Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the  N-mHtt interacts with proteins involved in the regulation of chromatin structure Reporter plasmids under the control of the CMV and TK promoters were co-transfected into both N548wt and N548hd cells. The luciferase activity driven by the two reporter plasmids was quantified 24 h following transfection using the Dual-Luciferase Reporter (DLR) Assay. Transcription driven by both the CMV and the TK promoters was significantly lower in N548hd cells relative to activity in N548wt cells . This reTo determine if the region of the CMV promoter required for transcriptional repression in the presence of N-mHtt could be identified, fragments of the CMV promoter spanning 772, 297 and 99 bp 5′ to the transcription start site  were transfected into N548wt and N548hd cells. The sequential deletion of the CMV promoter resulted in progressively lower luciferase activity , indicatOne of the hypothesized mechanisms regarding how N-mHtt inhibits transcription suggests that N-mHtt interacts with and sequesters gene-specific transcription factors. If transcription driven by the CMV promoter relied on the function of N-mHtt interacting transcription factors, functional impairment of that factor resulting from its interaction with N-mHtt could explain the decreased CMV activity detected in N548hd cells. The 227 bp of CMV promoter upstream of the transcription start site were analyzed using MatInspector software to identify putative transcription factor binding sites of proteins known to interact with N-mHtt. This CMV promoter fragment contained three putative NF-κB binding sites, three putative CREB response element (CRE) binding sites, one putative Sp1 binding site, one putative binding site for CCAAT-enhancer binding protein (C/EBP) and one putative TATA binding protein (TBP) binding site . Each siet al. (2005) demonstrated that N-mHtt interacts with the RAP30 subunit of the general transcription factor IIF (TFIIF), and that the N-mHtt/RAP30 interaction was involved in transcriptional repression of a 75 bp fragment of the D2 dopamine receptor promoter et al. (2005) showed that the N-mHtt interacted at the region of RAP30 required for its association with its TFIIF binding partner RAP74 A study by Zhai If levels of RAP74 were limiting, and N-mHtt interacted with RAP30 in a manner that did not inhibit the formation of TFIIF, overexpression of RAP30 or RAP74 on their own would not restore transcription in the presence of N-mHtt. In an attempt to increase the amount of functional TFIIF in N548hd cells, plasmids driving the expression of RAP30 and RAP74 cDNA were co-transfected into N548wt and N548hd cells along with reporter plasmids driven by the CMV and TK promoters. TK activity was significantly increased in N548wt cells following overexpression of both RAP30 and RAP74 . This reIn vitro promoter binding assays were performed to determine whether N-mHtt was present in the complement of proteins directly associated with the N-mHtt-affected CMV and TK promoters. A biotinylated 297 bp fragment of the CMV promoter, and a 768 bp fragment of the TK promoter were attached to streptavadin-coated magnetic beads and incubated with nuclear extract from either N548wt or N548hd cells. The proteins specifically associated with the promoters were isolated and probed with an antibody specific for Htt. Bands were detected in the complement of proteins specifically associated with both the CMV and TK promoters at the sizes predicted for the N-terminal fragment of Htt overexpressed in N548wt and N548hd cells  or its empty vector control (shNeg) were transfected into N548wt and N548hd cells along with reporter plasmids driven by the CMV and TK promoters. Transfection with shHtt reduced but did not eliminate N-terminal Htt expression in N548wt and N548hd cells  huntingtin with either 25 or 109 CAG repeats had decreased transcription in the presence of N-mHtt. These results suggest that N-mHtt may impair transcription through a mechanism involving interaction with proteins that bind near, or at, the transcription start site.The purpose of this study was to test and expand upon the current theories of N-mHtt-mediated transcriptional dysregulation using the highly active CMV and TK promoters. A promoter deletion experiment was performed to determine if a specific region of the CMV promoter was required for N-mHtt-mediated transcriptional repression. Although sequential deletion of the CMV promoter resulted in a progressive decrease in transcriptional activity, transcription driven by the smallest promoter fragment tested was significantly lower in N548hd cells compared to N548wt cells. Similar results have been reported in studies that utilized other N-mHtt-affected promoters. The minimally active fragments of the DARPP-32 (160 bp) Linker-scanning mutagenesis was performed to determine if transcriptional repression of the CMV promoter was the result of interference by N-mHtt with the function of gene-specific transcription factors. Elimination of putative binding sites of transcription factors known to interact with N-mHtt did not restore CMV activity in N548hd cells to levels observed in N548wt cells. This suggested that no known N-mHtt-interacting transcription factor was solely responsible for inhibition of CMV activity. A previous study successfully alleviated N-mHtt-mediated transcriptional repression using linker-scanning mutagenesis. Elimination of a C/EBP binding site resulted in the recovery of transcription at the argininosuccinate acid lyase (AAL) promoter in the presence of N-mHtt Hdh cell lines expressing mHtt following overexpression of TFIIF or its components. This result suggested either that TFIIF was not involved in N-mHtt-mediated transcriptional repression of the CMV and TK promoters, or that N-mHtt was interacting with TFIIF in a manner that could not be overcome by overexpression of TFIIF or its components.TFIIF (composed of RAP30/RAP74 subunits) is a member of the core transcriptional machinery. RAP30 has been shown to interact with N-mHtt in vitro promoter binding assay was performed to determine whether N-mHtt was present or absent from the complement of proteins directly associated with the CMV promoter. In contrast to the predictions made by sequestration models of N-mHtt-mediated transcriptional dysregulation, N-mHtt in nuclear extract obtained from N548hd cells was among the proteins specifically associated with the N-mHtt-affected CMV and TK promoters. It is not possible to determine based on the technique used whether N-mHtt was bound directly to the DNA, or whether the presence of N-mHtt in the promoter-bound protein fraction resulted from its association with DNA-bound transcription factors or co-factors. Knowing that N-mHtt is capable of interacting with numerous proteins that make up the core transcriptional machinery Numerous studies have suggested that N-mHtt interferes with transcription by binding to and sequestering gene-specific transcription factors from affected promoters Although TFIIF did not appear to be involved in transcriptional repression of the CMV and TK promoters, evidence remained to suggest that N-mHtt impaired transcription through interactions with the core transcriptional machinery. TBP is another member of the core machinery and has been previously shown to interact with N-mHtt. TBP was overexpressed to determine whether it played a role in the decreased activity of the CMV and TK promoters in N548hd cells. TBP overexpression was able to significantly increase activity of the TK but not the CMV promoter in N548hd cells. As TBP is a key component of the basal transcriptional machinery, the involvement of TBP in N-mHtt-mediated transcriptional repression at one gene would suggest it contributes to some degree of transcriptional repression at all genes. The ability of TBP overexpression to alleviate transcriptional repression at the TK but not the CMV promoter suggested either that an additional mechanism was involved in inhibition of CMV activity, or that through a similar mechanism, N-mHtt exhibited a stronger inhibition of CMV activity than TK activity.Hdh cell model. Transcription driven by the CMV, but not the TK, promoter was significantly lower in both Q7/111 and Q111/111 cells compared to activity in Q7/7 wild-type cells. In agreement with the results from the shRNA knockdown experiment, the TK promoter, which models a more resistant N-mHtt-affected promoter, was unaffected at the lower level of N-mHtt expression in the StHdh cells, whereas the more sensitive CMV promoter exhibited decreased transcriptional activity. These two experiments provided further evidence that the CMV and TK promoters had differential susceptibility to the inhibitory effects of N-mHtt.Genes are generally regarded as affected or unaffected with respect to their susceptibility to N-mHtt-mediated transcriptional repression. The possibility that the CMV and TK promoters had different degrees of susceptibility to N-mHtt was an interesting idea, and one that required further investigation. shRNA-mediated knockdown of N-mHtt was performed in N548hd cells to determine if transcriptional repression of the CMV and TK promoters could be alleviated by reducing the amount of N-mHtt in the cell. Reduction of N-mHtt expression increased transcription driven by the TK, but not the CMV, promoter in N548hd cells. This provided further evidence that two N-mHtt-affected promoters may have differential susceptibility to the effects of N-mHtt. The TK promoter, representing a more resistant promoter, was unaffected in the presence of the new, lower level of N-mHtt expression generated following shRNA expression. The CMV promoter, representing a more sensitive N-mHtt-affected promoter, remained susceptible to N-mHtt-mediated transcriptional repression at the new, lower N-mHtt expression level. Because the N548 cell model does not represent a physiologically accurate model of HD, the impact of mHtt on CMV and TK activity was tested using the STHdh cells along with the CMV reporter plasmid to determine if CMV activity could be restored in cells expressing physiologically accurate levels of mHtt. Increasing expression of TFIIF or TBP did not increase transcription driven by the CMV reporter plasmid in the Q7/111 or Q111/111 cells relative to cells transfected with empty vector control. This result suggested that alleviating N-mHtt-mediated transcriptional repression at certain promoters is more complex than simply increasing the pool of soluble N-mHtt interacting proteins.The inability of TFIIF or TBP overexpression to restore CMV activity in N548hd cells may have been due to the artificially high expression level of N-mHtt in this cell line. TFIIF and TBP were overexpressed in STThe primary conclusions of this study were that N-mHtt appeared to impair transcription near, or at the transcription start site, and that transcriptional repression may result from interactions between N-mHtt and DNA-binding transcription factors or co-factors occurring at the promoter. The differential susceptibility of the CMV and TK promoters to the concentration of N-mHtt present in striatal cells highlighted the fact that not all promoters are similarly affected. Increased expression and accumulation of mHtt, as well as increased nuclear accumulation of N-mHtt in animal models of HD have been correlated with disease onset and increased severity, suggesting that the symptoms of HD are directly associated with the concentration of mHtt One important question that arises from these assumptions is, how are some genes more susceptible to the concentration of N-mHtt than others? One explanation is that transcription factors known to interact with N-mHtt may function as vehicles for recruiting N-mHtt to the promoter. Once recruited to the promoter, N-mHtt either impairs or inhibits the formation, release, or function of the core transcriptional complexes. Recruitment of N-mHtt to a promoter by a protein can be modeled in terms of probability. The probability, therefore, that transcription of a given gene will be impaired in the presence of N-mHtt is directly related to the concentration of N-mHtt present in the nucleus, the number of N-mHtt-interacting proteins required for proper transcription of that given gene, and their relative affinities for N-mHtt. This model assumes that N-mHtt can interact with multiple proteins simultaneously and that interaction inhibits the transcriptional machinery that facilitates normal transcription.According to this model, promoters regulated by more N-mHtt-interacting proteins would recruit N-mHtt more frequently than promoters regulated by fewer N-mHtt-interacting proteins, and would display reduced transcript levels in the presence of lower concentrations of N-mHtt. This model would also explain why overexpression of a single N-mHtt-interacting protein would have limited efficacy in alleviating transcriptional repression at a promoter in the presence of N-mHtt. If a promoter is regulated by a large number of N-mHtt-interacting proteins, overexpression of one of those N-mHtt-interacting proteins would increase the non-N-mHtt-bound pool of that specific protein available for transcription, but would not change the frequency that the other N-mHtt-interacting proteins recruited N-mHtt to the promoter. In line with this assumption, very few studies have reported success in restoring transcriptional activity through overexpression of an individual N-mHtt-interacting protein. Conversely, two studies have shown success in overexpressing multiple transcription factors when expression of those proteins on their own proved unsuccessful Several avenues exist to alleviate N-mHtt-mediated inhibition of transcription as proposed by this model. Overexpression of multiple N-mHtt-interacting proteins would be expected to have more success than overexpression of individual N-mHtt-interacting proteins at alleviating promoter-specific transcriptional represession. Overexpression of fragments of N-mHtt-interacting proteins that were able to associate with N-mHtt but not integrate into transcriptional complexes would have the added benefit of occupying N-mHtt and impeding its ability to associate with transcriptional complexes. Removing fragments of a promoter would likely reduce the number of N-mHtt-interacting proteins regulating the activity of that promoter because smaller promoters have fewer transcription factor binding sites than larger ones. Although this would reduce the vulnerability of a promoter to the effects of N-mHtt, it would remove endogenous regulatory mechanisms. While such strategies may work experimentally, the most therapeutically beneficial strategies would be those aimed at reducing the amount of N-mHtt in the nucleus, either through knockdown of mHtt, inhibiting the cleavage of mHtt, increasing nucleus efflux of N-mHtt or through increased degradation of the fragment. Each of these strategies would decrease the frequency with which N-mHtt was recruited to all promoters and in turn would reduce the frequency that N-mHtt inhibited transcription. Importantly, several groups have demonstrated that inhibition of mHtt cleavage decreases the concentration of N-mHtt in the nucleus and reduced cell death 2 and 95% air. The cells were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% (v/v) fetal bovine serum  as described previously . The generation and handling of ST14A cells and their derivatives was in accordance with the guidelines of the Institute of Pharmacological Sciences, University of Milano, Milan, Italy .Immortalized striatal cells, produced and originally characterized by Dr. Elena Cattaneo, from embryonic day 14 rats (ST14A), as well as derivatives stably expressing the N-terminal 548 amino acids of human Htt with 15 (N548wt), or 128 (N548hd) glutamine repeats (gifts from Dr. Elena Cattaneo), were cultured at the permissive temperature of 33°C with 5% COHdh Q7/7, Q7/111 and Q111/111 cells were purchased from the Coriell Institute for Medical Research. Cells were cultured at the permissive temperature of 33°C with 5% CO2 and 95% air in DMEM media as described in the literature provided by Coriell.STCTCGAGCCAGTGCCAAGCTGAT was used in combination with the antisense primer CACAGGACGGGTGTGGTC to amplify a 772 bp fragment of the CMV promoter using the pCMV-luc plasmid as a template. PCR reaction conditions consisted of a 15 m incubation at 95°C followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 m. The reaction was completed with a 10 m extension at 72°C. The truncated CMV promoter fragments were produced using the antisense primer above in combination with the sense primer GCCCAGTACATGACCTTACGGG (to produce the −227 CMV fragment) or AAATGTCGTAATAACCCCGCCC (to produce the −99 CMV fragment). The resulting PCR products were run on an agarose gel, gel extracted, cloned into pGEM-T vector and subsequently subcloned into MluI/BglII-digested pGL3-basic plasmid. Each of these steps was performed according to manufacturer’s directions.Polymerase chain reaction (PCR) was performed to generate the CMV promoter sequences to create a CMV promoter reporter plasmid. The sense primer Plasmids used in this study that were not generated in the lab included pEGFP-N1 (U55762 BD Biosciences), pRL-TK , pCMV-luc , pCDNA-RAP30 and pCDNA-RAP74 (gifts from Dr. Dimitrius Kranic) and pProEx-Hta-TBP (a gift from Dr. Ulrich Hartl).Cells were transfected with 50 ng pEGFP-N1 plasmid, 50 ng of pRL-TK plasmid, and 200 ng of pGL3-CMV plasmid using Lipofectamine (Invitrogen) and PLUS reagent (Invitrogen) according to manufacturer’s directions. In overexpression experiments, 200 ng of expression vector, or the equivalent mass of empty-vector control was added to the mix. Transfections were performed in serum-free media. Serum was added to the media to a final concentration of 10% (v/v) 4 h after transfection mixes were added to the cells.n luminometer (Turner Biosystems). Following quantification of TK and CMV activity, the protein concentration in the collection sample was determined using a standard Bradford Assay.Twenty-four hours after transfection, promoter activity was measured using the DLR™ Assay System (Promega) according to manufacturer’s protocol using a 20/20CTCGAGCCAGTGCCAAGCTGAT and the antisense primer CACAGGACGGGTGTGGTC to produce CMV promoter fragments upstream and downstream of the sequence targeted for mutation. The resulting PCR products were cloned into pGEM-T vectors and then extracted using restriction digest. The two fragments were then ligated together using the complementary linker portion. PCR was used to amplify the ligated CMV promoter containing a mutated target sequence. The PCR product was isolated, ligated into pGEM-T, and then subcloned into a pGL3 reporter plasmid. DNA sequencing was performed to verify the presence and location of each mutation.The 297 bp of the CMV promoter 5′ to the transcription start site were analyzed using MatInspector online software, provided by Genomatix software suite . Putative transcription factor binding sites identified by the online matrix for transcription factors that have been previously shown to interact with mHtt were targeted for deletion. The primer pairs described in GCCCAGTACATGACCTTACGGG and CTTTATGTTTTTGGCGTCTTCC. A 3′ biotinylated fragment of the TK promoter spanning from 768 bp 5′ to 27 bp 3′, relative to the transcription start site was generated via PCR from the pRL-TK reporter plasmid using primer sequences CGGTGGTTAGGGTTTGTCTGACGC and GCAGGGTCGCTCGGTGTTCG. The promoter fragments were attached to streptavadin-coated magnetic M-280 dynabeads (Invitrogen) using a MPC®-S Magnetic Particle Concentrator  according to manufacturer protocol. Prior to the binding assay, the DNA-bound beads were incubated for 15 min in 150 µl blocking buffer  and then washed with transcription buffer (see manufacturers protocol).A 3′ biotinylated fragment of the CMV promoter spanning from 297 bp 5′ to 75 bp 3′, relative to the transcription start site was generated via PCR from the pGL3-CMV reporter plasmid using primer sequences 2, 2 mM EDTA, 2.5 mM DTT, 0.05% (v/v) NP40, 30 ng/µl poly dI:dC] to a final volume of 50 µl and added to the Dynabead-bound promoters. The tubes were incubated at room temperature on the rotisserie shaker for 30 m. The tubes were placed on the magnet for 2 m and the supernatant was removed and stored as the unbound fraction. The pellets were washed 3 times with 400 µl reaction wash buffer  and the supernatants were collected for analysis. The final pellet was suspended in 20 µl of Laemmli Sample Buffer (BioRad), fractionated on an polyacrylamide gel, and was probed with antibody MAB2166.Nuclear extract from N548wt and N548hd cells was extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to kit protocol, and the protein was quantified using a standard Bradford assay. Nuclear extract from N548wt and N548hd cells was combined with binding buffer [100 mM KCl, 20 mM HEPES (pH 7.6), 5 mM MgClhuntingtin mRNA (shHtt) or empty plasmid (shNeg) as described previously. The cells were grown for 48 h following transfection, at which point cell lysates were collected and the DLR™ Assay was performed as described. Western blotting was performed using antibody MAB2166 to probe a gel containing 5 µg of cell lysate.N548wt and N548hd cells were transfected with a mix containing 200 ng of pGL3-CMV plasmid, 50 ng of pRL-TK plasmid, 50 ng of pEGFP-N1 plasmid and either 30 nM plasmid driving the expression of RNA complementary to the sequence between nucleotides 413–438 of human Antibodies used in this study included anti-huntingtin antibody MAB2166 (Millipore), anti-RAP30 antibody ab28180 (abcam) and anti-RAP74 antibody ab28179 (abcam).t-test or one- or two-way ANOVA as indicted in each figure legend. Two-way ANOVA were followed by Tamhane’s T2 post-hoc test for unequal variance or Bonferroni post-hoc test as indicated. Data are shown as the mean ± the standard error of the mean (S.E.M.).Statistical significance was set at 0.05 and determined via one- or two-tailed


SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain – such as MKRN3 and NDN – are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the  SNURF-SNRPN, NECDIN (NDN), MAGEL2, and MKRN3 is associated with PWS. In contrast, AS is associated with loss of expression or mutation of the maternally expressed UBE3A gene SNURF-SNRPN gene  that appears to be required for establishment and/or maintenance of the paternal epigenotype across the imprinted AS/PWS domain SNRPN. Mapping of microdeletions associated with AS has identified a 0.88 kb region, termed the AS-SRO, that is located ∼ 35 kb upstream of exon 1 of SNRPN and appears to govern the maternal epigenotype Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are two distinct neurogenetic diseases associated with the same ∼2 Mb domain in human chromosomal region15q11–13  and YY1 within multiple regulatory regions exclusively on the paternally-inherited chromosome, suggesting a role for these factors in the coordinate parent-of-origin regulation of genes within this domain. The ubiquity of nuclear respiratory factors in this region also suggests a possible functional connection between regulation of genes in the AS/PWS domain and respiration. We also present evidence that the murine Mkrn3 locus lies in close proximity to the PWS-IC specifically on the paternal chromosome and discuss the implications of this allele-specific spatial organization of the AS/PWS domain in the context of transcription factor binding patterns.To identify elements that may mediate PWS-IC function and potentially regulate imprinted expression of Snrpn gene (and PWS-IC) employed mice carrying a 35 kb PWS-IC deletion on either the maternally- or paternally-inherited chromosome PWS(del) and TgAS(del) mice carrying a transgene-associated deletion of the entire AS/PWS region on either the paternally- or maternally-inherited chromosome, respectively, as well as immortalized fibroblast cell lines generated from these mice AS(del) mice were bred with C57BL6/J male mice to obtain TgAS(del) mice for analysis; male TgAS(del) mice were bred with C57BL6/J females to obtain TgPWS(del) mice. Both cell lines were grown at 37°C in DMEM medium supplemented with 10% FBS and penicillin/streptomycin in 5% CO2. Experiments using primary mouse tissues were carried out in strict accordance with and approved by the University of Florida Institutional Animal Care and Use Committee (protocol number D586). Animals were humanely euthanized according to the American Veterinary Medical Association Guidelines for Euthanasia and all efforts were made to minimize suffering.Allele-specific analysis of the Primary mouse brain cell suspensions were prepared from 1–2 day newborn mice. Pups were humanely sacrificed by rapid decapitation and brain tissue was dissected and placed in ice cold phosphate buffered saline (PBS) containing 0.5 M sucrose. Dissected brains were transferred to fresh ice cold PBS/sucrose solution where blood vessels and other fibrous tissue were removed with forceps. Remaining brain tissue was then minced into ∼1–2 mm pieces using scissors and triturated gently in PBS/sucrose with a Pasteur pipette. At regular intervals, the triturated cell suspension was monitored by light microscopy to determine when the tissue was optimally dispersed into single cells (but not lysed to nuclei). The resulting cell suspension was filtered though a cheese cloth into a conical tube on ice. The remaining tissue fragments were further triturated in fresh PBS/sucrose, monitored for tissue dispersion to single cells, and the supernatant filtered and combined with the initial supernatant; cells in the pooled supernatants were used for the analyses described below. Single cell suspensions from primary mouse spleen cells were generated from dissected adult spleens by cutting spleens into sections and gently squeezing the tissue to release a suspension of single cells into a cell culture dish containing PBS and 0.5 M sucrose.Snrpn gene synthesized by PCR and cloned. Probe B was a 115 bp bp Pst I (+2214 from transcription initiation site)–EcoRI fragment (+2329) from plasmid pGN72 containing a BamHI-EcoR I fragment of the Snrpn gene , then purified DNA from DNase-treated samples was digested with EcoRI and Southern blotted. Blots were hybridized with a α-32P-dCTP radiolabeled 0.7 kb Bgl II-EcoRI fragment from the Mkrn3 locus.Analysis of DNase I hypersensitivity by Southern blot analysis was performed as described previously In vivo footprinting by ligation-mediated PCR (LMPCR) was performed essentially as described previously Snrpn in primary mouse brain, aliquots of dispersed primary brain cells were treated with increasing concentrations of dimethyl sulfate (DMS) and genomic DNA was purified. For LMPCR, the primer extension reaction (using the first primer in each set shown below) was performed with Vent DNA polymerase (New England Biolabs) using a deoxynucleotide triphosphate mix that included 7-deaza-dGTP at a ratio of 7-deaza-dGTP/dGTP of 3∶1 as previously described 4, and 0.25 mM of each deoxynucleotide triphosphate) and cycling of the reaction at 59°C for 1 min., 53°C for 1 min., 57°C for 1 min., 60°C for 1 min., 68°C for 1 min., and 76°C for 1 min. After ligation of the linker primer, the ligated template was then subjected to PCR amplification of the appropriate region using Taq DNA polymerase with the gene-specific 2nd primer of each LMPCR primer set  and the LMPCR linker primer. PCR amplification with Taq polymerase (Invitrogen) was performed in 40 mM NaCl, 10 mM Tris-HCl, pH 8.9, 1.75 mM MgCl2, 0.25 mM of each dNTP (with a 3∶1 7–deaza-dGTP/dGTP mixture substituted for dGTP). Reactions were initiated by denaturation of each sample at 94°C for 3 min. Then each sample was subjected to 23 cycles of the following: denaturation at 94°C for 20 sec., annealing of the PCR primers for 1 min. at the temperature shown for each gene-specific 2nd primer , extension at 76°C for 1 min. with an incremental increase of 3 sec. per cycle, and a final extension at 76°C for 10 min. after the PCR final cycle. Gel electrophoresis of the LMPCR-amplified DNA was carried out on 5% denaturing Long Ranger DNA sequencing gels, and electrotransfer of the gel onto Hybond N+ nylon membrane (Amersham) was performed in an electroblotting apparatus  as described previously Snrpn gene from positions −305 bp to +308 bp (relative to the transcription initiation site) was cloned into M13 mp18 and M13mp19 to obtain single-stranded clones M13mp1873 and M13mp1973 containing the sense and antisense strands, respectively. These single-stranded DNA templates were used to prepare appropriate strand-specific probes of interest by primer extension using the second LMPCR primer from the region of interest. The probe for analysis of the lower strand was synthesized from template M13mp1973, while probes for analysis of the upper strand were synthesized from M13mp1873. Probes were synthesized by primer extension using the Klenow fragment of DNA polymerase in the presence of 10 uCi of α-32P-dCTP. Purification of the radiolabeled probe was carried out in 6% acrylamide gels in TBE  by excising the probe from the gel, then macerating the gel fragment in hybridization buffer  and added to the prehybridized blot. Hybridization was carried out overnight at 65°C. Following hybridization, the blot was washed at 65°C in wash solution  and exposed to Kodak X-OMAT film.Snrpn promoter region were:LMPCR primer sets for analysis of the upper strand of the CACTCCTCAGAACCAAGC) and TY382 ; Primer set 2: TY644 (ACTAACACACCCAAGGAGTC) and TY643 .Primer set 1: TY381  and TY558 .Primer set 3: TY557  and TgAS(del) mouse brain tissue was treated with 0.5% DMS at room temperature for 45 seconds, then cleaved with piperidine and subjected to ligation-mediated PCR as described for the experimental samples.This primer set covered a region on the lower strand from position −125 bp to +100 bp of the Mkrn3, DMS treatment was performed using approximately ∼2–3×107 TgPWSdel or TgASdel cells for each DMS treatment. LMPCR was performed essentially as previously described by Chen et. al. Mkrn3 5′ flanking region, the following four pairs of gene-specific primer sets were used:For analysis of CTTCAGCACCTGCCTCC for primer extension, and GTGGGCCTCAATGGGAGCTGTAGACT for LMPCR; 1Ua: CTCTTCCATCTTACAGCGTG for primer extension, and TGCCTCTAACACTGTCCAAAAACCCGAATA for LMPCR;1Ub: ATCCCAGTGTCTCAAGCAG for primer extension, and GAAACAGGCACGCGAAAAACATGGC for LMPCR;1La: ACCTGGAGAGTTTAAAACATCA for primer extension, and TGAGGGGAAACACTGTGGAAACGGG for LMPCR.1Lb: In vivo footprints were visualized with 32P-labelled strand-specific probes. Hybridization probes were prepared from a double-stranded plasmid template (plasmid EN40 generously provided by Robert Nicholls) containing a 3.9 kb EcoRI-NotI fragment (encompassing the Mkrn3 promoter region) cloned into a pZErOTM-2 vector. Plasmid DNA was first purified and linearized by double digestion with NotI and EcoRI, then strand-specific radiolabeled single-stranded hybridization probes were synthesized from the linearized plasmid template with the same gene-specific primer used for each LMPCR reaction. For each probe synthesis reaction, 4 µl H2O, 3 µl template (∼0.5 ug/ul), and 1 µl of the gene-specific primer (0.3 µg/µl) were first incubated at 95°C for 10 min. and snap frozen on dry ice. Then, 5 µl of 5X labeling buffer from the DECAprimerTMII  kit (without dCTP), 10 µl α-32P-dCTP , and 1 µl DNA pol I Klenow fragment  were added to the denatured template/primer solution and incubated at 37°C for 30 min. The reaction was stopped by adding 35µl formamide-dye solution  and denatured at 95°C for 10 minutes. The single-stranded radiolabeled probes then were gel-purified by electrophoresis in a 6% acrylamide gel and excised from the gel. Radiolabeled probes were hybridized to electrotransferred membranes to visualize in vivo footprints in autoradiograms as described previously Mkrn3 sequencing ladder, purified genomic DNA from each of the fibroblast cell lines derived from TgPWS(del) and TgAS(del) embryos was DMS and piperidine treated, then subjected to ligation-mediated PCR, as described above.PCR products from LMPCR were size-fractionated on DNA sequencing gels, and electrotransferred onto a nylon membrane as described previously Snrpn and Mkrn3 loci were performed as previously described Snrpn were as follows:ChIP assays for the TTGCAAAGTGCCCTCTCTGTCTG, GATGGGGAGATTTTGAAACAGGATG;Primer set 1 : AGACCCCTGCATTGCGGC, GCATCTCCGGCTCCCTCTCC;Primer set 2 : TGGTCTGGGGGATGGGAGC, CTGAACACACAAGCCATGGCAAC;Primer set 3 : AGGGGTCGTGTCGCATGTCA, AATGGGGAGGGGTCTACTGCC;Primer set 4 : CATTTGGATTCTGCTTTCGACATTC, CAGGGTGGCCATGTTAGGAACTG.Primer set 5 : Mkrn3 were as follows:PCR primers used for ChIP assays of CCTTAGGTTGTGAGACTCCTTCCAATA, TCTTCCTTTCCTCACCCACTAACTTGAA;Region 1 (−1091 to −971): CGGTGAAGCCCTAGGAATGGTGT, GGAGCGAAGTGCATCGATTTTTGT;Region 2 (−104 to +18): GGTGGTGGATCATCAAGCGCA, GGCCAGGCGAAGCACAGAATG;Region 3 (+1526 to +1639): GAACAGAGGATACTCCCACTAAAACCAA, TGTGCCAAGGGGTTCTTTGTTCA.Region 4 (+4635 to +4743): Ndn were as follows:PCR primers used for ChIP assays of CCTACCACCCTTCTGGCTTCCCATY1210: CTCGGTGGAGACCAGCAGAGGAA.TY1211: Magel2 were as follows:PCR primers used for ChIP assays of CGCCCCTCTGAACAATCCACTTGTY1212: TCACACAGAGTGTCGGCTGGCTCTY1213: Snrpn locus in cultured cells, a total of at least three independent immunoprecipitations were performed for each antibody, and quantitative real-time PCR with each primer set for each immunoprecipitation was performed in duplicate. ChIP analysis of YY1 and NRF-1 in mouse tissues consisted of two independent immunoprecipitations and duplicate quantitative PCR reactions for each primer set and immunoprecipitated sample. For ChIP analysis of Mkrn3 for Sp1 and NRF-2 binding in cultured cells, a total of four immunoprecipitations were performed for each antibody, and quantitative real-time PCR with each primer set for each immunoprecipitation was performed in duplicate. Mkrn3 ChIP analysis of YY1 in cultured cells, and YY1 and NRF-2 analysis in mouse tissues, consisted of two independent immunoprecipitations and duplicate quantitative PCR reactions for each primer set and immunoprecipitated sample. Antibodies for ChIP analyses were obtained from Santa Cruz Biotechnology.For ChIP analysis of YY1 and NRF-1 binding to the 7 cells, or primary brain cells from 4–6 mouse brains prepared as described above, were resuspended in 45 ml of fresh RPMI-1640 and cross-linked with 1% formaldehyde at room temperature for 10 minutes. Cells were lysed with 20 strokes of a Dounce homogenizer on ice . Lysed nuclei were washed as described then resuspended in 7.24 ml of 1X NEBuffer #2 (NEB) and digested with 8,000 U of EcoRI (NEB) at 37°C overnight. Ligation was performed in 160 ml of ligation reaction buffer; for the ligation reaction, the DNA was divided in half, with one half receiving T4 ligase and the other half serving as a non-ligated control which received the same volume of buffer rather than ligase. Then samples were heated to 65°C for 30′, treated with 10 mg Proteinase K (Invitrogen) overnight at 65°C, then an additional 10 mg Proteinase K was added and the samples were incubated for an additional 2 hours at 42°C. Samples were phenol extracted, ethanol precipitated, desalted, redissolved in 1 ml 1X TE buffer, and treated with 10 µg RNase A to yield final 3C templates. Novel ligation products formed via long-rang interactions in vivo were detected by PCR using the following 3C PCR primers: anchor, CATTTCCTTAACCTGGTGCTACGATAG; a, GTGCCACACAGTTTGAGAACAGGAT; d, ATATGTGCATTTGTCGTTTGTGTATGTATC; e, CACAGTTAAAACGAACACAATAGTCCCAA. For a 25 ul PCR reaction using 3C primers, 1–2 µl template DNA was mixed with 10 pmol of each primer, 5 nmol of each dATP, dTTP, dGTP, dCTP, 0.625 units of Hotstar Taq DNA polymerase (Qiagen). All PCR reactions were performed with 45 cycles using the following program: 95°C for 15 minutes, followed by 44 cycles of 95°C for 45 seconds, 57°C for 60 seconds, 72°C for 45 seconds, then an elongation of 10 minutes at 72°C. PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed. The DNA sequences of the novel ligated fragments were confirmed by sequencing; PCR-amplified DNA fragments were isolated directly from the gel with the Qiagen Gel Purification Kit and ligated into a Topo TA vector . For each PCR product of the 3C assays, at least 6 clones were sequenced to verify that the expected 3C PCR product was amplified.3C assays were performed as described previously Snrpn gene to identify potential regulatory regions and their associated cis- and trans-acting regulatory elements. We first mapped DNase I hypersensitive (DH) sites in the 5′ region of the Snrpn gene in primary brain cells. Then selected DH sites were further characterized to identify binding of specific transcription factors to sites within these DH regions. Distal paternally-expressed genes in the AS/PWS domain regulated by the PWS-IC  were also examined for association with specific transcription factors and physical proximity to the PWS-IC to provide insight into potential mechanisms of PWS-IC function.We have postulated previously that regulatory factors associated with the PWS-IC on the paternally-inherited chromosome may mediate PWS-IC function Snrpn and other genes in the AS/PWS region expressed exclusively from the paternally-inherited chromosome are generally expressed at elevated levels in the brain Snrpn locus, from position −6346 to +2373 (relative to the transcription initiation site), primary brain cells were subjected to DNase I treatment and Southern blotting using EcoRI and hybridization probe A. Results of the EcoRI Southern blot analysis are shown in Snrpn promoter region, and DHS3 marks the approximate 3′ edge of the differentially methylated 5′ CpG island that starts at the Snrpn promoter and spans exon 1 and part of intron 1. In contrast, no prominent hypersensitive bands were reproducibly detected in brain cells carrying a paternal PWS-IC deletion, though numerous faint bands could be detected in some EcoRI blots as shown in Because SNPRN gene in human lymphoblasts SNRPN gene Snrpn, further analysis was performed using NcoI and hybridization probe C. As shown in To confirm DH sites detected by EcoRI digestion, DNase-treated samples were digested with TaqI and the Southern blot was hybridized with probe B . In addiSnrpn gene in primary brain cells, all of which are specific to the paternal chromosome. Based on the sizes of the hypersensitive bands and the positions of the restriction sites used to detect them, the positions of these DH sites relative to the Snrpn gene are summarized in Snrpn gene We now have identified and determined the allele-specificity of six major DH sites in the 5′ region of the murine Snrpn promoter region (DHS2) by performing ligation-mediated PCR (LMPCR) in vivo footprint analysis on primary mouse brain cells. To examine the maternal and paternal alleles independently, in vivo footprinting analysis was performed on brain cells isolated from TgPWS(del) and TgAS(del) mice that carry a deletion of the entire AS/PWS domain on either the paternally-inherited or maternally-inherited chromosome, respectively To identify specific regulatory sites and factors within these major paternal DH sites, we first analyzed the in vivo footprint analysis in mouse brain cells. All of the footprints identified lie within a 100 bp region which overlaps the Snrpn minimal promoter defined by Hershko et al. in transient expression assays in vivo treated samples of intact TgAS(del) cells (lanes 6–8) relative to naked DNA from TgAS(del) cells (lane 5). This altered pattern of band intensity was not observed on the maternal allele (lanes 1–4) in TgPWS(del) cells, which indicates that footprint P1 is specific to the paternal allele. Analysis of the upper strand with primer set 1 (AS(del) cells). A second footprint specific to the paternal allele, P3, was detected with this primer set at position −61 as a band of decreased intensity (lanes 6–8) relative to naked DNA (lane 5). Neither footprint was detected in TgPWS(del) cells carrying the maternal allele (lanes 1–4). AS(del) cells (lane 5) and in samples containing the maternal allele in TgPWS(del) cells which were of the same intensity. This footprint (P4) was subtle in comparison to other footprinted sites, but was highly reproducible in multiple independent sequencing gels, LMPCR reactions, and preparations of DMS-treated samples. Footprint P5 (in vivo treated samples (lanes 6–8) of TgAS(del) cells relative to naked DNA from the same cells (lane 5).er set 1  identifiprint P5  was detein vivo DMS footprint pattern for the Snrpn promoter on the paternal allele and shows potential transcription factor binding sites that were identified by analysis with the TRANSFAC transcription factor database. The sequence associated with P1 is a potential binding site for the transcription factor CTCF which is a ubiquitous transcription factor involved in numerous mechanisms of gene regulation, including the regulation of other imprinted gene clusters such as the H19 and Igf2 cluster and long-range organization of the genome SNRPN promoter showed several potential CTCF binding sites, two of which were also associated with DMS in vivo footprints; however, ChIP assays were unable to confirm binding of CTCF to the human promoter SNRPN promoters. Footprints P3 and P4 lie within potential binding sites for nuclear respiratory factor-1 (NRF-1), which also has been shown to be associated with the human SNRPN locus SNRPN promoter region, no evidence for binding of YY1 was detected by in vivo footprinting in the minimal mouse Snrpn promoter.in vivo footprinting analysis was performed on spleen cells isolated from TgPWS(del) and TgAS(del) mice. However, in contrast to our results in brain cells, no in vivo footprints were detected in the promoter region in spleen cells (data not shown).A similar DMS in vivo footprint analysis for binding of NRF-1 and YY1 to the Snrpn promoter region (DHS2), we performed ChIP analysis using antibodies against NRF-1 and YY1. ChIP assays for NRF-1 and YY1 were also performed on the murine CAS  because the human CAS was shown to be bound by NRF-1 and YY1 on the paternal allele Snrpn transcription initiation site. ChIP analyses focused on NRF-1 and YY1 because: 1) NRF's and YY1 bind to other paternally expressed genes in the AS/PWS domain (see below), 2) binding of nuclear respiratory factors suggest a potential relationship with regulation of respiration, and 3) YY1 binding sites have been reported to be associated with regulatory regions of other imprinted loci To confirm Snrpn promoter region (primer set 2), a region ∼900 bp downstream of the transcription initiation site (primer set 3), within the murine CAS sequence (primer set 4), and the potential YY1 binding site at DHS6 (primer set 5). ChIP assays were performed on primary newborn brain and adult spleen cells prepared from C57Bl/6 mice. As shown in Snrpn promoter region as well as in the mouse CAS in brain cells. No evidence for binding of NRF-1 was detected using primer sets 1, 3, or 5 in brain cells. ChIP analysis detected binding of YY1 in the region the CAS as well as the potential YY1 binding site at DHS6 in brain cells, but not in the Snrpn promoter or regions associated with primers 1 or 3. Because these ChIP assays were performed on brain cells from C57Bl/6 mice, it was not possible to determine which allele(s) was bound by NRF-1 and YY1. Nonetheless, because Snrpn is expressed only from the paternally-inherited allele, all in vivo footprints in the promoter region were detected only on the paternal allele, and the binding of NRF-1 and YY1 in the human SNRPN CAS occurred only on the paternal allele, it is very likely that the results from these ChIP assays reflect binding of NRF-1 and YY1 specifically on the paternal allele in the Snprn locus in brain cells. ChIP assays using antibodies against CTCF detected little or no binding of CTCF within the promoter region in brain cells (data not shown), consistent with previous ChIP analysis of the human SNRPN promoter region in lymphoblasts which also showed no detectable binding of CTCF though the human promoter also exhibited in vivo footprints at or adjacent to potential CTCF sites in vivo footprint analysis of the minimal Snrpn promoter region in spleen cells which detected no in vivo footprints on either the paternal or maternal alleles.As indicated in Mkrn3 locus. This allowed us to compare regulatory elements between the murine PWS-IC region (and Snrpn gene) and one of its target genes. We first determined the location of DH sites in the Mkrn3 locus in TgPWSdel and TgASdel cells, immortalized cultured fibroblast cell lines derived from the mouse model of PWS in which a transgene insertion led to deletion of the AS/PWS region (the mouse line termed TgPWS/AS(del); PWSdel cells derived from these mice in which the deletion is paternally inherited allows for analysis of the maternal allele only, whereas TgASdel cells in which the deletion is maternally inherited allows for analysis of the paternal allele only. To verify that DNA methylation imprints in the Mkrn3 locus are maintained in cultured TgASdel and TgPWSdel fibroblast cells, the Mkrn3 promoter region in these cells was subjected to high resolution DNA methylation analysis by sodium bisulfite genomic sequencing. Analysis of the 5′ CpG island, including the promoter region, from positions −131 to +162 showed hypermethylation of the maternal allele in TgPWSdel cells and an essentially unmethylated paternal promoter in TgASdel cells (data not shown). Thus, the expected differential DNA methylation imprints are present at the Mkrn3 locus in the immortalized cultured fibroblasts derived from the TgPWSdel and TgASdel mouse lines. Maintenance of imprinting in the TgPWSdel and TgASdel cell lines also was verified by analysis of allele-specific gene expression within the AS/PWS domain (data not shown). Thus, the Mkrn3 locus is correctly imprinted in the TgPWSdel and TgASdel cell lines and that these lines can serve as a cell culture system to examine aspects of imprinted regulation of the Mkrn3 gene.To identify cis- and trans-acting regulatory elements in a distal gene expressed specifically from the paternally-inherited chromosome and regulated by the PWS-IC, we analyzed transcription factor binding to the murine PWSdel cells which contain the maternally-inherited allele, only the expected 5.2 kb genomic EcoRI DNA fragment was detected. In contrast, three subfragments generated by DNase hypersensitivity  specific to the paternally-inherited allele were detected in TgASdel cells. The strongest DNase-dependent subfragment, representing DH site 2 (DHS2), was centered ∼100 bp upstream of the transcription initiation site of Mkrn3, whereas DHS1 mapped ∼190 bp downstream of the transcription initiation site. The weakest hypersensitive site, DHS3, was located ∼600 bp upstream of the transcription initiation site. Because DHS2 was the strongest DH site in our analysis and localized to the Mkrn3 promoter, all subsequent studies were focused on this region.As shown in in vivo, LMPCR in vivo footprinting was performed across DHS2  from positions −200 to +150 on both the upper and lower strands using DMS. Four LMPCR primer sets were used, 1Ua and 1Ub to footprint the upper strand, and 1La and 1Lb to footprint the lower strand. Cell lines TgPWSdel and TgASdel were used to footprint the maternal and paternal alleles, respectively, as described above.DNA sequence analysis using TRANSFAC for the presence of potential transcription factor binding sites within DHS2 revealed binding sites for various factors including multiple nuclear respiratory factor-2 (NRF-2), YY1, and Sp1 sites, as well as a binding site for STAT1. Like NRF-1, NRF-2 is a transcription factor commonly found as an activator of nuclear genes associated with mitochondrial biosynthesis and oxidative phosphorylation in vivo footprinting pattern of the upper strand from positions −86 to +19 using LMPCR primer set 1Ua. Comparison of the DMS modification and cleavage patterns between in vivo-treated samples  and naked DNA treated with DMS (lanes 0) revealed footprinted sites only on the paternal allele ; no consistent footprints were observed in this region on the maternal allele (in TgPWSdel cells). Sites of DMS protection or enhanced DMS reactivity were clearly detected at guanines associated with potential binding sites for STAT1 , overlapping binding sequences for NRF-2 and Sp1 , NRF-2 , overlapping NRF-2 and Sp1 binding sites , and Sp1 . Footprinted guanine nucleotides at positions +4, +7, and +14 on the paternal allele located just downstream of the primary transcription initiation site are associated with a sequence that bears a resemblance to a binding site for YY1 but is not an absolute match for the consensus YY1 binding sequence. No other footprints were detected on the upper strand in the region from −120 to +46 using primer set 1Ua in either the TgASdel or TgPWSdel cells.Mkrn3 promoter region further upstream of position −120 on the upper stand, a second primer set, 1Ub, was used to detect footprints in the region from positions −50 to −220  across or nearby on the opposite strand, thereby confirming the presence of most footprints in the region. All of the footprinted sites in this 350 bp region are located within or adjacent to binding sites for the transcription factors NRF-2, Sp1, YY1, or STAT1 and generally in regions of the promoter that are strongly conserved in the mouse, rat, and human Mrkn3 promoter sequences. Our finding of in vivo footprints at potential YY1 and NRF-2 binding sites in the Mkrn3 locus indicate a notable similarity of transcription factors interacting with both Mkrn3 and the PWS-IC/Snprn gene.in vivo footprinting studies that suggested binding of YY1, Sp1, and NRF-2 in the Mkrn3 promoter, ChIP analysis was performed at four sites across the Mkrn3 locus (ASdel or TgPWSdel cells: ∼1 kb upstream of the transcription initiation site (region 1), in the immediate 5′ flanking region from positions −105 to −5 (region 2), within the body of the gene from positions +1639 to +1526 (region 3), and ∼4.7 kb downstream of the transcription initiation site (region 4). Mkrn3 locus using antibodies against NRF-2, Sp1, and YY1. NRF-2 is highly enriched in the promoter region on the paternal allele , with no other regions assayed within or flanking Mkrn3 showing significant levels of NRF-2 binding. This result is consistent with in vivo footprinting data that indicate transcription factor occupancy at multiple potential NRF-2 binding sites  reflected a similar pattern of factor binding within normal intact mouse tissues, ChIP analysis was performed on primary brain and spleen cell preparations from C57BL/6 mice. As shown in Mkrn3 promoter region  on the paternal allele because the Mkrn3 gene has been shown to be paternally expressed in mouse brain tissue Mkrn3 promoter in spleen cells compared to brain cells . Thus, both Mkrn3 and Ndn – two genes regulated by the PWS-IC – are bound by NRF's and YY1, suggesting these factors could contribute to the coordinate expression and regulation of Mkrn3 and Ndn on the paternal chromosome by the PWS-IC. However, similar ChIP analysis of the mouse Magel2 promoter region demonstrated little, if any, association of NRF-1 or YY1 with the promoter region in mouse brain, spleen, and TgASdel or TgPWSdel cells (data not shown). This is consistent with sequence analysis of the Magel2 promoter which showed an absence of canonical NRF-1, NRF-2, or YY1 sites.To examine the possibility that similar transcription factors may also be associated with other paternally-expressed loci regulated by the PWS-IC within the AS/PWS imprinted domain, we performed ChIP analysis of the mouse Mkrn3 promoter region led us to examine whether or not these two loci separated by >2 mb of genomic DNA are in close physical proximity specifically on the paternal chromosome  in conjunction with primers from the Mkrn3 and Snrpn loci. PCR products were run on agarose gels and stained with ethidium bromide to visualize bands derived from amplification across novel junctions of ligated EcoRI fragments that were in close proximity in vivo. As shown in Mkrn3 locus and upstream of the Snrpn promoter region were used in combination with the PWS-IC anchor primer. Primers a and e are located ∼1250 bp downstream and ∼3360 bp upstream of the Mkrn3 transcription initiation site, respectively, and are contained on the same 5.2 kb genomic EcoRI fragment . Primer d is located 17.9 kb upstream of the Snrpn promoter and ∼20 kb upstream of the anchor primer.The presence of NRF's and YY1 in both the PWS-IC and romosome . 3C anala and the 3C template containing the paternal allele, demonstrating that a long-range interaction is occurring between the PWS-IC and the Mkrn3 locus on the paternal chromosome . In addition, we detected a PCR product between the anchor primer and primer d on both the maternal and paternal chromosomes , or that on both the maternal and paternal chromosomes, the region containing primer d is engaging in a specific interactions with the PWS-IC and reflecting a previously unknown functional interaction; at this time, we cannot distinguish between these two possibilities. The result observed with primer d is also notable because it demonstrates that the lack of PCR products from the TgPWSdel  3C template using primer a was not simply due to a general inability to PCR-amplify 3C templates generated from the TgPWSdel cells. All negative control lanes  showed no detectable PCR product, as expected. All PCR products from 3C templates were extracted from gels and cloned, then multiple clones from each PCR reaction were sequenced to verify that the expected novel ligation products between two distant genomic EcoRI fragments were amplified from the 3C templates. All of the sequenced clones for each PCR product . These data suggest a long-range interaction in vivo between the Mkrn3 locus and PWS-IC exclusively on the paternal chromosome in mouse brain cells, results similar to that in mouse fibroblasts. Again, primer d in conjunction with the anchor primer generated a readily detectable PCR product from both the maternal and paternal chromosomes of brain cells  are located within the 6 kb interval that currently defines the murine PWS-IC. Thus, we hypothesize that DHS 1–5 are major components of the PWS-IC and play a role in mediating PWS-IC function. DHS2 and DHS5 in mouse brain cells correspond to the two DH sites found in human lymphoblasts . Comparing our data with Snrpn DNase I hypersensitivity data from the UCSC Genome Browser (http://genome.ucsc.edu), strong distinct peaks of hypersensitivity corresponding to DH sites 2–5 are reported in the Genome Browser in 8 week adult mouse brains, day 14.5 embryo brains, and mouse ES cells, and less distinct peaks at these DH sites in 8 week adult mouse cerebrum and cerebellum tissue. The parent-of-origin of the DH sites in the UCSC Genome Browser was not determined. Weak or no peaks of hypersensitivity are reported in the Genome Browser for DH sites 1 and 6 in most tissue and cell samples analyzed. However, analysis of ES cells shows two strong peaks of hypersensitivity upstream of the Snrpn promoter at positions −1.3 kb and −3 kb. The peak at −3 kb corresponds to DH site 1 in our newborn brain cells, but we have not detected a hypersensitive site at position −1.3 kb in our assays of newborn brain on either the paternal or maternal alleles. Furthermore, other brain samples reported on the Genome Browser also do not detect this −1.3 kb DH site. Thus, an apparent ES cell-specific DH site of unknown allele-specificity is located between DHS1 and DHS2. In addition, no other cell or tissue shown on the Genome Browser show a strong DH site corresponding to DHS6 in our primary brain cells, though multiple cells/tissues show a weak signal corresponding to the position of DHS6 from our study. We cannot explain why DHS1 is readily detected only in newborn brain and ES cells, or why no other reported cell or tissue other than newborn brain cells shows a major hypersensitive site corresponding to DHS6. Based on our finding of six DH sites in the PWS-IC of primary mouse brain cells, we would predict that the PWS-IC/SNRPN gene in human primary brain cells contains more than the two DH sites we identified in on the paternal chromosome in human lymphoblasts Previous studies of the PW-IC region in human lymphoblasts have shown it to be associated with two DH sites exclusively on the paternal chromosome, one located in the Snprn locus that defines the minimal PWS-IC in the mouse contains DH sites 1 through 5 and yielded a complete imprinting defect in the AS/PWS domain when paternally transmitted Snrpn promoter region and exon 1, and including only DHS2, showed no disruption of either imprinted gene expression nor DNA methylation patterns when paternally  inherited Snrpn promoter region (DHS2) is not essential for PWS-IC function in the mouse. However, a 4.8 kb deletion of the Snrpn 5′ region that included the same 0.9 kb region of the promoter as well as sequences ∼1.4 kb upstream and ∼2.5 kb downstream of the promoter yielded mice exhibiting growth retardation, a partial imprinting defect, and 40–50% perinatal lethality when paternally inherited Snrpn locus which extended from Snrpn intron 1 downstream to Ube3A and removed DH site 6 showed no apparent effect on imprinting The location of the six major DH sites we have identified here may provide insight into the effects of various targeted deletions of the PWS-IC in mice see  assumingMkrn3 and Ndn, two distal genes regulated by the murine PWS-IC located >2 mb from the IC. All factors are bound exclusively on the paternally-inherited alleles. Strikingly, all of these regulatory regions are associated with nuclear respiratory factors, either NRF-1 or NRF-2. NRF-1 is associated with the Snrpn promoter region, the CAS  have been shown to contain clustered YY1 binding sites Peg3 and Gnas domains, as well as the Snrpn locus The PWS-IC has been postulated to act as a positive regulator of paternally-expressed genes within the AS/PWS domain SNRPN gene as a conserved cis-acting regulatory region associated with DNase hypersensitivity and enhancer activity; however, it was located just downstream and outside of the human PWS-SRO Snprn locus We previously identified the intronic CAS in the human Snrpn identified here are associated with several cis-acting elements identified in earlier published reports that used the minimal Snrpn promoter in either transfection studies or in a mini-transgene Snrpn promoter activity Snrpn mini-transgene SNRPN promoter cis-acting elements  identified in these mini-transgene studies were not footprinted in our experiments; at this time we cannot determine if these latter cis-acting sites are simply not occupied by regulatory proteins in the cells we analyzed, or if trans-acting factors associated with these sites do not interact directly with guanine residues and, therefore, were not detected by DMS footprinting. Analysis of imprinting of the Snrpn mini-transgene also led to the identification of sequences in the 1st intron of Snrpn that are identical to the cis-acting elements in the Snrpn promoter that were required for correct imprinting of the mini-transgene In vivo footprinted sites in ASdel mice which carry a deletion of the murine AS/PWS region on the maternally-inherited chromosome ASdel phenotypes, or how mutation or disruption of imprinting in the AS/PWS domain could affect regulation of energy metabolism.The presence of both NRF-1 and NRF-2 in multiple regulatory regions and multiple genes within the AS/PWS domain suggests a functional relationship between regulation of the AS/PWS domain and regulation of cellular respiration and/or mitochondrial biosynthesis. This potential relationship between energy metabolism and AS/PWS is intriguing because of the obesity phenotype characteristic of PWS patients as well as the late onset obesity seen in TgMagel2 locus for which we find no strong evidence for binding of these factors to the promoter region on the paternal chromosome in mouse brain, spleen, TgASdel or TgPWSdel cells. It is possible that regulation of Magel2 may include additional unidentified regulatory region(s) outside of the promoter region that bind YY1 and/or NRF's, or that Magel2 is regulated by the PWS-IC via a different mechanism than Mkrn3/Ndn that does not involve YY1 and NRF's.It appears that an exception to regulation of paternally-expressed genes by nuclear respiratory factors and YY1 may be the mouse Mkrn3, Ndn) on the paternal chromosome because of the tissue-specific pattern of expression in brain cells exhibited by these target genes Snrpn promoter region in brain cells did not identify binding sites of known neuronal transcription factors.In addition to ubiquitous transcription factors , it is likely that tissue/cell type-specific transcription factors also interact with the PWS-IC and/or the regulatory regions of its target genes  into a structure similar to an active chromatin hub  an essentially equivalent result because of the low resolution of 3C assays, suggesting that a broad region containing and surrounding MKRN3/Mkrn3 lies in proximity to the PWS-IC on the paternal chromosome; 2) a three-dimensional organization on the paternal chromosome in which multiple regions (presumably regulatory regions) across the distal paternally-expressed gene cluster all lie in proximity to the PWS-IC (perhaps within the context of a transcription factory); and/or, 3) the possibility that interactions with the PWS-IC on the paternal chromosome are dynamic and that at different times within a given cell different regions within the distal gene cluster transiently interact with the PWS-IC (within a transcription factory). The fact that the region of the mouse Mkrn3 locus that interacts with the PWS-IC contains a DMR, while Rabinovitz et al. find the interacting region downstream of the human MKRN3 gene to be unmethylated on both alleles, further suggest that methylation patterns of the region interacting with the PWS-IC are unlikely to govern this interaction.Rabinovitz et al. used 3C analysis of the human AS/PWS domain to demonstrate an allele-specific long-range interaction between the PWS-IC and a region located between the Currently, a direct role for transcription factors in PWS-IC function has not yet been established. However, coordinate and developmental regulation of other multigene domains such as the β-globin domain and T helper type 2 cytokine locus have been shown to be dependent upon specific transcription factors that also mediate long-range intrachromosomal interactions within these domains


Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var) are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD) of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2) has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.Histone modifications are important regulators of gene expression in all eukaryotes. In  Plasmodium falciparum, histone modifications have been shown to play a significant role in controlling gene expression involved in many aspects of their lifecycle, including the complex gene expression patterns associated with antigenic variation. The various histone modifications that are found within the parasite's genome have now been extensively cataloged, and the enzymes that are responsible for adding and removing them have been identified. However, how these enzymes are recruited to specific regions of the genome to coordinate gene expression is not understood. In this paper, we provide the first evidence for recruitment of a unique histone methyltransferase to specific regions of the genome through its tethering to RNA polymerase II. We find that disruption of this interaction results in major changes in expression patterns of genes involved in antigenic variation, demonstrating the importance of regulated recruitment of histone modifiers for parasite biology.Chemical modifications to histones, the proteins that serve as the primary units of chromatin, often determine whether specific genes are actively transcribed or condensed into transcriptionally silent regions of the genome. In the malaria parasite  Plasmodium falciparum genome Plasmodium biology, and identified these enzymes as potential targets for disease intervention.The use of alternative histone modifications is an important mechanism utilized by eukaryotic cells to control many aspects of nuclear biology, including patterns of gene expression, DNA replication, chromosome segregation and overall genome organization. In malaria parasites, histone modifications have been shown to play an important role in regulating many features of host-parasite interactions, including red blood cell invasion Plasmodium.While the role histone modifications play in regulating gene expression is well established, an important and poorly understood aspect of this process is how various histone modifiers get directed to specific places within the genome. In some organisms, the production of noncoding RNAs (ncRNAs) has been implicated in the recruitment of various histone modifiers to specific loci Su(var)3–9, Enhancer of Zeste, Trithorax 2) class of methyltransferases. These enzymes deposit a tri-methyl group on the lysine in the 36th position of histone H3 (H3K36me3), which can subsequently be removed by its cognate demethylase JmjC1 One of the few well-characterized mechanisms of directed recruitment of a histone modifier is that of the SET2  and biochemical work demonstrated that this protein possesses histone methyltransferase activity et al. generated a knockout of PfSET2  and showed that this resulted in a profound reduction of H3K36me3, providing strong evidence that PfSET2 deposits this mark on P. falciparum chromatin var gene family. Moreover, knockout of PfSET2 resulted in disruption of mutually exclusive expression while displaying no other detectable phenotype. Thus in P. falciparum, SET2 appears to be devoted to regulating mutually exclusive expression and antigenic variation. This is in sharp contrast to higher eukaryotes where SET2 deposits H3K36me3 throughout the genome, within virtually all coding regions that are actively transcribed. Given these dramatic differences in H3K36me3 distribution and function, it is unclear how PfSET2 specifically marks only very narrow regions of the parasite's genome and whether RNA pol II plays a role in its recruitment, as it does in higher eukaryotes. Understanding how PfSET2 gets targeted to genes that undergo mutually exclusive expression will shed light on a key step in the regulation of antigenic variation by P. falciparum.Phylogenetic studies of histone methyltransferases in var gene regulation. The multi-copy var gene family encodes the primary antigenic determinant, PfEMP1, expressed on the surface of infected red blood cells and has been shown to be transcriptionally regulated epigenetically in vitro protein binding assays and the creation of dominant-negative transgenic lines, we confirmed that the histone methyltransferase PfSET2 is an important player in var gene regulation. More importantly, we identified a region of the protein that binds directly to RNA pol II in vitro and showed that disruption of this interaction in transgenic parasites drastically alters var gene expression patterns. Surprisingly, unlike the SET2 orthologs of higher eukaryotes, PfSET2 binds to RNA pol II when it is unphosphorylated, a characteristic inconsistent with its recruitment during the transcription of mRNAs and providing a possible mechanism for how recruitment of PfSET2 is limited to very narrow regions of the genome. These data suggest a model in which PfSET2 is recruited to var gene loci by RNA pol II during the production of previously described var-specific ncRNAs, and that the resulting histone mark plays a role in coordinating mutually exclusive expression and var expression switching.Examination of RNA pol II from malaria parasites previously identified an unusual expansion of the CTD in parasites that infect primates when compared to rodent malaria parasites Unmethylated, mono, di and trimethylated peptides that include and flank the H3K36 residue were synthesized and conjugated at Rockefeller University using the following epitope: RKSAPISAGI-K-KPHRYRPGT. Two rabbits were immunized with the trimethylated peptides and rabbit polyclonal antibodies were obtained via a 77-day protocol (Covance). To increase specificity, the anti-H3K36me3 serum was pre-absorbed by incubating with unmethylated, monomethylated, and dimethylated peptides. 1 ml of a 1∶10 dilution of rabbit serum was incubated with 15 µl of a 1 mg/ml solution of each peptide for 30 min at 37 degrees, rocking. Specificity of the final antibody preparation was determined using peptide competition assays and verified through assaying detection of 15 ug of purified unmethylated, monomethylated, dimethylated and trimethylated peptides through slot-blot assays.9 cells of the NF54 line of P. falciparum were lysed in RIPA buffer , plus mammalian protease inhibitor cocktail (Sigma) supplemented with 200 mg ml-1 PMSF and 4 milligrams ml-1 pepstatin. 1 to 15 micrograms of lysates were separated by SDS-PAGE using a 15% acrylamide gel and transferred to a PVDF membrane. Membranes were blocked overnight with 5% milk. Primary antibodies were detected with horseradish-peroxidase-conjugated sheep anti-rabbit antibodies. Primary antibodies were either the anti-H3K36me3 described above or commercially available antibodies against H3-core.A total of 2×10P. falciparum histones were extracted and purified by a modified protocol as previously described by Schecter, et al.2O for 2 h to overnight. The sample was retrieved and lyophilized in a SpeedVac overnight. The powder was diluted in ∼100 µL H2O. SDS-PAGE/Western was performed to detect the H3K36me3 mark.var expression profiles Chromatin Immunoprecipitation was performed as previously described P. falciparum and P. berghei were used as template for PCR amplification of the R2 and R3 regions (R2R3) of the C-terminal domain (CTD) of Rpb1. The sequence encoding a 3× Flag-tag was incorporated into the reverse primer in each reaction. The amplified products were cloned into the pSUMO expression vector, which includes an N-terminal His6 tag. Recombinant proteins were expressed in BL21(DE3) E. coli cells, which were subsequently purified using Nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen). Proteins were concentrated and stored in 50 mM Tris, pH 8.0, 0.1 M NaCl and 10% glycerol. Primers used for PCR amplification are described in Genomic DNA from (1–359), PfSET2(351–828), PfSET2(1060–1535), PfSET2(1540–2080) and PfSET2(2264–2548)) were amplified by PCR using P. falciparum NF54 genomic DNA as template. The DNA sequence encoding the ySRI was amplified by PCR using Sacharomyces cerevisiae genomic DNA. The sequence encoding an HA epitope tag was incorporated into the reverse primers to create HA-tagged versions of each fragment. The sequence of the PCR primers is provided in E. coli cells as described above. Cultures expressing the recombinant proteins were harvested and lysed as described above, with the exception that EDTA and MgCl2 were added to the lysis buffer to final concentrations of 0.5 mM and 1.0 mM respectively. Cleared lysates were used for co-immunoprecipitation assays and expression of truncates were monitored by SDS-PAGE and Western blot.The DNA sequences encoding five different PfSET2 fragments . Phosphorylation of the CTD was monitored by SDS-PAGE and Western blot.Purified Flag-tagged R2R3 portions of the CTD of Rpb1 from Lysates from bacterial cultures expressing HA-tagged PfSET2 fragments and the ySRI were incubated with purified, recombinant Rpb1 CTD in both phosphorylated and unphosphorylated forms in 3% BSA at 4°C for 16 h. Reactions were precleared on control agarose resin (Thermoscientific) and loaded onto Protein A agarose beads (Millipore) for two hours. Bead were washed extensively three times with 50 mM Tris, pH 8.0, 0.5 M NaCl, 0.5% Tween-20 and 10% glycerol. Polyclonal anti-HA antibody  was used to immunoprecipitate PfSET2 and any interacting proteins. Immunoprecipitated proteins were separated by SDS-PAGE and the CTD of Rpb1 was detected by Western blotting using anti-Flag antibody.Proteins were separated on 4–20% Tris-HCl acrylamide gels and analyzed by Western blot. Antibodies were detected using the SuperSignal West Pico Chemiluminescent substrate reagents from Thermoscientific. Monoclonal mouse anti-HA and anti-His antibodies (Sigma) were used at dilutions of 1∶2000 and 1∶6250 respectively. Monoclonal anti-Flag (Sigma) was used at a dilution of 1∶4000. All antibodies were detected using anti-mouse IgG (whole molecule) peroxidase, produced in rabbit (Sigma) at a dilution of 1∶100,000.et al.et al.The dominant negative experiments utilized clonally stable NF54 bulk cultures previously generated by Frank RNA was extracted as previously described var gene PF3D7-0421100 were used for qRT-PCR. The standard curve method was used to quantitate mRNA levels . All reactions were compared to control genes encoding acyl-tRNA synthetase, actin and a P. falciparum circumsporozoite protein. All reactions were performed using ITAQ SYBR supermix (Bio-Rad) in 10 uL reactions using an ABI Prism 7900 HT real time PCR machine. For gene expression studies, cDNA was evaluated for genomic DNA contamination prior to assessing var gene expression. To detect transcription of all var genes, we employed a real-time PCR set developed by Salanti et al.http://www.appliedbiosystems.com). The formula 2ΔCT was used to convert the ΔCT values to relative copy numbers. All reactions were performed in duplicate and averaged in Microsoft Excel. Transcriptional profiles of all the individual var genes are presented as pie graphs prepared as described by Frank et al.var gene was defined as the dominant gene.For chromatin immunoprecipitation studies, forward and reverse primer pairs with complementary sequences within the Plasmodium species found that the CTD is uniquely expanded in parasites that infect primates Previous examination of the CTD of RNA pol II from numerous primate and non-primate P. falciparum, P. vivax and P. knowlesi) with the genomes of three rodent parasites . Specifically, we used a collection of 202 transcription factors and histone modifiers identified in P. falciparum by Bischoff et al.Plasmodium species. Three protein encoding genes were identified that were present within the genomes of P. falciparum, P. knowlesi and P. vivax but missing in all three rodent lineages. These included the histone methyltransferase PfSet2 (PF3D7_1322100) and its complementary demethylase PfJmjC1 (PF3D7_0809900) (th position of histone H3 (H3K36) respectively, thus suggesting they represent a complementary pair of histone modifiers specific to primate parasites. Of particular interest for this study, SET2 orthologs from higher eukaryotes have been shown to directly bind to the CTD of RNA pol II, adding confidence to our hypothesis that the presence of these histone modifiers might be directly related to the expanded CTD found in primate parasites. The third gene found to be unique to primate parasites, a predicted zinc-finger transcription factor expressed in gametocytes (PF3D7_1134600), will not be described further here.As a first step to test this hypothesis, we compared the genome sequences of three primate parasites (0809900) . These tSet2 and JmjC1 are in fact absent from the rodent parasite genomes and not simply mis-annotated or not identified due to low levels of sequence similarity, we examined the genome assemblies for syntenic open reading frames that could represent the missing genes. All six genomes displayed near perfect synteny in the regions surrounding both PfSet2 and PfJmjC1, however these two genes are entirely missing from the genomes of all three rodent parasites (P. berghei genome flanking the syntenic positions where Set2 and JmjC1 were predicted to exist. PCR amplifications yielded the predicted product in both cases and confirmed that neither Set2 nor JmjC1 exist at these positions in the genome (not shown). Degenerate PCR primers to Set2 and JmjC1 also failed to amplify products using P. berghei genomic DNA as template, further suggesting that these two genes are likely not present within the rodent parasite genomes.To verify that orthologs of arasites . No predth position (H3K36me3) P. falciparum. Conflicting reports exist in the literature regarding the presence of this particular histone modification. Issar and colleagues were unable to detect H3K36me3 using a commercially available antibody preparation generated against yeast H3K36me3 et al. also did not detect this modification in a proteomic analysis of chromatin extracted from cultured malaria parasites et al. detected H3K36me3 within P. falciparum chromatin and also detected the appropriate methyltransferase activity using recombinant PfSET2 on H3 core et al. detected H3K36me3 using an alternative source of commercially available antibodies, and found that the level of this modification was greatly reduced with PfSET2 was knocked out, thus provided more convincing data for the presence of this histone modification in P. falciparum and its deposition by PfSET2 P. falciparum histone H3 has several amino acid polymorphisms close to K36 when compared to higher eukaryotes, making the use of commercially available antibodies generated against H3 peptides from heterologous organisms potentially problematic, and providing a possible explanation for the disparate results. Thus independent confirmation of the presence or absence of H3K36me3 within chromatin from P. falciparum was desirable.Methyltransferases of the SET2 class are predicted to transfer three methyl groups to histone H3 at the lysine in the 36P. falciparum histone H3. To determine the ability of these antibodies to distinguish between H3K36, H3K36me, H3K36me2 and H3K36me3, we utilized slot-blots of synthetic peptides in all four methylated states. Our custom antibodies only recognized H3K36me3  to that of the PfSRIR. Recombinant ySRI and PfSRIR were incubated with both the unphosphorylated and phosphorylated versions of the in vitro data suggested that PfSET2 binds to RNA pol II when the CTD is unphosphorylated, suggesting that it might be recruited when the polymerase complex is engaged in a function other than transcribing an mRNA. To investigate these potentially contradictory models, we performed chromatin immunoprecipitation (ChIP) experiments to examine the deposition of H3K36me3 in live parasites utilizing the custom antibodies generated against H3K36me3. Following standard ChIP procedures, we isolated chromatin from cultured asexual parasites and performed quantitative real-time PCR on the immunoprecipitated fractions. We examined two “housekeeping” genes that are known to be actively transcribed into mRNA as well as a stage specific gene (csp) that is transcriptionally silent during asexual development var genes. These genes serve as a particularly useful model since they are subject to both silencing and activation through epigenetic mechanisms var genes when they are “on” and actively transcribing mRNA or when they are “off” and no mRNA synthesis can be detected. We performed parallel ChIP experiments using different isogenic parasite lines that had been grown for a limited number of generations after cloning and that expressed different subsets of the var gene family. This enabled us to examine individual var genes in both their active and silent states.If binding of PfSET2 to the RNA pol II complex requires phosphorylation of the serines of the CTD of Rpb1 as has been shown in higher eukaryotes, we anticipated that the H3K36me3 mark would be found at actively transcribed genes within the parasite's genome, including most or all housekeeping genes. However, our csp locus  gene to enable selection of transfected parasites. The use of bsd as a selectable marker enables control of transgene expression levels by manipulating drug concentrations in the culture media var gene expression, we transfected two parasite lines  that were previously shown to exhibit relatively stable var gene expression patterns var gene expression was determined by quantitative realtime RT-PCR using a primer set designed to specifically detect each individual var gene in the parasite's genome var gene expression, and the dominantly expressed var gene remained dominant in parasites grown at each drug concentration  was strongly upregulated in response to over-expression of the dominant-negative construct. In A3, this gene became expressed at a level equal to the previously dominant transcript (MAL6P1.252) while in C3 it almost entirely replaced the previously active gene (PFD0625c). This var gene (PFL0030c or PFL3D7_1200600), also referred to as var2csa, is an unusual member of the var gene family that is under the control of a unique promoter type . In addition, its transcript appears to be under translational control through the presence of an upstream open reading frame within the 5′ leader of the transcript var2csa, or alternatively that this gene simply happens by chance to be preferentially activated in these closely related parasite subclones. A comprehensive set of experiments utilizing the generation of numerous additional transfected parasite lines derived from several distinct geographical isolates will be required to definitively address this question.Interestingly, in both transfected lines, a single et al. both support a key role for PfSET2 in var gene regulation and antigenic variation by P. falciparum, however the details of how this histone mark influences var gene expression remain to be illuminated. In their analysis of the PfSET2 knockout lines, Jiang and colleagues observed that H3K36me3 is greatly reduced or absent at the mRNA transcription start site of the active var gene while this mark covers the promoter regions of silent var genes var gene family in a transcriptional repressed state. However this hypothesis does not offer an explanation for the additional observation that H3K36me3 is heavily present throughout the coding regions of all var genes, regardless of their transcriptional state. This differs from other histone marks associated with var genes (for example H3K9me3 or H3K9ac) which seem to more strictly mark the promoter and 5′ upstream regions of either active or silent genes. The fact that all var genes, both active and silent, are marked by H3K36me3 suggests that this histone modification might have an additional or alternative function than simply being a mark for promoter silencing. One possibility is that H3K36me3 contributes to the recognition of individual var genes as members of the var gene family and therefore subject to mutually exclusive expression. It has previously been observed that when var upstream promoters are isolated away from the influence of an adjacent var intron, for example when isolated on episomes, they are no longer subject to mutually exclusive expression and become constitutively active var gene family. The regulatory activity of var introns has been shown to be dependent on their own promoter activity, a property responsible for the production of previously identified noncoding, or “sterile” RNAs var introns is disrupted if the sequence responsible for their promoter activity is deleted var intron is replaced by an alternative promoter sequence var introns exert their influence not through their specific DNA sequence per se, but rather through the recruitment of RNA pol II, which in turn recruits PfSET2 to the var locus, thereby resulting in the deposition of H3K36me3. Thus this model provides a possible mechanistic explanation for several previous observations regarding the role of var introns in coordinating expression of the var gene family.The work presented here and in the recent paper by Jiang var ncRNAs terminate at a different position than var mRNAs transcribed from the same DNA template, and they do not get polyadenylated var ncRNAs are likely transcribed by an RNA pol II complex in which the CTD is not typically phosphorylated. When these observations are taken together an attractive model emerges for how PfSET2 gets recruited only to specific regions of the genome. Only when transcribing a ncRNA does the CTD assume an atypically or unphosphorylated conformation. The recruitment of PfSET2 and the deposition of the H3K36me3 mark would thus be limited to these regions of the genome. Consistent with this hypothesis, recent genome-wide mapping of H3K36me3 also found this mark at telomeres var genes The recruitment of PfSET2 during the transcription of noncoding RNAs rather than mRNAs could also provide an explanation for the unusual observation that PfSET2 binds to the CTD when it is unphosphorylated. In model organisms, when transcribing mRNA the CTD undergoes sequential phosphorylation at the S2 and S5 positions of the heptad repeats. While phosphorylation of S5 is necessary for initiation of transcription, it is phosphorylation at S2 that is important for choosing the proper site of termination as well as recruiting factors required for polyadenylation var gene expression. Each var gene contains three RNA pol II promoters, one upstream of the coding region that is responsible for mRNA production and two located within the intron that result in production of both sense and anti-sense ncRNAs  that initiate from the introns of var genes. These promoters recruit RNA pol II during the late trophozoite and schizont stages and appear to be active at all var genes simultaneously var loci, given that all var loci are marked by H3K36me3 regardless of whether the other two promoters are active or silent. Previous studies have proposed a “pairing” mechanism by which var introns and upstream promoters directly interact in a one-to-one basis The role of RNA pol II in recruiting histone modifiers has become evident through studies of model eukaryotes and likely plays multiple roles in regulating e ncRNAs . The upsvar gene expression switching in our transfected lines. The construct was not designed to reduce the enzymatic activity of the endogenous protein, but rather to interfere with its recruitment to the RNA pol II complex, thereby down-regulating its ability to modify chromatin at var genes. We hypothesize that H3K36me3 is involved in recognition of var genes for inclusion in the mutually exclusive expression pathway. If correct, down-regulation of PfSET2 could “loosen” this control, thereby resulting in accelerated switching. This model also predicts that a full knock-out of PfSET2 would completely disrupt mutually exclusive expression, leading to expression of many or all var genes simultaneously. This is exactly what was observed in a recent study describing parasites in which PfSET2 was knocked out var2csa was preferentially activated in response to over-expression of the PfSET2 dominant-negative construct raises the question of whether this var gene occupies a unique position within the regulatory pathway that coordinates var gene expression. In addition to being regulated at the translational level and having an upstream promoter region unlike other var genes, this gene has also been reported to display an exceptionally high “on” switching rate in certain subclones var gene regulatory pathway. Our data might provide additional evidence of a unique role for var2csa in controlling var gene expression and antigenic variation.Over-expression of the PfSET2 dominant-negative construct led to accelerated in vitro binding assays, PfSET2 binds equally well to both the expanded CTD of P. falciparum as well as the much shorter CTD of P berghei, conceivably arguing that the CTD expansion in primate parasites is unrelated to interactions with PfSET2. Alternatively, the CTD is thought to interact with several proteins simultaneously during transcription, and thus an extended length could provide it with a greater capacity to recruit additional proteins to the transcriptional complex. Hence the expanded CTD could provide the transcriptional complex the added capacity to recruit PfSET2 in addition to other proteins required for its functionality. This property would not be evident in our in vitro binding assays, which utilized individual proteins. A similar interpretation can be made for the yeast system, in which a minimum of eight heptads are required for viability of the organisms, but binding of SET2 is evident with only three in an in vitro assay et al. found that knocking out PfSet2 does not result in a complete loss of H3K36me3 in P. falciparumThe lack of SET2 in the genomes of rodent parasites is perhaps more puzzling, although it is consistent with a possible role for PfSET2 in antigenic variation, a process that has been shown to be regulated somewhat differently in parasites of rodents var gene expression and antigenic variation in P. falciparum. Perhaps more importantly, we also propose a mechanism for its recruitment to var loci through interactions with RNA pol II, potentially during the production of ncRNAs. These observations provide an additional degree of understanding for how proper chromatin structure is assembled at various genomic positions, and may provide a new paradigm for understanding how other aspects of parasite biology are controlled.In conclusion, here we provide data to support the role of the histone methyltransferase PfSET2 in regulating Figure S1var loci and telomeres, which have previously been shown to localize at multiple locations within the nucleus. A possible explanation is that in fixed cells, the H3K36me3 epitope might only be accessible to the antibody when it is in open chromatin, and therefore the IFAs only allow visualization of the single active var gene. Additional work will be required to determine the cause of this pattern.Immunofluorescence assay (IFA) of rings and schizonts using anti-H3K36me3 antibodies. Localization of the antibody is shown in red, the parasite nuclei are stained with Dapi (blue). In both stages the antibody detects a single spot in which this histone modification appears to be localized. Similar results were recently reported by Jiang et al (2013). These results contrast with ChIP data that detect H3K36me3 at all (PDF)Click here for additional data file.Figure S2P. falciparum 3D7. The heptad repeats typical of Rpb1 are underlined, and the serine residues that are sites for phosphorylation are marked with asterisks.The amino acid sequence of the R2 and R3 regions of the C-terminal domain of Rpb1 from (PDF)Click here for additional data file.Figure S3var gene family transcription profile from A3 cultures  and 10 µg/ml blasticidin (B). var gene expression profiles in the presence of the dominant-negative, PfSRI at 2 µg/ml (C) and 10 µg/ml blasticidin (D). The p10 primer pair detects PFL0030c, or var2csa, at higher concentrations of blasticidin.cultures  shown as(PDF)Click here for additional data file.Figure S4var gene family transcription profile for C3 cultures from var gene in the cultures expressing Luciferase does not change at both 2 µg/ml and 10 µg/ml blasticidin (A and B respectively). Like the A3 experiments, var2csa also becomes the dominant expressing var gene in the presence of PfSRIR but only at 10 µg/ml blasticidin (C and D).(PDF)Click here for additional data file.Figure S5var gene family after two (A and B) independent increases from 2 µg/ml to 10 µg/ml blasticidin in A3 cultures overexpressing the dominant-negative, PfSRIR. The results in both experiments are similar to those shown in Transcriptional profile of the (PDF)Click here for additional data file.Figure S6var gene transcription profile at 10 µg/ml blasticidin.An independent transfection of C3 cultures with the constructs expressing Luciferase (A) and the dominant-negative, PfSRIR (B) shows a similar change in (PDF)Click here for additional data file.Figure S7var gene PF3D7_0421100. Black bars show results from chromatin extracted from the C3 line of NF54, in which PF3D7_0421100 is the actively expressed var gene. The gray bars show chromatin extracted from the A3 line of NF54 in which this var gene is transcriptionally silent. The bars display the mean +/− standard deviation of relative amounts of bound DNA (see methods) from four independent experiments.Chromatin immunoprecipitation data from (PDF)Click here for additional data file.Table S1Primers used for PCR amplification of different regions of PfSET2.(DOCX)Click here for additional data file.Table S2Primers used for Q-PCR amplification to determine expression levels of PfSET2 and the PfSRIR.(DOC)Click here for additional data file.