Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle. Kaposi’s Sarcoma (KS), first described in 1872 by the Hungarian dermatologist Moritz Kaposi, is defined as a multiple idiopathic sarcoma of the skin. The causative agent of Kaposi’s sarcoma was identified as the human herpesvirus 8 (HHV8), or Kaposi’s sarcoma-associated herpesvirus (KSHV) from the tissue biopsies of AIDS-associated KS by Chang and Moore in 1994, using the representational difference analysis approach . AdditioKaposiʼs sarcoma is the first tumor to be associated with HIV infection and remains the most common cancer in Sub‐Saharan Africa and the second most common cancer in HIV‐infected patients . KS is ain vitro infection model for understanding the molecular mechanisms of KSHV-related oncogenesis, in terms of cell transformation, signaling, cell growth, cell survival, angiogenesis and host-immune invasion, though how KSHV directly contributes to this B-cell malignancy is yet to be known [PEL, also referred to as body cavity-based lymphoma (BCBL), is an aggressive form of non-Hodgkin’s B-cell lymphoma and is linked to KSHV infection and commonly found in late stage immunocompromised AIDS patients . Evidencbe known .The plasmablastic variant of Multicentric Castleman’s disease (MCD) containing large plasmablastic cells is frequently linked to KSHV infection and is usually characterized by lymphadenopathy and immune deregulation ,26. MCD Epidemiology studies on KSHV have identified eight distinctive subtypes based on the sequence analysis of a well-conserved minor capsid gene, ORF26 . Though KSHV prevalence is found to correlate with geographic location. For instance, subgroup A1–4 and subtype C are predominant among individuals in North Europe, the United States and in some regions of Asia and Middle East. Subgroup B1–4 is located primarily in sub-Saharan Africa while D and E strains are found principally in Australia, the Pacific and Brazilian Amerindians ,37,38. SAlthough, the transmission modes and the risk factors for KSHV infection are not well understood, the virus is reported to transmit through both sexual and non-sexual transmission routes ,43. In lKSHV has a double stranded DNA (dsDNA) genome of 165–170 kb consisting of long unique coding region, which is ~140 kb in length and is flanked with highly GC-rich 801 bp long terminal repeat sequences (TRs) that encodes for nearly 86 viral open-reading frames (vORFs), 12 miRNAs and a number of non-coding RNAs (ncRNAs) and antisense RNAs ,49. The de novo infection  and establishment of latency, the incoming linear dsDNA is circularized using the cellular enzymatic machinery to generate a closed-circular DNA, which is further maintained in a circular episomal form tightly packed as nucleosome (Each KSHV virion consists of a linear dsDNA duplex. The virion particle binds to the host cell surface receptor and penetrates into the host cell cytoplasm by a complex multistep process (reviewed in ). Virioncleosome . Encapsiviral epigenome”, which significantly impacts both latency and lytic reactivation of the viral genome. Additionally, studies have indicated that viruses that enter and hide in the host nucleus have co-evolved with numerous cellular chromatin modulation mechanisms to ensure their survival and propagation [Though the processes of viral genome circularization and chromatinization are poorly understood, there are key regulatory steps in the life cycle of many viruses, which are crucial for the establishment and persistence of the viral quiescence or latency ,54. The Recent studies have uncovered a wealth of information regarding the dynamic aspects of chromatin structure and the mechanisms whereby chromatin modifications regulate the viral gene expression and viral-host chromatin interactions. Importantly, chromatin and epigenetic modulation of KSHV genome represents a novel antiviral target for blocking virus-mediated tumorigenesis. This compilation review summarizes some of the emerging concepts that will describe in the detail our current knowledge of chromatin assembly and remodeling factors, and epigenetic alterations, including DNA methylation, post-translational histone modifications, and nucleosome occupancy of KSHV genome, as the master controllers of KSHV’s biphasic life cycle, gene expression pattern and associated pathogenesis.The eukaryotic DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeating units of chromatin. The nucleosome consists of an octameric histone core containing two H2A-H2B dimers and one H3-H4 tetramer with 147-bp segment of DNA wrapped in 1.65 turns and ~50 bp of free DNA separating the neighboring nucleosomes . Togetheactively transcribed euchromatin or to highly condensed, transcriptionally silent heterochromatin and histone modifications can be found in both varieties of the chromatin. Euchromatin state is characterized by high levels of histone acetylation and methylation of lysine residues at 4, 36 and 79 of histone H3. Alternatively, the heterochromatin has low levels of acetylation and high levels of methylation of lysine residues at 9 and 27 of histone H3. Genes that are transcriptionally active possess high levels of H3K4me3, H3K27ac, H2BK5ac and H4K20me1 histone marks in their promoter region and H3K79me1 and H4K20me1 along the gene body [epigenome). Several well defined “epigenomic marks” on chromatin range from chemical modifications on DNA, histone proteins and 3D chromatin organization.Acetylation of histone tails, carried out by histone acetyltransferases (HAT’s) results in the relaxation of the chromatin structure through increased charge repulsions and is primarily associated with active gene expression . Higher-ene body . These pStructure of chromatin is highly variable and dynamic and their structures vary among different cell types. Analysis of the chromatin structure at the genomic level leads to characterization of the landscape of the epigenome that helps to define the gene expression pattern of any cell. Advances in next-generation sequencing technologies have enabled a high-resolution genome-wide investigation of the viral and cellular epigenomic landscape in various cell types. Powerful sequencing-based methods are used as excellent tools to interrogate the interplay between genomic locations of open chromatin regions, DNA-binding factors, nucleosomes and chromatin conformations at a single nucleotide resolution ,61.Formaldehyde-Assisted Isolation of Regulatory Elements) has been utilized as an alternative and robust approach than antibody-dependent ChIP-sequencing, to depict several regions of open chromatin in the latent KSHV genome. FAIRE, followed by next-generation sequencing (FAIRE-seq), together with previously identified nucleosome depletion and histone modification data, has led to the identification of the regulatory elements in KSHV genome, adding to our existing knowledge on the whole genome landscape of latent KSHV chromatin [Chromatin Immunoprecipitation (ChIP) analysis of DNA-histone complexes is one of the efficient methods for the elucidation of chromatin structure and histone modifications. ChIP assays combined with DNA microarrays (ChIP-on-Chip) or high-throughput sequencing of purified DNA fragments (ChIP-seq) and chromatin conformation capture assays (3C) are primarily used to characterize endogenous chromatin structure together with mapping the genomic locations of DNA-associated proteins. In the context of the KSHV genome, prior studies conducted on KSHV infected cell lines at nucleotide resolution using ChIP-seq established the presence of histones, DNA methylation, DNA-protein interactions such as RNA polymerase II binding, LANA and CTCF-cohesin occupancy and nucleosome depletion on the latent KSHV episome ,63,64. Mhromatin .minichromosomes or episomes” that transcribes and replicates simultaneously with the cellular chromosome thereby evades host’s immune surveillance [During latent infection, the KSHV genome persists in a relatively quiescent state as multicopy chromatinized “eillance . Major leillance ). Viral eillance ,67.Majority of the KS biopsy samples and PEL cell lines exhibits latent gene expression profiles, thus provides a remarkable tumor model for better understanding of the mechanisms that regulate the expression of viral genes during latency. These latency models express a restricted yet variable pattern of viral latency-associated genes that modulate both viral and cellular gene expressions to establish and maintain latency. KSHV latent genes or transcripts include the latency-associated nuclear antigen LANA encoded by ORF73, viral cyclinD homologue, v-Cyclin encoded by ORF72, a viral homologue of a FLICE-inhibitory protein v-FLIP encoded by ORF71, Kaposin encoded by K12, and a cluster of 12 viral microRNAs ,70,71,72For most of the KSHV positive PEL-derived cell lines studied so far the expression of the major viral latent proteins: LANA, v-Cyclin and v-FLIP is limited to a single multi-cistronic latent transcript . LANA, fLatency-associated Nuclear Antigen (LANA): LANA, a large nuclear protein of 220–230 kDa with DNA binding and chromatin binding domains in carboxy and amnio terminal domains, respectively is encoded by ORF73 and is critical for the establishment of latent infection [nfection ,82. LANAnfection . Additionfection ,87,88,89nfection . LANA prnfection . LANA plnfection . Additionfection .in vitro binding assays to be a 13 bp sequence within the TR [The role of LANA in the persistence of the KSHV genome was identified by using the 33-kb left-end of KSHV in a persistence assay in BJAB cells expressing LANA . Addition the TR . Addition the TR . The firn the TR . Bindingn the TR ,97.The DNA binding domain of LANA is mapped to the C-terminal domain between amino acids 996 and 1139 . Scannin1 and early S phase by licensing of the replication origin sites by sequential loading of ORCs, cdc6, Cdt1, and the heterohexameric complex MCM2-7 (minichromosome maintenance proteins 2 to 7) to the origins to be used during the S phase [A number of studies have demonstrated the role of LANA in modulating the activities of various cellular pathways by directly interacting with the major players of the pathways or complexing with additional proteins to favor cell growth (reviewed in ). LANA d S phase . Upon lo S phase ,110.MYC, JUN and CCND1 (Cyclin D1), which drive cell cycle progression [RB1) cells enter cell cycle arrest upon exogenous expression of pRb, but the interaction of LANA with pRb overcomes this phenotype, suggesting re-entry of these cells into the S-phase of cell cycle. LANA expressing cells inhibits pRb and p53 pathways that enable these cells to circumvent the G1/S checkpoint and the apoptotic pathway, respectively, which leads to the immortalization and tumorigenesis of LANA expressing cells [LANA has also been shown to upregulate the proteins important for the immortalization of infected cells, which includes modulation of hTERT and E2F1 responsive promoters ,112. LANgression . Additiong cells .-Cyclin: It is encoded by ORF72 and is the homologue of cellular D-cyclin which acts like a constitutive activator of cellular cyclin-dependent kinase 6 (CDK6) to regulate cellular proliferation and viral replication [vlication . The v-Clication . The exalication . Additiolication .-FLIP/K13: ORF71 encodes the KSHV homologue of cellular FLICE like inhibitory protein v-FLIP, also known as K13. KSHV v-FLIP is likely to contribute to latency by promoting cell survival [kB pathway [kB pathway activation by v-FLIP has been linked to KSHV lytic replication as the KSHV mutant deficient in v-FLIP inhibits ORF50/RTA lytic gene expression [vsurvival ,118. The pathway . NF-kB ppression . Additiopression ).Kaposins: The Kaposin locus (K12) and surrounding direct repeat regions DR1 and DR2 encode for 3 proteins, namely, Kaposin A, B and C [ B and C . Kaposin B and C . Kaposin B and C . All of  B and C .LANA2/vIRF-3: The KSHV genome encodes four viral homologues of cellular interferon regulatory protein (vIRFs) to counteract the interferon system (IFN) for evading the host’s immune response. One such protein predominantly expressed during KSHV latency is K10.5/LANA2 that is considered as a KSHV defense protein against IFN [kB, which suggests its role in KSHV infection and pathogenesis [inst IFN . LANA2 iinst IFN . LANA2 aogenesis .Viral microRNAs: KSHV encodes 12 pre-miRNAs, of which 10 miRNA (miR-K1-9 and -K11) are encoded between the kaposin and ORF71/K13 region while the other two miRNAs, miR-K10 and -K12 are located in the coding and 3' untranslated region of K12, respectively [ectively ,130,131.ectively ,134,135.ectively ,137. KSHectively ,138. Sevectively .LANA-dependent DNA Replication and Epigenetic Modifications at the KSHV TR: KSHV LANA, a DNA binding nuclear protein acts as an initiator of DNA replication by specifically binding to a ~60 bp motif in the KSHV TRs [KSHV TRs . A DNA-LKSHV TRs ,95,140. KSHV TRs ,141,142.KSHV TRs ,106,110.1/S transition phases of the cell cycle, chromatin structure at the TR (nucleosomes and two LANA binding sites) is modified to make the viral DNA more accessible to the host cell DNA replication machinery. Two independent new studies [During the G studies ,144 have studies ,144.Studies so far clearly show that latent KSHV genome is extensively chromatinized with both the activating (H3ac and H3K4me3) and repressive (H3K9me3 and H3K27me3) histone marks among them the Latency-associated genes possess activating histone marks as well as colocalize with transcriptionally active RNA polymerase II (RNAPII). Immediate early (IE) and early genes (E) have bivalent chromatin marks  or active chromatin marks (acH3/H3K4me3-rich) whereas all of the late genes have H3K9me3- and H3K27me3-marked heterochromatin. Also, two of the chromatin modifying enzymes: EZH2 (H3K27me3 histone methyltransferase) of the Polycomb Repressive Complex 2 (PRC2) family and JMJD2A (H3K9me3 histone demethylase) have been shown to associate with the latent KSHV genome ,145,146.de novo infection and typically represses the viral gene expression [Additionally, the DNA replication is governed by various epigenetic modifications including DNA methylation, post-synthesis histone modification patterns and nucleosome occupancy . DNA metpression   and cohesin plays an integral role in sister-chromatid cohesion and chromosome segregation during mitosis [ mitosis . CTCF/co mitosis . Depleti mitosis ,151. Thi mitosis . Genome- mitosis ,150. The mitosis . The mol mitosis .via extensive DNA-loop formation [Higher order DNA structure is likely to be a heritable epigenetic regulatory factor. Higher order DNA structures like those involving DNA looping have been identified for the CTCF binding sites in the KSHV LANA promoter region . A loop ormation . For KSHormation . Depletiormation .i.e., imperfect maintenance of the chromatin states was found to coordinate the latent and lytic gene control [Recently, MAPit  single-molecule footprinting assays were employed to characterize several chromatin states at selected loci within mammalian nuclei ,156. TheWithin the predominantly latent population of KS spindle cells a few percent of the latent cells express markers of lytic replication ,158. DurDuring lytic phase, viral gene expression is time-controlled and tightly regulated in order to allow a systematic synthesis of viral gene products. Lytic genes are widely distributed across the whole KSHV genome with their expression been controlled by several different promoters. Genes expressed during the lytic cycle can be grouped according to their timing and expression in response to the protein synthesis/DNA replication inhibitors as immediate early (IE), early (E) and late (L) genes ,162,163.Recently, anem’s group performed a comprehensive genome analysis of transcriptional and translational profiles of the KSHV genome during the productive/lytic infection in epithelial iSLK-219 cell line using a combination of mRNA-sequencing (mRNA-seq) and ribosome profiling (Ribo-seq) . This shThe Lytic reactivation is regulated by alteration in the histone modifications of the viral genome including acetylation of core histone tails by histone acetyltransferases (HATs) and making the chromatin transcriptionally active. On the other hand, deacetylation of histone tails by histone acetyltransferases (HDACs) condenses the chromatin to make it transcriptionally silent . During KSHV viral long non-coding PAN RNA and gene regulation: Among the early gene transcripts, PAN RNA is the most abundant lytic cycle transcript of KSHV [cis-acting RTA Response Element (RRE) present in the PAN RNA promoter region and by ORF57, which further stabilizes PAN RNA [ of KSHV ,161. New of KSHV . Long no of KSHV . KSHV ex of KSHV ,179. PAN of KSHV . The exp PAN RNA ,182,183.In order to elucidate the role of PAN RNA in the KSHV life cycle, recent studies on a recombinant BACmid with a deleted PAN RNA locus showed decreased RTA expression in the induced cells at both the early and late induction time points . The RTAde novo KSHV infection, viral replication or serial propagation. Similarly, many other standard established cell lines such as 293 cells have been reported to support a very low level of KSHV infection with limited release of infectious virus [PEL cell lines are latently infected by KSHV and can be induced to trigger lytic cycle and produce infectious virions. However, PEL cells being lymphoid in nature do not support us virus ,185. Thede novo or primary infection involves attachment of the viral envelope proteins to host cell receptors and entry in the target cells by a multistep complex process [The process of KSHV  process . Several process ,188,189. process ,190. KSH process .i.e., activated H3K4me3 and acetylated H3K9 (acH3) and repressive histone marks [Two independent studies have used ChIP-on-chip assays to provide the first unbiased and genome-wide views of the latent KSHV chromatin in infected BCBL-1 and SLK cells indicating a uniform distribution of total histone H3 and genomic localization of its modified forms, ne marks ,144. Latne marks .De novo acquisition of H3K27me3 marks is catalyzed by EZH2, which together with SUZ12 and EED form the subunits of the PRC2 complex. Both PRC1 and PRC2 are recruited to the KSHV genome leading to the temporally ordered biphasic euchromatin to heterochromatin transition in SLK and TIME cells, following de novo infection [versus lytic replication [In order to provide an overall description of the pre-latency phase of KSHV infection, a comprehensive epigenetic study was performed by Dr. Jae Jung’s group on SLK cells using ChIP-on-chip and FAIRE analysis . During nfection . In contlication .de novo infected SLK, established from tumor biopsy of oral mucosa, cells [i.e., the time period when H3K27me3 marks accumulate on the KSHV episome. However, KSHV infection is reported to influence relocalization of ND10s components especially Sp100 protein, which is efficiently and permanently relocalized from nucleoplasmic and chromatin-associated fractions into the insoluble matrix by LANA, which induces SUMOylation of Sp100. Depletion of ND10s core components, Sp100, PML or Daxx did not interfere with latency establishment, though depletion of Sp100 accelerates the occupancy of the repressive histone mark H3K27me3 on viral episomes indicating that Sp100 acts as a negative regulator of PRC2 recruitment onto the KSHV genome [During the past years, components of distinct nuclear compartments called Promyelocytic leukemia nuclear bodies (PML-NB) have been found to regulate viral chromatin and viral gene expression . PML-NB,a, cells . The KSHV genome .KSHV is a complex and sophisticated oncogenic virus that developed numerous regulatory mechanisms to modulate the host-cell proliferation, apoptosis and host immune evasion, enabling the prolonged survival of the infected cell with the following latent infection and lytic reactivation. Despite the enormous wealth of information available about the mechanisms of how gammaherpesviruses persist in tumor cells, we are far from fully understanding the mechanism of latency establishment by KSHV, and the key cellular and viral factors that are responsible for restricted lytic gene expression during primary infection. Undoubtedly, the chromatin remodeling and epigenetic modifications of both viral and host genomes play crucial roles in determining the expression pattern of genes, that will lead to abortive or persistent infection. These epigenetic modifications include DNA methylation, histones post-translational modifications and higher-order chromosome conformations, including long intervening DNA loops. More importantly, prior to the chromatinization of incoming viral genomes, function of the cellular chromatin modifying enzymes is deregulated by a combination of viral proteins in order to re-program the cellular gene expression profiles and generate an altered chromatin landscape of KSHV genome favoring latency establishment ,199. Alt


Escherichia coli has served as the archetypal organism on which the overwhelming majority of biochemical characterizations of bacterial RNA polymerase (RNAP) have been focused; the properties of E. coli RNAP have been accepted as generally representative for all bacterial RNAPs. Here, we directly compare the initiation properties of a mycobacterial transcription system with E. coli RNAP on two different promoters. The detailed characterizations include abortive transcription assays, RNAP/promoter complex stability assays and DNAse I and KMnO4 footprinting. Based on footprinting, we find that promoter complexes formed by E. coli and mycobacterial RNAPs use very similar protein/DNA interactions and generate the same transcription bubbles. However, we find that the open promoter complexes formed by E. coli RNAP on the two promoters tested are highly stable and essentially irreversible (with lifetimes much greater than 1 h), while the open promoter complexes on the same two promoters formed by mycobacterial RNAP are very unstable (lifetimes of about 2 min or less) and readily reversible. We show here that CarD, an essential mycobacterial transcription activator that is not found in E. coli, stabilizes the mycobacterial RNAP/open promoter complexes considerably by preventing transcription bubble collapse. Mycobacterium tuberculosis (Mtb), continues to pose a major health problem, particularly due to the increase in multidrug resistant strains . RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for the antibiotic rifampicin, a first line therapeutic treatment for tuberculosis , is competent for RNA synthesis (Escherichia coli (Eco) has served as the archetypal organism on which the overwhelming majority of biochemical characterizations of bacterial RNAP have been focused. The properties of Eco RNAP have been accepted as generally representative for all bacterial RNAPs.The catalytic core of the bacterial RNAP, comprising five subunits  and Thermus thermophilus (Tth) RNAPs  RNAPs –7 has prh) RNAPs –13. Studsubtilis , M. smegmegmatis  and Mtb tis —the splayed minor groove at the double-/single-stranded DNA junction at the upstream edge of the transcription bubble.CarD  was ideus; . The transcription activator CarD stabilizes the mycobacterial RNAP/open promoter complexes considerably by preventing collapse of the transcription bubble, thereby compensating for the enzyme's relatively feeble activity on aMtb rRNA promoter.Here, we directly compare the initiation properties of a mycobacterial transcription system with Eco core RNAP was overexpressed and purified from Eco BL21(DE3) cells co-transformed with pGEMABC  and pACYCDuet-1_Ec_rpoZ (encoding rpoZ) as described (Eco σ70 was overexpressed and purified as described (M. bovis (Mbo) core RNAP and σA were overexpressed and purified using methods modified from Czyz et al. (Mbo core RNAP subunits were co-overexpressed in Eco pRARE2 (Novagen) cells overnight at room temperature for ∼16 h after induction with 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Cell pellets were lysed with a continuous flow French press (Avestin) and the clarified cell lysate was treated by polyethyleneimine (PEI) precipitation to remove nucleic acids. Proteins eluted from the PEI pellet were then purified by Ni2+—affinity chromatography and the eluted sample concentrated by centrifugal filtration (VivaSpin) and further purified by size exclusion chromatography. Buffers and detailed methods are as described in detail in Twist et al. (Mbo/Mtb σA was overexpressed in Eco pRARE2 cells and purified as previously described (Mbo/Mtb CarD was overexpressed from Eco BL21(DE3) as previously described for Tth CarD , 1 mM MgCl2, 0.1 mM DTT, 50 μg/ml bovine serum albumin]. Reactions (20 μl) were carried out in a 37°C water bath with proteins using the following protocol: core RNAP (200 nM) and σA (1 μM) were combined and incubated at 37°C for 5 min to form holoenzyme. CarD  was then added to holoenzyme and incubated for an additional 5 min. Next, promoter DNA (10 nM) was added and RPO was allowed to form for 15 min at 37°C. Abortive transcription was initiated by the addition of an NTP mix containing the initiating dinucleotide , the next NTP  and FC-bubble competitor DNA . After 10 min, transcription was quenched by the addition of 2× stop buffer . Reactions were heated at 95°C for 1 min and immediately loaded on a 23% polyacrylamide gel (19:1 acrylamide:bis-acrylamide). Abortive products were visualized by exposing the gel on a phosphorimager plate overnight and digitized using a Typhoon phosphorimager. Data were quantified using Image J (Proteins used for the  Image J .32P]-end-labeled PCR primer. The resulting PCR products were then gel purified as described above. DNaseI (New England Biolabs) was diluted to 500 U/μl and kept on ice. Reactions (25 μl) were carried out in a 37°C water bath and in 1× transcription buffer. Core RNAP (400 nM) and σA (2 μM) were incubated for 5 min to form holoenzyme. CarD (4 μM), when used, was added to the holoenzyme and incubated for another 5 min, followed by the addition of the 32P-labeled promoter DNA (200 fmol). Formation of RPo was allowed to proceed for 15 min and competitor added for times indicated  promoter DNA as described above for DNAse I footprinting. Potassium permanganate (KMnO4) was added to a final concentration of 2 mM, incubated for 2 min, then the reactions were quenched by the addition of 25 μl of stop buffer . The DNA was precipitated with 200 μl EtOH, pelleted, then washed with 100% EtOH to remove all traces of KMnO4. The DNA pellet was then resuspended in 100 μl of piperidine (1 M) and incubated at 90°C for 30 min to cleave the DNA at modified thymine residues. After cleavage, the DNA was precipitated with 100% EtOH, pelleted, washed, air-dried, re-suspended in 2× loading buffer and subjected to electrophoresis and visualized as described for the DNase I assays.Open complexes were formed on 5′- is always very low. The primary anion is glutamate, which varies dramatically in concentration (30–260 mM) depending on the osmolarity of the surrounding medium . Saturating amounts of CarD boosted Mbo RNAP transcription almost 9-fold but did not alter the KCl-sensitivity .On the AP3 promoter, Eco and Mbo (±CarD) RNAPs showed very similar activites as in low [KCl]. However, both RNAPs were much less sensitive to increasing [KGlu]. Eco RNAP activity was completely resistant to increasing [KGlu] up to the highest [KGlu] tested, 250 mM .In low [KGlu] (≤50 mM) on AP3, both M Figure . Mbo RNAEco RNAP on AC50 was much less sensitive to increasing [KCl] (≥150 mM) than Mbo RNAP . For all subsequent assays (both Eco and Mbo RNAPs on both AP3 and AC50 promoters), transcription conditions used are 10 mM (for AP3) or 150 mM (for AC50) [Kglu], as noted.Like on the AP3 promoter, the activity of We tested the lifetime of competitor-resistant RNAP/promoter complexes using the abortive initiation assay in KGlu. For these and other assays where promoter complexes were challenged with competitor, we chose not to use heparin, which actively dissociates RNAP from promoters . InsteadMbo RNAP on both promoters, we observed biphasic decay with a rapidly decaying component (t1/2 ∼2 min) with a decay rate strongly dependent on the presence of CarD, and a very slow component (t1/2 ∼5 h) that did not seem to depend on CarD . On AC50, the slow-decaying component could be essentially eliminated by increasing [KGlu]. The experiments shown in Figure Mbo RNAP transcription activity in the absence of CarD was extremely weak and only decreased with higher [KGlu]  activity of Mbo RNAP without CarD .With ] Figure . We therEco RNAP showed high transcription activity that was essentially completely competitor resistant over the 60 min assay time . The presence of saturating amounts of CarD stimulated Mbo RNAP transcription by almost an order of magnitude (consistent with previous results) to nearly the same level as Eco RNAP (at time 0). CarD also dramatically stabilized the Mbo RNAP promoter complexes, increasing the t1/2 by more than 10-fold  showed that this residue is critical for optimal transcription of the rRNA promoters in both Tth and Mtb  but was still able to stabilize the Mbo RNAP promoter complexes to nearly the same level as wt-CarD .Substitution of conserved CarD W85 to A , but this activity decayed extremely rapidly upon competitor challenge (t1/2 = 0.92 min). Saturating amounts of CarD had only a slight effect on Mbo RNAP transcription at time 0 (1.3-fold activation) but, like on AP3, dramatically stabilized the complexes to dissociation (63-fold increase in t1/2 compared to no CarD). The CarDW85A mutant had little effect on Mbo RNAP transcription at time 0, and stabilized the promoter complexes at an intermediate level (13-fold increase in t1/2 compared to no CarD). As with the AP3 promoter, CarDW85F was able to stabilize the open complex on AC50 at levels similar to wt-CarD , while promoter complexes of Mbo RNAP were highly unstable . CarD had two major effects on Mbo RNAP initiation, to activate the overall level of transcription  and to stabilize the otherwise highly unstable Mbo RNAP promoter complexes (on both promoters). The CarDW85A substitution caused a partial loss of transcription activation (on AP3) activity as well as a partial defect in promoter complex stabilization (on both promoters) while CarDW85F showed stabilization similar to that of wt-CarD.In summary, under these assay conditions, Mtb AP3 promoter has a non-optimal 18-bp −10/−35 spacer   is not due to the non-optimal, 18-bp spacer.The s Figure . On the ] Figure , and for] Figure . To test3 Figure . In AP3 Figure . None ofMboAσ is very similar to Eco70σ and the residues contacting the −10 and −35 promoter regions are highly conserved σ to Eco core RNAP with MboAσ (Eco-core/A)σ on the AP3 promoter. MboAσ was able to direct transcription of AP3 by Eco core RNAP almost as well as 70σ, suggesting that the weak activity of Mbo holoenzyme does not originate from Aσ  nor Eco-core/Aσ holoenzymes (we have no evidence that CarD interacts with Eco RNAP).onserved ,33. In c factors , share vA Figure . As expeEco70σ and Mbo core RNAP was not detectable by native gel electrophoresis (22). Therefore, as expected, Eco70σ directed only very weak transcription of AP3 by the Mbo core RNAP  could clash with lineage-specific insert 2 of the mycobacterial RNAP β′ subunit  failed to protect most of the DNA from DNAse I cleavage. Evidence of Mbo RNAP interaction with the DNA was apparent through the presence of some hypersensitive sites, such as at −34 and +14/+15  produced footprints similar to wild-type CarD , and the Mbo RNAP footprint extended slightly further downstream (+17/+18) than the Eco RNAP footprint  bases, and the modified T's can be subsequently detected by strand cleavage. This approach can thus be used to probe transcription bubble formation in RPo (4 footprinting (template strand) to examine the transcription bubble formed by Eco and Mbo RNAPs, and to examine the effect of CarD on Mbo RNAP transcription bubble formation.KMnOn in RPo ,41. We uEco RNAP holoenzyme at promoters, as measured by KMnO4 footprinting, typically extends from about −11 to +2  where the sequence from +3 and downstream was altered, generating a template-strand T at +5 , increasing the half-life in the presence of competitor more than 60-fold , and addition of CarD had no effect. We conclude that the very short half-life of Mbo RPo on AC50  that shows transcription activity on the optimized AC50 promoter , while Eco RNAP formed essentially irreversible promoter complexes on both promoters  as expected, but did not alter the structure of the footprints  also did not alter the structure of the footprints  but still show transcription levels similar to Eco RNAP  despite the high off-rate, even in the absence of CarD , Figure Mbo RNAP transcription activity remain unknown. More extensive swapping of promoter regions, such as the discriminator  that is predicted to insert into the distorted minor groove at the upstream edge of the −10 element  residue inserted into the upstream edge of the transcription bubble is sufficient for nearly full CarD function.The CarD/DNA interaction involves a universally conserved Trp , β′In6 is absent Eco has squaticus  and M. smegmatis , have alomplexes . Our finy Figure  suggestsy Figure . For exaature of ; SI3 accature of ] decreasature of . In RNAPs absent , suggestM. smegmatis established that CarD is a global regulator, being present at essentially all promoter regions in the genome, suggesting that CarD is not a promoter-specific regulator (Mbo RNAP, even on an optimized promoter like AC50 (Figure ChIP-seq experiments in egulator . We have0 Figure . This suSupplementary Data are available at NAR Online.


However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at −165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.Human telomerase gene  Upon inst cells .+ and Tel− cells, but had no effect on hTERT transcription in the presence of HDAC inhibitor TSA, suggesting that EBPs relieved the promoter repression by HDACs, likely by recruiting histone acetyl transferases (HATs) to the promoter. c-Myc overexpression activated the wildtype, but not the mutant, hTERT promoter, despite of the presence of numerous potential c-Myc binding sites downstream of the promoter in the mutant BACs , we found that USF1, USF2 and c-Myc/Max bound to the chromatinized hTERT promoter via these two E-boxes. Mutations at these sites reduced the hTERT promoter activity in both Telant BACs , indicatBAC 117B23-cFtRvSVP  and Figu, referre+) and GM847.7 (Tel−) were cultured in Minimal Essential Medium (MEM) with 10% Fetal Bovine Serum (FBS) and RMBT was performed as previously described (Firefly luciferase (Fluc) activities were normalized to Renilla luciferase (Rluc) activities from cotransfected pRLSV40. In BAC reporter assays, Rluc activities from the hTERT promoter were normalized to Fluc activities from the CRR9 promoter. In addition, the data were also normalized to cell numbers, as determined by thiazolyl blue tetrazolium bromide (MTT) assays. In all cases, the ratios of Rluc to Fluc activities correlated well with Rluc activities normalized to MTT counts. All reporter assays were done in triplicates and repeated at least once.Acceptor cell lines, 3C167b3.1  were purchased from Sigma–Aldrich . Control pLKO.1 plasmids, vector and scrambled shRNAs and lentiviral packaging plasmids were obtained from Addgene . Lentiviruses were packaged in 293T cells co-transfected with a lentiviral shRNA plasmid and a cocktail of the packaging plasmids pGAG-POL, pREV and pVSVG. Lentiviral infection was performed at approximately multiplicity of infection (MOI) = 5.cDNAs synthesis and quantitative polymerase chain reaction (PCR) were performed as previously described . Primer ChIP was performed as described previously . Antibod+) and GM847.7 (Tel−) cells using the RMBT technique . Both ce Figure .CLPTM1L , hTERT and SLC6A18 (or Xtrp2 gene) . The difference paralleled endogenous hTERT expression in these two cell lines  BAC contains 160 kb of human genomic sequence encompassing three complete genomic loci, ) Figure . A Fluc ectively . Becausexpressed , Fluc aceviously , when a c Figure  or by Rls Figure , but wass Figure  23,24).,24.CLPTMhTERT gene  and 1-kb downstream of the promoter (RlucO), positioned within the Rluc ORF in H(wt) BAC, were used to as negative controls. The amplicon Up5k detected both endogenous and transgenic sequences, whereas RlucO recognized only the BAC sequence.Chromatin plays a critical role in the regulation of ERT gene . As prevs Figure , indicat+ cells than in Tel− cells. This difference between the endogenous and transgenic sequences could be attributed to the presence of a translocated hTERT allele in Tel+ cells (3C167b), with the breakage point at 6-kb upstream of its promoter  at both transgenic and endogenous hTERT sequences were significantly higher in Tel+ cells than in Tel− cells. H3K4 trimethylation was also more abundant in Tel+ cells than in Tel− cells at both transgenic and endogenous hTERT promoters, but not at Up5k and RlucO sites. Overall, these data were consistent with the finding that, while the hTERT promoter was transcriptionally active in Tel+ cells, the hTERT gene was repressed in both Tel+ and Tel− cells.As shown in Figure promoter . The trahTERT gene  reporter. As shown in Figure + cells than in Tel− cells. Conversely, Mxd1, a repressive Myc family protein, was not detected at either endogenous or transgenic hTERT promoters in these cell lines, although, as a positive control, Mxd1 signals were detected at the E-box containing NPM1 promoter in both lines (data not shown). Thus, the data showed that the hTERT promoter were occupied by multiple EBPs, contributing to its activation in Tel+ cells.To determine which EBPs bound to the hTERT promoters, ChIP experiments were performed in Tel+ and Tel− via RMBT. As shown in Figure + and Tel− cells. However, EboxD mutation in H(EboxD) and H(EboxDM) reduced hTERT promoter activities by 8–9-folds in Tel+ cells. This mutation also reduced hTERT promoter activity in Tel− cells, although less evidently, due to the much lower hTERT expression in these cells. There seemed to be no synergistic effect of the double mutations in H(EboxDM) in either Tel+ or Tel− cells. The data indicated that EboxD was more important for hTERT regulation while EboxU played a minor role. Upon TSA treatment, the differences between wildtype and mutant promoters disappeared , H(EboxD) or H(EboxDM). In this experiment, in addition to the transgenic promoter region, adjacent genomic sites at Up5k, Up2k, Up1k, RlucO, Dn1k and Dn2k, were also assessed to determine the specificities of promoter recruitment of these factors. As shown in Figure Next, to determine which TFs were recruited by these E-boxes, ChIP was performed in Tel+ H(wt) cells were infected by individual lentiviruses and KD efficiencies were assessed by western blot analyses 4 days post infection. As shown in Figure To dissect the functions of individual EBPs in hTERT regulation, RNA interference was carried out using lentiviral shRNAs against each protein. In this experiment, TelAs expected, lentiviral shRNAs against c-Myc, Max, USF1 and USF2 significantly reduced the binding of each of these proteins to the hTERT core promoters in the transgenic Figure  and endoTo understand the interplay among these EBPs at the hTERT promoters, promoter recruitment of all proteins was measured upon the KD of each EBP. Not surprisingly, Max KD attenuated c-Myc binding to the transgenic hTERT promoter Figure  as c-MycOn the other hand, KD of USF1, USF2 or both proteins led to a decrease in the binding of c-Myc, but not Max, to transgenic and endogenous hTERT promoters Figure , althougTo determine chromatin changes associated with reduced recruitment of EBPs to the hTERT promoter, covalent modifications of histones H3 and H4 were examined by ChIP/qPCR analysis. As shown in Figure Because c-Myc/Max was found to function as activators of the hTERT promoter ,39, it w+ cells containing H(EboxD) or H(EboxDM) were infected with lentiviral shRNAs against c-Myc as well as other Myc family proteins Max and Mxd1. As shown in Figure Previous studies, using transiently transfected promoter fragments, have shown that the two proximal E-boxes were critical in c-Myc mediated hTERT activation . To dete+ and Tel− cells. c-Myc expression was 3–5-fold higher in c-Myc virus infected cells than in control cells as determined by western analysis . c-Myc overexpression in Tel+ H(wt) cells led to a significantly increased recruitment of c-Myc/Max, but not USF1 or USF2, to the hTERT promoter  by 2–3-folds, but failed to activate the hTERT promoters in H(EboxD) and H(EboxDM) reporters  cells  cells containing LNCX-c-Myc or LNCX vector were infected with lentiviral shRNAs. As shown in Figure To further demonstrate that low c-Myc expression resulted in the activation of hTERT promoter, Tel+ and Tel− cells  and H(EboxDM), in which the E-boxes were mutated, were similarly activated. c-Myc has been previously shown to repress several negative regulators of cell proliferation, including c/EBPα, p15Ink4b, p21Cip1 and p27Kip1, by interacting with zinc finger TFs Sp1 and Miz1 that bound to initiator elements of TATA-less promoters of the target genes . Thus, wMost studies have shown that Myc family proteins regulate gene expression via direct promoter binding. However, this notion was challenged by the finding that loss of myc function resulted in changes in global chromatin structure, accompanied by histone hypoacetylation and altered methylation . While Kin vitro cell proliferation, whereas the transgenic loci were established by de novo assembly of chromatin on the integrated BAC DNAs. For example, H3K9 trimethylation was somewhat higher at the endogenous promoter than the transgenic reporter (Figure hTERT loci. In addition, most of the epigenetic differences between transgenic and endogenous loci are difficult to detect, due to their identical DNA sequences. Nevertheless, while potential differences between epigenetic states of endogenous and transgenic loci might impact on their regulation, our study demonstrated that the chromatinized hTERT reporters are much-improved models for studying chromatin-dependent hTERT regulation than transient reporter assays.Whereas the activity of transgenic hTERT promoter paralleled that of the endogenous promoter, chromatin changes in response to c-Myc/Max KD were greater at the transgenic promoter. This was not a complete surprise because the endogenous genes have been subjected to epigenetic modifications during developmental processes as well as r Figure  and thisIn summary, we report here the first mutational analysis of DNA elements in a chromatin context using the RMBT technique. These experiments demonstrated, for the first time, that c-Myc/Max had dual functions in hTERT regulation, that is, its involvement not only in activating hTERT transcription but also in maintaining the repressive states of hTERT promoter in somatic cells.Supplementary Data are available at NAR Online.


Many noncoding genomic loci have remained constant over long evolutionary periods, suggesting that they are exposed to strong selective pressures. The molecular functions of these elements have been partially elucidated, but the fundamental reason for their extreme conservation is still unknown.Drosophila melanogaster. At the sequence level, HCNEs are GC-rich and have a characteristic oligomeric composition. They have higher levels of stable nucleosome occupancy than their flanking regions, and lower levels of mononucleosomes and H3.3, suggesting that these regions reside in compact chromatin. Furthermore, these regions showed remarkable modulations in histone modification and the expression levels of adjacent genes during development. Although HCNEs are primarily initiated late in replication, about 10% were related to early replication origins. Finally, HCNEs showed strong enrichment within lamina-associated domains.To gain new insights into the extreme selection of highly conserved noncoding elements (HCNEs), we used a systematic analysis of multi-omic data to study the epigenetic regulation of such elements during the development of HCNEs have distinct and protective sequence properties, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. These observations indicate that such elements are likely to have essential cellular functions, and offer insights into their epigenetic properties. Genomic DNA is subject to diverse mutations caused by chemicals, replication errors, and mobile genetic elements. Coding sequences are generally under higher selective pressure than noncoding sequences, due to the essential roles that proteins play in the cell Indeed, many highly conserved noncoding elements (HCNEs), noncoding loci that maintain high level of similarity across different species, function as developmental enhancers In eukaryotic cells, histones pack DNA into nucleosomes to form the chromatin structure; this protects DNA from damage, and offers an additional layer of regulation via the chemical modification of histones Drosophila melanogaster. Our results indicate the following: HCNEs intrinsically favor stable nucleosome occupancy at the sequence level; HCNEs reside within nucleosome-enriched and mononucleosome- and H3.3-depleted regions in S2 cells; the chromatin regions around HCNEs undergo significant changes in epigenetic modification during development, and such changes are correlated with the transcription levels of flanking genes; most HCNEs fire later in replication, however some serve as early replication origins; and HCNEs are significantly associated with lamina-associated domains (LADs). Our results collectively indicate that HCNEs are under special evolutionary control at the levels of chromatin and nuclear structural organization.Here, we performed an integrative analysis of the epigenetic properties and regulations of HCNEs throughout the development of D. melanogaster genome  are broad genomic regions of conserved synteny that harbor dense distributions of HCNE loci, developmental and regulatory genes. GRBs are thought to have emerged due to evolutionary pressure to maintain HCNEs and their target genes in Drosophila core promoters are classified into five types: Inr-only, Inr/DPE, TATA box/Inr, Motif1 followed by Motif6 (Motif1/6) , 2,612 of which had reliable predictions available for their core promoter types. The distribution of core promoter types among the HCNE-proximal genes was significantly different from expected  enrichment between genes of distinct promoter types Nucleosome occupancy and positioning is intimately related to the regulation and protection of genetic material D. melanogaster embryonic S2 cell line To assess the intrinsic information embedded in HCNEs, we analyzed their nucleosome occupancy in the H3.3, which is a non-canonical histone that replaces H3.1 during chromatin-disrupting processes, such as transcriptional regulation Taken together, these findings indicate that HCNEs are characterized by a high nucleosome density, a low mononucleosome density, and a low H3.3 density in the S2 cell line. Thus, HCNEs appear to exist in a more compact chromatin environment compared to their flanking regions.nejire  for any association with the regulation of transcription in our system.Chemical modifications of histones can determine the state of chromatin and regulate gene expression D. melanogaster. However, H3K9me3 and H3K27me3 had broad peaks that spanned many kb while the HCNEs covered only small parts of these regions , and found that the stage and tissue specificities of HCNE-proximal gene expression followed a bimodal distribution . This inWe next explored the expression profiles of the HCNE-proximal genes that showed stage/tissue-specific expression based on a stringent cutoff inferred from their bimodal distributions . Clusters of stage-specific genes with Inr-motif promoters showed high levels of expression across most developmental stages . In contHistone modifications around genes are often correlated with their transcriptional regulation. Thus, we studied whether histone modification and/or CBP binding at HCNEs could be associated with the transcription levels of nearby genes. For all promoter types, we grouped the stage-specific HCNE-proximal genes by the presence or absence of active markers  at the nearest HCNE (A marker is considered present at HCNEs if at least 10% of HCNE length overlap with marker peak). Interestingly, genes with Inr only, TATA box/Inr and DRE promoters near active HCNEs showed higher expression levels than those near inactive HCNEs , Kc-167 (Kc), and S2-DRSC (S2) cells. We found that HCNEs fire later during replication compared to other genomic loci in Bg3 (P = 8.9e-9), Kc (P = 1.4e-9), and S2 (P = 5.45e-21) cells  cells . This re1) cells , indicatD. melanogaster genome The nuclear lamina, which is an important part of the nuclear structure, functions in important cellular processes of metazoan cells, including chromatin organization and DNA replication To gain new insights into the overall properties of HCNEs, we performed cluster analysis  using the identified HCNEs and the examined genomic and epigenomic features . To reduA number of HCNE clusters were observed. The members of the most prominent cluster were tri-methylated on H3 during development  Cis-regulation can control distant genes, but it is generally difficult to discriminate between target and bystander genes due to the lack of comprehensive transcriptomic and epigenetic data D. melanogaster genome.Histone modification is often associated with the transcriptional activation or repression of nearby target genes. Consistent with this, many HCNE-proximal genes, most notably genes with Inr-motif and DRE promoters, showed higher expression levels when their associated HCNEs were in the active chromatin state . This suD. melanogaster, and thus should be under strong negative selection. Future work is needed to identify the molecular mechanism(s) through which the origin-recognition complex recognizes these specific sequences.The orchestrated and properly timed initiation of replication from multiple origins during cell division is essential for the vertical transfer of genetic materials. Thus, most species maintain common genomic and epigenomic features at their replication origins. Replication origins are GC-rich, but their nascent strands are AT-rich, allowing the DNA to open easily Previous studies have shown that HCNEs, including ultraconserved elements, are not mutational cold spots Studies have shown that genomic elements and nuclear structures frequently undergo crosstalk and dynamic regulation. The nuclear scaffold/matrix provides both a mechanical anchor and distinct territories for genomic elements and proteins during various processes, such as replication and transcription D. melanogaster genome 1, which is the primary component of the nuclear lamina, binds to matrix-attachment regions Drosophila Kc cells 1 predominates in the early chicken embryo and decreases thereafter, whereas lamin A shows the opposite expression pattern LADs have been relatively well characterized in the Because HCNEs do not represent mutational cold spots D. melanogaster genome  and extracted highly conserved elements using the ECE algorithm (https://github.com/Magdoll//ECE) We downloaded the DM3 compilation of the To investigate possible TFBSs within HCNEs, we identified overrepresented heptamers in HCNEs sequences and queried the top 50 against FlyReg motifs Drosophila genus, along with their microsyntenic blocks mapping We downloaded a list of 1,321 genes showing conserved microsynteny in the http://tools.genome.duke.edu/generegulation/McPromoter006/mcpromoter.rel5.thres0.03.gff], using a stringent threshold of 0.03. Each gene was assigned the promoter type prediction found within 500 bp upstream of the TSS and a minimum of 500 bp and the length of 5′UTR downstream of the TSS. When multiple predictions were made for a given gene, we chose the one with the highest score. The 5′UTR coordinates were obtained from FlyMine To determine the promoter type of the HCNE-proximal genes, we download McPromoter ftp://data.modencode.org/D.melanogaster/Histone-Modification/ChIP-seq/computed-peaks_gff3/]. Some developmental time points were missing for some of the histone markers. For example, the larva 3, adult male and adult female stages were missing data for H3K9me3 and the adult female stage was missing data for H3K27me3. We obtained ChIP-Seq peaks for CBPs throughout the same developmental stages, with the exceptions of the embryonic 8 to 12 h and larva 2 stages, which were missing. Missing data were ignored in our analysis. The histone modification and CBP binding data can be found in the modENCODE depository under the following IDs (DCCids): modENCODE_862, modENCODE_863, modENCODE_854, modENCODE_856, modENCODE_857 modENCODE_855, modENCODE_859, modENCODE_860, modENCODE_861, and modENCODE_858. The data for our genome-wide replication timing characterization and the early origin of replication peaks can be found under the following DCCids: modENCODE_668, modENCODE_66 and modENCODE_670 (for replication timing) and modENCODE_3441 (for the early origin of replication peaks).We obtained the preprocessed nucleosome occupancy data (profiling by genome-wide tiling array) deposited by Henikoff et al. Due to the lack of raw data for many of the studied epigenomic modifications , we analyzed the frequencies of the histone modification ChIP-Seq peaks on HCNEs rather than the enrichment levels of these modifications. We considered a marker present in an HCNE if the overlap between the peak and HCNE covered at least 10% of the HCNE length.D. melanogaster in reads per kilobase per million (RPKM) for 30 developmental stages and 28 tissues, as compiled in FlyBase. The utilized data are available at FlyBase [ftp://ftp.flybase.net/releases/FB2014_02/precomputed_files/genes/gene_rpkm_report_fb_2014_02.tsv.gz]. We changed values greater than 100 to 100 .We downloaded the normalized read counts of ftp://data.modencode.org/D.melanogaster/mRNA/RNA-seq/alignment_sam/]. We computed normalized read counts (in RPKM) for each gene.To assess the relationship between histone modification at HCNEs and the transcriptional activity of proximal genes, we downloaded files of aligned reads from the modENCODE ftp server [We computed the Shannon entropy for the expression of each gene using the following formula:n as the base of the log in order to keep the value between 0 and 1. Genes with uniform distribution of expression had entropy of 1, while those expressed under only one condition had entropy of 0.Where Figure S1PhastCons score distributions for exons, intergenic regions, and introns.(TIF)Click here for additional data file.Figure S2Boxplot illustrates HCNE coverage for various histone modifications and CBP peaks.(TIF)Click here for additional data file.Figure S3Heatmaps illustrating the expression levels of tissue-specific HCNE proximal genes across 28 different tissues (similar to (TIF)Click here for additional data file.Table S1Table listing the HCNEs defined in this study.(XLSX)Click here for additional data file.Table S2Table mapping the top 50 overrepresented heptamers to known transcription factor binding sites.(XLSX)Click here for additional data file.Table S3Table listing the Gene Ontology enrichment  among the HCNE-proximal genes.(XLSX)Click here for additional data file.Table S4Table listing the protein domains found to be enriched among the HCNE-proximal genes.(XLSX)Click here for additional data file.Table S5Table listing P-values associated with (XLSX)Click here for additional data file.Table S6Table contains the values of the analyzed features used in the clustering analysis .(XLSX)Click here for additional data file.


Saccharomyces cerevisiae, the transcription factor Cst6p has been reported to play important roles in several biological processes. However, the genome-wide targets of Cst6p and its physiological functions remain unknown. Here, we mapped the genome-wide binding sites of Cst6p at high resolution. Cst6p binds to the promoter regions of 59 genes with various biological functions when cells are grown on ethanol but hardly binds to the promoter at any gene when cells are grown on glucose. The retarded growth of the CST6 deletion mutant on ethanol is attributed to the markedly decreased expression of NCE103, encoding a carbonic anhydrase, which is a direct target of Cst6p. The target genes of Cst6p have a large overlap with those of stress-responsive transcription factors, such as Sko1p and Skn7p. In addition, a CST6 deletion mutant growing on ethanol shows hypersensitivity to oxidative stress and ethanol stress, assigning Cst6p as a new member of the stress-responsive transcriptional regulatory network. These results show that mapping of genome-wide binding sites can provide new insights into the function of transcription factors and highlight the highly connected and condition-dependent nature of the transcriptional regulatory network in S. cerevisiae.In the model eukaryote  Saccharomyces cerevisiae, the transcription factor Cst6p has been reported to regulate several biological processes, while its genome-wide targets remain unknown. Here, we mapped the genome-wide binding sites of Cst6p at high resolution. We show that the binding of Cst6p to its target promoters is condition dependent and explain the mechanism for the retarded growth of the CST6 deletion mutant on ethanol. Furthermore, we demonstrate that Cst6p is a new member of a stress-responsive transcriptional regulatory network. These results provide deeper understanding of the function of the dynamic transcriptional regulatory network in S. cerevisiae.Transcription factors regulate the activity of various biological processes through binding to specific DNA sequences. Therefore, the determination of binding positions is important for the understanding of the regulatory effects of transcription factors. In the model eukaryote  Saccharomyces cerevisiae, about 209 sequence-specific TFs have been characterized or predicted (Transcription factors (TFs) bind to specific DNA sequences to affect the transcription status of nearby genes and, thus, regulate the activities of related biological processes. In the model eukaryote redicted , and theredicted , it is predicted . The binS. cerevisiae, Cst6p is an ATF/CREB family TF with a basic leucine zipper (bZIP) domain. All three members in this family, Sko1p, Aca1p, and Cst6p , bind to the 5′ TGACGTCA 3′ sequence in vitro , which allows for identifying the location of DNA-binding proteins at high resolution  than on glucose   , an addiACGT 3′) . For groACGT 3′) , indicatP value ≤0.01) identified by ChIP-chip for growth in yeast extract-peptone-dextrose (YPD) medium .The exonuclease digestion in ChIP-exo allowed high-resolution mapping of Cst6p locations. Overall, the binding sites have an average distance of 591 bp from the start codons . The prepromoter  was cleapromoter . Multiplch other  or, perhch other . Using tCBF2, GAT2, HAP4, PHD1, RMI1, ROX1, SIR4, SOK2, YAP1, and YAP6) encode DNA-binding or transcriptional regulatory proteins, and (ii) 16 of the targets encode mitochondrial proteins, including six located in the mitochondrial envelope. In terms of cellular processes, the target genes are categorized into cellular respiration, gluconeogenesis, stress response, and pseudohyphal growth. Interestingly, in addition to NCE103, Cst6p binds to the promoters of ACC1 and PYC1, whose protein products are major HCO3−-consuming enzymes in S. cerevisiae.Gene ontology analysis of the 59 target genes (cst6Δ strain were compared with their expression levels in the wild type. The CST6 deletion mutant in the CEN.PK genetic background is viable on ethanol (see below), allowing the measurement of gene expression under this growth condition where we found Cst6p to be most active. As expected, the expression of CST6 itself was not detected in the cst6Δ strain (data not shown). The expression of NCE103 had a 92.6% decrease in the cst6Δ strain relative to its expression in the wild type, while a smaller decrease of 50.2% was observed during growth on glucose  had significantly decreased expression (approximate 15% to 45%) relative to their expression in the wild type for growth on ethanol. RPS3 had a 30.9% increase in expression, while the other eight genes tested did not show significant changes in expression between the cst6Δ strain and the wild type. No relation could be found between the extent of the expression changes of the target genes and the binding strength  of Cst6p on their promoters.To investigate the regulatory effects of the Cst6p binding events identified, the expression levels of 17 target genes in the  glucose . For thecst6Δ strain had a longer lag phase, whereas the specific growth rate was only slightly lower later in the cultivation (cst6Δ strain was similar to that of the wild type and respiratory growth after the diauxic shift was not affected (In a medium with 1% (vol/vol) ethanol as the sole carbon source, the tivation . This grtivation , probablaffected .NCE103 was decreased to a greater extent than the expression of the other target genes with growth on ethanol , uracil, and l-arginine . Si. SiNCE10d growth .in vitro, they were at first supposed to be involved in response to stress. However, phenotypic and gene expression analyses showed that Cst6p and Aca1p are not involved in the responses to various stresses  for Sko1p  was identified as the only TF activating NCE103 expression by screening a TF knockout mutant library .The binding to stress-related genes and the results of stress resistance tests establish Cst6p as a stress-responsive TF. As seen by the results in f stress , 22. Frof stress . Except Δ strain , indicatin vitro, and the activating effects of Cst6p and Aca1p on basal expression of stress-responsive genes are only observable in the sko1Δ background  it is important to use functionally active conditions to map the binding sites of TFs, (ii) genome-wide mapping of targets can reveal novel functions of TFs and suggest additional conditions for target mapping, and (iii) the transcriptional regulatory network may be far more complex than previously imagined due to the extensive combinatorial regulation and existence of TF cascades. Integrative analysis of data from different types of study is therefore critically needed in order to reconstruct complete transcriptional regulatory networks.S. cerevisiae strains used in this study (cst6Δ strain was constructed by transforming CEN.PK 113-5D with a Kluyveromyces lactis URA3 (KlURA3) marker gene . Similarly, the prototrophic wild-type strain was constructed by integrating KlURA3 into the ura3-52 locus of CEN.PK 113-5D by homologous recombination. The tagged strain CST6-TAP was constructed by integrating a tagging cassette containing the tandem affinity purification (TAP) tag CBP-ProtA coding sequence and KlURA3 . For constitutive expression of NCE103 in the CST6 deletion background, CST6 in CEN.PK 113-5D was first replaced by a 159-bp fragment downstream from CST6, followed by KlURA3 to obtain strain cst6ΔU, and then the KlURA3 marker gene in cst6ΔU was looped out by the homologous recombination of the two direct-repeat 159-bp fragments on 5-fluoroorotic acid medium to obtain strain cst6ΔL. Plasmid pRS416-NCE103, constructed by inserting the NCE103 gene flanked by the TEF1 promoter and its own terminator between the XbaI and KpnI sites of a centromere plasmid, pRS416 (cst6ΔL to get the cst6Δ(NCE103) strain. All the primers for cassette constructions and PCR identifications are listed in All the is study  are deriis study , provide pWJ1042 ) flanked, pRS416 , was tra−1) 7.5 g (NH4)2SO4, 14.4 g KH2PO4, 0.5 g MgSO4·7H2O, vitamins and trace metals as used in reference l-arginine were added to a final concentration of 20 mg liter−1 of each compound. For growth assays on agar plates, cell suspensions were adjusted to an optical density at 600 nm (OD600) of 1.0 and diluted four times in 10-fold series. Three microliters of each dilution was spotted onto SC−Ura plates  supplemented with carbon sources as indicated above, and photographs were taken after 2 to 3 days of growth at 30°C.Single colonies from fresh agar plates were inoculated into 3 ml YPD medium or minimal medium (as described below) with 2% (wt/vol) glucose in tubes and grown for 12 to 24 h. Cells were harvested by centrifugation and resuspended in sterile water to obtain precultures. For growth in liquid medium , strains were cultivated on a rotary shaker at 200 rpm and 30°C in minimal medium containing (liter600 of 1.5 to 1.8 (mid-log phase). Formaldehyde with a final concentration of 1% (wt/vol) and distilled water were added to the cultures to cross-link protein-DNA complexes with an OD600 of 1.0 and a total volume of 100 ml. Cross-linking was performed for 12 min at room temperature with shaking and quenched by adding 2.5 M glycine to a final concentration of 125 mM. After 5 min, cells were washed twice with 20 ml cold TBS  and frozen with liquid nitrogen. ChIP-exo was performed according to the methods in references f  were used for on-bead end repair, incorporation of 3′-d(A) DNA tails, first adaptor ligation, nick filling, and chromatin trimming, respectively. The first adaptors contain unique 6-bp index sequences  glucose or 1% (vol/vol) ethanol as the sole carbon source to an ODS. cerevisiae S288C with Bowtie2 (http://genome.sph.umich.edu/wiki/BamUtil:_trimBam) to increase the resolution. To identify peaks and compare biological duplicates, the program GEM  in minimal medium with 2% (wt/vol) glucose or 1% (vol/vol) ethanol as the sole carbon source, harvested by centrifugation after mixing with crushed ice, frozen in liquid nitrogen, and then stored at −80°C. A FastPrep-24 homogenizer  was used to disrupt cells, and total RNA was isolated using the RNeasy minikit (Qiagen). cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen). For quantitative PCR (qPCR), the DyNAmo flash SYBR green qPCR kit (Thermo Scientific) was used with a reaction mixture volume of 20 µl. Previously published primers for CST6 and NCE103 (ACT1 was used as the reference gene for gene expression level comparison using the cycle threshold (ΔΔTC) method (t test in Microsoft Excel 2013.Cells were cultivated to an ODd NCE103  were use) method . StatistGSE76154.The ChIP-exo data have been deposited in the Gene Expression Omnibus database under the accession number Figure S1 Spot assay of the growth of strains. Download Figure S1, DOCX file, 0.3 MBTable S1 The binding events of Cst6p identified by ChIP-exo.Table S1, XLSX file, 0.01 MBTable S2 The Cst6p-binding target genes identified by ChIP-exo.Table S2, XLSX file, 0.02 MBTable S3 GO annotation of Cst6p target genes.Table S3, XLSX file, 0.01 MBTable S4 Transcription factors having shared binding targets with Cst6p.Table S4, XLSX file, 0.01 MBTable S5 Primers used for strain construction and qPCR.Table S5, DOCX file, 0.02 MBTable S6 Adapters and primers used for ChIP-exo.Table S6, DOCX file, 0.01 MB


Igf2r, Slc22a2, and Slc22a3 genes and the paternally expressed long non-coding RNA (lncRNA) Airn. Although it is known that Igf2r and Airn are reciprocally imprinted, the timing of imprinted expression and accompanying epigenetic changes have not been well characterized in vivo.Appropriate epigenetic regulation of gene expression during lineage allocation and tissue differentiation is required for normal development. One example is genomic imprinting, which is defined as parent-of-origin mono-allelic gene expression. Imprinting is established largely due to epigenetic differences arriving in the zygote from sperm and egg haploid genomes. In the mouse, there are approximately 150 known imprinted genes, many of which occur in imprinted gene clusters that are regulated together. One imprinted cluster includes the maternally expressed Igf2r/Airn locus correlating with differential establishment of imprinted expression during gastrulation. Our results show that Igf2r is expressed from both alleles in the E6.5 epiblast. After gastrulation commences, the locus becomes imprinted in the embryonic lineage with the lncRNA Airn expressed from the paternal allele and Igf2r restricted to maternal allele expression. We document differentially enriched allele-specific histone modifications in extraembryonic and embryonic tissues. We also document for the first time allele-specific spreading of DNA methylation during gastrulation concurrent with establishment of imprinted expression of Igf2r. Importantly, we show that imprinted expression does not change in the extraembryonic lineage even though maternal DMR2 methylation spreading does occur, suggesting distinct mechanisms at play in embryonic and extraembryonic lineages.Here we show lineage- and temporal-specific regulation of DNA methylation and histone modifications at the in vivo.These results indicate that similar to preimplantation, gastrulation represents a window of dynamic lineage-specific epigenetic regulation The online version of this article (doi:10.1186/s13072-015-0003-y) contains supplementary material, which is available to authorized users. A regulatory theme has emerged at many imprinted clusters in which a single long non-coding RNA (lncRNA) is thought to repressively regulate genes in cis through direct transcriptional blocking and/or recruitment of repressive chromatin remodeling complexes such as G9a and PRC2, resulting in differential allele-specific histone modifications [Genomic imprinting is an epigenetic phenomenon that results in mono-allelic gene expression in a parent-of-origin manner. Imprinted expression has been identified at approximately 150 mouse genes, which often occurs in clusters containing multiple imprinted transcripts . Expressications ,4.Igf2r, Slc22a2, and Slc22a3 genes and the paternally expressed lncRNA Airn [Slc22a1, Mas, and Plg). The Airn promoter lies in the second intron of Igf2r, and Airn transcription occurs from the opposite strand overlapping Igf2r exons 1 and 2 [Airn expression may participate in imprinting of the maternally expressed genes by blocking access of the transcriptional machinery to the Igf2r start site [Airn has been shown to be required for silencing of Igf2r [Airn recruitment of repressive complexes such as G9a to their promoters [Igf2r is observed in ES cells and only becomes imprinted upon differentiation in vitro [Igf2r and Airn has been documented in preimplantation and late stage embryos [Igf2r/Airn in vivo remain unknown. Here we characterize tissue-specific dynamics of expression and epigenetic modifications that occur at Igf2r/Airn during normal gastrulation. We show that significant epigenetic regulation occurs at imprinted loci during epiblast differentiation in vivo.One cluster on mouse chromosome 17 includes the maternally expressed RNA Airn , and sev 1 and 2 -7. Paterart site , and traof Igf2r ,9. Paterromoters . Biallelin vitro . Althoug embryos -12, line embryos ,8,13, buIgf2r/Airn imprinted cluster contains the maternally expressed Igf2r, Slc22a2, and Slc22a3, the paternally expressed Airn, and the non-imprinted Plg, Slc22a1, and Mas1 genes  and visceral endoderm (VE) at E6.5 and the embryonic (EM) and extraembryonic (EX) tissues of E7.5 embryos . Single-strand confirmation polymorphism (SSCP) revealed that Igf2r is expressed from both alleles in the EPI of E6.5 embryos  of Airn  are required for imprinted expression at many loci -16. The pression . In all pression  . Importantly, the epiblast of embryos 3 and 4 express both alleles of Igf2r and paternal Airn, suggesting that transcription from both loci can occur on opposite strands of the same paternal chromosome. Analysis with single cell-resolution will be important to support this finding.To more closely examine the timing of r Figure A. The sps Figure B. HoweveAirn transcript is 108 kb, we designed amplicons along its length to assess if the entire lncRNA is detectable in embryonic lineages at various developmental stages .Since the s Figure E. Qualits Figure F. At E7.s Figure F. By E9.s Figure F, +. AltAirn transcription. This could explain activation of only the paternal unmethylated allele in the embryo. Furthermore, the difference in Igf2r and Airn expression between EPI  and VE  indicate a distinct mechanism during epiblast differentiation that activates Airn . Alternatively, there may be regulation on the paternal allele that initially inhibits Airn transcription in epiblast. Either scenario indicates that neither DNA methylation nor Airn expression is responsible for silencing paternal Igf2r in the epiblast. Taken together, the observation that DNA methylation dynamics are the same in embryonic and extraembryonic tissues but that allelic expression patterns are different, require an embryonic lineage-specific mechanism responsible for establishment of imprinted expression.These findings raise the possibility that maternal DNA methylation spreading is required to inhibit maternal Igf2/H19, Ctcf binds to the unmethylated maternal ICR preventing Igf2-enhancer interactions [Igf2r/Airn. At E6.5, very little Ctcf transcript is detectable in EPI or VE  but is dispensable until the next major lineage decisions are made during gastrulation. This possibility supports the idea that genome-wide epigenetic alterations are required during gastrulation lineage decisions, similar to preimplantation. In the future, conditional deletion strategies may make it feasible to functionally test the requirement of Ctcf in specific tissues during gastrulation.The methylation-sensitive insulator Ctcf participates in chromatin looping at imprinted loci -20. At Iractions . We therE Figure A, but exCtcf in the epiblast at E6.5 and the biallelic binding of Ctcf at E8.5 suggest that while Ctcf may play a role in maintaining imprinted expression at later stages, it is not involved in the initiation of paternal Igf2r silencing or Airn activation in the embryonic lineage during gastrulation.Ctcf chromatin immunoprecipitation (ChIP) indicates Ctcf binds to DMR2 in both embryonic and extraembryonic E8.5 tissues Figure B. SequenIgf2r imprinting in the central nervous system [Allele-specific histone modifications (HMODs) have been shown to correlate with s system ,22. We tAirn), and H3K9me3 is enriched on the maternal allele .Similar to binding of Ctcf, we observe allele-specific enrichment of HMODs at DMR2 in extraembryonic tissues but not in chromatin derived from the embryo Figure E. In extIgf2r is imprinted at least 2 days prior. This indicates differential methylation  and X-inactivation may utilize a common mechanism. Identification of the lineage-specific machinery that enables epigenetic changes specifically in the embryo will lead to a more complete understanding of the events underlying normal gastrulation and epigenetic reprogramming.Our results indicate lineage-specific regulation of iewed in ) suggestPWD/PhJ/ForeJ (JAX 005267) and C57BL/6J (JAX 000664) males. Embryos were microdissected for DNA and mRNA extraction. MII oocytes were collected from superovulated B6D2F1 females to confirm ICR methylation.All procedures were approved by the University of Massachusetts Amherst Institutional Animal Care and Use Committee. Embryos were derived from C57BL/6J (JAX 000664) and PWD/PhJ (JAX004660). Reciprocal F1 embryos were derived from female C75BL6/J Chr17Airn RT-PCR are shown in Additional file Airn restriction fragment length polymorphism (RFLP) was performed with AvaI. SSCP was performed on Igf2r PCR products with MDE polyacrylamide gel electrophoresis . PCR products were visualized by ethidium bromide illumination and imaging.Total RNA was isolated using the Roche High Pure RNA Isolation Kit . cDNA synthesis was performed using Bio-Rad iScript cDNA synthesis kit (170-8891) . Primers for allele-specific expression and full-length Bisulfite sequencing was performed as previously described  with theE8.5 C75BL/6JxPWD/PhJ-F1 embryos were dissected, and embryonic and extraembryonic tissues were separated and immediately processed using instructions in either ChIP-IT High Sensitivity kit  or Zymo-Spin ChIP kit . Samples were kept on ice and either sonicated twice for 20 s with the Heat Systems Sonicator/Ultra Processor (output 3) or sonicated for 30 s on/20 s off for 3 min using a cup horn adaptor for the QSonica A500 . After sonication, 1% of each sample was removed for input control. Immunoprecipitation was carried out using Active Motif Protein G agarose beads or magnetic Protein G Dynabeads  and either anti-Ctcf , anti-H3K4me3 , anti-H3K9me3 (Abcam ab8898), or H3K27me3  along with normal rabbit IgG. After antibody incubation, beads were washed and DNA was collected using manufacturer’s protocol. ChIP-PCR primers found in Additional file


Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. Nucleosome positioning and chromatin accessibility are important contributors to the regulation of gene expression. Here the authors describe a method that allows the simultaneous measurement of nucleosome occupancy and chromatin accessibility in the same assay, revealing new features of chromatin organization linked to gene regulation. Nuclear factors facilitate the compaction of genomic DNA into chromatin2347891011Current approaches for mapping nucleosome occupancy often rely on digestion with nucleases, most commonly micrococcal nuclease (MNase); however digestion assays are prone to technical issues that can hinder interpretation and sample-to-sample comparison of the results1315161820212223We have developed a new methodology to quantify chromatin accessibility across the genomes of various complexity by utilizing different MNase concentrations. Differences in nucleosome occupancy measured under light and deep-digestion conditions have been used previously to map ‘fragile' nucleosomes, that is, nucleosomes seen well only under light-digestion conditions, in distinct regulatory regions in yeast19Drosophila melanogaster S2 cells using four different concentrations of micrococcal nuclease (MNase titrations). An exponential titration series of MNase was used  that was chosen to generate a small amount of mononucleosomal fragments at the lowest MNase concentration and predominantly mononucleosomal DNA at the highest concentration of MNase  as an initial test of MNase titration. Previous studies have shown a nucleosome ‘free' region at the TSS surrounded by nucleosomes at −1 and +1 positions, and defined arrays of nucleosomes propagating into the gene. We found  that the26These data demonstrate two opposing scenarios of chromatin response to MNase titration: the number of sequenced DNA fragments from a given locus might increase or decrease with increasing MNase concentration. For example, under light-digestion conditions, regions with accessible DNA are preferentially released by MNase (such as the ‘−1' nucleosome); however these regions get digested to fragments of sub-nucleosomal sizes (which are excluded in our library construction protocol) under deeper digestion conditions (scenario 1) . In contTo measure the response of a genomic locus to MNase titration, we calculate a metric that reflects the rate of the change in signal at a particular locus in response to decreasing MNase concentration  is complicated. The correlation between MACC and DHS reaches maximal positive values at enhancers, indicating that both metrics are suitable for identification of these regions, and reaches lowest values at the 3′-ends of the genes . Thus, MThe relation between MACC and other chromatin features is further exemplified in We conclude that MACC provides both a quantitative and qualitative description of chromatin accessibility and that it captures the expected trends for ‘active' and ‘repressed' regions of the genome. It provides additional information by assigning an accessibility metric to all parts of the genome.Protection from MNase digestion can result both from histones in nucleosomes and from non-histone protein (NHP) factors. Thus, c-MACC scores are expected to reflect a combination of these types of protection. Consistent with this expectation28Overall, h-MACC and c-MACC show a high degree of similarity . Given tcf. c- and h-MACC distributions at TSS, Deviation between h- and c-MACC values is mostly observed for sites with positive c-MACC values . The available data sets focus on components of regulatory factors that are used broadly  and are underrepresented in data on binding of gene-specific DNA-binding factors. This limits our ability to determine the extent to which MACC might reflect binding by gene-specific factors, which is anticipated from previous studies to play a key role in accessibilityTo test the hypothesis that certain of these peaks are associated with NHP binding, we examined their overlap with known NHP-binding sites using publicly available data (see Methods section)To explore the relative contribution of histones and NHP binding to the sites in groups 1 and 2, we quantified H3 levels for these sites. As expected by their definition, loci from group 1 are almost uniformly depleted of H3, loci from group 2 are associated with a peak in the H3 profile . This suThese sites exhibit distinct patterns of enrichment in relation to regulatory features : group 2We used a number of experimental and computational approaches to validate the group 1 and 2 sites. To this end we ruled out that these sites are artifacts of the experimental platform used in our study by performing MNase-qPCR experiment . FinallyA detailed analysis of the chromatin organization around sites from group 1 and group 2 revealed different patterns of nucleosome accessibility (h-MACC) for these groups . AccessiWe determined how nucleosome occupancy relates to the accessibility metric provided by h-MACC. Nucleosome occupancy obtained in assays using one mononucleosome pool generated by MNase digestion differs from that obtained with distinct MNase concentration (see above). To measure nucleosome occupancy, we therefore used the averaged frequency of paired-end reads in 300-bp bins over all MNase titration points. This approximates the signal that could be obtained if the libraries corresponding to the individual titration points were pooled together to produce a single map of nucleosome occupancy. Thus, occupancy and h-MACC are measured using the same data.We tested the correlation between the ‘pooled' nucleosome occupancy and h-MACC over the genome . SurprisOne interpretation of this finding is that the nucleosomes at the genomic locations with both high accessibility and occupancy , and on NPCs isolated from mouse embryonic brain (NPC2). We found that regions of c-MACC were consistent between the two pluripotent lines and between the two NPC lines, but differed considerably between the pluripotent and differentiated lines. For example, genes active in NPCs but inactive in ESCs had higher c-MACC in NPCs , on neuronal precursor cells (NPCs) derived  in NPCs  and vice in NPCs . To dete in NPCs . Most ofWe introduce an integrative approach to investigate chromatin structure that uses measurements of sensitivity to MNase titration across the genome. Traditionally MNase assays have been used to profile nucleosome occupancy. Because apparent nucleosome occupancy varies with the level of digestion, comparing and interpreting individual experiments performed with a single MNase concentration is difficult to do with confidence. The level of chromatin digestion using MNase is sensitive to even slight variation in enzyme activity, and different nucleosomes are released by MNase cleavage with different efficiency. We present an approach to MNase digestion that is self-normalized and focused on DNA accessibility rather than on nucleosome occupancy. This approach is more robust to experimental variability and its outcome is easier to interpret.The MACC protocol directly addresses the relationship between nucleosome occupancy and DNA accessibility since both of them are profiled in the same assay. We demonstrate that in addition to the expected scenario, where high occupancy corresponds to low DNA accessibility, regulatory loci often have chromatin arrangements characterized by relatively high nucleosome occupancy with high accessibility. We infer that this arrangement is facilitated by nucleosomes of low stability. These regions are often enriched in marks of active chromatin , which might contribute to high accessibility. Another factor contributing to high accessibility of such regions can be an increase in inter-nucleosomal spacing (linkers)cf. DHS, H3.3, ATAC-Seq)835in vitro methylation of genomic DNA (MeDIP footprint)MACC provides advantages over other methods for studying the physical state of chromatin. First, it allows profiling of the entire genome, rather than focusing on local regions of ‘open' chromatin  with the number of tags used in the ATAC-Seq profiling of GM12878 cells (∼200 millions of paired-end tags)A comparison of MACC profiles computed for the whole-chromatin digest and for the samples enriched in a core histone reveals sites where chromatin accessibility is affected by the factors other than nucleosomes. Analysis at such sites allowed detection of different modes of protein binding, with and without predominant nucleosome displacement. These modes have patterns that differ with regulation, suggesting that MACC profiling may have a wide range of applications in generating hypotheses for regulatory mechanism. For instance, binding of pioneer transcription factors to nucleosomal DNA during initiation of cell-fate changes and subsequent chromatin rearrangements may involve different modes of protein binding detectable by the MACC approach7 S2 cells were pelleted and rinsed twice in phosphate-buffered saline (PBS). Cells were crosslinked in 1.1% formaldehyde in PBS for 10 min at room temperature, tumbling end over end in a volume of 10 ml. After addition of 50 μl of 2.5 M glycine, the cells were tumbled at room temperature for 2 min to quench the reaction. Cells were next rinsed twice with cold PBS before pelleting and flash freezing in liquid nitrogen. For MNase digestion, the cell pellet was resuspended in PBS with 0.1% Triton X-100 (PBS–TX). Digestion of 106 cells per titration point took place in a volume of 400 μl PBS–TX supplemented with 1 mM CaCl2. Either 1.5, 6.25, 25 or 100 U of MNase  were added to pre-warmed cells and incubated at 37 °C for 3 min. Digestion was halted by moving to ice and adding 10 μl of 250 mM EDTA, 250 mM EGTA. Before DNA clean-up, the digestions were adjusted to 0.5% SDS and 10 mM Tris pH 8. For DNA cleanup, digestions were incubated with RNase (Roche) for 30 min at 37 °C, with proteinase K (Roche) for 60 min at 55 °C, and incubated at 65 °C for 60 min to reverse crosslinks. This was followed by phenol–chloroform extraction and ethanol precipitation. In some experiments, the purified DNA was subjected to a low-stringency size selection to remove high-molecular weight DNA, although excluding this step does not affect the results (data not shown). For size selection, digestion products in a volume of 100 μl of water were incubated for 5 min at room temperature with 60 μl of Agencourt AMPure XP beads (Beckman Coulter). After bead separation, the supernatant was moved to a new tube and 120 μl of new bead suspension was added. DNA was eluted from these beads and used as input into the library preparation protocol described in Bowman et al.Drosophila S2 cells were grown in Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 28 °C. For fixation, 10For histone ChIP, after halting the MNase digestion, adding EDTA/EGTA, and adding SDS, 135 μl was removed from the 400 μl digestion as an input fraction and kept overnight at 4 °C. The remainder of each digestion was split in half (135 μl each). Each chromatin aliquot was independently adjusted to ChIP buffer conditions in a volume of 500 μl ). After adding 1 ml additional ChIP buffer to each chromatin aliquot , this was tumbled end over end for 10 min at 4 °C and then subjected to a high-speed spin in a microcentrifuge for 10 min at 4 °C. Supernatant was taken to a new tube for precipitation. Antibody (2 μl) was added to each tube . Chromatin precipitation was performed as described7) were harvested on ice, washed in cold PBS and their RNA was extracted using the RNeasy Kit (Qiagen) with on column DNAse digestion according to the manufacturer's protocol. Ribosomal RNA was depleted from the total RNA (5 μg) by using the RiboZero gold magnetic kit (Epicentre/Illumina) according to manufacturer's instructions. cDNA was generated with the TruSeq non–stranded kit (Illumina) according to manufacturer's instructions. cDNA Libraries for next-generation sequencing were assembled by blunt end repair and A-tailing of the cDNA, followed by adapter ligation and PCR with bar-coded sequencing primers according to Bowman et al.Drosophila S2 cells  were used in a double size selection with ratios of 0.7 × and 1.7 × to obtain a range of fragment sizes from ∼100 to 1,000 bp. The resulting fragments from four MNase concentrations in the range  were prepared individually for bar-coded sequencing−1 of leukemia inhibitory factor (EMD Millipore). ESC cultures were depleted of feeder cells before use in experiments. Neural progenitors (NPC1) were derived by in vitro differentiation from J1 ESC and maintained using previously described methodsL-ornithine coated plates in DMEM:F12 containing B-27 and N-2 supplements (Gibco) and EGF+FGF-2 (10 ng ml−1). One million cells were crosslinked at room temperature for 10 min with 1% formaldehyde and crosslinking stopped by the addition of 125 mM glycine. Nuclei were isolated and MNase digestion performed on either 200,000 or 250,000 nuclei per reaction. Digestions were performed using 64, 16, 4 or 1 U MNase (Worthington) in 10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2 for 10 min  or 15 min  at 25C. Reactions were stopped with EDTA/EGTA and 0.5% SDS and 125 mM NaCl added to the samples. RNase A and proteinase K treatment along with cross-link reversal at 65 °C were performed. DNA was purified by phenol–chloroform extraction and column purification. MNase digestion was evaluated and the recovered DNA from 1 to 64 U MNase digested samples was used to generate sequencing libraries as described, without fragment length selection.J1 ESCs (ESC1) and E14-derived ESCs (ESC2) were maintained on mitomycin-C-inactivated embryonic fibroblast feeder layers in DMEM supplemented with 15% FBS (Hyclone) and 1,000 U mlD. melanogaster, Homo sapiens and Mus musculus data respectively using Bowtie aligner v. 0.12.9 .The sequenced paired-end reads were mapped to dm3, hg19 and mm9 genomes in the cases of 2.9 ref. . Only unet al.http://genome.ucsc.edu/ENCODE). The fly genome annotation based on the combinatorial patterns of chromatin modifications (‘chromatin states') was taken as defined by modENCODE consortiumet al.http://genome.ucsc.edu/ENCODE and http://intermine.modencode.org). The coordinates of Psc and Trx binding sites were identified by Enderle et al.et al.29The coordinates of the genes were taken according to the annotations for dm3, mm9 and hg19 versions of the fly, mouse and human genomes, respectively. The coordinates of the active enhancers were taken according to modENCODE annotation for S2 cellsMACC values were obtained for each 300-bp (fly) or 500-bp (mouse and human) bin in the genome by fitting linear regression on the normalized read frequencies computed for each titration point  so that the bins with high P values (low significance) would have near-zero weighted MACC scores. The MACC scores computed with and without correction correlated well (r=0.96 and higher); thus this correction had only a limited effect on the results and was not used for final scores. To test how the number of used titration points affects the MACC scores we repeated MACC estimation for individual replicates using 4, 3 and 2 titration points. This analysis confirmed that using complete set of the titration data (4 points) results in the strongest correlation between replicates . For generation of HMM based on randomized data, shuffling of MACC profile within each chromosome was used. Hierarchical clustering was performed using unweighted pair group method with the distance between profiles computed as (1—Pearson's correlation coefficients). To ensure that our findings are not biased by variability in the fragment size distributions, we reproduced crucial observations with MNase and MACC profiles computed only with fragments of mono-, di- and tri-nucleosomal lengths .Z-scores. Bins with Z-score above a given threshold were selected as NHP-binding sites. When a particular protein was represented by more than one profile we preserved the profile with lowest variability in the number of peaks obtained under different Z-score thresholds ).NHP-binding data produced by modENCODE consortium and individual studies (ChIP-chip and ChIP-Seq) were collected from public sources (as described above). The protein enrichment, that is, ChIP signal over input, was computed in 300-bp bins for each of collected profiles. The enrichment was further transformed into i−1<c-MACCi>c-MACCi+1). H3 enrichment was computed in 300-bp bins as an H3-ChIP/input ratio between fragment frequencies pooled over all MNase titration points. To further validate MACC peak features we also performed c- and h-MACC comparison at higher resolution using 100-bp bins, and this analysis reproduced our major findings  and group 2 (high c-MACC and high h-MACC): c-MACC higher than 80% of all positive c-MACC scores and h-MACC lower than 10% of all positive h-MACC scores (group 1); c-MACC higher than 80% of all positive c-MACC scores and h-MACC higher than 90% of all positive h-MACC scores (group 2). MACC peaks represent bins with c-MACC values higher than those in the adjacent bins  under accession number GSE78984.How to cite this article: Mieczkowski, J. et al. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat. Commun. 7:11485 doi: 10.1038/ncomms11485 (2016).Supplementary Figures 1-26 and Supplementary Tables 1-2