A library of active genome regulatory elements (putative promoters and enhancers) from MIA PaCa-2 pancreatic adenocarcinoma cells was constructed using a specially designed lentiviral vector and a massive parallel reporter assay (ChIP-lentiMPRA). Chromatin immunoprecipitation of the cell genomic DNA by H3K27ac antibodies was used for primary enrichment of the library for regulatory elements. Totally, 11,264 unique genome regions, many of which are capable of enhancing the expression of the CopGFP reporter gene from the minimal CMV promoter, were identified. The regions tend to be located near promoters. Based on the proximity assay, we found an enrichment of highly expressed genes among those associated with three or more mapped distal regions (2 kb distant from the 5′-ends of genes). It was shown significant enrichment of genes related to carcinogenesis or Mia PaCa-2 cell identity genes in this group. In contrast, genes associated with 1–2 distal regions or only with proximal regions (within 2 kbp of the 5′-ends of genes) are more often related to housekeeping functions. Thus, ChIP-lentiMPRA is a useful strategy for creating libraries of regulatory elements for the study of tumor-specific gene transcription. Promoters and enhancers are among the most important elements of the genome that ensure gene transcription ,2. In reBoth promoters and enhancers can serve as RNA synthesis start sites , often in a bidirectional manner ,10,11,12Active enhancers and promoters are typically flanked by nucleosomes that have similar post-translational modifications of histones, such as H3K27Ac (histone H3 acetylated at lysine 27), H3K9Ac, H3K4Me1, or some others ,19,20,21In recent years, a number of functional approaches, including MPRA , have been developed to identify sequences with enhancer and/or promoter activity in complex mixtures of DNA fragments (DNA libraries) ,28,29,30The use of lentiviral vectors in the creation of libraries for MPRA  makes itThe success of MPRA approaches depends on a variety of parameters, including the source, size, and quality of the input DNA, as well as cloning efficiency.In this work, we obtained an enriched whole genome library of enhancer and promoter elements active in cells of an aggressive ductal adenocarcinoma pancreatic cell line MIA PaCa-2. We identified more than 11 thousand  DNA sequences representing potential enhancers and promoters in the obtained library. The mapped DNA sequences tend to be located in areas with super-enhancer properties nearby to the key genes determining a given cell type. Therefore, ChIP-lentiMPRA with inhibition of RNA interference is a useful strategy to obtain highly representative libraries of regulatory elements for the study of tumor-specific gene transcription.We perfected the lentiMPRA technique by combining it with specific chromatin immunoprecipitation and with inhibition of RNA interference (ChIP-lentiMPRA). The general scheme for obtaining highly representative libraries of active promoters and enhancers is shown in For immunoprecipitation, we used antibodies to H3K27ac, which is the major chromatin tag of active promoters and enhancers. This allowed us to obtain an input (primary) library of ChIP DNA presumably enriched with active regulatory elements. Then we selected a fraction of the ChIP DNA fragments that are capable of enhancing the expression of the reporter gene. For this, we amplified ChIP DNA by PCR and cloned it to the lentiviral vector pLVPGm.1-mP, which was specially designed for lentiMPRA in this work. This vector contains the gene of CopGFP fluorescent protein under the control of the cytomegalovirus minimal promoter and the cloning site for enhancer analysis. pLVPGm.1-mP showed high efficiency in cloning and selection of genome regulatory elements. During cloning, we used inhibition of RNA interference by the Nodamura virus protein B2. Such an approach has been shown to dramatically increase the titer of lentiviral vectors ; howeverIn the first stage, we constructed a basic second-generation self-inactivating lentiviral vector pLVPGm.1-mP  controlling the reporter gene of the fluorescent protein CopGFP . The vector also includes the mPgk-1-PuroR cassette, which provides transduced cells resistance to puromycin. On the basis of pLVPGm.1-mP, a positive control vector pLVPGm.1-EmP was also obtained, which contains a cytomegalovirus enhancer inserted in the cloning region near the mP promoter.To test the functionality of the obtained lentiviral vectors, we used MIA PaCa-2 pancreatic ductal adenocarcinoma cells. MIA PaCa-2 cells transduced with a basic vector without an enhancer or a control vector containing a cytomegalovirus enhancer were obtained c. After To evaluate the performance of the proposed approach, we analyzed viral titer using the FACS-based titration on MIA PaCa-2 cells  and evaluated the dependence of transduction efficiency (percentage of transduced cells) and average lentiviral DNA copy number in transduced populations on virus dilutions during transduction .At a titer of 2.3 million transducing units (TU) per ml and 15 mL of viral medium volume, we obtained about 35 million functional viral particles (TU). We showed that transduction with undiluted viral stock of the control vector (MOI 7.2 TU per cell), 61% of GFP-positive cells in the population were detected with a relatively low number of integrated lentiviral DNA of 2.3 copies per cell . These dIt was previously shown that antisense transcripts in lentiviral plasmids lead to the destruction of viral pre-mRNA in the packaging cells because of RNA interference and, consequently, to a decrease in viral titer . Since eTo test this assumption, we examined the production of viral particles in 293T cells transduced with an equimolar mixture of pLVPGm.1 plasmids containing the CopGFP gene under the control of a strong cytomegalovirus (CMV) promoter/enhancer in sense or antisense orientations . Indeed,Addition of a plasmid expressing the RNA interference suppressor protein NovB2 (Nodamuravirus B2 protein) during co-transfection of the packaging cells resulted in almost complete restoration of the viral titer values, which is consistent with the results of other studies . TherefoE. coli library of 20 million clones with close to 100% clones with inserts. This indicates a high representation of ChIP DNA fragments in the pool of pLVPGm.1-L-mP plasmids.We performed chromatin immunoprecipitation (ChIP) of MIA PaCa-2 cells with antibodies to H3K27ac to obtain a primary DNA library enriched with promoters and enhancers. The library of ChIP DNA fragments was ligated to the adapter containing the library primer annealing site and the restriction site required for cloning . After PCR amplification, the fragments , its enrichment with functional sites did not change qualitatively.5 GFP-positive cells with the highest fluorescence intensity were selected using FACS. This experiment was repeated, and 25 × 105 cells were obtained additionally. Genomic DNA was isolated from both populations, and preparative PCR amplification of the selected fragments was performed . As a result, L1 and L2 libraries, presumably enriched with functional elements, were obtained from the two populations of selected cells.A pool of lentiviral plasmids pLVPGm.1-L-mP from the previous stage was used to produce viral particles in the 293T packaging cell line. After transduction and selection of transduced cells with puromycin, 12 × 10To estimate the proportion of functional elements in the obtained libraries, the activity of 28 randomly selected DNA fragments from the L1 library was analyzed in the transient expression system. The fragments were cloned into the pGRm.1-mP plasmid a 5′-end ATP5MK and PDCD11 . The lentiviral DNA content in the L1 population was 2.4 copies per cell on average, which is comparable with the results obtained previously for the control populations . This meThe DNA fragments of the obtained libraries were sequenced using massive parallel paired-end sequencing. The resulting reads were mapped to the human reference genome (GRCh38/hg38). In total, 6244 and 5245 fragments were mapped for the L1 and L2 libraries, respectively. Analysis of the overlap of the mapped fragments with different functional regions showed that both libraries are equally enriched in functional sites  compared to the library of random fragments obtained in silico . TherefoThe obtained map was compared with the maps of regions with elevated H3K27ac modification levels in MIA PaCa-2 and Capan-2 cells, as reported by Diafferia et al. . Of the The mapped fragments tend to co-localize with regions with the highest content of the H3K27ac chromatin tag b. High HAs noted above, a total of 11,264 selected fragments were mapped in the genome. Our goal was to search for functionally active elements of the genome, so we further considered only the H3K27ac enriched regions. We found that the mapped regions are concentrated near gene promoters . We diviA total of 2537 proximal and 2801 distal regions were identified. Using the GREAT service , we deteUsing Expression Atlas data , we showWe divided the D genes into genes associated with 1–2 mapped regions  and genes associated with three or more distal regions  . InteresThe vast majority of genes belong to one of the three categories reviewed, though some genes may belong to more than one category . As showHMGA1, MYC, ID1, NR2F1, CEBPB, STAT3, TP63, SOX9, ZEB1, and others [D2-genes of the categories “EMT-related genes” (54 genes) and “Genes significantly upregulated in pancreatic cancer” (53 genes) are, on average, expressed at higher levels in MIA PaCa-2 cells than other genes in this group . Many ofd others . These rFor a more detailed analysis of the functions of the genes associated with the mapped regions, we investigated their features using the Gene Ontology database. We showed that the D2 gene group is enriched in genes that are involved in processes relevant to carcinogenesis, in particular, in the regulation of cell death (GO:0010941) and apoptosis (GO:0043065) , cell miPancreatic cancer is one of the most aggressive and malignant tumors. As with other tumors, successful treatment requires early diagnosis, correct subtyping, knowledge of general patterns of the disease, and morphological and molecular characterization of a particular case ,56,57,58In this work, we constructed a library of active genome regulatory elements (promoters and enhancers) from MIA PaCa-2 pancreatic ductal carcinoma cells using an improved approach combining chromatin immunoprecipitation with a lentivirus vector-based massive parallel reporter assay (ChIP-lentiMPRA).The initial set of DNA fragments for cloning was obtained by immunoprecipitation of MIA PaCa-2 cell chromatin with antibodies to the acetylated histone H3K27ac. This chromatin modification represents one of the most important but not the only characteristic of active enhancers , which iThe isolated H3K27ac-ChIP DNA fraction was cloned into the lentiviral vector pLVPGm.1-mP, which we constructed for efficient cloning and selection of active promoters and enhancers. This vector contains a CopGFP reporter gene under the control of the minimal CMV promoter and a site for cloning the DNA elements under study. Earlier, other authors used lentivirus vectors in the creation of libraries for MPRA ,31,32,33In addition, we applied inhibition of RNA interference by the Nodamura virus protein B2 (NovB2) , which hE. coli library of immunoprecipitated DNA fragments we obtained from MIA PaCa-2 cells contained about 20 million clones. Up to 100% of the clones contained a vector with an insert, with at least 21% of the inserts representing active functional genomic elements.As a result, the Massive parallel sequencing of the clones obtained after FACS of the primary library allowed us to identify 11,264 unique genome regions enriched with the H3K27ac tag characteristic of promoters, enhancers, and super-enhancers. The result obtained significantly exceeds the number of regulatory elements previously studied by other authors in MRPA, where the initial DNA was obtained by other methods. For example, in the work of Inoue et al. , 2464 prHMGA1, MYC, ID1, NR2F1, CEBPB, STAT3, TP63, SOX9, and others [Among the genes associated with the regions mapped in this work, we identified genes related to epithelial–mesenchymal transition and a group of genes for which increased expression has been shown in pancreatic cancer. Most genes of these groups are associated with three or more mapped distal regions (D2 genes). This may indicate a relationship between the density of enhancer-like elements and the expression levels of genes associated with them. We also identified genes encoding transcription factors specific to the cell line under study—d others . The assThe distribution of mapped regions is consistent with the findings of other researchers that many enhancers of “housekeeping” genes tend to concentrate near the 5′-ends of genes, while enhancers of genes with complex specific regulation (such as those regulating organism development) are more often located at a considerable distance from the 5′-ends of genes, in introns or intergenic space . In addiThus, our approach to the construction of a library of functional genome elements and the principle of selection of cloned DNA fragments allowed us to identify important genome regulatory elements available for further studies. Many of these elements may act like master regulators because they are associated with groups of genes (three or more genes) that are responsible for tumorigenic or tumor-like cell behavior. Mutations of regulatory elements or changes in their epigenetic landscape due to various reasons can contribute to tumor progression even without damaging the target genes themselves ,64.It should be noted that histones on active regulatory elements can also carry other epigenetic tags, including both acetyl (H3K64ac and H3K122ac) and methyl groups (H3K4Me1), as well as having low H3K27ac ,65. In tA detailed description of the experimental design can be found in the methods in 7 logarithmically growing MIA PaCa-2 cells were fixed, pelleted, and lysed as detailed in the methods in PSMB5, PSMB2, and COPZ1 were amplified. Non-promoter regions were selected from the regions that do not overlap with potential cis-regulatory elements (UCSC Browser track “ENCODE Candidate Cis-Regulatory Elements (cCREs) combined from all cell types”). Primers are listed in Approximately 1 × 10293T cells were transfected with 12 μg lentiviral plasmid, 6 μg pMD.G , 12 μg pThe MIA PaCa-2 cells were transduced with both non-diluted virus and with dilutions 1/32, 1/64, 1/128, and 1/256. After incubation, the viral-containing medium was replaced with a fresh medium. Then, the numbers of GFP-positive cells in each well were determined by flow cytometry, and the titer was calculated using a percentage of GFP-positive cells.The End Repair/dA-Tailing Module  was used to convert ChIP DNA to repaired DNA having 5’ phosphorylated dA-tailed ends. Repaired DNA fragments were ligated to adapter, obtained by annealing equimolar amounts of the two oligonucleotides  with ChIP DNA library fragments was isolated. An aliquot of the library was used to assess cloned ChIP DNA enrichment by functional elements relative to Input DNA qPCR as described above. In total, 30 colonies grown on agar plates were analyzed by PCR with primers P1 and P2  cell sorter, and approximately 3.7 × 10E. coli DH-5alpha was transformed by the ligated vector. Plasmid DNAs containing inserts were isolated from 28 clones and used for transfection of MIA PaPa-2 cells. Populations of transfected cells were analyzed by a Nikon TE2000U microscope equipped with epifluorescence optics  and flow cytofluorimeter FACScan (BD Biosciences).Genomic DNA was isolated from GFP-positive cells and amplified by nested PCR using primers P3, P4, and nested primers P1 and P2 . AmplifiSamples of genomic DNA isolated from GFP-positive clones were amplified using primers and conditions recommended by Illumina protocol “16S Metagenomic Sequencing Library Preparation” . The first PCR was performed using external primers P3 and P4 , flankinR1 and R2 files of each library were transformed to fastqsanger format using FASTQ Groomer   . Adapterhttp://genome.ucsc.edu (accessed on 7 April 2022)). A BED file with human 93,873 super-enhancers derived from H3K27ac ChIP-seq data for various cell types was downloaded from the SEA version 3.0 database (http://sea.edbc.org (accessed on 7 April 2022)). The coordinates of the MIA PaCa-2 super-enhancers were extracted from file SE_02_0723_SE_hg38.bed (hg38), downloaded from the SEdb 2.0 database (http://www.licpathway.net/sedb (accessed on 21 November 2022)). Coordinates of the CAGE peaks were obtained using the Table Extraction Tool of the FANTOM5 project (https://fantom.gsc.riken.jp/5/ (accessed on 7 April 2022)). Random genomic regions were generated using the ShuffleBed tool [https://usegalaxy.org (accessed on 7 April 2022)) [We analyzed the intersection of mapped regions and random genomic regions with known ChIP-seq H3K27ac peaks obtained for MIA PaCa-2 and Capan-2 cells , with wiBed tool  on the wl 2022)) . The numhttps://www.bioinformatics.babraham.ac.uk/projects/seqmonk/ (accessed on 7 April 2022)) was used to analyze and obtain data on the relative distribution of mapped regions around the 5-ends of genes.The program SeqMonk version 1.48.1 (https://david.ncifcrf.gov/home.jsp (accessed on 7 April 2022). Obtained GO terms with corresponding p-adjusted values (p-adj < 0.05) were used to visualize GO terms clusters with REVIGO [http://revigo.irb.hr/ (accessed on 7 April 2022)) using the following options: remove obsolete GO terms, choose species Homo sapiens (9606), SimRel (default) semantic similarity measure.GREAT  was usedh REVIGO  (http://Our study demonstrated that ChIP-lentiMPRA approach using pLVPGm.1-mP lentiviral vector with the RNA interference inhibition is a useful strategy to create representative libraries of regulatory elements of the genome. Further study of cloned enhancers and promoters from MIA PaCa-2 cells, as well as the construction of libraries of regulatory elements based on other principles, will allow us to understand the specific role of such elements in cellular processes including carcinogenesis.


Histoplasma capsulatum adapts to this decreased copper through upregulation of the Ctr3 copper transporter. In this study, we show that Histoplasma recognizes the characteristic low-copper phagosomal environment of activated macrophages through the copper-dependent transcriptional regulator Mac1. Multiple cis-acting regulatory sequences in the CTR3 promoter control upregulation of CTR3 transcription under low-copper conditions, and the loss of Mac1 function prevents induction of Ctr3 under low-copper conditions. During adaptive immunity, this loss of copper sensing by Mac1 attenuates Histoplasma virulence more severely than loss of Ctr3 alone, indicating that Mac1 controls the expression of additional mechanisms important for pathogenesis. Transcriptional profiling of Histoplasma yeasts identified a small regulon of Mac1-dependent genes, with the most strongly regulated genes encoding proteins linked to copper, iron, and zinc homeostasis and defenses against reactive oxygen . Accordingly, the loss of Mac1 function increased sensitivity to copper and iron restriction and blocked low-copper-induced expression of extracellular catalase activity. Thus, Mac1-mediated sensing of low-copper signals to Histoplasma yeasts their residence within the activated macrophage phagosome, and in response, Histoplasma yeasts produce factors, including non-copper-dependent factors, to combat the enhanced defenses of activated macrophages.Intracellular pathogens residing within macrophage phagosomes are challenged with recognizing the phagosomal environment and appropriately responding to changing host defense strategies imposed in this organelle. One such phagocyte defense is the restriction of available copper as a form of nutritional immunity during the adaptive immune response to fungal pathogens. The intracellular fungal pathogen  Copper is an essential trace nutrient for virtually all cells, but it can be toxic in high concentrations. In fungi, copper is required as a metal cofactor for many enzymes, including the reactive oxygen-detoxifying superoxide dismutases, melanin biosynthetic enzymes, and mitochondrial cytochrome oxidase. However, excess copper is disruptive to iron-sulfur clusters and contributes to the production of damaging reactive oxygen species through Fenton reactions . Due to Histoplasma capsulatum must acquire nutrients, including copper, to survive and replicate within a canonically microbicidal environment. The temperature-mediated transition to the pathogenic yeast phase defines much of Histoplasma’s virulence program  reporter gene. These transcriptional reporters, consisting of 485, 335, 285, 166, 133, and 62 bp upstream of the CTR3 start codon, were transformed into Histoplasma yeasts and the GFP fluorescence measured under low-copper (20 nM CuSO4) and high-copper (5 μM CuSO4) conditions  to control for any copper-dependent differences in growth or general transcription. A GFP-negative control (strain G217B) was also included to indicate background levels of yeast fluorescence . Therefore, the 285-bp promoter was defined as the minimal CTR3 promoter for further experiments on transcriptional regulation.To identify the nditions  and B. Grescence . The 485CTR3 promoter truncations focused attention on potential regulatory elements within the 285-bp upstream region. Since copper regulates CTR3 expression, we used the defined S. cerevisiae copper-response element (CuRE) motif to search for the presence of potential copper-response elements within the 285-bp Histoplasma CTR3 promoter region. Three putative CuREs were identified within the CTR3 promoter at bases −273, −260, and −176 relative to the CTR3 start codon and were designated A, B2, and C, respectively. A fourth putative CuRE (designated B1) was manually identified as overlapping with site B2 at bp −263. CuREs A and B2 were on the strand opposite CTR3 transcription, whereas CuREs B1 and C were on the sense strand  and manually (dotted lines). Native and scrambled sequences are represented by black and blue text, respectively. Red arrow denotes the start codon of the CTR3 coding sequence (CDS). The Saccharomyces cerevisiae Mac1 binding motif  is included at top left. Download FIG S1, EPS file, 2.8 MB.Copyright © 2022 Ray and Rappleye.2022Ray and Rappleye.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. cerevisiae, CuRE motifs are bound by the Mac1 transcription factor (–Podospora anserina that does not produce the multicopper oxidase laccase (Cryptococcus Cuf1) that appears to be a hybrid between AceI and Mac1 and modulates transcriptional responses to both high and low copper  within a clade distinct from the Mac1 orthologs. Notable exceptions to the dual copper-responsive transcription factor situation are fungi that only have a single copper-fist domain protein . A few fungi  have at least three copper-fist domain-containing proteins, although the reason for the expansion of these proteins is unknown. The Histoplasma genome encodes a single Mac1 ortholog (Hca_04395) and an AceI/Cup2 ortholog (Hca_03650), and this dual copper-fist regulator situation is present in related dimorphic fungi .In n factor –14. Mult laccase , represe laccase . In many laccase . Howeverw copper . To deteatedness . Most euHistoplasma Mac1 protein regulates expression of the CTR family of copper transporter-encoding genes, low-copper induction of CTR gene transcription was surveyed following depletion of Mac1. Mac1 was depleted from Histoplasma yeasts by RNAi (4) or low copper . Expression of the CTR1, CTR2, and CTR3 genes was quantified via qRT-PCR. Depletion of Mac1 had no significant effect on expression of CTR1 and CTR2  of the copper-chelator BCS by >1,000-fold compared to wild-type Histoplasma yeasts  or high-copper (5 μM CuSO4) conditions, which are sufficient for up- and downregulation of CTR3 expression  motifs within the promoter region of the Ctr3-encoding gene to promote CTR3 transcription under low copper availability. In this regard, Histoplasma Mac1 is similar to Mac1 orthologs in other Ascomycetes, and the required CuREs present within the Histoplasma CTR3 promoter are similar to the CuRE consensus motif defined for S. cerevisiae , Mac1 is required for the induction of CTR3 when copper becomes limiting as a part of the mammalian immune response to infection. Together, the actions of Ryp1 and Mac1 contribute to activation of the Histoplasma pathogenesis program, as well as adaptation to changing phagosomal conditions due to activation of the host immune response.The regulation of gene expression in response to environmental conditions often includes both revisiae , 14 , which is similar, but not identical to the published S. cerevisiae CuRE (data not shown). We speculate that this may represent regulation by the high-copper-sensing AceI transcription factor, which shares 55% identity and 70% similarity with Mac1 in the copper fist DNA-binding domain and could therefore have a similar binding motif. Given the limitations of transcription factor binding site prediction using the S. cerevisiae CuRE, future DNA binding experiments with Histoplasma Mac1 and AceI will be required to elucidate their true binding motifs.In addition to induction of catalases and a superoxide dismutase, Mac1 regulates expression of immunity . Anothertoplasma , but thetoplasma . Interesrophores . Howeverrophores . In contin vitro . Since cresponse . We suspresponse  overcomeHistoplasma virulence beyond simple acquisition of copper. In low copper, Mac1 induces the expression of Ctr3, as well as CatB, CatP, and Sod4 antioxidant genes, none of which use copper as a cofactor or have obvious connections to copper. In addition, 10 other genes of unknown function are induced, including three putative transcription factors  and to provide increased ROS detoxification are stimulated. While temperature is the primary regulator of Histoplasma virulence which enables colonization and proliferation within host cells during innate immune responses, sensing of copper limitation by Mac1 allows Histoplasma to further adapt to the enhanced antifungal defenses imposed by the host macrophage during the adaptive immune response.Our results indicate that Mac1 coordinates multiple aspects of Histoplasma capsulatum strains used in this study were derived from the G217B clinical isolate and are listed in Histoplasma-macrophage medium  or with RPMI depleted of any trace metals through treatment with Chelex resin. Media were buffered to pH 5 and supplemented with FeSO4, ZnSO4, or CuSO4 where necessary. For growth on solid media, strains were plated on HMM solidified with 0.6% agarose supplemented with 25 μM FeSO4. For growth of uracil auxotrophs, HMM was supplemented with 100 μg/mL uracil. Yeast liquid cultures were grown at 37°C with continuous shaking (200 rpm). For growth experiments and infections, Histoplasma yeast cultures were grown to late exponential phase. Growth was quantified by measuring culture turbidity using optical density at 595 nm using a BioTek Synergy 2 plate reader or by enumeration of CFU via serial dilutions on solid HMM.All 10.1128/mbio.03773-21.1TABLE S1Histoplasma strains utilized in this study. (B) CTR3 promoter transcriptional reporter constructs. Download Table S1, DOCX file, 0.01 MB.(A) Copyright © 2022 Ray and Rappleye.2022Ray and Rappleye.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Histoplasma strains, plasmids were introduced via an Agrobacterium tumefaciens delivery system and recovered using uracil selection   were isolated previously from an Agrobacterium-mediated mutagenesis screen . The nucleotide sequence corresponding to 1 kb upstream of the CTR3 coding sequence was extracted from the Histoplasma strain G217B genome and searched for instances of the S. cerevisiae CuRE using the FIMO program from the MEME suite  and HMM supplemented with 10 μM CuSO4. In addition, a strain expressing GFP driven by the constitutive TEF1 promoter was included as a nonregulated GFP positive control, as well as the GFP negative strain G217B. Images were obtained for each colony using a modified transilluminator system with GFP emission filters (CTR3 promoter reporter strains was reported relative to control spots of the constitutively expressed GFP under the control of the TEF1 promoter (TEF1-GFPP). To test the necessity of Mac1 binding motifs for the copper response of the CTR3 promoter, the Mac1 binding motifs were scrambled individually or together using overlap PCR and then cloned into the same GFP expression vector used for the promoter truncations. Constructs were transformed into Histoplasma yeasts, isolates were plated in high- and low-copper media, and the GFP fluorescence was quantified as described above.To determine the minimal  filters , and the filters . GFP fluH. capsulatum strain G217B, by BLAST search using the copper-fist DNA-binding domain sequence (pfam00649), as well as the known copper-dependent DNA-binding proteins Cup2 (S. cerevisiae), Cuf1 (C. neoformans), Cuf2 (S. pombe), Mac1 (S. cerevisiae), and AceA (A. nidulans). Protein sequences were trimmed to the N-terminal 70 amino acids that includes the copper-fist DNA-binding domain. Sequences were aligned using CLUSTAL-Omega  functions.Protein sequences for copper-dependent transcription factors were retrieved from fungal genomes, including AL-Omega , and theHistoplasma yeasts from exponentially growing cultures were collected, washed, and resuspended in RPMI medium containing serial dilutions of the copper chelator bathocuproinedisulfonic acid (BCS), the iron chelator bathophenanthrolinedisulfonic acid (BPS), or the zinc chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or, alternatively, in HMM medium containing supplemental copper(II) sulfate. Histoplasma cultures were incubated at 37°C with continuous shaking (200 rpm), and yeast growth was quantified by measuring the culture turbidity  with a BioTek Synergy 2 plate reader. The Histoplasma growth in each medium is reported relative to the growth in basal medium alone.Histoplasma culture supernatants were collected from 5-day-old stationary-phase cultures grown in either standard HMM (10 nM CuSO4) or HMM supplemented with 5 μM CuSO4. After collection, BCS was added to a final concentration of 10 μM to chelate any trace copper that might interfere with catalase activity. To monitor CatB extracellular catalase activity, 15 mM H2O2 was added to culture supernatants, and the degradation of H2O2 was monitored by assaying samples at 0, 5, 10, 15, and 20 min with a cobalt/bicarbonate-based colorimetric indicator of peroxide . The absorbance of the colorimetric indicator was measured at 440 nm, as described previously (eviously .MAC1-RNAi Histoplasma yeast cultures grown in either HMM (low copper) or HMM with 5 μM added CuSO4 (high copper), with samples prepared from two biological replicates for each condition. Yeasts were collected via centrifugation , and RNA was released from cells by resuspension of yeasts in TRIzol reagent (Thermo Fisher) and mechanical disruption of yeasts with 0.5 mm-diameter glass beads. Cellular RNA was purified with Direct-zol RNA miniprep columns (Zymo Research). RNA samples were treated with Turbo DNase (Invitrogen) to remove contaminating DNA. For RT-qPCR, cDNA was generated from total RNA using Maxima reverse transcriptase (Thermo Scientific) primed with random pentadecamers. Quantitative PCR was performed using gene-specific primer pairs, and amplified products were quantified via SYBR green-based detection . After normalization to the Histoplasma actin (ACT1) gene, changes in gene expression relative to TEF1 were determined using the cycle threshold (ΔΔTC) method  method . For RNA) method . Reads w v2.5.0a , and rea v2.5.0a . Differe v2.5.0a , and genin vitro infection experiments. Macrophages were cultivated at 37°C with 5% CO2/95% air in Ham F-12 media (Corning) containing 10% fetal bovine serum . Macrophages were infected with Histoplasma yeasts at a multiplicity of infection (MOI) of 1:1 (yeasts:macrophages) for intracellular yeast enumeration or an MOI of 1:3 for macrophage survival assays. Noninternalized yeasts were removed after 2 h by shaking plates  and replacement of the medium on cells with fresh Ham F-12 with 10% FBS. Cultures of infected macrophages were mixed daily by shaking  to disperse yeasts from lysed cells. For intracellular proliferation assays, macrophages were lysed with H2O and scraping of wells with a pipette tip. Serial dilutions of lysates were plated on solid HMM for enumeration of CFU. For macrophage survival assays, surviving macrophages were quantified at 0, 3, 4, and 5 days postinfection by measuring the LacZ activity relative to that of uninfected macrophages , and the β-galactosidase activity in each well was then measured using a BioTek Synergy 2 plate reader (absorbance at 420 nm with correction at 600 nm).A LacZ-expressing P388D1 murine macrophage cell line was utilized for all rophages . To quan4Histoplasma yeast were introduced intranasally into isoflurane-anesthetized wild-type C57BL/6 mice. At 6, 12, or 14 days postinfection, mice were euthanized, and lungs (and spleens when indicated) were collected, homogenized in HMM, and then serially diluted and plated onto solid HMM for CFU enumeration. Animal experiments were performed in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University (protocol 2007A0241).Suspensions of 2 × 10P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).Data were analyzed by Student’s t-test  to determine statistically significant differences, which are indicated in the figure graphs by asterisks (*, PRJNA812927. All strains are available from the authors upon request.RNAseq data has been submitted under BioProject ID: 10.1128/mbio.03773-21.2TABLE S2Table S2, DOCX file, 0.01 MB.Primers used in this study. Download Copyright © 2022 Ray and Rappleye.2022Ray and Rappleye.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


During mammalian neurodevelopment, signaling pathways converge upon transcription factors (TFs) to establish appropriate gene expression programmes leading to the production of distinct neural and glial cell types. This process is partially regulated by the dynamic modulation of chromatin states by epigenetic systems, including the polycomb group (PcG) family of co-repressors. PcG proteins form multi-subunit assemblies that sub-divide into distinct, yet functionally related families. Polycomb repressive complexes 1 and 2 (PRC1 and 2) modify the chemical properties of chromatin by covalently modifying histone tails via H2A ubiquitination (H2AK119ub1) and H3 methylation, respectively. In contrast to the PRCs, the Polycomb repressive deubiquitinase (PR-DUB) complex removes H2AK119ub1 from chromatin through the action of the C-terminal hydrolase BAP1. Genetic screening has identified several PcG mutations that are causally associated with a range of congenital neuropathologies associated with both localised and/or systemic growth abnormalities. As PRC1 and PR-DUB hold opposing functions to control H2AK119ub1 levels across the genome, it is plausible that such neurodevelopmental disorders arise through a common mechanism. In this review, we will focus on advancements regarding the composition and opposing molecular functions of mammalian PRC1 and PR-DUB, and explore how their dysfunction contributes to the emergence of neurodevelopmental disorders. During mammalian development, signaling pathways act in concert with tissue-specific transcription factors (TFs) to establish appropriate gene expression patterns. Transcription does not, however, occur on naked DNA, but instead in the context of the nucleoprotein complex chromatin; DNA wrapped around basic histone proteins. This configuration provides a scaffold to package the genome into the nucleus and a substrate for a plethora of posttranslational modifications. In addition, chromatin can undergo changes to its packaging state across orders of magnitude—from local regulatory domains to interactions that span entire chromosomes. These chemical and physical modifications can be permissive for, or refractory to the molecular processes that operate on DNA. Indeed this ‘epigenetic’ information ensures the appropriate and proportionate temporal and spatial transcriptional response during cellular specification ,2,3. In One of the best characterised of the epigenetic regulatory systems is that of the polycomb group (PcG) family of co-repressors. PcG proteins assemble into multi-subunit complexes that alter both the chemical and structural properties of chromatin to antagonise transcription. These complexes sub-divide into distinct functionally related families. Polycomb repressive complexes 1 and 2 (PRC1 and 2) modify the chemical properties of chromatin by covalently modifying histone tails. PRC2 contains EZH1 or 2, SET domain methyltransferases which can mono-, di- and tri-methylate lysine 27 of histone H3 (H3K27me1-3) ,5,6,7. PIn mice, loss of core PcG proteins leads to early embryonic lethality with failure occurring at, or shortly after gastrulation ,17,18. IPRC1 complexes contain a highly conserved catalytic core . In mammDrosophila polycomb  and polyhomeotic  proteins. The chromodomain of CBX subunits has affinity for histone H3 lysine methylations (H3K27me3 and H3K9me3) and these have been shown to be important for PRC1 targeting to chromatin [In mammals, cPRC1 complexes assemble around a dimer of RING1A/B and either PCGF2/MEL18 or PCGF4/BMI1. This assembly associates with additional subunits, including homologs of the hromatin ,30,31. Chromatin ,27,29. Chromatin ,32  techniques have revealed that distal chromosomal regions, separated by great genomic distances, cluster within the nucleus in a PRC1-dependent manner. Disruption of RING1B or subunits of cPRC1 lead to the loss of interactions and marked alterations to nuclear organisation ,37,38,39cPRC1 exhibits relatively low E3 ubiquitin ligase activity in vitro and contributes minimally to the deposition of H2AK119ub1 in vivo, suggesting that this may not be its primary molecular function ,54,55,56ncPRC1 complexes comprise dimers of RING1A/B and PCGF1, 3, 5 or 6, associated with either RYBP or its homolog YAF2 in place of CBX ,76 Figu. The incThe lack of CBX subunits means that ncPRC1 complexes do not recognise H3K27me3 and therefore deposit H2A ubiquitination in a PRC2-independent manner . In addiI53A/I53ARing1b) present with substantially reduced global H2AK119ub1 levels  die perinatally, however, as for Drosophila bearing a functionally equivalent mutation in the RING1B homolog Sce (I48A/I48ASce), they do not present with the earlier embryonic developmental deficits associated with RING1B deficiency [I53A/I53ARing1b cells [I53A/D56KRing1b or I53S/I53SRing1a−/−Ring1b) present with a more complete loss of H2AK119ub1 and leads to substantial gene misregulation [I53A/I53ARing1b) does not impair gross epidermal development but does lead to ectopic expansion of non-skin cell types [H2AK119ub1 is highly abundant in mammals, with approximately 10% of total H2A in mESCs carrying the modification ,54. Howeld-type; ,79 and uld-type; ,37,79,80ficiency . Global 3A cells ,78,79. Igulation ,78. Thesll types . This suDrosophila embryos carrying mutations in H2A or H2Av that block PRC1-mediated ubiquitination (K118RH2A and K120R/K121RH2Av respectively) and modestly reduce H3K27me3 can still support normal embryonic development [Drosophila operates through a well-defined DNA sequence-dependent recruitment mechanism, yet no equivalent system has been identified in mammals (reviewed in [Drosophila and human respectively) may therefore have led to an increased dependence upon H2AK119ub1 for robust targeting of polycomb complexes in the absence of sequence specific recruitment. Further work is required to disentangle the direct versus indirect contribution of H2AK119ub1 in gene repression in mammals.Recent observations suggest that PRC1 reduces gene expression through alterations in transcriptional burst frequency, however, the biophysical contribution of H2AK119ub1 to this is currently unknown . H2AK119elopment . The reliewed in ). The exDrosophila). In flies, the functional core of PR-DUB comprises the C-terminal hydrolase Calypso and its obligate partner Additional Sex Combs (Asx), that together constitute the active enzymatic core of the complex [The PR-DUB complex cleaves ubiquitin conjugates from both chromatin and soluble protein targets, but is best characterised for its erasure of H2AK119ub1  that pass through sequential, spatiotemporally restricted phases of neural precursor cell (NPC) expansion, neurogenesis and gliogenesis. Accumulating research highlights the importance of epigenetic regulation in this process, with PcG-mediated transcriptional regulation in particular being critical to ensure the fidelity of cortical growth and specification. In mice, targeted ablation of EZH2 prior to the onset of neurogenesis leads to an accelerated neurogenic phase, a premature switch into gliogenesis and a smaller cortex at birth . In contnd Foxg1 . Finallynd Foxg1 . These fCongenital defects in various PRC1 subunits leads to developmental deficits reflecting their role in a range of disparate developmental processes. A subset of PRC1 subunits are either upregulated during neural development and/or have context-specific functions in the developing nervous system. Perturbations in these components leads to defined NDDs, the characteristics and aetiologies of which will be explored further below.Ring1B expression [Ring1B knockout results in early embryonic lethality, presumably due to RING1B’s more ubiquitous expression compared to RING1A, which is primarily detected in the central nervous system [RING1A is one of two E3-ubiquitin ligases that constitute the core enzymatic subunit of PRC1. Whilst the role of the paralogous RING1B subunit is well documented for its involvement in NPC fate transitions, the role of RING1A is less clear ,108,111.pression . In conts system ,20. Reces system .R95Q to ubiquitinate histone H2A, consistent with significantly reduced levels of H2AK119ub1 found in cells derived from the affected individual. In contrast, RING1A protein levels were unaffected suggesting that the observed phenotype was a result of a change in RING1A function rather than its abundance. Engineering this mutation in SPAT-3, the RING1 orthologue in C. elegans, resulted in reduced ubiquitination and abnormal neuronal cell migration and axon guidance patterns in agreement with previous observations [C. elegans has also identified a role for SPAT-3 in neuronal cell fate specification in a manner that protects the differentiation programme from environmental stress [A heterozygous de novo mutation in RING1A (p.R95Q) was identified in a child who displayed characteristics of perturbed neurodevelopment and microcephaly in early childhood. The individual presented with delayed acquisition of language and adaptive social skills, ID and psychotic symptoms that developed at adolescence . Modellirvations ,114. Recl stress .The overwhelming burden of evidence suggests that RING1A is functionally subordinate to RING1B in mammals; however, abrogated RING1A function leads to reduced H2AK119ub1 and disordered neurodevelopment. This observation suggests that the RING1 paralogues are not entirely redundant, hinting at unappreciated context specific functionality for RING1A during brain development.As previously discussed, PHC proteins are important constituents of cPRC1 and play a central role in its capacity to coordinate both local and distant chromosomal interactions ,39,42. DIn 2013, Awad and colleagues identified an individual bearing a novel missense mutation (p.L992F) in the SAM domain of PHC1 that was implicated in primary microcephaly pathogenesis . This muBCORL1 underlie the rare X-linked recessive NDD—Shukla-Vernon syndrome (SHUVER) [One of the most prominent non-canonical PRC1 subunits is BCL6 corepressor-like 1 (BCORL1). BCORL1 is linked to PRC1.1’s enzymatic core through PCGF1 and is thought to repress transcription through interactions with C-terminal binding proteins (CtBPs) and Class II histone deacetylases (HDACs) ,121. BCO(SHUVER) . SHUVER (SHUVER) ,123. In (SHUVER) ,123,124.None of the known pathogenic BCORL1 variants impair protein domains that are required for gene repression, including both the C-terminal binding protein domain (CBD) and PCGF Ub-like fold discriminator (PUFD) domain ,124. ConA second ncPRC1 subunit implicated in neurodevelopmental disorders is autism susceptibility candidate 2 (AUTS2). AUTS2 is expressed at high levels throughout the brain during early development and is linked to PRC1.3 and PRC1.5 through interaction with PCGF3 and PCGF5 respectively ,126,127.AUTS2 was originally linked to neurodevelopmental disorders in a study that described a pathogenic mutation found in a pair of monozygotic twins with ASD . Since tAUTS2 gene encodes two alternative protein isoforms that are involved in a range of physiological roles in neural development and gene regulation [Experimental exploration into the mechanism of this family of disorders has shown that AUTS2 deficiency leads to defects in neuronal development, neurite formation, neural migration and perturbed synaptic function; characteristics which correspond with AUTS2 disruption in humans ,140,141.gulation ,138. Thegulation . Despitegulation . This sugulation ,142,143.gulation .Disruption of PR-DUB function is implicated in multiple pathological contexts, including malignant cancers and neurodevelopmental conditions. In addition to its roles in development, PR-DUB function in DNA repair, and indeed, mutations of PR-DUB subunits are frequently seen in melanomas and mesotheliomas ,146,147.Drosophila Asx–ASXL1, 2 and 3, the products of which form mutually exclusive heterodimers with BAP1 to establish the catalytic core of the PR-DUB complex [Drosophila Asx, although the overall sequence has diverged [Humans and mice encode three homologues of  complex ,150,151.diverged . Consistdiverged . Despitediverged ,153. In diverged . Experimdiverged . Interesdiverged .ASXL1 and 2 are expressed during embryogenesis; however whilst ASXL1 transcript levels remain high in the forebrain into adulthood, ASXL2 levels decrease in most tissues perinatally [ASXL3/Asxl3 expression levels are low in neural progenitors but high in post-mitotic neurons in both humans and mice [Some of these differences could be attributed to context specific expression rather than functional differences; indeed the ASXLs have distinct expression profiles during mammalian development. In humans, inatally . In contand mice ,157. At and mice .ASXL1, 2 and 3 cause Bohring–Opitz , Sh, ShASXL1tz  and Baitz  syndromtz  family of transcriptional co-regulators, and interacts with the PR-DUB complex in a mutually exclusive manner to its paralogue, MBD6 ,87,167.  complex ,169. The complex . In line complex ,103,170. complex ,172.The relative proportion of MBD5 that associates with BAP1 (PR-DUB) is higher in differentiated NPCs relative to mESCs . CoupledThese observations suggest that proper dosage of MBD5 is vital for normal PR-DUB function and consequently for neurodevelopment, but that some aspects of MAND are specific to the nature of the underlying causal genetic lesion. Even though it is known that MBD5 is necessary, its function and biochemical properties are poorly understood. Further investigation is required to understand how improper MBD5 dosage impacts on PR-DUB function, H2AK119ub1 levels and the cellular and developmental characteristics of MAND disorders.Genetic lesions that impair the polycomb system, one of the paradigms of epigenetic regulation, are a prominent cause of a range of NDDs. In this review, we have explored the impact of perturbations in two of these complexes—PRC1 and PR-DUB whose opposing actions control the deposition and abundance of the H2AK119ub1 mark.Protein ubiquitination is used as a control mechanism for a wide array of cellular processes, the mechanics of which relate, in part, to the linkage chemistry of the underlying ubiquitin conjugate . With reTo determine why perturbations in H2AK119ub1 levels lead to neuropathologies, we must understand its contribution to gene regulation. PRC1 is a multi-functional family of complexes; however, recent investigations have provided conclusive evidence that H2AK119ub1 is central to PRC1-mediated gene repression ,78,187. Drosophila development [Despite possessing counteracting functions, mutations in PRC1 and PR-DUB give rise to phenotypically related neurodevelopmental disorders, suggesting that an ‘optimal’ level of H2AK119ub1 is required for healthy brain development ,174,177.elopment . Whetherelopment ,196,197.There is currently insufficient evidence to determine if dysfunction of individual NDD-associated PRC1 and PR-DUB components contribute to a shared pathological gene expression signature in neural cell types. However, mutations in RING1A and ASXL1-3 would be predicted to directly alter H2AK119ub1 levels and mutations in AUTS2, BCORL1, PHC1 and MBD5 to alter the distribution and occupancy of their respective complexes. It is also interesting to note that both reduced and increased levels of the chromatin proteins MBD5 and MeCP2 lead to phenotypically related disorders ,198,199.It is important that we remain open to alternative functions of the polycomb system; both in terms of context and mechanism of action. The largest body of evidence relating to the genetics, underlying molecular basis and phenotype of any PRC1-associated NDD is that of AUTS2 syndrome ,136,137.RING1A and PCGF4 are implicated in the ubiquitination of chromatin-associated proteins with a speculated role in mitotic bookmarking ,201. FurThe commonalities between the NDDs discussed here are suggestive of a unifying disease mechanism. However, context-specific expression and function, variable phenotypic penetrance, unappreciated functionality and complex genetic underpinnings make determining such relationships challenging. With respect to this last point, it is interesting to note that whilst the vast majority of the known NDD-associated mutations occur de novo in the affected individuals, examples of inherited mutations associated with milder phenotypes exist for both ASXL3 and AUTS2 ,207. In


Mycobacterium tuberculosis (MTB) is the main source of tuberculosis (TB), one of the oldest known diseases in the human population. Despite the drug discovery efforts of past decades, TB is still one of the leading causes of mortality and claimed more than 1.5 million lives worldwide in 2020. Due to the emergence of drug-resistant strains and patient non-compliance during treatments, there is a pressing need to find alternative therapeutic agents for TB. One of the important areas for developing new treatments is in the inhibition of the transcription step of gene expression; it is the first step to synthesize a copy of the genetic material in the form of mRNA. This further translates to functional protein synthesis, which is crucial for the bacteria living processes. MTB contains a bacterial DNA-dependent RNA polymerase (RNAP), which is the key enzyme for the transcription process. MTB RNAP has been targeted for designing and developing antitubercular agents because gene transcription is essential for the mycobacteria survival. Initiation, elongation, and termination are the three important sequential steps in the transcription process. Each step is complex and highly regulated, involving multiple transcription factors. This review is focused on the MTB transcription machinery, especially in the nature of MTB RNAP as the main enzyme that is regulated by transcription factors. The mechanism and conformational dynamics that occur during transcription are discussed and summarized. Finally, the current progress on MTB transcription inhibition and possible drug target in mycobacterial RNAP are also described to provide insight for future antitubercular drug design and development. Mycobacterium tuberculosis (MTB) bacilli from an active TB patient. According to a previous hypothesis, the Mycobacterium genus was supposed to have originated more than 150 million years ago [Tuberculosis (TB) is a communicable disease that infects patients through inhalation of the expelled droplets containing the ears ago . In pastears ago ,4,5. Beyears ago . TB treaears ago ,8. Treatears ago ,10. Due ears ago .E. coli RpoB [Knowing how the mycobacterial cell works is a prominent point of departure for drug discovery attempts for designing TB treatments. Every cell function of an organism is dictated by proteins and protein synthesis in a living system in a highly regulated manner ,13. In toli RpoB . RIF posoli RpoB . The actoli RpoB ,22.During the transcription process, RNAP undergoes a sequence of events to activate its catalytic-competent form and begin the RNA synthesis. This process involves engagement with the DNA promoter, unwinding the double-stranded DNA, loading the template into the active site, NTP substrate uptake, and the nucleotidyl transfer to synthesize the phosphodiester bond in the nascent RNA. As targeting the transcription process is still one of the attractive strategies to develop a new inhibitor to combat TB, this review aims to discuss the nature of MTB RNAP as well as the mechanism of the bacterial transcription process and current drugs to target MTB RNAP. The current progress of drug discovery and development related to bacterial transcription inhibition is also summarized to provide an insight into the advancement on antitubercular drugs.RNAPs are found as highly conserved molecular machinery, which is important for the transcription process as a part of the gene expression system in living organisms. In both eukaryotes and prokaryotes, RNAPs work as multi-unit enzymes. Eukaryotic cells undergo a more complex transcription process, as they possess three different RNAPs  with a distinct number of subunits, in which each RNAP is assigned for a specific transcription material . In contE. coli RNAP and Thermus RNAP—ranging from the regulatory system, kinetics during initiation, and the presence of crucial transcription factors such as CarD and RbpA protein [Despite sharing the same lineage and structural similarities, mechanistic studies found that the MTB RNAP exhibited several differences compared to two of the most studied RNAP— protein . The ~40 protein . MovemenThe core enzyme assembly a starts 2+-containing active site and acts as a tether between the RNAP and promoter DNA template. In contrast, RbpA binds to the other side of the β subunits using residues 478–677, contributing to the stability of the initiation complex [The function of β and β′ subunits is for the claw-shaped core of RNAP. The β subunit has a 150 kDa size, which is slightly smaller than a 155 kDa β′ subunit. Both of these large proteins bind with the α subunits dimer in an organized manner, where the C-terminus of the β subunit is positioned near the N-terminus of the β′ subunit. Each of these subunits have a double-psi beta-barrel motif as a part of the RNAP active site; the active site coordinates with the Mg (II) ions utilizing the aspartic acid triad to facilitate the nucleotidyl transfer reaction . Residue complex ,38. Asid complex ,40.E. coli β′-ZBD resulted in weaker promoter binding and lowered the ratio of stable initiation complex formation [MTB RNAP is a zinc metalloenzyme, and the zinc-binding domain (ZBD) possesses a prominent role in the transcription process. The ZBD is located inside the β′ subunit and is characterized by four cysteine residues that coordinate with the zinc ions. Another characteristic is that this domain has positively charged residues. The transcription factor RbpA binds to this domain, indicating its importance in transcription regulation. Other than the regulatory effect, this domain might interact directly with the DNA promoter within the spacer region to strengthen the interaction between the -35 element and the σ4 domain . This isormation . In agreormation ,44.The catalysis of RNA synthesis involves transfer of the nucleotidyl from the NTP substrate that is bound to the active site using the 3′-OH ends of the newly synthesized RNA. After the transfer is complete, RNAP moves to accommodate the transfer of the next complementary nucleotide in the template sequence. The mobile domain of the β′ subunit regulates this cycle. This mobile domain can switch between the ‘trigger loop’ and ‘trigger helix’ conformations. The loop conformation allows NTP entry to the active state; after the change to the helix conformation, the channel is closed, and the transfer reaction starts. Other domains of the β′ subunit, called ‘fork loop’ and ‘bridge helix’, can make contact with the mobile domain to modulate coordination of the nucleotide addition cycle .E. coli ω-coding gene (rpoZ) once revealed that the core RNAP without ω subunit appeared to be morphologically the same [M. stegmatis and E. coli, where the stabilization role of this subunit can be substituted with another factor such as GroEL homologs in E. coli [During the RNAP core enzyme assembly, the ω subunit is the last and smallest subunit added to the complex. The ω subunit remains as the least-studied subunit; it was first thought that this subunit is not necessary for the transcriptional activity. A knockout study of the the same . It was the same . The desthe same . This ph E. coli ,50. It iE. coli transcription model, due to the difficult handling of the mycobacterial pathogen. However, the usage of an E. coli model for antimycobacterial study was found to be ineffective since the structural shift might make a significant difference in the activity study. For example, RIF sensitivity towards MTB RNAP is 1000-fold higher compared to E. coli RNAP, although RIF was found to bind tightly to the same binding site in E. coli RNAP [Initially, studies on mycobacterial transcription were conducted based on an oli RNAP . StructuRegulating the transcription process helps bacteria to defend themselves from the host’s defence mechanisms that prevent their growth during the infection. As one of the organisms with complex infection and adaptation mechanisms, MTB has distinct and highly coordinated regulatory features . The traSigma (σ) factor proteins play a significant role in the MTB transcriptional process. These proteins bind reversibly to the RNAP core and form the holoenzyme during the initiation step. The specificity of RNAP is determined by the type of σ factor protein to which it is attached; various σ factor proteins direct RNAP to recognize a specific set of DNA promoters . Based osigA), and 12 accessory of σ factors , which are associated with different tasks in the same signalling network [SigA serves as the principal σ factor, and its role is for the transcription regulation of housekeeping genes. Moreover, sigA was found to be overexpressed in in vivo TB-infected pulmonary macrophage environments, indicating its role in regulating virulence-related genes during the early phase of infection [SigB is closely related to sigA (62% homologous) and is believed to regulate more generalized stress conditions. A recent study indicated that sigB can recognize some housekeeping genes’ promoter regions during the exponential phase. This serves as evidence that sigB can act as a counterpart to sigA during this phase [SigF was first thought to be upregulated in the case of nutrient starvation; however, global gene expression profiling revealed that sigF regulates the transcription of cell wall-associated genes such as MmpL, PE, and PPE families for survival during the host–pathogen interactions [sigE and sigH are downregulated, indicating their importance in maintaining the MTB persistence in the non-replicating/slow-replicating state [Each type of bacteria expresses a different number of σ factor proteins, with most having a primary and multiple accessory σ factors . MTB pos network . SigA senfection . SigB isis phase . SigF waractions ,62. The ractions ,63. During state . Relativng state .Despite the disparity in function, the σ factors work in a collective manner, forming an intricate and multi-layer signalling network to regulate the RNAP. An integrated study to reconstruct this regulatory network revealed that there is a high connectivity between the components that are built through 41 direct interactions . These iSome RNAP-binding proteins are known to regulate the MTB transcription process, such as CarD, RbpA, and NusG. CarD is conserved and essential in mycobacteria as a global transcription regulator. Structural analysis revealed that CarD stabilizes the open promoter complex formed between the holoenzyme and σA during the initiation step ,68. In tE. coli. E. coli and MTB share a similar transcription system, but the stability of their open promoter complexes is distinctly different. A single round abortive initiation assay for E. coli and MTB transcription revealed that mycobacterium open promoter complex decayed rapidly in the absence of CarD, unlike in E. coli, which still showed high transcription activity [CarD presence is essential in MTB, although this protein is interestingly absent in activity .Inhibition of CarD/RNAP β subunit interaction was reported to influence the viability and susceptibility of the pathogen towards various antitubercular agents. A higher response to RIF was observed when the CarD/RNAP interaction was targeted, which encouraged the synergistic treatment for TB . AlthougE. coli [M. stegmatis and RbpA binding site (residue R381 on the β subunit) to the RIF binding site on the β subunit [50 of RIF from transcription initiation assay with and without the presence of RbpA shows constant inhibition (IC50: ~10 nM) [As another transcription factor, RNAP binding protein A (RbpA) is firstly found to be specific to Actinomycetes. Like CarD, RbpA is essential for MTB growth, but it is absent in E. coli . MTB Rbp subunit ,78. In M ~10 nM) . Structu ~10 nM) .During the initiation step, it was found that RbpA synergistically works with CarD to stabilize the open promoter complex formation through a different mechanism of action. Both RbpA and CarD bind to a different binding site on the complex , which wIn opposition to the BL and SID, other domains of RbpA such as NTT and CD were found to act antagonistically towards the open promoter complex stabilization. The deletion of both domains was proven to increase the complex’s stability, but it is believed that NTT and CD could contribute to RNAP activity through a distinct mechanism due to the strategic position they hold on the RNAP-σA complex. A recent study showed that RbpA NTT influences Fidaxomicin (Fdx) activity towards RNAP. Fdx inhibits MTB RNAP by blocking the clamp movement and holds it in the open clamp formation, preventing the initiation step. It was revealed that the E17 and R10 residues on RbpA NTT provide additional contact with Fdx, resulted in stronger interaction on the Fdx binding site . This reE. coli NusG revealed that this protein can interact with RNAP at the central cleft, with the N-terminal domain bound at the upstream fork junction. This position ensures clamp closure and is hypothesized to promote the elongation complex stabilization [After the initiation process is completed, the σ factor detaches from the initiation complex to form the elongation complex. During elongation, RNAP moves along the DNA template to synthesize the new RNA. This dynamic is regulated by some elongation factors, one being the group of N-utilization substances (Nus) protein. Four proteins belong to this class, including NusA, NusB, NusE, and NusG. This class is known to promote antitermination processes in transcription and remains as a potential target for antibiotics. The Nus proteins are potential targets because they bind to the elongation complex and remain there until the elongation is completed. Aside from playing a part in anti-termination, they are capable of regulating the elongation pause and termination processes ,85,86,87lization . Targetilization ,90.Bacterial transcription is a multi-step process that consists of initiation, elongation, and termination. These processes are initiated by the formation of the RNAP holoenzyme, where the core enzyme (α2ββ′ω) binds to the σ factor protein. Afterwards, the initiation step takes place, where the RNAP holoenzyme recognizes a specific DNA promoter sequence and triggers the DNA strand to unwind. After the template DNA is exposed, nucleotide addition occurs during the elongation phase producing the nascent RNA strand. This process is terminated (termination step) once the enzyme recognizes a termination sequence in the template, releasing the newly synthesized RNA product. In this section, the dynamics and kinetics characterization of MTB RNAP during the transcription process are discussed.Binding of the sigma factor protein to the RNAP enables this enzyme to form specific binding with the promoter DNA and initiates the transcription process. During the initiation, MTB RNAP undergoes a number of conformational changes to form a closed promoter complex (RPc). This further stabilizes into an open promoter complex (RPo) to finally generate the initiating complex (RPitc). RPc formation is induced when the promoter DNA is attached to the RNAP. In this step, the σ factor protein and the α subunit of RNAP both play important roles in holding the promoter DNA together close to the RNAP β/β′ cleft to facilitate the transcription bubble formation in the next step . The σ pE. coli RNAP during the transcription initiation [The RPc formation induces a series of changes to generate the catalytically competent complex RPo. These changes aim to unwind the dsDNA strand and place the template strand on the active site of RNAP to initiate the RNA synthesis. A recent study revealed the mechanism of DNA unwinding during the RPo formation by observing a single-molecule fluorescence resonance towards itiation . From thitiation .The MTB RPo is known to dissociate rather fast, after ~1 to 2 min; the stabilization is needed to maintain an effective transcription rate. As mentioned before, transcription factors CarD and RbpA have been revealed to work together to restrict the movement of RNAP/promoter interactions and avoid the collapsing of the transcription bubble. During RPo formation, RPc undergoes isomerization, forming two intermediates before it forms the stable RPo. CarD and RbpA regulate this process by increasing the second intermediate formation rate, bypassing the energy barrier to form more stable RPo. This is different from other transcription activators such as CRP that play a part during the initial promoter-recognition step ,95,96,97Transcription bubble formation in the RPo triggers RNA synthesis from the template DNA. This process generates a covalent phosphodiester bond between the 3′OH end of the newly synthesized RNA and the α-phosphate on the added nucleotide to increase one nucleotide to the chain, followed by the release of pyrophosphate as a side product. At the beginning, interaction between RNAP and promoter is still maintained. More of the template strand is dragged inside the transcription bubble, resulting in the expansion of the strand and the addition of tension to the ‘scrunched’ promoter DNA. As a result, the promoter DNA is detached from the initiation complex to release the tension, and the σ factor is also known to be dissociated from the complex, triggering the formation of an elongation complex. Elongation involves continuous DNA duplex melting and the addition of subsequent base pairs to the opened strand. A productive and undisrupted elongation step ensures that the pathogen synthesizes the full-length sequence to make a functional protein, which supports the pathogen’s living functions. In the case of non-abortive initiation, the promoter escape and the σ factor dissociation act as the hallway for the stable ternary elongation complex formation. During the elongation, MTB RNAP moves through the single-stranded template in the 3′ to 5′ direction, adding one complementary nucleotide from the secondary channel each to the long nascent RNA product. The new RNA transcript is unloaded from the complex through a narrow hole formed by the β-flap domain and zinc-binding domain. This movement should be energetically balanced, as well as highly coordinated with the incoming NTP substrate. In MTB RNAP, auxiliary transcription factors such as the Nus protein control this process to avoid elongation failure ,100,101.The elongation complex continues to move through the template until it recognizes a termination sequence. Bacteria have two types of transcription termination: intrinsic termination and factor-dependent termination. Intrinsic termination occurs due to the instability of the ternary elongation complex caused by the weak RNA/DNA hybrid, which is called ‘U-tract’. ‘U-tract’ is a 7–9 nucleotide long sequence that is composed by a GC-rich hairpin, followed by uracil-rich tract. This specific sequence results in the synthesis of the stem-loop structure and induces transcript release by the TEC to terminate the transcription process. Another mechanism involves the Rho termination factor, an ATP-dependent RNA helicase. Rho factor binds to the Rho utilization (rut) site on the newly synthesized RNA, moving through the RNA chain and dissociating the TEC ,103.Understanding the mechanism, structural characteristics, and conformational dynamics of the transcription process provides the insights needed to develop a potential inhibitor for MTB. In this section, several compounds that have been known to inhibit bacterial RNAP are summarized in Fidaxomicin, also known as Lipiarmycin A3, Tiacumicin B, or OPT-80, is a glycosylated macrocyclic lactone compound that was approved for treating gastrointestinal infection caused by Clostridium difficile . It is kThe potency of Fidaxomicin as an antitubercular agent was initially characterized during a natural product screening for antitubercular agent, where this antibiotic was identified as a hit compound . In an iMyxopyronin and corallopyronin are α-pyrone antibiotics derived from myxobacteria ,131. BotE. coli RNAP.Several clinical challenges have been addressed regarding the usage of these α-pyrone compounds as antitubercular agents. The bactericidal activity was found to dramatically decline in the presence of serum albumin due to hydrophobic interaction. It was later studied that myxopyronin also interacts with RNAP, mainly through hydrophobic interaction, which eliminates the possibility of developing a less hydrophobic analogue of this compound ,133. AnoRipostatin is a polyketide-derived macrolide with no structural similarity to the α-pyrone RNAP inhibitor . RipostaSorangicin is a macrolide polyether natural product isolated from Sorangium cellulosum . SorangiStreptolydigin is a tetramic acid antibiotic, which has been found to inhibit nucleotide addition in the initiation and elongation step of bacterial transcription . The binE. coli Gre factor. An affinity pulldown assay and in vitro transcription assays confirmed the ability of this protein to bind with RNAP and the transcript cleavage activity. Another gene, Rv3788, was also found to share a lower degree of similarity with E. coli GreA compared to Rv1080c [E. coli. Structural study showed that Rv3788 inhibited transcription through its N-terminus domain that fit in the narrowest region of the secondary channel and blocked the nucleotide entry to the RNAP active site. The inhibitory activity of Rv3788 was significantly reduced in the presence of MTB Gre, showing that they competed for the same binding site in MTB RNAP [Prokaryotic Gre proteins are transcription factors that work by stimulating the endogenous cleavage of aberrant 3′ end transcript . In MTB  Rv1080c . Rv3788 MTB RNAP .Another compound known to bind to the secondary channel of MTB RNAP is D-AAP1 . This compound belongs to a novel class of RNAP inhibitor found from the high-throughput screening of synthetic compounds using promoter-dependent fluorescence-based transcription assay. D-AAP1 binds the bridge helix binding site deep inside the secondary channel, and conformational change in this site is essential to accommodate NTP uptake during the transcription . While iRifamycin is an important class of antibiotics with a wide spectrum of activity. Rifamycin compounds are known to target bacterial RNAP with a well-established inhibitory mechanism (detail reviewed in ). RIF, tKanglemycin A is a natural rifamycin isolated from soil, and its antimicrobial activity was identified first through a disk diffusion assay. This compound has a distinct structural feature compared to the existing semisynthetic rifamycins, with modifications on the ansa chain instead of the naphthoquinone moiety. Kanglemycin A has 2,2-dimethyl-succinic acid substitution on the C20 position and a unique sugar β-O-3,4-O,O′-methylene digitoxose on the C27 ,115. TheDespite sharing the same binding site with RIF, kanglemycin A is active against RIF-resistant mutants of MTB RNAP. Structural study showed that the unique substitution on the ansa chain mediates the binding towards the mutant by forming new interactions with residues that do not interact with RIF. The sugar side chain interacts with residues R173 and T433 of MTB RpoB, and the succinic acid side chain forms a salt bridge with R604. Derivatization to the succinic acid chain has been performed in an effort to improve the bioavailability and in vivo efficacy of this compound, which resulted in better potency against WT bacteria, but loss of activity against the RIF-resistant mutants .E. coli RNAP and exhibits a narrow spectrum activity towards other bacteria, possibly because of poor penetration [50 value, while alpha, β -diaminopropanoic acid (Apa) was found to be not critical for binding, as the GE23077 binding site is adjacent to the RIF binding site. The bipartite inhibitor GE23077-RIF was evaluated in the previous study, and the hybrid inhibitor was found to be more potent compared to GE23077 and RIF alone [GE23077 is a naturally occurring cyclic heptapeptide, isolated from Actinomadura sp. . This coetration . In vitretration . This sietration . AlteratIF alone .E. coli transcription [Microcin J25 is a ‘lasso’ peptide antibiotic that showed wide-range activity towards the cription ,147. Thicription ,149. Miccription . A systecription . Gly1 ancription ,153.From The transcription process is complex and essential in mycobacteria. This process is highly regulated to ensure a successive and accurate transcript production is translated into a functional protein for this pathogen’s survival. The multi-subunit MTB RNAP acts as the machinery for this process, bearing the transcription site at its active site. MTB RNAP undergoes several conformational changes during the initiation, elongation, and termination processes. These changes involve attachment or detachment to some transcription factors to coordinate the DNA-promoter melting and insertion to the binding site to start the RNA synthesis. Some compounds have been found to inhibit this complex process at a certain point, while an understanding of the mechanisms revealed the gaps in the development of a novel compound targeting a different step. Most RNAP inhibitors are known to target the initiation process. While proven effective, targeting PPI between the subunit and transcription factor or modulating the regulator function to delay promoter escape/create more stalled complex might also increase the abortive transcription rate. Other gaps may exist in the elongation complex, where the induction of pausing or backtracking could lead to incomplete transcription or missense mutation. Disrupting the termination step through misrecognition of the termination factor rho also disturbs the accuracy of protein synthesis.