostly distinct target genes.DCI1 is one of the Set3-repressed genes overlapped by lncRNA transcription from a distal promoter (SET3 deletion, lncRNA transcript levels were not changed, but DCI1 mRNA transcripts were elevated during galactose incubation. Before induction, the DCI1 mRNA promoter had a peak of H3K4me2 as a result of the overlapping lncRNA transcription. The DCI1 promoter showed increased acetylation upon loss of Set3, explaining its elevated transcription upon induction.62 No derepression of this gene was observed in mutants for the Set2-Rpd3S pathway. In contrast, AAD10 is also overlapped with lncRNA transcription from an upstream promoter, but is repressed by Set2 (AAD10 promoter was enriched with H3K36me3 and derepressed in mutants for the Set2-Rpd3S pathway but not Set3C.promoter . Upon SE by Set2 . InteresDCI1 are ~0.55 kb apart, but the distance between the two AAD10 promoters is ~1.5 kb. Furthermore, the average distances between the lncRNA and mRNA promoters of Set3-repressed and Set2-regulated genes were ~0.9 kb and 2.0 kb, respectively. These findings support the idea that the distance between mRNA and lncRNA promoter may specify the Set2-Rpd3S or Set3 HDAC-mediated repression.74What determines the specificity for these two pathways? An interesting difference between Set3- and Set2-repressed genes is the distance between mRNA and lncRNA promoters . For exaAAD10 caused expression to become insensitive to the Set2-Rpd3S pathway. H3K36me3 was significantly reduced at the AAD10 mRNA promoter and histone acetylation was not changed in mutants for the Set2-Rpd3S pathway. In addition, AAD10 transcript levels were very similar in both wild type and SET2 deletion cells.74 These results indicate that shortening the distance between the two promoters of AAD10 alleviates repression by the Set2-Rpd3S pathway. Furthermore, although H3K4me2 was low at AAD10 promoter in the normal context, an elevated level was seen when the distance was reduced. Correspondingly, histone acetylation and AAD10 transcript levels were now increased in mutants for Set3 HDAC. Thus, alteration of the distance between the two promoters switches the repression of AAD10 by H3K36me3-Rpd3S to the H3K4me2-Set3 HDAC repression pathway.Consistent with this, reducing the distance between the two promoters of 13 Our recent findings suggest that although lncRNA can be transcribed from any site of a genome, positions of overlapping lncRNA promoters may be important for targeting distinct histone methylations and chromatin regulators to promoter regions of an associated mRNA gene.Transcription of lncRNA as well as lncRNA itself can play important roles in gene regulation.74 However, the HATs responsible for acetylation at these regions are not completely known. Some factors associated with distinct HATs have been shown to bind H3K36me3. For example, Nto1 and Pdp3 of NuA3 preferentially bind to H3K36me3.56 Furthermore, Eaf3 is a component of both NuA4 HAT and Rpd3S HDAC. Future works will determine if NuA3 or NuA4 HAT can acetylate histones at Set2-repressed promoters and increase the rate of mRNA and lncRNA induction upon environmental changes.Co-transcriptional histone methylations and their downstream effectors have critical roles in almost all steps of gene regulation including initiation, elongation, RNA processing and termination. Despite their importance, global gene expression is often not significantly affected by loss of these factors in optimized or laboratory growth conditions. Instead, stronger effects can be observed in more realistically dynamic conditions where gene expression is continuously changing. H3K4me2-Set3 HDAC and the Set2-Rpd3S pathways deacetylate histones at certain mRNA or lncRNA promoters where H3K4me2 and H3K36me3 are enriched, respectively, and negatively regulate the rate of mRNA or lncRNA induction.76 Instead, greater effects are observed upon loss of chromatin regulators when cells are triggered with stresses or undergo cellular differentiation, suggesting that the maintenance of gene expression dynamics during this period is critical for fate decisions of stem cells. Future works need to identify the genes that are dynamically regulated during stem cell differentiation, and study the function of chromatin regulators in this process.Regulation of gene expression dynamics is likely to be essential for normal development process, adaptation to new environmental conditions and proper cellular responses to extracellular stresses. A previous report revealed that knockdown of many chromatin regulators had no obvious effect on stem cell maintenance.79 Altered expression, translocation, and amplification of genes for H3K4 HMTs and H3K36 HMTs have been reported in distinct types of cancers.79 Overexpression or mutations of genes encoding HDMs for both methyl marks are also observed in cancers.79 In addition, ING family proteins that directly bind methylated lysines function as tumor suppressors or oncogenes.80 Current studies tend to directly compare global gene expression in normal and cancer cells to identify cancer-associated genes. However, since mutation or overexpression of the factors related to co-transcriptional methylations will have greater effects on gene expression changes in a more dynamic condition, studying their functions during cancer development and progression will be much more important and allow us to identify novel genes that are differentially expressed in cancer cells.The factors involved in co-transcriptional histone methylations and their downstream effectors have been directly linked to many cancers.


Genomic imprinting in mammals was discovered over 30 years ago through elegant embryological and genetic experiments in mice. Imprinted genes show a monoallelic and parent of origin–specific expression pattern; the development of techniques that can distinguish between expression from maternal and paternal chromosomes in mice, combined with high-throughput strategies, has allowed for identification of many more imprinted genes, most of which are conserved in humans. Undoubtedly, technical progress has greatly promoted progress in the field of genomic imprinting. Here, we summarize the techniques used to discover imprinted genes, identify new imprinted genes, define imprinting regulation mechanisms, and study imprinting functions. Mammals have two sets of chromosomes, one inherited from each parent. With the exception of postmeiotic germ cells, mammalian cells maintain a diploid genome during embryonic and postnatal development, and normally both alleles are equally expressed in cells. Intriguing observations from mouse genetic studies in the 1970s showed functional differences between paternal and maternal chromosomes in specific regions  and raisSeveral years later, in 1984, the most direct evidence of functional nonequivalence of mammalian parental genomes was provided by the Solter and Surani laboratories: reconstructed diploid embryos containing two maternal or two paternal genomes were generated through improved nuclear transfer technology –10. SpecIgf2r)  and genome-wide DNA methylation sequencing tools) using reciprocal hybrids of two mouse strains have been employed to identify imprinted genes based on parental-specific expression patterns or regions containing parental-specific DNA methylation , 18. MeaIgf2, a paternally expressed imprinted gene , and inositol polyphosphate-5-phosphatase F [Inpp5f]) in neonatal brains [Pde10], PHD finger protein 17 [Phf17], phosphatase and actin regulator 2 [Phactr2], zinc finger protein 64 [Zfp64], and HtrA serine peptidase 3 [Htra3]) in embryonic day (E)17.5 placentae -AG-haESCs) could efficiently support SC mice generation, with a birth rate of approximately 20% of transferred embryos , Peg3, small nuclear ribonucleoprotein-associated protein N [Snrpn], potassium voltage-gated channel subfamily Q member 1 [Kcnq1], Grb10, Igf2r, and Gnas) were used to replace the genome of oocytes for full-term development of bipaternal embryos [Previous observations in bimaternal mice generated from fusion of fg oocytes with ng oocytes carrying eletions , combine oocytes , 87 H19-DMR aRian long noncoding RNA (lncRNA) in vivo, which abolished the expression of the corresponding Rian gene from the maternally inherited allele [For over 30 years, the homologous recombination-based “knockout” method has been the standard approach to determine the function of imprinted genes in vivo , 76; losd allele , a manipd allele , acts asd allele . Fusion d allele , 101.H19-DMR and IG-DMR deletions can be used for efficient and stable generation of SC mice through ICAHCI. These “artificial spermatids” can be used to make multiple genetic modifications in vitro using CRISPR-Cas9 technology and generate mouse models with new genetic characteristics in a single step [Xist) locus [Xist that regulate X chromosome inactivation during mouse embryo development. In addition, genetically labeling an imprinted gene with a fluorescent or affinity tag in “artificial spermatids” would result in the ability to visualize, localize, and identify protein interactions in tagged mice in vivo [HaESCs have been successfully derived from different species, including rat , monkey gle step , 106–108t) locus , 110 in  in vivo . Moreove


Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed. Cholesterol transport from the outer to the inner mitochondria membrane by steroidogenic acute regulatory protein (StAR) represents a rate-limiting step of steroidogenesis . Ho. Ho2S2 cregnancy –117. in vitro that the activity of purified CYP17A1 was increased by the addition of POR proteins in a dose-dependent manner, resulting in increased androgen production [DNA microarray analyses demonstrated that POR expression is increased with the differentiation of MSCs into steroidogenic cells by SF-1/Ad4BP and cAMP . As in toduction –122. The POR [ POR gene spans more than 70 kb, including coding 1-15 exons and an untranslated exon 1 (exon 1U) residing about 38.8 kb upstream of the coding exon 1 [ POR gene. Consistent with these experimental results, some ABS patients had a deletion of this region, and POR gene was never transcribed from this allele [Although the mechanisms of transcriptional regulation by pituitary hormones remained unknown yet, Ogata and colleagues have analyzed the pivotal elements of the proximal promoter of human POR . The PORg exon 1 . The regg exon 1 . In addis allele , 125.We have shown that MSCs-derived steroidogenic cells provide an important tool for investigating the transcriptional regulation of ovarian steroidogenesis-related genes. Especially, it contributes to understand the function of SF-1/Ad4BP and LRH-1 by revealing the regulation of their transcriptional activity and their novel target genes. This system is also useful for studying other steroidogenic phenomena. Ovarian-specific LRH-1 isoform was also identified based on above studies , 127. Ho