The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes upregulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood.To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion.We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers, and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an upregulation of ALKBH3 non-bound inflammatory genes.The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression.The online version of this article (doi:10.1186/s13073-015-0180-0) contains supplementary material, which is available to authorized users. Genomic DNA is continuously subjected to various harmful insults, such as UV light, ionizing radiation, or nucleic-acid modifying compounds, resulting in thousands of DNA alterations in each cell every day . Such leE. coli, where it demethylates 1-methyladenine (1meA) and 3-methylcytosine (3meC) by oxidation of the N-linked methyl moiety. This reaction creates an unstable methyl-iminium intermediate that spontaneously hydrolyzes into formaldehyde and the non-alkylated base [DNA alkylation can be removed by base-excision repair (BER), direct reversal by methylguanine methyltransferase (MGMT), and dealkylation via the AlkB family , 7. The ted base –17. In mted base . Notablyted base , 18, 19.ted base , 20. Incted base , 21. Indted base . Enzymested base .in vivo genomic targeting of ALKBH3 are not yet fully understood.Recently, we described the cooperativity of ALKBH3 and the ASCC3 DNA helicase complex to promote DNA alkylation damage repair in various cancer cells. ALKBH3 knockdown causes elevated levels of 3-meC accompanied by increased DNA damage response (DDR) and reduced cell proliferation . HoweverHerein, using chromatin immunoprecipitation experiments followed by massively parallel sequencing analysis (ChIP-seq) we find that in PC3 prostate cancer cells ALKBH3 binding is enriched at transcription associated genomic loci, where ssDNA is accessible. Specifically, we find ALKBH3 bound at active gene promoters, enhancers, and regions with putative quadruplex DNA. Unexpectedly, ALKBH3 binds strongly to the initiation sites of some particularly highly expressed gene promoters. Interestingly, these promoters are bound by an unusually large number of transcriptional regulators, indicating a highly regulated ‘hyperactive’ promoter class. However, we find that loss of ALKBH3 does not directly affect expression of ALKBH3 occupied genes, suggesting a transcription unrelated function of ALKBH3. Instead, upon ALKBH3 knockdown we observe an increased expression of genes involved in inflammatory pathways, which could be a downstream effect of elevated DNA damage after ALKBH3 depletion , 25. TheU2OS, 293 T, NCI-H23, and PC3 cells were obtained from the American Type Culture Collection (ATCC) and maintained as previously described . ShRNAs Rabbit anti-ALKBH3 antibodies were obtained from Millipore .3), then incubated with blocking buffer , and finally stained with anti-rabbit-ALKBH3 (Millipore) diluted in blocking buffer. Secondary antibodies (goat anti-rabbit Alexa Fluor 488) were from Millipore.U2OS and PC3 cells were used for IF and were maintained as described previously . The celLentiviral shRNA against ALKBH3, ALKBH2, and GFP as control were used as described before . BrieflyChromatin was prepared from PC3 cells as previously described , except P value 1e-05) of H3K4me1 and H3K27ac (from LNCaP cells), which do not overlap with promoters . ETS transcription factor bindings sites were called using MACS with a cutoff P value of 1e-05. Promoters overlapping with an ETS binding site were considered as ETS factor bound promoters. Predicted G4 DNA sites were downloaded from [Microarrays were normalized and analyzed using the limma package for Bioconductor , 28. ChIded from  and convChIP-Seq and Microarray data are available at the GEO repository with the accession numbers GSE57568 and GSE57591.t-tests, or has been evaluated using hypergeometric probability tests.The significance of the data was either calculated by Cistrome, via unpaired Student’s P value: 1e-04) within the Cistrome platform [ALKBH3 gene promoter is bound by the ALKBH3 protein  analysis of the upregulated genes and found that many genes are involved in the innate/inflammatory immune response Fig. . InterfeSince most inflammatory genes are not directly bound by ALKBH3, we speculate that the inflammatory response may be an indirect consequence of DNA damage accumulation in the ALKBH3 deficient cells , becauseTogether these data suggest that ALKBH3 does not function to regulate transcriptional activity. Instead, the induction of inflammatory genes supports a potential role of ALKBH3 at its genomic targets to remove DNA alkylation adducts, such as 3-meC or 1-meA , 19, 24,Next we wished to determine if ALKBH3 was bound to specific DNA regions. Previous work showed that for DNA demethylation ALKBH3 prefers to demethylate ssDNA over dsDNA substrates , 18, 19.The group II promoter profile is similar to the RNA polymerase II profile - both peak at +60 bp and possess a ‘shoulder’ upstream of the transcription start site Fig.  and b. TInterestingly, the ALKBH3 group I promoter profile does not overlap significantly with RNA polymerase II. Instead, the ALKBH3 binding profile culminates slightly upstream of the TSS, at the site of transcription initiation Fig. . We specSingle-stranded DNA occurs not only at promoters, but also at other places in the genome. During transcription elongation, the transcription bubble moves along the gene. Interestingly, highly expressed genes, which have elevated transcription elongation, have elevated ALKBH3 levels in the gene body  Fig. . The prePrevious work suggested a pivotal role of ALKBH3 in multiple cancer types, such as prostate , 51, panWe initially performed genome localization studies to identify genomic regions bound by ALKBH3. We found that ALKBH3 preferentially occupies locations where ssDNA is occurring, such as promoters, enhancers, and G4 DNA (Fig. ALKBH3 was most enriched at the initiation site of a small number of highly expressed genes Fig. . Furtherab initio would neither be timely nor financially feasible.Characterization of the ALKBH3 bound promoters was carried out using publicly available data from different research groups and different cell types. Despite use of this relatively heterogeneous dataset, we were still able to detect a correlation between strong ALKBH3 binding at group I promoters and enrichment of factors involved in gene activation. This study highlights how publicly available datasets can be utilized to develop novel hypotheses, while creating these data Since we see this correlation of highest ALKBH3 binding with enriched binding of other factors across multiple cancer cell types we conclude that these hyperactive promoters exist in a relatively cell type independent manner. However, we cannot exclude that a transition between group I and group II promoters can occur and that a group II promoter might get promoted to a hyperactive promoter, and vice versa, once the level of activating factors at the promoter exceeds or falls below a certain threshold, respectively. Since we hardly see promoters where the ALKBH3 binding is in an intermediate state between group I and group II promoters, we hypothesize that these transitions – if they take place – are rare. It also remains to be determined whether these hyperactive promoters are restricted to highly proliferative cancer cells or if they occur in all cell types.ALKBH3 binding to group I promoters is about eight-fold stronger than to group II promoters. We hypothesize that an elevated formation or an increased accessibility of ssDNA at the initiation site of group I promoters is the source for the increased ALKBH3 binding (Fig. We performed microarray analysis to elucidate whether ALKBH3 affects transcription of its target genes (Fig. Knockdown of ALKBH3 in PC3 cells leads to an induction of inflammatory response gene expression Fig. . If trueSince ALKBH3 demethylates RNA in addition to ssDNA , 18, it Alkylating agents that methylate DNA and disrupt genomic integrity of fast proliferating cells are widely used in cancer treatment . Due to in vitro, in vivo, and via bioinformatics will help to better understand their mechanisms as well as their role in transcription regulation and during cancer progression.The genome-wide binding pattern of ALKBH3 revealed a strong binding to the initiation sites of a small number of highly active promoters. We hypothesize that these promoters are a new class of ubiquitously expressed promoters, which may have a specific initiation process, allowing ALKBH3 to ‘mark’ these promoters. Follow-up investigation of those hyperactive promoters


Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify  The gene expression program of stem/progenitor cells and their progeny is temporally controlled by the coordinated action of transcription factors (TFs) that bind to DNA regulatory elements, including promoters and enhancers. TFs regulate gene expression and at the same time shape the epigenetic state of chromatin by recruiting DNA methylation and histone modification complexes playing essential roles during commitment134568Genome-wide approaches allow to analyze the functional relationships between chromatin dynamics, gene expression patterns and cell phenotype with unprecedented levels of detail and integration. Most of the available knowledge on the molecular mechanisms driving stem cell development, commitment and differentiation comes from the study of murine models10121314151651819202122232425181927282930103233bona fide cell-specific enhancers and genes defining cell identity.This study describes the transcriptional and epigenetic profile of human HSPC and their early committed erythroid and myeloid progeny, as determined by CAGE, ChIP-seq for histone modifications, and retroviral scanning, a novel tool to map active regulatory sequences based on the integration properties of the Moloney murine leukemia virus (MLV). MLV integrates almost exclusively in active promoters and enhancers in mammalian genomes3536383940+/CD133+ population by FACS (low/CD36high and CD34−/CD13+ populations (+ and expressed low levels of glycophorin A (GYPA), indicating that they are mainly composed by erythroid progenitorsWe prospectively enriched human HSPC as CD34 by FACS . HSPC sh by FACS . Committulations . Over 95NOG, CHRDL1, TGFB1I1), or to the tumor necrosis factor superfamily, involved in T- and B-cell functions , while genes involved in leukocyte biology  were down-regulated upon erythroid commitment. Genes up-regulated in EPP are involved in erythrocyte differentiation and homeostasis and include the master regulators GATA1 and KLF1. Instead, genes up-regulated in MPP are mainly involved in immune defense function of neutrophils and macrophages   of gene expression profiles showed that biological replicates of each cell population were reproducible, and that HSPC, EPP and MPP exhibit distinct gene expression profiles . Supervi1, CTSG) .6 TSSs in each cell population, typically scattered over short genomic regions due to the inherent variability of transcription initiationHBB) in EPP, which started at low frequency at 13 nucleotide positions in a 58-bp region encompassing the 5′ UTR of HBB , a technique that identifies active transcription start sites (TSSs) at single base-pair resolution and measures the expression level of each transcriptR of HBB . We mappR of HBB . InteresR of HBB . NotablyR of HBB . About 3R of HBB .We defined >13,000 CAGE promoters of similar average length (160 ± 101 bp) in each cell type as clusters of nearby TSSs , >96% of which overlapping with epigenetically defined promoters . We assiP < 0.0001) (P < 0.01)   promoters between HSPC and EPP (306 down-regulated and 408 up-regulated) and 1,127 between HSPC and MPP (576 down-regulated and 551 up-regulated). We observed a high degree of correlation between DU CAGE promoters and DE genes: >40% DE genes were associated with DU CAGE annotated promoters. Moreover, we validated differential RNA expression by qRT-PCR in a sample of 18 DU promoters . Only 78 0.0001) , and 30% < 0.01) . DU prom 0.0001) , as well 0.0001) .LMO2 gene, coding for a developmentally regulated TF, is transcribed from 3 different promoters, of which Promoter 3 was active only in HSPC, Promoter 1 mainly in HSPC and MPP, and Promoter 2 predominantly in EPP  within the proximal regions of cell-specific promoters. HSPC-specific promoters were fairly enriched for binding motifs of the ETS family of TFs, which regulate development and maintenance of HSCs and their differentiation along multiple lineages . EPP- anNext, we analyzed CAGE promoters not assigned to known genes. Around 22% of these unannotated promoters harbored an epigenetic enhancer signature, a frequency that increased up to 45% for DU promoters , suggest+) from weak/inactive (H3K27ac−) regulatory elements. We identified >12,000 promoter regions in HSPC, EPP and MPP, with a similar average size, 60% of which carried the H3K27ac mark, and >44% were associated with CAGE TSSs (+ promoters (>72%) were actively transcribed, and of these, 12% were not annotated, suggesting the presence of yet uncharacterized genes. In parallel, we defined >49,000 putative enhancers in both multipotent and committed progenitors , and a low proportion of the weak/inactive ones, were shared with HSPC . On the + cell-specific enhancers on nearby genes, we analyzed the expression level of the closest CAGE promoters. We observed a mild increase in the expression level of promoters close to active enhancers in all three cell population compared to unrelated cell populations, and at least in EPP and MPP, a significant increase compared to total CAGE promoters used by these populations  have been described as clusters of enhancers, involved in the specification of cell identitysee+ non-targeted promoters and enhancers, respectively; vs. 12%, 57 vs. 11% and 73 vs. 17% in HSPC, EPP and MPP, respectively, p < 0.0001).We mapped and analyzed the distribution of >27,000 MLV integration sites in each cell population. About 22% of MLV integrations occurred in TSS-proximal regions and the remaining ones were equally distributed in intergenic and intragenic regions . Statistbona fide cell-specific and developmentally regulated enhancers. HSPC-specific, MLV-targeted enhancers were associated with genes expressed in hematopoietic organ development and in the immune system, while EPP-specific enhancers were associated to erythrocyte differentiation and MPP-specific enhancers to regulation of the immune system and leukocyte differentiation  of unannotated DU CAGE promoters were marked by histone modifications typical of enhancers. These results support the hypothesis that a consistent fraction of novel CAGE promoters drives the expression of eRNAs, possibly involved in the regulation of proximal genes4950515253555657Analysis of histone modification signatures allowed the identification of >49,000 putative enhancers in each cell population, indicating that most promoters interact with multiple enhancers. Differently from promoters, enhancers consistently changed upon commitment: >50% of the active enhancers and SEs mapped in EPP and MPP were not shared with HSPC, and 60 to 80% of the active enhancers and SEs mapped in HSPC disappeared in EPP and MPP. These data indicate that enhancers are dramatically redefined during lineage commitment, and that differential enhancer usage is responsible for the differential regulation of promoter activity underlying lineage restriction. Activation of the set of lineage-specific enhancers is most likely responsible for both activation of lineage-specific promoters and fine tuning of the non-specific ones.bona fide cell-specific enhancers. The analysis of a larger dataset of integrations (>1 million) in HSPCTo provide additional clues to the genomic regions defining cell identity, we exploited the integration properties of MLV, a retrovirus that targets chromatin regions, epigenetically marked as active promoters and enhancers35383940Overall, through a combination of transcriptional, epigenetic and MLV signatures, this study provides a genome-wide description of chromatin and transcriptional dynamics and a collection of unannotated regulatory elements differentially used during commitment of human hematopoietic progenitors. In particular, the MLV integration preferences provide a biological scanning function for regulatory elements specifically active during cell state transition and involved in the specification of cell identity.We obtained human cord blood from healthy donors. Informed consent was obtained from all subjects. All experiments were performed in accordance with the Declaration of Helsinki (TIGET01 protocol). San Raffaele Scientific Institute Ethical Committee approved this study. Culture conditions and purification are described in AffyQControl package. All arrays in the dataset have good and reproducible quality metrics according to standard guidelines. To convert CEL file fluorescence signals to log2 expression values, we used the robust multi-array average procedure RMA of the Bioconductor affy package and the HG-U133 Plus 2.0 custom Chip Definition Files (CDF) based on GeneAnnotprcomp function of the R stats package to verify the reproducibility of the 3 biological replicates. We used DNA-Chip Analyzer (dChip) software61http://www.hsph.harvard.edu/cli/complab/dchip/ for details).We determined the transcriptional profiles of multipotent and lineage–committed progenitors using Affymetrix HG-U133 Plus 2.0 GeneChip arrays  . We performed quality controls in R using the Bioconductor We extracted RNA from multipotent and lineage-committed progenitors obtained from a pool of 3 donors. DNAFORM Inc. (Japan) performed DeepCAGE library preparation and data analysis. We defined TSSs by summing the weighted number of CAGE tags at each genome position. Then we clustered TSSs to define promoters if they were within 20 bp of each other. Using a custom R-script, we annotated promoters using publicly available data sets. We validated CAGE data by qRT-PCR. The details of promoter construction and annotation, statistical analyses and qRT-PCR are described in We performed ChIP-seq for H3K4me3, H3K4me1 and H3K27ac using a pool of 3 donors. ChIP library preparation and sequencing are described in 8We transduced EPP and MPP obtained from a pool of 3 donors, mapped MLV integration sites by LM-PCR and pyrosequencing, and defined clusters of recurrent integrations, as previously described−2 were excluded.We performed gene functional annotation using Ingenuity Pathways AnalysisAccession codes: The datasets supporting the results of this article are available in the Gene Expression Omnibus repository under the accession number GSE70677.How to cite this article: Romano, O. et al. Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment. Sci. Rep.6, 24724; doi: 10.1038/srep24724 (2016).


Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M. tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M. tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M. tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.Expression of SigH, one of 12  Mycobacterium tuberculosis is a slow-growing but deadly pathogen that is able to cause a chronic infection through its ability to adapt to the multiple environments that it encounters in the human host during infection. During the course of M. tuberculosis infection, ranging from primary infection with active bacterial replication, to latent infection with restricted replication within granulomas, to cavitary tuberculosis (TB) where many bacteria are extracellular, M. tuberculosis is subject to an extensive and varying array of host-generated stresses. Among these are hypoxia, and several forms of oxidative and nitrosative stresses that can damage a broad range of bacterial macromolecules that are essential for viability , hydrolyzed casein (1g l-1), 0.2% glycerol, and 0.05% Tween 80. At OD600 = 0.5, aTc was added to the culture at a final concentration of 200 ng/ml and cells were grown for 24h, followed by cross-linking with formaldehyde, quenching, pelleting, cell lysis, DNA shearing, immunoprecipitation with anti-FLAG antibody and further processing as described [ChIP-Seq was performed as previously described with minor modifications . The sigpromoter . Prior tdextrose  g l-1, NGenomic DNA enriched for SigH binding was then sequenced on the Illumina platform using a GAIIx instrument (Boston University sequencing core facility). Significantly enriched peaks in the sequence data from the SigH-FLAG-expressing strain relative to the vector control strain were identified as previously described . BrieflysigH-FLAG fusion was expressed in either wild type M. tuberculosis H37Rv or a ΔsigH strain derived from H37Rv [sigH-FLAG were compared to results from the ΔsigH strain containing the vector only and significantly enriched sites were identified as described above. In addition to the SigH binding sites identified using the statistical approaches described above, additional candidate SigH binding sites were identified by visual inspection of tdf files displayed using the Integrative Genomics Viewer [The om H37Rv . Data frs Viewer . PutativsigH strains were grown to mid-log phase (OD600 = 0.4–0.5) at which point the cultures were subjected to heat stress at 52°C for 15 min. To assess transcription following oxidative stress, both strains were grown to mid-log phase and exposed to 50μM plumbagin for 20 minutes. Cells were then harvested by centrifugation, re-suspended in TRI Reagent (Molecular Resource Center) and mechanically disrupted . RNA was extracted according to the manufacturer’s protocol and purified using an RNA cleanup kit (Qiagen), followed by two rounds of DNAse treatment. The quantity and quality of RNA was determined by measuring absorbance at 260 and 280 nM using a Nanodrop instrument (Thermo Fisher). Reverse transcription was performed using the qScript cDNA synthesis kit (Quanta Bioscience).To evaluate whether binding sites identified by ChIP-Seq were linked to genes that showed SigH-dependent transcription, we performed quantitative reverse transcription PCR (qRT-PCR). H37Rv and ΔS1 Fig. qRT-PCR of cDNA was performed by using the PerfeCTa SYBR Green Supermix (Quanta Biosciences) on an Applied Biosystems 7300 real time PCR system using the following protocol: denaturation at 95°C for 3 min., followed by 40 cycles of denaturation at 95°C for 15 s and annealing/elongation at 60°C for 1 min. Data were analyzed using the ΔΔCT method using the housekeeping gene sigA as the control [sigH) >2 were analyzed in two biological replicates. The primers used for qRT-PCR are shown in S1 Table.Genes were selected for transcript quantification if they were adjacent to and oriented away from the SigH binding sites identified in the ChIP-Seq experiments. If a binding site was within the 5’ half of the annotated coding sequence of a gene the expression of the 3’ region of that gene was quantified. For some intragenic binding sites, we also determined whether a SigH-regulated antisense transcript was present. A map of all evaluated genes is shown in supplemental  control . The inisigH strain following heat stress were selected for experimental determination of transcription start points (TSPs). RNA Ligase Mediated-Rapid Amplification of cDNA Ends (5’-RLM-RACE) was performed on RNA extracted from H37Rv and the ΔsigH strain following 52°C heat stress as described above. First, Terminator 5’-phosphate dependent exonuclease (Epicentre) was used to digest the total RNA sample containing mRNAs, rRNAs and tRNAs to produce an mRNA-enriched preparation. RNA 5’-polyphosphatase was then used to convert 5´-triphosphorylated RNA (mRNA) into 5´-monophosphorylated RNA, which was then used for 5’ RACE adapter ligation. The resulting 5’ adapter-ligated mRNA was then used for cDNA synthesis  and this cDNA was then used as the template for amplification of specific genes. Two rounds of PCR were performed, using outer and then inner 3’ primers specific for each gene, with an adapter-specific 5’ primer in each reaction. Amplicons from the inner PCR were run on an agarose gel. PCR products present in reactions using RNA from wild type, but absent in reactions using RNA from the ΔsigH strain were gel-purified and cloned into pGEM-T (Promega). In cases where PCR products from H37Rv and ΔsigH only had amplicons of similar size, the amplicon from the wild type reaction was selected. The cloned PCR products were sequenced to identify the TSP as the locus-specific base immediately 3’ to the adapter sequence. The adapter sequence and the primers used for 5’-RACE experiments are shown in S2 Table.Genes that were determined to have >2-fold higher expression in wild type relative to the ΔThe 50 bases 5’ of each TSP identified experimentally by 5’-RLM RACE, together with SigH-regulated promoters that had been previously identified experimentally, were analyzed using the MEME algorithm , to deteM. tuberculosis genome that are bound by SigH. These experiments were performed using SigH fused to the FLAG epitope tag, expressed from an inducible TetR-regulated promoter [sigH-FLAG was introduced into both H37Rv (wild type) M. tuberculosis and a ΔsigH strain derived from H37Rv [Fig 1A). As measured by qRT-PCR, the level of sigH-FLAG expression in H37Rv following aTc-induction is approximately 60% of the expression of the level of sigH from the chromosomal copy of this gene following stimulation with heat stress, suggesting that the level of induced expression would not likely result in binding of chromosomal DNA at sites that are not bound by native SigH in response to stress . To perform the ChIP-Seq experiments, wild type M. tuberculosis H37Rv or a Δ sigH strain was grown to mid-log phase at which point expression of sigH-FLAG was induced by adding aTc and incubating at 37°C for 24h. DNA sites that were bound by SigH in these strains, but not in a strain in which the vector only was present, were identified using an optimized ChIP-Seq protocol as described in the materials and methods [ChIP-Seq was performed to identify the DNA sequences throughout the promoter . A repliom H37Rv . SigH-FL methods .S1 Fig and S3 Table). The ChIP-Seq binding data, represented graphically, are shown in Fig 2. The upper panel shows a representative binding peak for the region 5’ of sigE, which has a known SigH binding site, with the characteristic shift in the position of the sequences bound in the forward versus the reverse DNA strands. The distribution and read coverage for binding sites throughout the genome are shown in the lower panel. The ChIP-Seq experiments identified binding sites 5’ of all but one of the 7 genes previously confirmed to be directly regulated by SigH in our previous work. The exception was clpB, which is co-regulated by a heat responsive repressor whose binding site overlaps the SigH-regulated promoter of this gene [sigH strains relative to wild type [udgB, which encodes a DNA repair enzyme, rpmE, which encodes a zinc-binding ribosomal protein, msrB, which encodes an oxidized methionine repair enzyme, molybdopterin sulfuryltransferases, a set of genes encoding a cysteine biosynthesis pathway, and several genes of unknown function.62 sites showed significant SigH binding and 7 additional candidate binding sites were identified by visual inspection of the data . This is a higher proportion of transcription factor binding sites associated with regulated transcription compared to many other ChIP studies, e.g. 25 of 67 (37%) M. tuberculosis SigF binding sites were linked to SigF-dependent transcription in a ChIP-chip analysis, and a ChIP-Seq study of >100 M. tuberculosis transcription factors found transcription factor overexpression resulted in changes in expression of genes linked to <10% of transcription factor binding sites [sigH overexpression. We identified 26 of these binding sites in our experiments. It is not clear why we identified substantially more binding sites in our study, however, our optimization of SigH-FLAG expression together with our inclusion of a small number of manually identified sites likely contributed to the greater number of binding sites identified in this work. The several binding sites that are uniquely present in our ChIP-Seq results that are linked to SigH-regulated expression in our data and in previous studies supports the validity of our binding data  were linked to transcripts that had more than 2-fold greater expression in wild type compared to the Δng sites , 32. ThaH strain . This isFig 1B), indicating that false positive binding sites are not likely to result from excessive induced expression of SigH-FLAG in these experiments. This result suggests that SigH binds to sequences in the M. tuberculosis chromosome in a manner that does not lead to transcription initiation in the conditions we tested.Though over half of SigH binding sites in this study were linked to SigH-regulated transcription, a large proportion were not, indicating that a substantial number of the SigH binding sites that we identified do not function as SigH-regulated promoters under the heat stress condition we tested, where SigH is strongly expressed. Two potential reasons for this finding are i) off-target (non-promoter) SigH binding in the ChIP-Seq experiments, and ii) effects of additional regulatory factors, e.g. repressors or activators that may inhibit or enhance transcription from specific genes, and whose activity may be affected by different stresses. To address the first issue, we determined that expression of SigH-FLAG in response to aTc induction in the ChIP experiments was lower than expression of SigH from the native chromosomal gene following heat stress (sigH strains were grown to mid-log phase and subjected to heat stress or to oxidative stress with 50 μM plumbagin for 20 minutes. We verified increased sigH expression (17.3-fold) in response to plumbagin. The control genes Rv0759c and Rv2466c showed 2.4-fold and 118.9-fold induction, respectively, in response to plumbagin. Of the 15 genes that had not shown heat-induced increased expression, 7 showed low-level induced expression (2.3–3.6-fold), but none showed high-level (>5-fold) increased expression  contain sequences that match the SigH -10 and -35 consensus promoter sequences , 4 were linked to SigH regulation of an adjacent gene and 19 were not associated with SigH-regulation of transcription in the same or an adjacent gene in the conditions we tested . We also performed qRT-PCR targeting the region upstream of the binding site in a subset of genes in which intragenic binding sites were present in the 3’ half of the gene, to see if binding is associated with antisense transcription. Of the 8 genes analyzed, we were able to detect an anti-sense transcript in only one, Rv3056, which showed modestly (3.0-fold) increased heat stress-induced expression in wild type M. tuberculosis compared to the ΔsigH strain.We identified 33 SigH binding sites that were wholly within annotated coding sequences, which we defined as intragenic. This proportion is similar to what was observed for the tor FliA . SeveralsigH strain. Using 5’ RLM-RACE we successfully identified 25 TSPs that had not been previously identified. Of these, 18 were between 5 and 250 bp 5’ of the annotated initiation codon, 3 were within 5 bp of the initiation codon and 4 were >5 bp 3’ of the initiation codon. The first group of TSPs would produce transcripts with 5’-untranslated regions of RNA of various lengths consistent with typical leadered mRNAs, the second group are consistent with leaderless mRNAs but may also result from incorrect annotation of the initiation codon, while the third group suggests the possibility of alternative forms of the protein versus mis-annotation of the translational start site. All 7 previously identified TSPs are located in a position that would produce leadered mRNAs [To precisely determine the sequence requirements for SigH-dependent transcription, we attempted to identify the transcriptional start point (TSP) for genes linked to a SigH binding site for which a 3’ gene showed at least 2-fold greater expression in wild type compared to the Δed mRNAs .17)-GTT (where Y is C or T and R is A or G) . 25 of these promoters, including 18 newly identified in this work, have sequences that match both the -10 and -35 consensus sequence elements with appropriate spacing between these sequences. The remaining 7 promoters newly identified in this work do not match the consensus . In some cases, partial sequences corresponding to the -10 or -35 consensus are present at the appropriate position relative to the transcription start point. In addition to the sequence requirements for SigH binding indicated by these data, a key feature of the consensus is the restricted spacing between the -10 trimer and -35 hexamer. Of the 25 experimentally identified promoters, in 19 there are 17 bases between the -35 hexamer and the -10 trimer. In two promoters there are bases and in 4 promoters there are 18 bases separating these promoter elements.Using the MEME suite of software, we analyzed the 40 base pairs 5’ of these TSPs, together with the TSPs of the 7 promoters previously shown to be directly regulated by SigH , to idenFig 4). Among the 8 promoters that do not match the consensus sequences, the median induction is 7.1 fold with only 3 showing greater than 3-fold induction. Similarly, the median binding enrichment in the ChIP-Seq experiments was lower at sites containing promoters that lacked both the consensus promoter sequences (11.6-fold) compared to sites that do match both of these sequences (18.6-fold).Comparison of SigH-dependent transcription following heat stress of the experimentally validated promoters that contain the both the -10 and -35 consensus promoter sequences and those that do not, shows a striking difference between these groups. The genes 3’ of the 25 promoters that match the consensus show a mean 173-fold SigH-dependent induction following heat stress, with only 1 showing less than 8-fold induction  and Rv3206c (moeZ or moeB1) [msrB (Rv2674), which encodes an enzyme that repairs oxidized methionine residues.The newly identified SigH-regulated genes markedly expand the functional roles of SigH-regulated gene expression in response to stress. Prior results had demonstrated that SigH is important for aperones , 12. Ourgue SigR , Also str moeB1) . FurtherThough 41 of 69 of SigH-binding sites were linked to SigH-dependent gene expression, the expression of genes adjacent to many binding sites did not show evidence of SigH-regulation in response to heat stress. Testing the expression of several of these genes in response to plumbagin-induced oxidative stress showed low level SigH-dependent expression for 7, however most did not show increased expression in response to either stress. The lack of gene regulation linked to a subset of binding sites of sigma factors or transcription factors has been a consistent observation in many ChIP-Seq studies –34. In s17-18)-GTT, it is clear that variation that was not previously known can be tolerated at the 2nd, 5th and 6th positions of the -35 hexamer and at each position in the -10 trimer. In addition, while our previous results showed that SigE prefers a pyrimidine at the 6th position of the -35 hexamer [In previous work, based on a small number of genes directly regulated by SigH, we identified a SigH binding consensus and subsequently performed extensive mutagenesis of the -10 and -35 sequences , 19. ThiIn this work we have identified a greatly expanded direct SigH regulon that markedly broadens the role of SigH in stress response and recovery. In particular newly identified SigH-regulated genes indicate a direct role for SigH in DNA repair, sulfur metabolism, synthesis and salvage of sulfur-containing amino acids and recovery of translation. In addition to these specific new insights into SigH function, this research demonstrates the value of genome-wide approaches to understanding bacterial gene regulation, while highlighting the importance of targeted follow-up experiments to more fully understand the implications of the genome level data.S1 FigsigH strain) is shown for genes adjacent to each binding site. Where an expression ratio >2 was observed, the arrow is black; where an expression ratio is <2 the arrow is grey. Arrows corresponding to genes that were not tested are white.For each SigH binding site, the region containing a binding site is shown. Black bars under each locus indicate the location of the binding site. The expression ratio (fold induction following heat stress in wild type divided by fold induction following heat stress in the Δ(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


DNA interactions are important in the adult, where their perturbation can lead to disruption of homeostasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein–DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin‐binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology. WIREs Dev Biol 2016, 5:25–37. doi: 10.1002/wdev.205The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin‐binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein–WIREs website.For further resources related to this article, please visit the  Regulation of gene expression is primarily coordinated by interactions between proteins and DNA. Understanding the mechanisms by which these proteins mediate transcriptional regulation is fundamental to the study of genetics, cell, and developmental biology. The interactions of myriad DNA‐binding proteins and accessory factors are variable and complex, so the techniques used to interrogate them must be comprehensive and versatile. One such technique is DNA adenine methyltransferase identification (DamID).Drosophila, Caenorhabditis elegans, and ChlamydomonasEscherichia coli DNA adenine methyltransferase catalyzes the addition of a methyl group to the N6 position of adenine in the sequence, GATC.E. coli Dam as a fusion protein with a chromatin interacting protein of interest . Dam is recruited to specific loci by virtue of being tethered to a protein with an affinity for a particular sequence or chromatin environment, and is able to deposit methyl groups on nearby GATC sequences. After extracting methylated DNA from transgenic cells, the methylation sensitive restriction endonuclease, DpnI, can be used to fragment the genomic DNA. Methylated sequences may then be amplified by PCR and detected by various sequencing or array‐based methods to produce a map (chromatin profile) of loci at which the protein of interest has been in close proximity to, during the period in which the Dam‐fusion was expressed . The methylation at a particular loci is often expressed as the ratio of methylation for Dam‐fusion: Dam, thereby, normalizing the background methylation.Drosophila, which remains the model in which DamID has been most extensively applied.C. elegans.Since its inception by Bas Van Steensel and Steven Henikoff in 2000, DamID has been used to profile chromatin interactions in multiple organisms. The technique was first demonstrated in When preparing to interrogate chromatin associations of a protein of interest, there are several experimental approaches which can be considered, each with relative advantages and disadvantages Table .20 For aOne of the main drawbacks of ChIP, in comparison to DamID, is that it requires a highly specific antibody that binds with high affinity to the protein of interest.DamID and ChIP contrast in their relative suitability depending on the temporal expression or abundance of the protein of interest. If detecting interactions between DNA and a protein thought to be present at very low abundance, it may be difficult to obtain sufficient binding and purification even with a verified ChIP‐grade antibody. As Dam‐fusion proteins are required to be expressed at extremely low concentrations anyway, low endogenous protein abundance is not an impediment to DamID. For highly expressed proteins, either technique may be appropriate. In contrast, if the experiment requires profiling for a very short‐time interval, ChIP may be a more suitable choice, as Dam‐fusions are typically expressed for a minimum of several hours to achieve robust methylation. These differences in temporal suitability emphasize a fundamental difference between the two protocols, namely that ChIP measures where a protein is bound at any one instance, whereas DamID gives an indication of where a protein has previously been in proximity to the DNA sequence, even if only transiently.It is possible to use both ChIP and DamID to generate chromatin interaction profiles for individual cell or tissue types of a whole organism. However, for ChIP, this requires separation of the cells or nuclei of interest from the starting material, often using flow cytometry or dissection, both of which are time‐consuming processes. In contrast to this, genetic techniques can be exploited to give Dam methylation only in a tissue of interest, negating the need for cell separation (see ‘Cell type‐specific profiling with DamID’ subheading).bona fide chromatin interactions.Interpretation of data obtained from any genome‐scale experiment usually requires careful and considered implementation of bioinformatic techniques, specifically tailored to the experiment in question. DamID and ChIP are no exception to this. As ChIP is a longer established technique, the computational tools used to analyse ChIP data are subsequently better developed than for DamID. Furthermore, the majority of studies reporting DamID data have used microarray‐based approaches, while DamID‐seq has only been more widely adopted recently. However, computational pipelines have recently been published for the analysis of DamID‐seq data.de novo identification of important genes in various biological processes. Orian et al., have used this approach to generate chromatin profiles for the Myc/Mnt/Mad network of transcription factors, showing that these factors interact with around 15% of all coding regions.Drosophila genome, demonstrating the presence of transcriptional ‘hotspots’ at which many proteins may be recruited independently of their DNA‐binding specificity.Drosophila species.Of all the protein–DNA interactions that occur, the interaction between transcription factors and promoter or enhancer regions of genes is among the most studied, due to the fundamental role of these proteins in transcriptional regulation. DamID has been extensively utilized to uncover the mechanisms of transcriptional regulation by transcription factor binding in multiple organisms and cell types. DamID has been used to produce chromatin‐binding profiles for a diverse range of transcription factors with various physiological roles Figures a.33, 34,Drosophila genome, using a truncated form of the protein lacking a functional DNA‐binding domain, has been shown to have no effect on its targeting to genomic hotspots, indicating that recruitment of Bcd by external factors is sufficient for the interaction of Bcd with some loci.Despite intense study, the manner in which DNA‐binding proteins, such as transcription factors, interact with DNA is little understood in many cases. The fact that all the components needed for a DamID experiment are encoded within the experimental organism allows for the effects of precise manipulations of either the DNA‐binding protein, or target sequences of interest, to be monitored. For example, a Dam‐fusion protein may be expressed in an organism in which the consensus site at a particular locus has been modified, in order to determine the effect of specific mutations on transcription factor binding. Alternatively, mutations can be made in the sequence for the DNA‐binding protein itself to determine which specific residues or domains are required for interactions with genomic loci or consensus motifs. The feasibility of this approach has been demonstrated by Vogel et al., who monitored the effect of genome‐wide chromatin binding when specific point mutations were introduced to CBX1, a human orthologue of heterochromatin protein 1 (HP1).Chromatin can exist in a variety of configurations within the nucleus, defined by histone occupation or post‐translational modifications, which are thought to closely correlate with the regulation of gene expression. Traditionally, these regions of chromatin have been broadly categorized into euchromatin and heterochromatin .Drosophila and Arabidopsis to help elucidate the mechanisms of heterochromatin formation.Drosophila. Interestingly, this study revealed that HP1 is significantly associated with X chromosomes in males only. In Drosophila, dosage compensation upregulates transcription from the male chromosome, therefore, these data indicated a previously unknown role for HP1 in the activation of gene expression. The association of HP1 with transcriptionally active genomic loci has subsequently been more extensively characterized using DamID.DamID with proteins known to be involved in chromatin structure has been employed extensively to understand how chromatin regions correlate with their underlying DNA sequences Figures . One sucDrosophila identified extensive regions of polycomb binding and implicated polycomb mediated repression in several previously unidentified biological processes.DamID can also be used to profile chromatin binding of proteins known to induce chromatin modifications such as acetylation or methylation of histones. Such an approach can give an indication of genomic regions that have undergone chromatin modifications, . DamID has been used to generate binding profiles for a histone deacetylase (HDAC4) in rat cardiomyocytes.Drosophila cultured cells.In a recent study, DamID was used to profile binding for over 50 different proteins known to be associated with chromatin in C. elegans genome.Although DamID is predominantly used to target methylation to specific genomic loci, the relative promiscuity of Dam has also been exploited to characterize chromatin states more broadly. Sha et al. have used Dam expression in the absence of a fusion protein to probe accessible regions of the cis‐methylation) is also seen on distinct DNA strands at sequences in close proximity to a Dam‐targeted locus (trans‐methylation).The spatial organization of chromatin within the nucleus is highly regulated in order to achieve efficient packaging of DNA and to mediate transcriptional regulation. DamID has proved to be an invaluable resource in understanding how chromatin is arranged with respect to itself and the nuclear environment. Dam‐fusion proteins have been shown to have the ability to methylate GATC sequences up to 5 kb away from a Gal4‐targeted locus.trans‐interacting loci are already known, but not for the identification of novel interacting loci. Alternatively, Dam may be tethered to a specific locus by the manipulation of the sequence of interest to include a binding site for an exogenous DNA‐binding protein. This method has the advantage of being able to target one distinct locus, as well as providing the option of a control in which no exogenous sequence has been inserted for the Dam‐fusion to bind.In order to examine the nature of such chromatin looping, it may be necessary to target Dam to a specific locus. This can be achieved by a number of different strategies. First, Dam‐fusions can be created for proteins known to bind at a specific locus. Although this approach may appear straightforward, it is unlikely that any given transcription factor, insulator, or other chromatin‐binding protein will bind to a single locus. Therefore, this strategy may be applicable if Drosophila this has been demonstrated using the well‐characterized yeast transcription factor, GAL4. In this case, a GAL4 upstream activation sequence (UAS) was inserted into the genome adjacent to the site of interest to which the Dam‐GAL4 fusion protein could bind  are readily available,Saccharomyces cerevisiae. Fusion of Dam to the DNA‐binding domain of TetR was used to target Dam to Tet operators inserted at the HML locus. In this study, high levels of methylation were detected in trans at telomeric sequences, including those of different chromosomes, indicating a spatial relationship between the HML locus and telomeres within yeast nuclei.In  Figures e. This tRecent advances in genome‐targeting technologies may provide an even more efficient way to target Dam to specific loci. Recombinant proteins with affinity for specific loci, such as TALEs (transcription activator‐like effectors) may now be relatively easily produced.Drosophila cells.DamID has also been used to understand the interactions between chromatin and its surrounding environment. By fusing Dam with components of the nuclear lamina, it has been possible to determine the sequences that are located at the nuclear periphery or interact with the nuclear envelope Figures f.56 Thisin vivo.in vivo. This technique has been used to track the movements of LADs through mitosis in a single cell in real time, providing valuable insights into chromatin dynamics throughout the cell cycle.Chromatin is known to be dynamic within the nucleus, with genomic regions undergoing dramatic spatial rearrangements concomitant with cell differentiation or transcriptional state.Drosophila have demonstrated that the low‐level induction of Dam in a tissue specific and temporally controlled manner can be achieved to produce DNA‐binding profiles with spatial and temporal resolution without the need for cell purification , an expression system was developed in which Dam‐fusion proteins are encoded as a secondary open reading frame (ORF) in a bicistronic expression construct downstream of a UAS enhancer. This system takes advantage of the phenomenon of ribosome reinitiation to express the product of the secondary ORF at very low levels.Drosophila neural stem cells in order to assay the genome‐wide occupancy of Pol II on chromatin.The ability to easily perform DamID experiments in a cell type‐specific manner has drastically increased the scope of experiments possible to help understand chromatin biology in multicellular species. One particularly useful application of TaDa that has been demonstrated is its ability to profile the transcriptional state of specific cells. For this purpose, Dam was fused to the core subunit of RNA polymerase II (Pol II) and expressed in two separate populations of Drosophila will allow for extensive characterization of tissue‐specific transcription factor occupancy.Aside from transcriptional profiling, TaDa also has the potential to be employed for a range of novel applications. The binding of transcription factors to DNA is likely to be highly context specific, therefore, data produced from DamID experiments conducted in whole organisms may mask variation in transcription factor binding in individual cell or tissue types. TaDa may be employed to assess the extent to which there exists diversity of binding for ubiquitously expressed transcription factors across cell types. It is likely that the wealth of enhancer trap Gal4 lines available in Advances in chromatin profiling technologies have greatly improved our understanding of many fundamental biological processes including transcriptional regulation, DNA replication, and cell division. The use of DamID for profiling the interaction of proteins with chromatin has proved to be a valuable resource in the molecular biologist's toolkit, either as a complementary technique to existing technologies or as a powerful discovery methodology in its own right. The examples highlighted here illustrate just some of the applications for which DamID may be utilized, and it is likely that novel uses will emerge as the technology matures.Despite the many advantages of DamID, the technology is not without shortcomings which may have prevented DamID from becoming as widely used as its alternatives. For example, the lack of resolution in comparison to ChIP may put off some researchers. However, the simplicity, versatility, and potential for increased throughput may be sufficient to convert even the most ardent ChIP enthusiast. The fact that DamID requires handling of only DNA and not relatively unstable or difficult to handle substances such as RNA or protein, will also be appealing to researchers with limited proteomics experience. Furthermore, the ever decreasing cost of sequencing makes DamID accessible to more and more researchers. It is likely that the next few years will yield developments in the methodology and application of DamID, some of which will come from unexpected directions. However, several improvements to the method are desirable and are therefore likely to emerge due to necessity. First, it is probable that DamID will be adapted for use in even more diverse model systems, especially considering recent advances in genome editing technologies such as CRISPR/Cas9. Currently, DamID has only been demonstrated in mammalian cell lines; however, the development of DamID in mice, especially in a cell type‐specific manner, is an attractive prospect for understanding mammalian chromatin biology. TaDa also has the potential to be applied to organoids derived from human stem cells, which could be a powerful tool for investigating development and regulation of gene expression in human tissues. Second, improvements to the resolution of DamID will be sought after by some researchers, especially those who wish to explore the precise nature of interaction of DNA‐binding proteins with their underlying DNA sequences. It is conceivable that modifications of the Dam enzyme itself could result in more well‐defined methylation profiles. Some progress has been made in this area with the development of DamIP, a technique in which expression of a tethered Dam methylase is combined with immunoprecipitation using an antibody targeting N6 methylated adenine.The ‘proximity labeling’ approach at the heart of DamID has already inspired the development of similar methods in other areas of cell biology such as BioID. In BioID, a protein of interest is tagged with a promiscuous biotin ligase , which is able to biotinylate nearby proteins, which can then be detected using proteomic methods.


The G9a/GLP complex mediates mono- and dimethylation of Lys9 of histone H3 at specific gene loci, which is associated with transcriptional repression. However, the molecular mechanism by which the G9a/GLP complex is targeted to the specific gene loci for H3K9 methylation is unclear. In this study, with unbiased protein affinity purification, we found ZNF644 and WIZ as two core subunits in the G9a/GLP complex. ZNF644 and WIZ interact with the transcription activation domain of G9a and GLP, respectively. Moreover, both ZNF644 and WIZ contain multiple zinc finger motifs that recognize consensus DNA sequences. ZNF644 and WIZ target G9a and GLP to the chromatin and mediate the G9a/GLP complex-dependent H3K9 methylation as well as gene repression. Thus, our studies reveal two key subunits in the G9a/GLP complex that regulate the function of this histone methyltransferase complex.DOI:http://dx.doi.org/10.7554/eLife.05606.001 Genes encode instructions for processes within cells, but only a small subset of the genes within a cell will be switched on (or expressed) at any given time. The other genes are kept switched off until their instructions are needed. For example, some genes are switched on when it is time for a cell to divide or in response to changes in the environment.In humans and other eukaryotes, DNA is packaged within cells in proteins called histones. The level of gene expression can be altered by how tightly the DNA is packaged; if the DNA is more tightly packed around the histones, the gene will be expressed at lower levels than if the DNA is only loosely packed.A group of proteins called the G9a/GLP complex can alter histones to reduce the expression of some genes during embryo development, immune responses, and the formation of tumors. The complex works by attaching ‘methyl’ tags to the histones associated with particular genes, but it is not clear how it is able to specifically target these histones.Bian, Chen, and Yu used a technique called unbiased protein affinity purification to search for other proteins that can bind to the G9a/GLP complex. The experiments found two proteins called ZNF644 and WIZ, both of which are required for the G9a/GLP complex to be able to add methyl tags to histones.Further experiments revealed that ZNF644 and WIZ both contain regions called zinc finger motifs that enable them to identify and bind to specific sequences of DNA. Therefore, these proteins can guide the G9a/GLP complex to specific sites in the genome to switch off the expression of particular genes. A future challenge will be to try to modify these zinc finger motifs and guide the G9a/GLP complex to switch off other genes. This may allow us to develop therapies that could alter the expression of genes involved in cancer and other diseases.DOI:http://dx.doi.org/10.7554/eLife.05606.002 Post-translational modifications of histones, especially lysine methylation, play important roles in diverse biological processes, such as gene transcription, chromatin packaging, and cellular differentiation. Different lysine residues of histones are methylated by different histone lysine-specific methyltransferases (HKMTs). Among HKMTs, G9 and GLP specifically catalyze H3K9 mono- (H3K9me1) and dimethylation (H3K9me2) .G9a and GLP are paralogs with similar domain architecture. Both contain a transcription activation domain (TAD), a glutamate-rich domain, a cysteine-rich domain, 7 tandem ankyrin repeats (ANKs), and a methyltransferase domain . InteresRecent study suggests that the G9a/GLP complex is associated with Polycomb Repressive Complex 2 (PRC2) . EZH2, tAlthough the G9a/GLP complex plays an important role in epigenetic modification and gene transcription, the molecular mechanism by which the G9a/GLP complex is regulated in vivo remains elusive. Although several partners of G9a have been identified, it is unclear whether these partners form a stable complex with G9a and GLP, and directly control the G9a/GLP-dependent H3K9me1 and H3K9me2. In this study, we searched other possible subunit(s) in the G9a/GLP complex. With unbiased protein affinity purification, we found that ZNF644 and WIZ, two zinc finger proteins, interact with G9a and GLP, respectively. These two zinc finger proteins target G9a and GLP to genomic loci for the regulation of gene transcription.To explore the regulation mechanism of the G9a/GLP complex, we have searched the functional partner(s) of G9a using tandem protein affinity purification. Cell lysates of 293T cells stably expressing SFB-tagged G9a were subjected to two rounds of affinity purification. Since G9a and GLP form a heterodimer in vivo , we coulTo further confirm the interaction between these proteins in vivo, we first raised antibodies against endogenous ZNF644 or WIZ. Anti-ZNF644 and WIZ antibodies specifically recognized bands around 150 kDa and 175 kDa, respectively. Moreover, siZNF644 and siWIZ treatment diminished the expression of these two proteins, indicating that both antibodies specifically recognize the endogenous proteins . We nextNext, we examined the interaction domain in each subunit in this complex. Based on the domain architecture , we geneWe also mapped the interaction regions on WIZ and ZNF644 by generating series of internal deletion mutants of ZNF644 and WIZ, respectively . The D1 Since zinc finger motif is a DNA-binding module , we ask Since ZNF644 and WIZ are subunits in the G9a/GLP complex, we ask if ZNF644 and WIZ target the G9a/GLP complex to chromatin. We used siRNAs to knock down ZNF644 and/or WIZ. Lacking either ZNF644 or WIZ impaired the chromatin association of G9a and GLP . MoreoveCWH43, DIP2C and ROCK1 loci, G9a, ZNF644 and WIZ co-localized together at the promoter regions  to examine the genome-wide localization of G9a, ZNF644, and WIZ in 293T cells. We identified 14,153 G9a enriched regions, 12,777 ZNF644 enriched regions, and 11,853 WIZ enriched regions, respectively. The ChIP-seq results were validated using ChIP-qPCR to examine 30 randomly picked loci that represent a broad range of ChIP-seq fragment counts . To anal regions . At CACN regions . In contwith G9a . Taken thttp://floresta.eead.csic.es/rsat/peak-motifs_form.cgi), we examined only ZNF644-enriched regions (lacking WIZ), by which we excluded the possible loading of ZNF644 onto chromatin via WIZ. A specific DNA-binding sequence was concluded by the software  vector, respectively, the full-length cDNA of ZNF644, G9a, and GLP was cloned into pCMV-Myc vector, and the full-length cDNA of WIZ was also cloned into pCMV-HA vector. For protein co-immunoprecipitation experiments, G9a deletion mutants, WIZ deletion mutants, and GLP deletion mutants were cloned into SFB vector, respectively. ZNF644 deletion mutants were cloned into the pCMV-Myc vector.Primary antibodies used in this study include: mouse anti-G9a monoclonal antibody , mouse anti-HA and anti-Myc monoclonal antibodies , rabbit anti-H3K9me2 polyclonal antibody , rabbit anti-human ZNF644 antibody (raised against N-terminus a.a. 50–602), rabbit anti-human WIZ antibody (raised against N-terminus a.a. 220–750).The siRNAs targeting G9a, GLP, ZNF644, and WIZ were ordered from Dharmacon .2 plates of 293T cells stably expressing SFB-G9a or ZNF644 and washed cells with PBS. Cells were lysed with 30 ml ice-cold NETN300 buffer . The soluble fraction was incubated with 0.5 ml streptavidin-conjugated agarose beads. The beads were washed with NETN buffer three times. Associated proteins were eluted with 2 mM biotin in PBS and further incubated with 50-ml S beads .The bound proteins were eluted with SDS sample and analyzed with 10% SDS-PAGE and mass spectrometry.Purification of SFB triple-tagged protein was described previously . To searFor immunoprecipitation assays, 293T cells or U2OS cells were lysed with ice-cold NETN400 buffer  containing 10 mM NaF and 50 mM β-glycerophosphate. The soluble fractions were collected and diluted to 100 mM NaCl, then directly subjected to electrophoresis or immunoprecipitation with indicated antibodies followed by Western blotting analysis with indicated antibodies. For the SFB-tagged protein, streptavidin beads were used to perform the pull-down assay followed by Western blotting analysis.Chromatin immunoprecipitation assays (ChIP) were performed according to the protocol described by Upstate . The genomic DNA isolated from 293T cells was sonicated to an average size between 300 and 600 bp. Solubilized chromatin was immunoprecipitated with the antibody against WIZ, G9a, or ZNF644. Antibody–chromatin complexes were pulled-down using protein A-sepharose, washed, and then eluted. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol-chloroform, ethanol precipitated, treated with RNase, and dissolved with TE buffer. ChIP DNA was qualified using PicoGreen.http://galaxyproject.org/). Parameters settings were as follows: IgG ChIP-seq aligned reads were used as control file, tag size with 25 bp, band width with 300 bp. When comparing peaks from different samples, peaks were considered to be overlapping if they were within 2 kb of each other. The peaks obtained from ChIP-seq were matched to the annotated reference genome (human hg19) using Cisgenome 2.0. To view the peak density and position, Cisgenome 2.0 was used. To obtain the binding motif of ZNF644 and WIZ, the online software Peak-motifs http://floresta.eead.csic.es/rsat/peak-motifs_form.cgi) was used. A set of 30 PCR primer pairs . A single ‘A’ base was added to 3′ end with Klenow. Double-stranded adaptors (75 bp with a ‘T’ overhang) were ligated to the fragments with DNA ligase. Ligation products between 200 and 600 bp were gel purified to remove unligated adaptors and subjected to 20 PCR cycles. Completed libraries were quantified with PicoGreen. The DNA libraries were analyzed by Solexa/Illumina high-throughput sequencing. The read quality of each sample was determined by FastQC software. After prefiltering the raw data by removing sequence adaptors and low quality reads, the tags were mapped to the human genome (hg19) by Bowtie software. Parameters settings were listed as follows: -v, 3 , -5, 3 and -3, 7 . Peak detection was performed using MACS software from Galaxy browser , and distal intergenic region that does not encode any genes. Genes not uniquely mapped to the genome were excluded. To avoid redundancy, only the longest transcript variant of each gene was used to define chromosomal locations of the intragenic region, promoter region, and intergenic region.The read counts around the center of G9a-enriched peaks in promoter region were analyzed by SEQMINER software . The cenFor ChIP-seq data have been deposited in the Gene Expression Omnibus under accession number GSE62616.Recombinant proteins were purified from Sf9 insect cells. For generating baculovirus, DNA fragments containing full-length human ZNF644, N-terminus of ZNF644 (a.a. 1–300) (ZNF644N300), full-length human WIZ, and N-terminus of WIZ (a.a. 1–200) (WIZN200) were subcloned into pFastBac Vector with a GST tag. Baculoviruses were generated in accordance with the manufacturer's instructions . After Sf9 cells were infected with baculoviruses for 48 hr, the cells were harvested, washed with cold PBS three times and lysed with ice-cold NETN100 buffer . The soluble fraction was incubated with Glutathione–Sepharose beads and eluted with Glutathione.GGAATCATCGAACACAGAGTGAGGCTGGG -3′ (ZNF644 ‘WT’ DNA target);Oligonucleotide substrates were obtained from IDT  and were purified by polyacrylamide gel electrophoresis (PAGE). The following oligonucleotides containing ZNF644 binding motif and WIZ binding motif were used, 5′- GAGTAAGATCATGCCACTGCATTCCATTCCATTCAGATACTAGTACGGTCAG -3′ (WIZ ‘WT DNA target’). The oligonucleotides containing ZNF644 binding motif mutation and WIZ binding motif mutation were used as control, 5′- GAGTAAGATCATGCCACTGGCATTGTTGACTCACAGAGTGAGGCTGGG -3′ (ZNF644 ‘mutant’ DNA target);5′- GAGTCTCACTCACGCGCTGCAATCAGGAACAGATACTAGTACGGTCAG -3′ (WIZ ‘mutant’ DNA target). 48-mer oligonucleotides were annealed at 1:1 molar ratio to its complementary oligonucleotides to generate the dsDNA and then radio-labeled with 32P at the 5′-end. GST-ZNF644, GST-ZNF644N300, GST-WIZ, or GST-WIZN200 was incubated with 0.2 nM (molecules) radio-labeled DNA substrates for 2 hr at 4°C in buffer D (20 mM HEPES-KOH (pH 7.9), 20% glycerol (vol/vol), 0.2 mM EDTA, 0.1 M KCl, 0.5 mM PSMF, 1 mM DTT) with 1.25 μg/μl Bovine serum albumin, 1 mM DTT, 5 mM MgCl2. The samples were resolved by electrophoresis on a 7.5% polyacrylamide gel in TBE buffer for 70 min at 60 V. The gel was then dried and exposed to autoradiography film overnight.5′- GAGTCTCACTCACGCGCt-test, and ANOVA was used for more than two groups. For all analyses, a p value of less than 0.05 was considered significant. Results are given as means ± s.d.In all cases, multiple independent experiments were performed on different days to verify the reproducibility of experimental findings. Two-way comparison was performed using the  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see eLife. Your manuscript was reviewed by three experts in the field and by a member of the Board of Reviewing Editors (BRE). After a full discussion of the study and the reviews, we are happy to report that the reviewers and the BRE member found the study of interest to the journal and therefore, we will be pleased to consider a revised manuscript addressing the following issues:Thank you for sending your manuscript titled “ZNF644 and WIZ regulate the G9a/GLP complex for gene repression” for consideration for publication in 1) Provide statistical analyses and significance for the quantitative PCR studies and for the data on changes in gene expression when the G9a-GLP complex components are depleted.2) Provide comprehensive genome-wide analysis including metagene, whole gene distributions, and average promoter analysis for the observations made in the manuscript.3) Provide experimental evidence for direct interactions of ZNF644 and WIZ to the DNA sequence studied in the manuscript.4) Provide a deeper discussion between the current analysis and the previous studies in terms of domains of interaction between WIZ and G9a-GLP. Also clarification on the difference between Feinberg's studies on the requirement of megabase size domains of histone H3K9me2 Vs the current study on the role of promoters in this process.5) The manuscript needs to be read carefully for grammar and clarity.Further details can be found in the comments below.Reviewer #1Significance: It was previously known that WIZ associates with and stabilizes the G9a-GLP heterodimer, but this paper identifies a new binding partner ZNF644. Furthermore, in a major breakthrough the paper demonstrates that WIZ and ZNF644 bind directly to specific DNA sequences and thereby target G9a to specific chromosomal locations. Previously, it was known that a few repressive DNA binding transcription factors can recruit G9a to specific chromosomal loci, but this paper describes a major new mechanism for targeting G9a and GLP to chromatin. The scientific quality of the data presented is generally quite good, but with a few exceptions noted below. There are significant English problems with the manuscript.Major concerns: The quantitative PCR data for ChIP and for RNA expression throughout Overall recommendation: This is an exciting paper because it reports a new mechanism for targeting of G9a-GLP to specific chromosomal locations. Statistical analyses need to be provided for the quantitative PCR data. Furthermore, the data on changes in gene expression when G9a-GLP complex components are depleted is weak and of questionable statistical significance. This data should either be improved  or eliminated. While it would be nice to have an indication that G9a-GLP is having an effect on gene expression at the sites where they are recruited, the novel targeting mechanism would still, in my opinion, be important enough to merit publication. English needs to be improved throughout the manuscript.Reviewer #2G9a-GLP is important for assembly of H3K9 dimethylated facultative heterochromatin. A major question is how does the complex localize to its sites of action? This is a key question in the field of chromatin biology. The authors show that the zinc finger TFs WIZ and ZN644 associate with G9a-GLP in vivo. They map interacting domains by co-IP with deletion mutants and present genome wide ChIP data on co-localization of the complex and locus-specific ChIP in various RNAi mutants. The interaction with WIZ was previously shown so the novelty here is the association with ZN644, the identification of binding motifs and validation of co-localization genome wide. These are important findings of broad interest in the field.The major drawbacks are the lack of a comprehensive genome wide analysis . Additionally, while the authors show strong evidence for targeting of G9a-GLP by WIZ and ZNF644, this is not linked globally with H3K9 dimethylation. There is an overemphasis on locus-specific ChIP studies that lacked measures of statistical significance. Moreover, the paper lacked a deeper discussion of and comparison with other discoveries in the field. For example, there is some disagreement between the authors' analysis and previous studies in terms of domains of interaction between WIZ and G9a-GLP. Additionally, Feinberg showed megabase size domains of histone H3K9me2, while the authors' studies focus on the promoters. Is WIZ-ZNF644-G9a-GLP binding inside as well as at gene promoters? Finally, there are a huge number of disappointing grammatical issues.In general, I felt that the paper was a bit underdeveloped throughout but has the potential to make a significant contribution.Reviewer #3The manuscript from Bian and colleagues describes the interaction of G9A/GLP heterodimeric methyltransferase complex with two zinc finger containing proteins, Wiz and ZNF644. G9A/GLP activity results in heterochromatin formation and gene silencing. Affinity purification of G9A and ZNF644 was used to determine that G9A/GLP, Wiz and ZNF644 form a core complex. Wiz was shown previously to associate with G9A/GLP and alter its stability ; however, ZNF644 is a new component identified by this manuscript. Using immunoprecipitations of deletion mutants the authors determine that the TAD Domain of GLP and G9A interact selectively with WIZ and ZNF644, respectively. The authors go on to identify the genomic location of WIZ and ZNF644 and that the sites occupied by these proteins accounts for the majority of G9A occupied sites in the genome. Recruitment of G9A and the downstream readers of H3K9 methylation are effected by suppressing WIZ expression. Overall, the data make a compelling case for an important role of WIZ and ZNF644 in recruiting G9A/GLP to their genomic locations. To fully support the proposed role of these proteins in recognition of select genes through a defined DNA sequence, the following points should be addressed:1) This implication of the requirement of ZNF644 or WIZ for GLP/G9A recruitment is that the zinc-finger domains of these proteins mediate sequence specific DNA interactions. Although the authors identify motifs within genes that are bound by the GLP/G9A/WIZ/ZNF644 complex, the ability of ZNF644 and WIZ to directly bind these DNA sequences is not addressed. The authors should determine if WIZ or ZNF644 selectively and directly recognize the motifs identified in 2) The authors identify deletion mutants in ZNF644 and WIZ that fail to bind GLP and G9A. They go on to use these mutants in  After a full discussion of the study and the reviews, we are happy to report that the reviewers and the BRE member found the study of interest to the journal and therefore, we will be pleased to consider a revised manuscript addressing the following issues:1) Provide statistical analyses and significance for the quantitative PCR studies and for the data on changes in gene expression when the G9a-GLP complex components are depleted.P-value with less than 0.05 was considered as statistically significant difference. Results are given as means ± s.d. The description of statistical analysis was also included in the revised Materials and methods.Thank you for your suggestion. We have performed the statistical analyses for the quantitative PCR studies and for the data on changes in gene expression, including 2) Provide comprehensive genome-wide analysis including metagene, whole gene distributions, and average promoter analysis for the observations made in the manuscript.As suggested by the reviewers, we re-analyzed the ChIP-seq data. The whole genome was partitioned into three regions: intragenic region, promoter region (5kb upstream or downstream of the TSS), and distal intergenic region that does not encode any gene. As shown in revised 3) Provide experimental evidence for direct interactions of ZNF644 and WIZ to the DNA sequence studied in the manuscript.Following the suggestions of the reviewers, we performed electrophoretic mobility shift assays and examined the interaction between the recombinant ZNF644 or WIZ proteins and oligonucleotides with consensus sequences. Oligos with non-consensus sequence were served as negative control. We also used GST proteins without zinc finger motifs as the pull-down control. ZNF644 and WIZ clearly bind to the specific DNA motif in vitro . The res4) Provide a deeper discussion between the current analysis and the previous studies in terms of domains of interaction between WIZ and G9a-GLP. Also clarification on the difference between Feinberg's studies on the requirement of megabase size domains of histone H3K9me2 Vs the current study on the role of promoters in this process.Thank you for the suggestion. In the previous study , WIZ wasIt has been shown that G9a regulates gene transcription via catalyzing H3K9me2 at promoter regions . Consist5) The manuscript needs to be read carefully for grammar and clarity.Thank you for the reminder, the revised manuscript has been proofread by a native English speaker in our institution.


J. Biol. Chem. 2005, 280, 42477–42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP , describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase.  The common core of multi-subunit RNAP in cellular organisms is composed of five subunits that are conserved in all three domains of life. Bacterial RNAP core enzyme is the simplest and best characterized form, consisting of α (two copies), β, β', and ω subunits a. The coThermusthermophilus was first reported in the late 1970s . Th. Th52]. Y, 4GZZ) .2+, which is essential for RNA cleavage (PDB: 4WQT).Misincorporated nucleotides in RNA transcripts slow down the rate of transcription and result in the backward movement of RNAP (backtracking), which ejects the 3' RNA end into the secondary channel and later cleaves misincorporated nucleotides by RNAP endonuclease activity . This acThe transcription factor RapA is an ATP-dependent DNA translocase, which reactivates the stalled elongation complex . The strA , isolated from T. aquaticus and T. thermophilus, showed that three σ domains  arranged on the surface of the core enzyme whose function was to recognize the −35 and−10 elements (separated by ~17 bp DNA)  [Bacterial RNAP recognizes promoter DNA and initiates transcription as the holoenzyme form containing the catalytic core enzyme plus the promoter recognition σ factor . StructuU, 1IW7) ,33  . UniversT. thermophilus RNAP holoenzyme in complex with the promoter DNA fragment (−12 to +12 on DNA)  [Atomic details of −35 element recognition by σ domain 4 (σ4), as revealed by co-crystal structure, showed that the helix-turn-helix motif of σ4 reads the hexameric DNA sequence (PDB: 1KU7) . DetailsH, 4G7O) . The holde novo RNA priming  [Cellular RNAPs start transcription by  priming . StructuZ, 4OIO) ,41. The Z, 4OIO) . The str70 in E. coli and σA in other bacteria) for transcribing housekeeping genes required for log-phase growth. Other classes of σ factors, including the group 2 σ factors , the extracytoplasmic function (ECF) σ factors (σE and FecI in E. coli), and σN , direct RNAP to genes for induction of stress responses, flagella synthesis, and spore formation in spore-forming bacteria, such as Bacillus. Structures of these alternative σ factors were determined as stable domains  [E. coli RNAP can be readily prepared using an overexpression system, which allows RNAP structural study to move in a new direction using RNAP mutants.Crystal structures of bacterial RNAPs have been determined using the B: 4YG2) . E. coliE. coli RNAP has created new possibilities for the structural study of bacterial RNAP. Sixteen structures of E. coli RNAP with transcription factors or small molecules have been determined already, including the long-awaited structure of E. coli RNAP in complex with (p)ppGpp, a master regulator of stress response in bacteria  [T. thermphilus RNAP was also determined from the crystal structure of the T. thermphilus RNAP–ppGpp complex (PDB: 1SMY) [E. coli transcription regulation by ppGpp [Characterization of 2, 4JKR) ,75 (TablB: 1SMY) , but it by ppGpp .Binding of alternative σ factors to the RNAP core enzyme is regulated by anti-σ factor, which forms a stable complex with its target σ factor and blocks core RNAP binding. Structures of the σ/anti-σ factor complex have shown various ways that the core enzyme-binding interface of σ factor can be masked b. The σ/Bacterial RNAP physically interacts with protein factors during gene regulation. Most bacterial transcription factors bind upstream of promoter DNA and recruit RNAP to target promoters and/or facilitate formation of the open complex. Three crystal structures of a transcription factor, RNAP domain, and DNA ternary complex have provided insights into how these factors influence transcription activation through different mechanisms a.E. coli RNAP, CAP, and promoter DNA has been determined by cryo-electron microscopy  [Catabolite activator protein (CAP)  is the most well characterized bacterial transcription factor, providing insights into the interaction between the helix-turn-helix motif and DNA, the allosteric control of DNA binding, and transcription activation. In classical lactose operon regulation , CAP binB: 3IYD) .Bacteriophage λ cI protein (λcI) plays a central role in the lytic to lysogenic growth switch and is able to both activate and repress transcription. For activation of transcription, λcI binds just upstream of the −35 element (class II transcription factor binding site) and interacts with σ domain 4 (σ4). The crystal structure of the λcI–σ4–DNA ternary complex showed that these proteins also use a small interface for recruiting σ4 to the −35 element (PDB:1RIO) .PhoB is a classical two-component response regulator, which interacts with σ4 to activate gene expression. In contrast with λcI-dependent transcription activation, PhoB-dependent promoters lack a canonical −35 element. The structure of the PhoB–σ4–DNA ternary complex showed that contact between σ4 and the DNA major groove is less extensive; however, interaction between σ4 and PhoB compensates for this, allowing recruitment of RNAP to a target promoter lacking the −35 element (PDB: 3T72) .Bacillus subtilis that interacts with a large interface on αCTD, thereby forming a stable Spx–αCTD complex without a DNA platform. Spx regulates gene expression under conditions of disulfide stress, which is sensed by disulfide bond formation between two cysteine residues in Spx. Crystal structures of both oxidized and reduced forms of the Spx–αCTD complex provided insight into how the disulfide bond affects DNA binding  [Spx is a global transcription regulator in K, 3IHQ) ,63,64.Bacteriophages produce transcription factors that bind directly with host RNAPs and hijack host transcription machinery to transcribe phage genomes. Several structural models of bacterial RNAP in complex with phage proteins have been used to explain how phage protein remodels RNAP to redirect transcription machinery to the phage genome a 17,36,,71,72,73® and lipiarmycin), have been used in clinical practice. Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Structures of the RNAP–rifampin complex  provided a detailed view of the interaction between rifampin and the β subunit and explained how rifampin blocks RNA extension [T. thermophiles holoenzyme, PDB: 2A68, 2A69; E. coli holoenzyme, PDB: 4KN4, 4KN7) showed an additional interaction with the σ region 3.2 (σ finger) that may influence binding of template DNA at the active site, thereby reducing the efficiency of transcription initiation [Bacterial RNAP is essential for cell growth and viability, and lack of sequence and structural similarities between certain areas of bacterial and eukaryotic RNAP make this enzyme an attractive target for antibiotic development. Crystal structures of bacterial RNAP in complex with small molecule transcription inhibitors have been used to characterize binding sites, mechanisms of action, and mechanisms of resistance . Currentxtension ,81. The xtension . Chemicaitiation . The stritiation .Streptolydigin binds to key elements of the RNAP active site (bridge helix and trigger loop) and inhibits all reactions involved in RNAP transcription by blocking conformational changes in these elements that occur during transcription. Structures of the RNAP–streptolydigin complex revealed that it traps the bridge helix and trigger loop in straight and unfolded conformational states, respectively, by direct interaction with these elements  ,42,84.T. thermophilus RNAP–myxopyronin complex  have proposed two models of inhibition: myxopyronin inhibits transcription (1) by preventing the opening of the RNAP clamp that permits entry and unwinding of DNA (hinge jamming mechanism) [Myxopyronin binds to the RNAP switch region, which is a hinge of the RNAP clamp whose function is to open and close the DNA-binding channel. Upon binding, myxopyronin inhibits transcript initiation by preventing formation of the promoter open complex. Structural and biochemical studies on the chanism)  or (2) bchanism) .T. thermophilus RNAP–myxopyronin complex [E. coli RNAP in complex with squaramides were determined, structurally confirming that squaramides bind to RNAP switches  [A new structure of a bacterial RNAP–inhibitor complex was obtained by using salinamide, which binds the N-terminal end of the bridge helix and prevents nucleotide addition (PDB: 4MEX) . Squaram complex . CrystalK, 4YFN) .During the past decade, our understanding of bacterial transcription processes has drastically improvedas a result of structural studies on bacterial RNAP. I hope this review will be a useful reference for researchers who study the mechanism, structure and function of bacterial RNAP transcription and that structures presented in this review will provide guidelines for designing new experiments.